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CEI1/HSSUE CULTURING DEVICE, SYSTEM AND METHOD
FIELD OFTHE INVENTION
Tbeinyetifion is of a device, system and method for cell/tissue culture, and in particular, of such a deviee,. system and method for plant cell culture.
BACKGROUND OF THE INVENTION
Cell and tissue culture techniques have been available for many years and are well known in the art. Theprospect of using such culturing techniques economically is for the extraction of secondary metabolites, such as phaimaceutically active compounds.; various -substances to be used in cosmetics, hormones, enzymes, proteins, antigens, food additives and natural pesticides, from a harvest of the cultured cells= or ^tissues. .While potentially lucrative, this prospect has nevertheless not been effectively. exploited with industrial scale bioreactors which use slow growing pl&t^d Animal cell cultures, because of the high capital costs involved.
Backgrbuntl'art technology for the production of cell and/or tissue culture at industrial scale,' to. tie! used' for the production of such materials, is currently based on . glass bioreactojns-.and stainless steel bioreactors, which are expensive capital items. Furthermore- these- types of industrial bioreactors comprise complicated and expensive mixing-technologies such as impellers powered through expensive and complicated sterile seals;.soirie expetisive fennentors comprise an airlift multipart /construction;^. Suc^essfiil, operation of these bioreactors often requires the implementation
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capacity biofeacf6t?: namely, to -provide a number of smaller glass or stainless steel
bioreactors :>syfioseVtOteiI yplume capacity-matches requirements, while offering a
degree of flejaljiSt^for increasing orreducing.overall capacity, is nevertheless much
more e^en^e'\0^n.th[e provision" of a single larger bioreactor. Furthermore,
T^ESs^%p^^4^^^^^^VBip^^s& and stainless steel bioreactors are also high,
due to ■ low^ji'j&i^^^ sterilize the bioreactors after every
culturing cycle.. Cqnseqtientiiyj the products extracted from cells or tissues grown in such bioreactors.i^.exp0iisiye, and cannot at present compete commercially with comparable, .prodnpte.produced with alternative techniques. In fact, only one Japanese compm^'is-foown to use the aforementioned cell/tissue culture technique a>mmertiaUy^i§mg starless; steel bioreactors. This company produces Shikonin, a compound wM^h.iS-used'^hnbst exclusively in Japan.
Indiistrial-'Scale, aixd even large scale, bioreactor devices are traditionally permanent;or'set^-pehnaient> components, and no disclosure nor suggestion of the concept of a disposable bioreactor device for solving Ihe aforementioned problems regarding large' scale, cell/tissue culture production is known of. On the contrary, -disposable feimefitors and bioreactor devices are well known and exclusively directed to very small scale production volumes, such as in home brewing and for laboratory work. \ These bioreactor devices generally comprise a disposable bag which is. typipally cut open in.prder to harvest the cell/tissue yield, thus destroying any further ;us^]gte$s of the bag. One such known disposable bioreactor is produced by Osmotic, I^agl; (Agritech Israel, issue No. 1, Fall 1997, page 19) for small-scale use such as in ia|):0rat6ry research-. This bioreactor comprises a conical bag having an inlet through i^hich culture medium, air, inoculant and other optional additives may be introducedi/and h,as a volume of only about 1.5 litexs. Aeration is performed by introducing very stnall air bubbles which, in many cases, results in damage to cells, particularly'jri the. case.of plant cell cultures. In particular, these bags are specifically 4e?5igJi€d for asingle ciilture/harvest cycle only, and the bag contents are removed by cujHijig^ off the bottom of the bag- These bags are therefore not directed towards an: ecbnpinCal ?olutipn to Jhe question of providing industrial quantities of • the materials/fcbe extracted ffom.the culture, as discussed above.
The te^n ^disposable" in the present application means that the devices (bags,
biore^dors etp.)^dfe' designed to be discarded after use with only negligible, loss.
Thus devices ^aldefiorn stainless steel or glass are necessarily expensive devices and
do not cdn?titute;a?ii^gjigiblelo,ss for the operator of such devices. On the other hand,
devices, mad^iife;^^ flexible plastics, for example, are relatively
inexp^iave-^^j^Y^ttie^fpip.be,-and are, disposed of after use with negligible economic loss^T%^ttie^disposability of these bioreactor devices does not generally present an. ec^npi^e.:.disadvantage to the u^er, since even the low capital costs of
these items&'Ofisef against ease of use, storage and other practical considerations. In fact, at the small: scale production levels to which these devices are directed, such is the ecoiaom^ Of :the devices that there is no motivation to increase the complexity of the device bj it$Gpea#tion_in orderto allow such a device to be used repeatedly for more than one c^ti^^h^vesting cycle.
Furthdri:&e*ile■'. oonditions outside the disposable bioreactor devices are neithern^eff ii6i^-possible:in many cases, and thus once opened to extract the harvestable )d?WJHf is,neither cost-effective, nor practical, nor often possible to maintaiu- the opting, stenle,* leading to contamination of the bag and whatever contents may.yemaiitinsidfe. Thus, these disposable devices have no further use after one culturing cydie: * - .
Disposable bioreactor devices are thus relatively inexpensive for the quantities andipfo^uction-volumes which are typically required by non-industrial-scale users, afl^. are relatively easy to use by non-professional personnel. In feet it is this aspect ,6Bsii^U
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production vbluffiesof tiie'disposable devices, that is a major attraction of disposable
bioreactor dpvfcesi. Thils,,.the prior art disposable bioreactor devices have very little
in coniinon y^tJi'^iidustnal.sc^le bioreactors—structurally, operationally or in the
economics of ^scde-^and,mfact teach* away from providing a solution to the problems
associated with industfiil sca^le bioreactors, rather than in any way disclosing or
suggesting suph 4 solutiojy '
Anothe^ Mpid^.jn.whidh some advances have been made in terms of
experim^t^.cJr;l^tatory wb^whilb still not beang. useful for industrial-scale
iroc^sse^ i^jpJ^;;^U>c^ for pharmaceutical use have been
traditionally pfoduccd in mammaliaa' or bacterial expression systens. In the past decade a new>xptessiQ!a ^stem has been,developed in plants. This methodology utilizes..AgroJ)alicity of introdudiig %pfe&^foirvma^ ppdiiction of protons and pepiides, ihis methodology is 1 becoming increasifigly. gc^pufejas an/alternative protein expression system (Ma, J. K. C^ Drate, P^^^^ri^ou, P.; (2003) Nature reviews 4,794-805).
SUMMARY OF t5E INVENTION
The background art does not teach or suggest a device, system or method for industrial-scale production of materials through plant or animal cell culture with a disposable device. The background art also does not teach or suggest such a device, system or method for industrial-scale plant cell culture.
The present- invention overcomes these deficiencies of the background art by
providing: a;deyipei?yst^m.aiid method for axenically culturing and harvesting cells
and/or tissues^; including- biojreactors and fermentors. The device is preferably
disposable but nevertheless iqaay be used.continuously for a plurality of consecutive
cnilturing/haryfestttig.cycles prior to disposal of same. This invention also relates to
batteries of such-deyices \vhich may be used for large-scale production of cells and
tissues. • • "., / • ; .
According to preferred embodiments of the present invention, the present invention is:adapted for use with plant cell culture, for example by providing a low shear force whil$ still maintaining the proper flow of gas and/or liquids, and/or while maintaining the proper mixing conditions within the container of the device of the present invention!. For example, optionally and preferably the cells are grown in suspension,- anjd aeration, (flow- of air through the medium, although optionally any other gas of.g^ wmbination could be used) is performed such that low shear force is present. To ^§sfet the. maintenance of low shear force, optionally and preferably the container for wnfaiping tfie cpll culture is made from a flexible material and is also at least rounded^ inv shape, and is more preferably cylindrical and/or spherical in shape, ^ese^harac^pristifc^ also optionally provide an optional but preferred aspect of the Container, whi^h is maintenance of even flow and even shear forces.
It should be^noted that the phrase "plant cell culture" as used herein includes any type of iaative (naturally occurring) plant cells or genetically modified plant cells (e.g., transge^ic. and/or Otherwise genetically engineered plant cell that is grown in culture). wM^^^ production thereof or of an active ingredioit expressed therdn is coinmteroiafly 4^sired for use in the clinic (e.g., therapeutic), food industry (e.g., flavor, aroinaai^ai^c^lture (elg., pesticide)^ cosmetics, etc. The genetic engineering . may optipnrfl^ }f&'stable; pr.;transiCTLt. hi stable transformation, the nucleic acid molecule of flie present.invention is integrated into the plant genome and as such it . representsf*a ^t^ble'-ahd.inherited trait In transient transformation, the nucleic acid
molecule is. expressed by fhe cell transformed but it is not integrated into the genome aiid as such it represents a transient trait
Preferably^ the culture features cells that are not assembled to form a complete plant ^cfcihst.at least one biological structure of a plant is not present Optionally aiitf j^feably^-#ie 'odtura may feature a plurality of different types of plant ceils, Mt^pfefejabljtthe culture features a particular type of plant cell. It should be noted thai c§>llcMiafly?plant cultures featuring a particular type of plant cell may be originally deyiv^'ftom a plurality of different types of such plant cells.
The pfa^t^jl'ttiay; optionally be any type of plant cell but is optionally and preferably apja^iipotcell ^:p-.a cell derived from, obtained fiom, or originally based upon* a^^pl^tfoot^^jipre.preferably a plant root cell selected from the group consisting 6£ -d ley US patent No. 4^88,693 to Strobel et al). Thus, as described heareinabove, todi detailed in tiie Examples section below, ttie plant root cell may be an Agrobqctenim
Optionally and preferably, the plant cells are grown in suspension. The plant cell may ppti.onaEliy' ajfepybe^a plant leaf cell or a plant shoot cell, which are -respectively celfe;d^ved rfroxn, obtained from^ or originally based upon, a plant leaf or a plant shq0t■*".-?• ;>.: \ :'} -t-..-;...
In.a trof^n^'e^oowi^nV $& plant root cell is a canot cell. It should be
noted that ^.tf^k^njicjctisatorot pells of the invention are preferably grown in
. suspension; AS.^pimtipnEe^; £^pye ^d desciibed in the Examples, these cells were
transfoiyned ^^ M^4g^p
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embodiment of the present invention,* any suitable type of bacterial cell may
optionally, b^^usied^ifor s»ch .^ transformation, but preferably, an Agrobacterium tumefaciens. c$l &-We& &r infecting the preferred plant host cells described below-
'AItenuriive!y,-]a^
virus, for exan£ple*a viral yector and/or viral infection.
Ac^r^g^toipr^fCT^ embodiments of the present invention, 4here is
provided a'*jd^^vjlOT^])l^ disposable container for
coltnring pl^fc^gl^e ^f^osable* container is preferably capable of being used
continuously for at least one'&rfher" consecutive culturing/harvesting cycle, such that "disposable" does not restrict the container to only a single culturing/harvesting cycle. More preferably,- the device ftirther comprises a reusable harvester comprising a flow controfler, fpp enabling barvestiag of at least a desired portion of the medium containing cells ancl/or tissues ;when desired, thereby enabling the device to be used continuously ■• io% l&t least, *>iie further consecutive culturing/harvesting cycle. Optionally?*nd -i^^fis^ly^iSe'fiow^ wnfroller maintains sterility of a remainder of the mediuirt containing^cells:and/or.tissue, such that the remainder of the medium remaining from a;girevious harvested cycle, serves as inoculant for a next culture and harvest s^e^.v^.i :: rv^.. /-'
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Acc^rding^.QtiCTS^MinjQits of the present invention, there is provided a device/ sy^em:.a^d:in^iii6d "^hich are suitable for culturing any type of cell and/or tissue. Preferably^ ffiepres^tit invention is used for culturing a host cell. A host cell according td, tlie-:pr^ntmy^tion may optionally be transformed or transfected (penna^ently-and/ot transienjly) with a recombinant nucleic acid molecule encoding : a proteid of /interest^ o£;with an. expression vector comprising the nucleic acid molecules Si?ch:-niicldic acid molecule comprises a first nucleic acid sequence encoding the protein of interest,- optionally operably linked to one or more additional nucleic acid sequfences^encodirig a signal peptide or peptides of interest It should be noted that W:V^e
physicd Udca^fe^Vv!.v ■'•4*5,-/.^'. '■ \ ..
"C^U^H^ost^i^g". or "recombinant host cells'* are terms used
Mercharige^fe^^ihr^If.is.und terms refer not only to the
particular ratij^;pdils.but;4?o to tiie progeny or potential progeny of such a cell.
Beraxise/ebiiam.^Qj^fic^ibS^ may pccur in succeeding generation.due to either
mutation'or^:enyi^6ito^talii^u.enc6s, such progeny may not, in fact, be identical to
the parent qeli; ^tiare stfllinbluded within the scope of the term as used herein.
"Host cell" as'ai$^:terem.;refes to cells whidi can be recombinantly transformed
with naked ^KA.or^expr^ion vectors constructed using recombinant DNA
techniques.. A^ us^d .ierein, .ttie term "transfection" means the introduction of a
nucleic add/fevg^-A^^.P^A or.an expression vector, into a recipient cells by
nucleic acid^m^SM'gfene frahsfeiv 'Transformation", as used herei^ refers to a
process in'vW^^^^eU!*S/gdn6^e as a result of the cellular uptake of
exogenous j)|JAVJC* RNA, aid, for example the transformed cell expresses a Tecombinant form of the desired protein.
BotH it&nocotyledbnous and dicotyledonous plant cell cultures are suitable for use with the methods and devices of the present invention. There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, L, koau. Rev. Plant PhysioL, Plant. MoL BioL (1991) 42:205-225; Shimamoto et^N^ure (19$9) 338:274-276).
. Thft'pt&i^l^ methods of causing stable integration of exogenous DNA into plant genoinip$W$rifibbxle t^o main approaches:
0) : ^A^obact^um-inediated gene transfer Klee et al. (1987) Anna Rev. Plant .Pliysiolrt 2$i0jM6y. Klee. .and R&gers in Cell Culture and Somatic Cell Genetfcs of Pl^ntgj-^oL 6,Molecolar Biology of Plant Nuclear Genes, eds. Schell, J., and V^? L/^;^^deinic PubKsbers, San Diego, Calif. (1989) p. 2-25; Gateaiby, inPladtBiofeiGteplpgy, eds.•' Kung,.S. and Aintzeo, C. J., ButterworthPublishers, Bostoii, Mas^(t989)|p. 93-112.
(ii)*d^ect5I>NA upitake: PaszkowsM et al., in Cell Culture and Somatic Cell
Genetics of PlantsrVtiL; 6y Molecular Biology of Plant Nuclear Genes eds. Schell, J.,
and Vasil,L: ^; Academic Publishers, San Diego, Calif. (1989) p. 52-68; including
methods for dtr^ct uptake of DNA into protoplasts, Toriyama, K. et al; (1988)
Bio/Techtiold|y^;;l 072-1074. DNA uptake induced by brief electric shock of plant
cells; Zha^g d^^a^;Gett Rq>.- (1988) 7:379-384. Fromm et al! Nature (1986)
319:701^793..^^^ iiij^pn:into plant cells or tissues by. particle bombardment,
Klein dt al;'B^f|i&iolQg5r(.1988) 6:559-563; McCaibe et al. Bio/Technology (1988) .
6:923-926-;•fSfaB$8$&y$b& -Plant (1990) 79:206-209; by the use of niicropipette
systmis:\^^k^^^;jAeor. ' AppL GeneL (1987) 75:30-36; Neuhans and .
Spangenb^.l^^o?v.:.:R 79:213-217; glass fibers or silicon-carbide
whi&a;4itfp^^ icdl cultures, einbryos or callus tissue, U.S. Pat No..
5,464,76^ oz by; tfie &xect incubation of DNA with germinating pollen, DeWet et al. . in Expefimehfel ^tamputajfion of OVole Tissue, eds. Cha$>man,.G. P- and Mainteil, S. a and^Daniejs, W.:>I^n^naa, London, (1985) p- 197-209; and Ohta, Proc. Nafl. AcadJ '^Jp^^^^^nS^7t9l\\ . .
The Agrobaeterium system includes flie use of plasmid vectors that contain defined t^A^e^ienits-'ifiafc^ integrate into, fbe plant genomic DNA, Methods of
inoculation of.the .plant tissue vary depending upon the plant species and the Agrobacterixitp delivery system. A widely used approach, is the leaf disc procedure which can be; performed with any tissue explant that provides a good source for initiation of iyhqie plant differentiation- Horsch et al. in Plant Molecular Biology . Manual'AS, KKiwer Academic Publishers, Dordrecht (1988) p, 1-9, A supplementary approach employe the Agrobacterium delivery system in combination with vacuum infiltration.^ ■- jThg Agrobacterium system is especially viable in the creation of transgemc dicpt^iedjandtis plants.
There;^r&^arious; methods of direct DNA transfer into plant cells. In electroporatiqi£ &d. J>rotd^lasts are briefly exposed to a strong electric field. In microinjectid^ thei^NA is mechanically injected directly into the cells using very small micr;opi|)ettes^ ;fo\.rrieroparticle bombardment, the DNA is adsorbed on microprojectiles jsbch as. ina^nesium sulfate crystals or tungsten particles, and the microprojectfles g^e physically accelerated into cells or plant tissues.
Following stable tr^nsfonnation plant propagation can be exercised. The most cotnmon. method: of plant -propagation is by seed, or by micropropagation, which involves tissue cultoing, tissue culture multiplication, differentiation and plant formation.
Although stable transfonnation. is presently preferred, transient transformation
of leaf, cells, jroQt. cells, rperistematic cells or other cells is also envisaged by the
presentinventtpji^7, -, . •'';• '■•. ._-.-/. . .
Tiansi^t|ife^f^imaii6n can be; effected by any of the direct DNA transfer methods d^cijScsl d&ove;or by viral itifection using modified plant viruses.
Vim^matikyeb^eri shown to be useful for tiie transformation of plant hosts ■t-fsK3x^'C^i^^-^^^/s^:By.- Transformation of plants using plant viruses is described" to;j&:s for ^e'rih dcpiiessnig foreign DNA in many hosts, including plants, is descnT>^m^87V^26i\ -
-GpJast^tciSqar^f pl^it RNA viruses for the introduction and expression of non-viral exQgeho^s^mxcleic. acid^ sequences in plants is demonstrated by the above
references asvwefl -afrb^Rawspn, W.-'O. et aL, Virology (1989) 172:285-292; Takamatsu etak".. TSMB& I".\ (1987) 6307-311; French et aL Science (1986) 231:1294-1297; and Takamatsiiet al. FEBS Letters (1990) 269:73-76.
When the virus is a DNA-:virus, suitable modifications can be made to the
vims itself . Alternatively, the virus can first be cloned into a bacterial plasmid for
ease of constructing the desired viral vector with the foreign DNA. The virus can
then be excised from theiplasmid.. If the virus is a DNA virus, a bacterial origin of
replication can be .attached to/the viral DNA, which is then replicated by the bacteria.
Transcription: £nd frataslstiori of this DNA will produce the coat protein which will
emcapsidateihe, vf^iDNA; If the virus is an RNA virus, the virus is generally cloned
as a cDIJA and ^ei^^to a plasmid. The plasmid is then used to make all of the
constructions;/ATteKFtflA? viriis.is then produced by transcribing the viral sequence of
the plasiiud;;tod^imsl^ genes to produce the coat protein(s) which
encapsidate ffyh viral KNA: :[
Gonstracfibn-of plant KNA viruses for the introduction and expression in
. plants of nohrykal Exogenous nucleic acid sequences such as those included in the
construct of the present invention is demonstrated by the above references as well as
in U.S. Pat Nt>. 5,316,931: '
The viral Vectors are encapsidated by the coat proteins encoded by the recombinant plant- viral nucleic acid to produce, a recombinant plant virus. The recombinant pjant; viraliiiucleic add. or recombinant plant virus is used to infect ■ appropriate Ho^t'^aats. : The recombinant plant viral nucleic.acid.is capable of replication in^lip^systemic sp^ in the host, and transcription or expression of foreign gen^V(^feedmid^c acSd) in the host to produce the desired protein.
A;p)yp(^tide cto; ^so be eipressbd in the chromoplast A technique for introducirig^^ogaipus imeleip .^cid sequences to the genome of the chromoplasts is known. Thi&v^^fljre^involvesi.the following procedures. First, plant cells are
. chemically treatettso M tp^ refltice the numbo* of chromoplasts per cell to about one.
. Then, flie exogenous nucleic acid is. introduced via particle bcwnbardment into the cells with thtJ^idm^ofiritrbdiidpLg a£ least one exogenous.nucleic acdd molecule into the chn>|tooplaste?^?Hhie exogenous nucleic add is selected such fhat it is integratable into the diroiippl^s-genome via homologotis recombination, which is readily effected by eriigah^s inherent i$ the ^nromoplast To this aid, the exogenous nucleic
acid inc^e^%Sd3ition io a geaie of interest, at least one nucleic add stretch which
is derived froin the chromoplastfs genome. In addition, the exogenous nucleic acid includes a selectable marker; which serves by sequential selection procedures to ascertain that-all: or substantially all of the copies of the chromoplast genomes following suclv selection will include the exogenous nucleic acid. Further details relating to thfe;technique are found in U.S. Pat Nos. 4,945,050; and 5,693,507 which areiricciporated herein by reference. A polypeptide can thus be produced by the protein expression system of the. chromoplast and become integrated into the chromoplast1 s inner, membrane.
It shotud be appreciated that a drug resistance or other selectable marker is intended in"Mrtoto"facilitate the selection of the transfonnants. Additionally, the presence- of ar^el^abl^marker, such as drug resistance marker may be of use in detecting ;the ^es^iee of contaminating microorganisms in the fculture, and/or in the case of a r^^tmeVmarker ba^ed upon resistance to a chemical or other factor, the selection conditions), mayalso optionally and preferably prevent undesirable and/or contaminatinglriri^porg^sins froin multiplying in the culture medium. Such a pure culture of the%aiisfonned host: cell: would be obtained by culturing the cells under conditions which are.required for the induced phenotype's survival.
As indicated above, the host cells of the invention may be transfected or transformed with a nuclfeic acid molecule. As used herein, the term "nucleic acid" refers toi polynUcleqtides such as Vdeoxyribonucleic acid (DNA), and, where appropriate;. i^Qjmcleic: $cid (RNA)* The terms should also be understood to
include,. ^/^jwyaleiife,^^ arfejogs of ;eifhdr RNA.or DNA made from nucleotide
- ■ ■■•^.^■^;^^-v-:V.:. ■-.■■■■" - . *
analogs, an^>;%a|>pBca^.tp;.tne embodiment being desaibed, single-stranded (such
as .sense or anfi^dc^e)^ arid^abtible-stranded polynucleotides. .
M. yetianotiife. embpdiment, the host cell of the invention may be transfected or transfpkpaed: with an expfibssioii vector comprising the recombinant nucldc acid molecule?.,ccE^pr^ioii Ve&Qfs!?, as used h^ein, encompass vectors such as plasmids, viruses, .bact^io^iigej tnt^ratabIe:DNA .fragmoits, and o&ea: veliicles, whidi . enable the infpg^iion :pf.pNA: fragments into the genome of the host Expression vectors are ^ic^y^elSt3>Ucating:DNA or RNA constructs containing the desired gene Or. its /fra&nients, :'spd[. operdbly linked genetic control elements that are iecogmz^;M-^'"^t|^l&*lidsi cell andL" effect expression of the desired genes. These
control eletn^ts^e; capable of effecting expression within a suitable host
Generally, the genetic cohfrol elements can include a prokaryotic promoter system or
a eukaryotic promoter expr&siqn control system. Such system typically includes a
transcnptionalpromoter, an optional operator to control the onset of transcription,
transcription Enhancers to -eleyate the level of RNA expression, a sequence that
encodes, a.suiiafele nbosome binding site, RNA splice junctions, sequences that
terminate transcription and!translation and so forth. Expression vectors usually
contain an origin of replication that allows the vector to replicate independently of
the host cell. : .
Plagnrids^are! the- most wmmonly used form of vector but other forms of
vectors wMchsa^pi ^egpd^mtfi^ction an4 which are, or become, known in the
• ■'. *. * *'?-\' "-*• ■ .* -. •*" **"**.'.* art are suitaBl^^r^e hergtri^See, e.g., Pouwels et al. Cloning Vectors: a Laboratory
- ■ ;".'V^r;'•-■'-- *'.* - "
Manual (19.85:;and suppj^mettts), Else?viar, N.Y.; and Rodriquez, et al. (eds.) Vectors: a Survey of $lp|©caij^r &$ning. Vectors and their Uses, Buttersworth, Boston, Mass (1988), whichfare ;&TOj^MM^erednby reference.
hi g^^^,>sufchi^fei^ contain, in addition, specific genes which are capable of providing phenoty^ic.sdectidn' in transformed cells. The use of prokaryotic and eukaryotic viral expressipS-vectors:'to.express the genes coding for the polypeptides of the present iayOTtion arfe also contemplated.
In one: preferred embodiment, the host cell of the invention may be a eukaryotic or prpkafyptic cell.
In a prefCTripd ^l>p
.rtejtqx^^i^ei^y &ipdv is used herein for indicating that a first nucleic
acid sequ^^fe^e^l^Tj^^d wijth£ a. second nucleic acid sequence when the first
micleic:ad3'3^^ relationship with the second nucleic
add soqomdci v^^^fe^^Sa^rbmoter is opei^biy linked to a coding sequence if the . promoter aBFecis-^^ih^n^^tion or repression of the coding sequence. Optionally and i^efeiably- ppEr^iblyvlip^ed DNA sequences are contiguous (e.g. physically linked) and, wfe^eiftece^&y to^joinj?vyo protein-coding regions, in the same reading frame. Titus; ^Df^seqi^eQ and .a'regulatory sequencers) are connected in such a way as- to-r.^>enmt>.geae. :iexpressiQn when the appropriate molecules (e.g.,
transcriptioh^^^vator.]^^ are bound to the regulatory seqoence(s)-
In anothesrv ^boi&aeht, this recombinant nucleic acid molecule may
optionally 'further^ "comprise- an operably linked terminator which is preferably
functional in tfae&dst cetl^ stiph as a terminator that is functional in plant cells. The
recombinantn^cVeiQ: acid.molecule of the invention may optionally further comprise
additional ponfrol^.^ and/or selectable markers. It
should be noted ffi&tihese-regulatory-elements are operably linked to the recombinant molecule, /■■*"■'■- .
Regulatory^ elements that may be used in the expression constructs include promoters w^ch^ay bp either, heterologous or homologous to the host cell, preferably a ;pl^i;c«ll*>The promoter may be a plant promoter or a non-plant . promoter "which 'fe'Capable.of driving high levels of transcription of a linked sequence in the host: celi^fsticjbt'asyin plant cells and plants. Non-limiting examples of plant promote^ ;.-feat;'may beVmed effectively in practicing the invention include . cauliflower mosai^;\aras;(Ca^^ 35S, rbcS, the promoter for the chlorophyll a^ binding protem^AdH,-NOS aiid HI^[G2, or modifications or derivatives thereof. The promoter mayji>e^thisr: constitutive, or inducible. For example, and not by way of limitatioii, an\indudble promoter can be a promoter that promotes expression or increased expressipmqf^ttelysosomal-enzyme nucleotide sequence after mechanical gene activation (IvlGA) of the plant, plant tissue or plant celL
The expression vectors used for transfecting or transforming the host cells of the iriyention^an bd^additionally njodified according to methods known to those skilled in the. iaiiUio* ^nhapce or optimize heterologous gene expression in plants and plant celkc SB^^odifiGatibiis include but are not limited to mutating DNA regulato^.el^^fe$p:Mcife^eproiiioto strength or to alter the protein of interest
^e!?pi^^t^aaVejitiQn therefore represents a revolutionary solution to tiie aforemtotipiiMi^bl^^ 7of ihe. background art, by providing a disposable bioreactor device fox thQil^^s^e ptc^uction of cell/tissue cultures. The device of the present
•' '- ■* V" V\ ■ - * *'". tz~ - *■■ L'- • - * *
invention, ^Tm^^^^l^^.-^sposM^ is charactacized in comprising a reusable
harvesttng^.o^^^ of at least a portion of the medium
contah^g;cel&*§^^ enabling the device to be used
continaoosly^rohe^of moxesubsequaat consecutive culturing/harvesting cycles. In
an indu^d^f^fcohm^V'st^ the harvesting outlet during and after
harvesting majjf be assured to a significantly high degree at relatively low cost, by providing, for;-ekample, a sterile hood in which all Ihe necessary connections and disconnections; of;. service^ to and .from the device may be performed. "When eventu^yihej'dpvicedoes become contaminated it may then be disposed of with relatively little economic loss. Such devices may be cheaply manufactured, even for production vp^ittiies. of 50. or 100 liters or more of culture. Further, the ability to perform.-animbeirof cultofmg/haryesting cycles is economically lucrative, lowering even ftttthetthe effective cost per device.
A battery of such devices can be economically arranged, and the number of devices in the^batteiy may be controlled to closely match production to demand Thus, the transition from pilot plant bioreactors to large scale production may also be achieved in^-rel&tfrely simple and economic manner by adding more defaces to the batt^y.Futfliei^^e relatiyely low production volume of each device, coupled with the lackMsSKi'jmx^,^re^tilts in relatively higher yields as compared to typical stainless st^l$ioreacfor& t;
lie de^viqejv^f the present invention therefore has a number of advantages over the bacjfcground: art, ^including but not limited to, being disposable; being economical ta:pro^uce.and.simple to use; being disposable, but also being usable continuously for &rjplurality; of consecutive cycles of culturing and harvesting desired . cells and/or ..tissueis; and optionally being suitable for operation according to a method in whiclt, inoci^aiit i> only.i;fequired :to be provided for the first culturing cycle, while inoculant for spbs^qiient cydes is provided by a portion of the culture broth which remains in t^g3€^te,aft^harvesting same in a preceding cycle*
AcoordSi^io thejaresrait invention, th^e is provided a disposable device for axeaiic^lly c^t^fe^ ^^Th?ovesiing cells and/or tissue in at least one cycle, the device . comprising:, a i#OTpJ5^1e: disposable container having a top end and a bottom end, which CPdtsl^^^&^jit I^?t partially filled with a suitable sterile biological cell
and/or/ti^u^^M^ inoculant and/or sterile air and/or
-■"^V^rt^- :"v.":-->"•.- "'- . ■ '
required/Qffi§g,i|^dlj^ at^iftves^ the container comprising: © a gas outlet for
nass^^:6ii^^^^^lA'!sfn^ gases from the container; (if) an additive inlet for introdnrajg /flie ^66AsxA{s^f.ot flie culture medium and/or tihe additives into the container;.'-sDD^.jGii^^bibtizted'- m, £mther comprising (iii) a reusable harvester comprising^ a-flowrcoijtroilb: for.enabling harvesting of at least a desired portion of *
the medium" containing ^d/oa: tissues when desired; thereby ambling the device to be used'WBtiaiidusly. for at l&ast one further consecutive culturing/harvesting cycle, wheteiix/a^jrernamdqr. of the medium containing cells and/or tissue, remaining from a prev£ous;vharyested .cycle, may serve as inoculant for a next culture and harvest cycle^%Kerein1iie culture medium and/oT the required additives are provided. . OptioiiaUy^tHe disposable container is transparent and/or translucent. Also optionally the, 4wce furtljer comprises an air inlet for introducing sterile gas in the form ofbubble$ iMo.the culture medium through a first inlet opening, wherein the air inlet is cpnnectable to a suitable gas supply. Preferably, the air inlet is for introducing sterile gas. nk>re than once during culturing. More preferably, the air inlet is for contiguously introducing sterile gas. Optionally, a plurality of different gases are introSuc^JM different times and/or concentrations through, the air inlet
Preferably,;, the harvester comprising a contamination preventer for
- --■ ■ if -"*.'■•" substantially"'preventing ^introduction of contaminants into the container via the
'"} -r . * - i . ■ , ' , i
harvester. /;•;;■;,¥.-/'. ■■';'.■ ■■;
Optionally* ihe cbntaiiier is iion-rigid. Preferably, the container is made from a non-rigid plastix? inateriai. ;'More preferably, the material is selected from the group comprising polyethylene, polycarbonate, a copolymer of polyethylene and nylon, PVCahdEVA^ V: ' .:
Optiori'ally, the eoiitainei: is made from a laminate of more than one layer of
thematerials... .
Also- pjitiOnally, the container is formed by fusion bonding two suitable sheets ; ofthe.matOT^f^oiig:preii0termined^^^
^efgr^ly,:tb6.^:Met comprises an air inlet pipe extending from the inlet
opening to a Ideation inside the container at or near the bottom end thereof.-
■ - ■ .• -,.■■ *" •■ " -"• ■ ".
: Al'sq'^ air inlet comprises a least one air inlet pipe
. connectabl^fe>.*.^uital>ie air supply and.in commtmicatfon with a plurality of secondary M^pip^s;. e^K:the secondary inlet pipe extending to a location inside the contain^.*.Via?a ^liitable/Hilet'op^niik therein, for introducing sterile air in the form of bubbles. Mto'^e'^toeimedium. More preferably, the device comprises a substantially bpxflike g^inetej^cemfiguraiion, having an overall length, height and width. "Most j^f&ablyi the£eight-to-length ratio is between about 1 and about 3,
and preferdbiy?aKpiit i.85i Optionally, the, height to width ratio is between about 5
**
and about 30* andnreferahlv about 13.
Preferably^ the device comprises a support aperture substantially spanning the depth of tiie device, the apertpre adapted to enable the device to be supported on a suitable pole stippprt
Optionally thQ device further comprises a support structure for supporting the device; '^referabtyj.&e supiport structure comprises a pair of opposed frames, each of the frames ccxrapiising upper, and lower support members spaced by a plurality of substantially parallel vertical support members suitably joined to the upper and lower support members. More preferably, the plurality of vertical support members consists of.at. Jeast oiie th$ vertical support member at each longitudinal extremity of the uppep.aiid jower support members;
Als6 .the"prefera]bly5 the frames are spaced from each other by a plurality of spacing bars ^eagSbly of integrally joined to the frames.
Also^inor^prefeaBly, the spacing bars are strategically located such that the device may^beinketted afid removed relatively easily from the support structure.
Optiaii^By; the fewer siq^ort member of each the frame comprises at least one lower support ^adapted for receiving and supporting a corresponding portion of the bottom end of the device..
Preferably^" ,ea*ch the lower support is in the form of suitably shaped tab projecting frbiii each of the lower.support members in the direction of the opposed. • frame. ' :- ■ / ; - . ' :
OptibnJSUy, the frames each comprise at least one intetpartitioner piojectihg
from each frame fit ihe direction of the opposed frame, for to pushing against the
sidewall of ttfe deyice at a predetennined position, sudt that opposed pairs of the.
intetpartition^; effectively reduce the width of the device at the predetennined
position. . t*-: *-}.*' > ! ; . . -
.P^fei^lj^^ comprises suitable substantially vertical
- • * * '* **. ^t' •* r' ' '- Zs ' u -. - - ' ' '
membas. spa^|5^\tlie upper and Iowa: siqjport members in a direction towards tiie opposed fiam%mtn stutefele uppa: and lower struts.
C^tiG^^Uy^ft^ 5i?pport structure may comprise a plurality of castors for transporting.^ devices^ ;
Qp&oi^Xky^at least some of the air bubbles comprise a mean diameter of . between about) mm and about 10 mm.
Also optionally, 'at lea^t some of the air bubbles comprise a mean diameter of about 4 mm. .
Optiofcal^Vthe container comprises a suitable filter mounted on the gas outlet
.■' -'"■«•■■ * _■
for substantially ■ preyehtxiig :intxoduction of contaminants into the container via the gas outlet. ■ .'
Preferably* the container further comprises a suitable filter mounted on the additive inletvfo£substantially preventing introduction of contaminants into the* container; via the additive, inlet -
Also- preferably, there is a contamination preventer which comprises a U-sitaped fluid trap; wherein one ami thereof is aseptically mounted to an external outlet of the harvester by suitable aseptic connector.
Pref^bly/the harvester is located at the bottom of the bottom end of the container. . ..
Also-preferatly, the? harvester is located near the bottom of the bottom end of the coritain6ry^efi ;that;4f the-end:of each harvesting cycle the remainder of the medium coiitaiiiirigcens and/or tissue automatically remains at the bottom end of the container up to a level below the level of the harvester.
Optionally; and preferably, the remainder of the medium containing cells and/or tissue.is determined at least partially according to a distance d2 from the bottom of the corifeinei; to'the harvester.
Prefe^ly^i^eifemainder of the medium containing cells and/or tissue comprises, fipm.^bpuit 23% tp about 45% of the original volume of the culture medium. apd;:&e intpcularit More preferably, the remainder of the medium containing cells ^3/or tissue, comprises from about 10% to about 20% of the original volume of tht^Viiltdre medium and the inoculant
(^&ji^^^G^\iom€sid is substantially convex. .
AJ^.t)gtioiMy,1he^btltfom end is sobstaatially frusta-conical.
Prefej^ly^vtbe container comprises an internal tillable volume of between
about 5 -ttteis-^piiri^^ about 50 liters and. 150 liters,
andprefa^it'al^^li^jitdra:. '
> ■ Optionally? the device further comprises suitable attaches: for attaching the device to asiikable support structure. Preferably, the attacher comprises a loop of suitable material, preferably integrally attached to the top end of the container.
Aceor^ng;to preferred embodiments of the present invention, the device is adapted to.jjlafit cfell culture. Preferably, the plant cell culture comprises plant cells obtained froma plaiit jooL More preferably, the plant root is selected from the group consisting. of Jkgrobaetenum rihzogenes transformed root cell, celery cell, ginger cell, horseradish cell and carrot cell.
Optionally^ there is provided a battery of the devices, comprising at least two the disposable-, devices as previously described. Preferably, the devices are supported by ^suitable support structure via the attacher of each the device. Also preferably,.thefgas outlet of each the device is suitably connected to a common gas outlet piping Which optionally comprises a blocker for preventing contaminants from flowing into ^re devices.. Preferably, the blocker comprises a suitable filter.
Optipnalfy* the additive; inlet of each the device is suitably connected to a
* #* * * ^ . ' >. - ^
common additive; inlet,piping*having a free end optionally comprising suitable aseptic -confiecftpr thereat ..•
Optioriall^,.the fnpe end is oohnectable to a suitable supply of medium and/or
additives. , ... " . ■
•*■"*'". * . ■ * . Preferably, the. harvester of each the device is suitably connected to a
common harv^tmgpipin^laying a,free end optionally comprising suitable aseptic connector thereat V/ .
More pre^ibly,1 tibie battery further comprises a contamination preventer for
substantially .J?feventmg".m^ contaminants into the container via the
common h^^tfaig piping. .;Prefen*bly, the contamination preventer comprises a U-shaped fluid.txap, .^hgreipr pile arm hereof is free having an opening and wherein the other end thereof is aseptically mountable to the free ^id of the common harvesting piping via spft^a aseptic-connector. ...
.M(»rBlgre^rably;"ihei.fifee pad of the U-tube is connedable to a suitable receivingtofik-- V ? - . * v (^SoEpl^.fli&air'irii^ of ^ch the device is suitably connected to a common air inlet jpqmgl^^^ a ftjki ^od optionally comprising suitable aseptic connector thereat T^Gfej^l^'^e-^B&ead is eonnectable to a suitable air supply.
According to: other preferred embodimeats of fee present invention, there is provided a^metboii for axenically cultiiring and harvesting cells and/or tissue in a disposable device comprising: providing the device which comprises a sterilisable transparent and/or-transluceDLt disposable container having a top end and a bottom end, which-container may bp at least partially filled with a suitable sterile biological cell and/oi-tis^e^culture.medixun and/or axenic inoculant and/or sterile air and/or other sterile required additives, the container comprising: (i) gas outlet for removing excess air and/ot^waste gases from the container, (ii) additive inlet for introducing the inoctfant^^pr the culhire medium and/or the additives into the container; (iii) reusable han^este^ comprising suitable flow controller for enabling harvesting of at least a portions of 3he m^im containing cells and/or tissue when desired, thereby enabling-the cfeyrqe ib.be used continuously for at least one further consecutive cycle, wherein a remainder of the medium containing cells and/or tissue, remaining from a previously ti&^GStbd- cycle., may serve as inoculant for a next culture and harvest cycle, whereni th£ culture .medium and/or the required additives are provided; providing * axeip^-inopul^jit via. the harvester; providing sterile the culture medium and/or, ste^eytfte'^^tiyelyia the additive inlet; optionally illuminating the container.wi^,;«?rte^nal light; and allowing the cells and/or tissue to grow in the mediumto.adfejfc^'yielcl! .- .
Prefe^lyi.;%e;xnethod_ further comprises: allowing excess air and/or waste gases to leayaJhe; eontaiiigr, continuously via the gas outlet
Mpre.prefe^jly,/^ comprises: checking for contaminants
and/or the qi^ity.jof tKe. cells/tissues wMch are produced in the container, if contaminmts-^'found or .the cells/tissues which are produced are of poor quality, the device ata^ite dojateixts-'are disposed of; if contaminants are not found, harvesting the desired pottibilof the medium containing cells and/or tissue.
Most ^efei^ly,;winle harvesting the desired portion, leaving a remainder of
medium wnta&mg ^cells', antj/or tissue in the container, wherein the remainder of
medium sdfv^^inbcqlatit for a ne$t cultura^harvest cycle. Also most preferably. .
the method_ jS^^oDlnp^es; providing sterile the culture mediiim and/of sterile the
additives fijr. ffi^b^ ^Jpr^arve^/cycle via the additive inlet; and repeating the
growth cy^e :^^^e
are of poof go^^;^Iier^tq)on ttie iieyice andits contents are disposed of
Preferably, tfce device farther comprises an air inlet for introducing sterile air
in the form of bubbles into the culture medium through a first inlet opening
connectable to'a statable, sterile air supply, the method further comprising the step of
providing sfedle.air to .the air Met during the first and each subsequent cycle. More
. preferably; th£.stegle air is supplied continuously throughout at least one culturing
. cycle
Also^ E^repr^erably»vthe sterile air is supplied in pulses during at least one
culturing cycle; ;;. . ;
Accor^ngvlo still other preferred embodiments of the present invention, there
• '.":'/ . ' .
J^ ■ ■*' . * . ■ ■--•"
is prodded a inethod for axenically culturing and harvesting cells and/or tissue in a
battery of disposabledevices, comprising: providing a battery of devices as described .
above, and fq£aV least one the device theareof: providing axenic inoculant to the
device via tfaie common harvesting piping; providing sterile the culture medium
and/or sterile^.th^'additiVes to the device via the common additive inlet piping;
optionally flhimipati^ with external light; and allowing the cells and/or
tissue in the deyipe to grow in the medium to a desired yield.
Prefe^Iy,vthe,m6Siod further comprises: allowing excess air and/or waste
gases to leavejthe device■ .^contiQUOUsly via the common gas outlet piping; checking
for contaminm%and/or^tfie - quality; of the cells/tissues which are produced in the .
device: if HI :$txp>:;3evice contaminants are found or the cells/tissues which are
produced 9re?of;p^ of the device is closed off preventing
contaminafiptt^^^^e-d^c^ of the battery; if in all of the devices of the battery
. rontamin^iM^v^^ijnd'ot W6 cells/tissues which are produced therein are of poor
quaKiy/^jtlx£^ are disposed o^ if contaminants are not .
foimd ahd th^xiai^;t>f ffie prt
- - .-*" *'-** ~,•*?* ****** "*,**. •" •
device, harvesting a idesifed portion of the medium containing cells and/or tissue via
the commoii: harvesting prping and the contamination prev^ita: to a suitable
* ■ * ' *
receiving;tanl&;/r-\"J;1: .-.';; ...
.Prefe^ly>7^rem&md^x>f medium containing cells and/or tissue remains in
. the c»ntamdv"^^^;t&Q xCT^ii^er serves as. inoculant for a next culture/harvest
, cycle; an&f^fastti^.^ providing sterile the.culture medium and/or
sterile the a(^ti^;^r ih^iiekt cultiire^Aiarvest cycle via the addifive inleL
, Also preferably, the gfowth cycle is repeated until the contaminants are found
or the cells/tissues, which are produced are of poor quality for all of the devices of the battery, whereupon' the contamination preventer is disconnected from the common harvester aadth&~device$ arid flieir contents are disposed o£
According to yet other preferred embodiments of the present invention, there is proAided,a.jnd4ic)d'for axenically culturing and harvesting cells and/or tissue in a battayofdisj^sable devices comprising: providing a battery of devices as described above, andfor at least one "the device thereof; providing axenic inoculant to the device via the common, harvesting piping; providing sterile the culture medium and/or sterile >the:: additivesto; the device via the common additive inlet piping; providinig^^^v^ir to. the^ device via the common air inlet piping optionally illumina&g ^ede^ce wth external light; and allowing the cells and/or tissue in the
• device ftx grow in the medium to a desired yield.
Prefdtably, the. method further comprises: allowing excess air and/or waste gases to. leave the.- device contmuouisly via the common gas outlet piping; and checkinjg'fpr contgminante and/or the quality of the cells/tissues which are produced in the deviber'tf in the. device contaminants are found or the cells/tissues which are produced' are^of j)por quality, the harvester of the device is closed off preventing contaroinatioBi;jb£otherQxe devices of the battery; if in all of the devices of the battery contanunantsv&e^foimdpr. the cells/tissues which are produced therein are of poor quality, alt t^e* devices kiad flieir contents are disposed o^ if contaminants are not found and: tl^\cp^^ 6f th6:produced cells/tissues is acceptable, the device is considered H^^^bie.-; -. -; . . MqreBfe^i^ly,:tlie;me&^ comprises: harvesting at least a desired .
portion i>f |he^pa^urn containing cells and/or tissue for each harvestable device via
■ the cx)minon;:ji£^yestifig= -piping and the contaxnination preventer to a suitable
";v .o'K^: ?;V' y" . ■■■ .;.' ': ■
receivingtanfe^'.':o -.V; . .'.:'■'
Most ^ef^raibly/ a: rCToiaindea' of medium containing cells and/or tissue
iemam&%H^\^ remainder serves as inoculant for a next
c^tare/har/^cyGle; and tfie-inethod &rther comprises: providing sterile the culture medium aad/OT. s^Ie; tte -itdBitives for Ae next culture/harvest cycle via tiie* additive inlet
Also lipst.prafeTably, the growth cycle is repeated until the c^tamiBaaats are found or the c£lls/tissues which are produced are of poor quality for all of the devices of the battery* ^hereupon the contamination.preventer is disconnected from the common harvester and the devices and their contents are disposed ot
Accoi%ig/to still other embodiments of the present invention, there is provided a]d$y\ti§:foi..plant cell culture, comprising a disposable container for culturing pjaStVcfeHsn/ Preferably, the disposable container is capable of being used continuously.^M least-"'one further consecutive culturing/harvesting cycle. More preferably, the device further comprises: a reusable harvester comprising a flow controller for^: enabling Jiarvesting of at least a desired portion of the medium containing; .c
According \ to;. yet\ other embodiments of the pres^it invention, there is
provided, a mfetihbd for culturing plant cells, comprising: culturing plant cells in a
disposable qofiferner. .. . ■
Prefea^ly^ the disposable container comprises an air inlet for introducing sterile gas or ipofiibination of gases.
More |^^cably^ftfe sterile gas comprises air. Most preferably, the sterile gas combination fcbjmprises.£ combination of air and additional oxygen.
Prefei^ijf^^a^gen is added separately from the air.
'•M^'-yp^^A^j^j&fygeiiM added a plurality of days after initiating cell
cultured .." -; ;*
Eie&l^l^.-&Q;ist€atile;.gas or combination of gases is added more than once during oatiM^-kV *'•>( ' ,
Also p^fe^bly, fb& air islet is for continuously introducing sterile gas.
Also j^fed&iy, a plurality of diffeceat gases are introduced at different times and/or concesA^&h^ flirougli flie air inlet
Bsdga^^S^^'^^bcd'fiirfiier comprises: aerating the cells through, the inlet More i^era^^e^eaa&^compnses administering at least 1.5 L gas per minute.
)ptionadly and preferably, the method further comprises: providing sufficient medium for growing the cells. More preferably, sufficient medium is at a concentration of at least about 125% of a normal concentration of medium.
Preferably, themethod further comprises: adding media during growth of the cells but before? harvesting. More preferably, the method further comprises adding additional media at least about 3 days-after starting culturing the cells.
Prefe^ably^'ihemethod farther comprises: replacing media completely at least about 3 days afe starting bulturing the cells;
Alto pfefe^ly^theinedium comprises a mixture of sugars.
Also ^fei^btyj^tiie:medium comprises, a largo: amount of sucrose than normal for dell'culture;";, ' ;
Preferably^ the plant eells produce a recombinant protein.
BRIEF DBSCRIPTIONOF THE DRAWINGS
The invention is herein described, by way of example only, with reference to the accompanying drawings, wherein:
FIGS, la-c illustrate the main components of a first embodiment of the device of the present. invention in front elevation and in cross-sectional side view, .respectively fox'-figures'.. lAahd IB, and an exemplary system according to the present invention? for; Figure JC;
FIGS- 2^^tt'2B.;illustrate the main components of a second embodiment of the device of &e^reseqit invention in front elevation and in cross-sectional side view, respectively;-'-.
FIG. 3?ffli^at^ihe,main components of a third embodiment of the device of the present inv^t&n in croSs-sectionial side view;
FIG;. 4-lllusb^tes tli^ s^^iri lines of the first embodiment of the device of the present iavtoQw^ fr6hf/elevation^;
FiGS.;5a;and 5b .iftiistrate the .'main components of a fourth embodiment of the device • of-fhe- preseftt iiiventibn in side view and in cross-sectional top view, respectively;.. *
HGS.:5c |ndSdiUxisftate trSnsy^se cross-sections of the fourth embodiment.
FIGS.6a mi'0b illustrate tfae main components of a fifth embodiment of the device c>f the present invention in side view and in cross-sectional top view, respectively;
FIGS. 6e and 6d illustrate transverse cross-sections of the fifth embodiment
taken along lirie&B^B and €-C in FIG; 6(a);
FIG. 7:flh3&trates the embodiment of FIG. 5 in perspective view; FIG, Sffli^traEtes ^eembodrnxeat of FIG. 6 in perspective view; FIG; 9oUtistrates a! support structure for use with the embodiments of FIGS. 5
to8; -.' ' ::■'*"■ M-i". :('■■' .-;'-.■
FIG, i^-'fltDSi^&fh^main-OTmponeiits of a preferred embodiment of the
battery oftftepf^^tmvi^on comprising a plurality of devices of any one of FIGS. Ito8; /:^:^y'::\l , FIGS, llariaiid ljl> sHow. an expression cassette and vector for use with the present invention;" -*/•" ; -
FIG. l^shpws growthi of transfdrmed (Glucocerebrosidase (GCD)) carrot cell suspension in a. jdeyice according to the present invention as opposed to an Erlenmeyer flask; .
FIG. 13 shows the relative amount of GCD produced by the device according to the present ipvepition as opposed to, an Erlenmeyer flask;
FIG. 14* snows Xh$ start point of 7% and 15% packed cell volume with regard to the gfowfll^ q^^whiph are parallel;
FIG:/i5:^owsthfe amount of GCD protein fiom a quantitative Weston blot forthe^j^o.jM^m^fiditions; :
VB3^^^gs^ ^ymjsyec.^ an ext^ided p^iod of time. (14 days) to find the stationaEypoifit^vV *' •-•""•v.-*hv
FIG; 17,$sbf& thaitlie'm^iintiiii amount of GCD (relative to other proteins) is pro
iax)diK^d.st9rti;M.3ediiiK "- :.
• ■ -.'-*" * *- .t. " -■. ■* ■ ■
£1G» i&. sKows tfsat && replacement of media and/or the addition of fresh media OA ti&e fowffiday maintains high growth levdi of cells beyond day 8.
FIG, 19^^w"ffie*^mbunt of GCD produced under the conditions described in
• «*
Figure 18; -,
FiG..20 show the amoimt of GCD produced under the conditions described in Figure 18;. -""• ' •
FIG. 21shcws theeffect of different sugar regimes on cell growth;
FIGS: :22a and 22b show the effect of different sugar regimes on production of GCD; v- FIGS. J23a, an4 23b show the effect of aeration rate on cell growth in a 10 L device acodrdtog^.flie present invention;
FTC}- 24 £)^6y^s^:oSQci.of adding more oxygen to the device according to
the pres^ mYenfign;:.\ v
FIQ. ^5; sBpwS.jthe electrophoretic separation of Human Factor X coding
sequence (arrow) following amplification by PCR;
FKJf. |6-shQWS 1fie";.li|ated CE-FX-KDEL construct, comprising the Factor X
seqron^'K^ti^ omega and OCS Terminator sequences.
Locatioa of fce;r6Qbghitioii sites for restriction enzyme is marked;
FIG: 2% isV^nap'of thepBluescript SK vector, into which the ligated cassette CE-FX-KDEI^ ;w"as introduced;-
FIG. 2B .is a restriction analysis of the clones transformed with the plasmids pzp-FX-ER and p.GREENnos-kaha-FX-ER, showing the cassettes, and plasmids used in cloningand expression of the Human Factor X in plant cells. Lane 1 is clone
3 transfonned';Wifti;.the.coiistruct pzp-FX-ER, before restriction enzyme digestion.
Lane Z.is cl6n^"3r.aa|ekEcx>Ri and Hihdni digestion. Lane 3 is clpne 4 transformed
with the c^n^j^Jg^F^ERi- before restriction enzyme digestion. Lane 4 is clone
4 after, E^|^^fe|ii|^^gesti6n, Lane 5 is the CaMV35S+pmega-FX-ER
expresaon^setfS^]^^;:^
before rest^ctio^;^ Lane 7 is clone 3 aftrar Asp718 and Xbal
digestion. Ii^/§^clQ^ pGREEN nos-kana-FX-ER, before
. J^stnc&onppi^m^Mg^p^.l^ clone 8 after Asp718 and Xbal digestion. Note Hie banfl .^jhp; pa^35^6mega-EX»"ER expression cassette in all the- -traEsfomed;'dQnes,r S^^ij&lecdlfflrwei^tsbaidards;
^^/^^^y^f^^ipi^'of'&c pGREEN-nos-kana-FX-ER construct, cou^Md^ng jtte F^^r rX'^sequeoceJigated between the CaMV35S+Omega, OCS TetminaJor arj& NFIH .seouencra. -location of the recognition sites for restriction enzyme is ihaafced; .^
FHX 30 jjjbws a Westein.blot analysis oitne ceiiuiar contents of anumber of transformed carrot cell lines; Factor X expression was detected on the Western blot by purified polyclonsl rabbit anti-Human Factor X IgG (Affinity Biologicals, Hamilton, Oiflari0? Canada). Note the strong expression of Factor X in the fine transformed with ;pGREEN-nos-kanaTFX-ER (lanes 1 and 2). MW = molecular weight standards; . .
. FIG*-3-1 shows the accurate cleavage of the recombinant Human Factor X
expressed implant, cells;- .Tie endopeptidase furin, which is responsible for
propeptide re^ioyM.and single chiain to light/heavy chain processing of Human
Factor X, a6q^fely;^g^te4 the recpmbinant Human Factor X (see lanes 4 and 5)
expr^sed.m^l^.^Q^ size of the active Xa. MW — molecular weight
- standards;: ':$*. C ^r^l' -ii'".'.'/. '
- - - . t.' fi . - ., '•*» ™"- '••-'i ■ ' ■ .
FIG.; 32 i^^grap^: sHpwing the catalytic activity of the recombinant Human Factor X ex^K?ssed:.nf plant "cells. Cell extracts from transformed carrot cells ( •, A and ■) mid ^tra^foroied controls (+ > * and •) were reacted with the chromogemc ;f ^fcsfcr^te Pefachrome, and the products monitored by ^ectrophqtoi^ety^at OE>4ti5 i^;
FIG. 3^ stows the electrophoretic separation of Human 1&/3 coding sequence
. (arrow) foll6\^gatoplifi6^tidn by PCR. Lane 1 is the ifbKDEL sequence (targeting
to the ER). Lane 2 is the ifiiSTOP sequence (targeting to the apoplast). MW =
molecidar.TY.ei&^standar^sj • / .
FIQ. 34;s^o^ the:j?lectrpphpretic separation of amplified Human Ifh/8 coding
sequaice cXbjiii&tjtifa E coli^QHSc^.xisiiig tiie CE-K expression cassette.. Positive
clones y/^p.^i^dl^y:$GWf^sly^s of the inserts lising the CaMV35S forward and
the TainiQat^r jpyp&p:Riders (see. Figure 29). Lanes 1-7 are positive clones
showing, ^^^j)StGP;^er^ flie positive control CE-fe-his,
without^.the,ife|^^ *;^ihe.'SI>NAw is a negative control PCR reaction without
FIG; 35;sliow^ theelectroplioretic separation of amplified Human Ifhjf? coding sequence clpn^v^toJBi^UrDH5o; using the CE-K expression cassette. Positive clones were ^e^Ae&^y^PCR analysis of the inserts using the CaMV35S-K>mega forward axri-ffi^O^.Td^iiniaaor reverse primers (see Figure 37), Lanes 1-4 and 6
are positive, clones showing the CE-ifo-KDEL insert Lane 5 is a clone not m expressing Hum&.lfh/?. M = molecular weight standards;
FIG. 36 sKows the-electrophoretic separation of restriction analysis products of the ife-poatiye clones;. The left panel shows the electrophoretic separation of restriction aiiafysifc products of the positive clones bearing CE-ifii-STOP and CE-ifii-KDELinserts;(airpw), using the restriction enzymes EcoRI+Sall (lanes 1-5). Lane 1 is CT-ifii-ICbEL-poative clone 1 (see FIG. 35). digested with EcoRRSalL Lane 2 is CE-ifa-KDEI^p6sitive clone 2 (see FIG. 35) digested with EcoRI+SaU.JLane 3 is CE-ifii-STOP^poaitiye clone.l (see FIG. 34) digested with EcoRI+SalL Lane 4 is CE-i%STO^6§ffiye clone2.(see.FIG. 34) digested with EcoRI+SalL Lane 5 is CE-Fx (lackm^ffiej"ife^Vin3^t) digested with EcoRI+SalL M = molecular weight standards^ -^ej^ght pan^l; shows the electrophoretic separation of restriction analysis ^products; of &e positive clones bearing CE-ifc-STOP and CE-ifh-KDEL inserts (arrow) ■*xi^gther^Wctipn enzymes KpnI+Xbal (lanes 6-9). Lane 6is CE-ifa-KDEL-positiV^cldne l.(see FIG. 35) digested with KpnI+XbaL Lane 7 is CE-ifii-
KDEL-positiy?;ciptie:2 (see FIG. 35) without restriction enzyme digestion. Lane 8 is
. ■ - • -* :J ■ x ■ • ■
CE-ifiirSTOP-positiye."clone.l (see FIG. 34) without restriction enzyme digestion.
Lane 9 is GE-ifii-STOP-positive clone 1 (see FIG. 34) digested with KpnI+XbaL M — molecular; weight standards;
FIG. 37 shows the ligated CE-ifh-KDEL construct, comprising the Human I&|3 coding'sbqueicp.ligated between the CaMV35S-Khnega and OCS Terminator sequences.-.. I^&itiori of the recognition sites for restriction enzyme is marked;
FIG/3^iSvam:^p- of the pzp ill binary vector used for preparation of the pzp-ifii-KDEL ^a^ip^ifii^SXOP. plasmids, with the restriction ^lzyme recognition sites
marked;. -;-.^:■■:•?!;:'- ■ '• ■: '-'*'
EIG^;;39 ds.^a;-Western,blot showing the immune detection of recombinant Human Ifcp eypfessed in cairpt cell clones transformed with agrobacterium LB4404
bearin^ftep^^J^EiVwd.pzih^^^ Calli weare grown firpm the
- •" ' \"-' ■/vV-^/.v.-' . *.:; s. "... - ■. • .
transformed .^l5:itt;agar>vvith antibiotic selection, and then transferred to individual.
plates for fl^eertfonths. Cellular contents of the transformed calli (lanes 1-10) were extracted an&^tfoted 66-P AGE,,blotted, anithe recombinant human infjS. detected with affinity;jpiMfi^l rabbit^ti-iterferon£ antibodies. MW = molecular weight
standards. Sfc* Eoiative control: 3ng recombinant Human interferon 0 stressed in
CHO cells; ..-'
3FIG. 40 sbj>wsihe electrophoretic separation of infectious bursal disease virus viral prof^ui 2^VPII) coding sequence (arrow) following amplification by PCR. Lanes 1, 2 dn& ? are the VPII sequence. Lanes 4 and 5 are negative control PCR reactions, without DNA- and without polymerase, respectively. MW1 is XHE molecular weight standards, and MW2 is Ibp ladder molecular weight standards;
FIG. 41 siiows the electrophoretiG separation of amplified VPII coding sequence cloned into. E 'coli -JSHSa, using the CE-K expression cassette. Positive clones were ^leptfed.by PCR.analysis, of the inserts using the CaMV35S+Omega forward an^tKb^^^.Xe^ttttnator reverse primes (see Figure 37). Lanes 1-6 are the tested clones*^jj^angs 2,3:&nd5 show positive clon^ with the VPII insert. Lane 7 is a positive cofltfol:; PORi^oduct of VPm. Lane 8 is PCR products with DNA of an
empty CE cassette.-: Laa^ 9and\10 are negative control PCR reactions, without
.' *'r' T"* * .* * -■ - • • *. *
DNA and withoi?t'pol)$Qerase, respectively. M == molecular weight standards;
FIQ. '42 i^,a 6iap of tbe GE binary vector used for preparation of the CE-VPII plasmids, wi&ihe-rfetription enzyme recognition sites marked; and
FIG.. 43ai-'arid-'43b are aPAGE analysis (43A) and Western blot (43B)
showing. elepjrophoretic separation and immune detection of recombinant VPII
expressed in carrot cell clones transformed with agrobacterium LB4404 bearing the
pGA492-CE-VPn. pliasmjd. Calli were grown from the transformed cells in agar
with antibioti^selectiQiii. and^thea transferred to individual plates for three, months.
Cellular wiit^>o|the;transfonned calli (lanes 2,3,5,6,7,10,11,13,14, and 15) w^:^
extracted. and^e»2rate$d pji PAGE, blotted, and the recombinant VPII detected with
chicked ahtfrJ^^(^atib^di^ (Fi^ + = Positive controls (VPII protein).
Lanes I arid ^&r^.!VPII tfeU sii^)dision (a mixture of transformation events). Lanes 4
,*"*.*■ •■■"**.•.» - ■ * -. .* *-'-.
and 12 E^.n&aitft^/w^ transformed with fibe "empty" vector alone, and
lanes 8 and l5.^rW;i£^ contents, of untransformed carrot cells.
DETAttfflpESaKTOOHO EMBODIMENTS
Tbb jtt^Ktit inydtt^opis -of a device, systetn and method for axenically
colturing ai^^^e^tiag*;tells ^ind/pr- tissues, including bioreactors and fennoitojs.
Ite device is jpr^fex^bly disposable but nevertheless may be used continuously for a
plurality ofronspeutive b^foiiing/harvesting cycles prior to disposal of same. This invention also relates to batteries of such devices which may be used for large-scale production of cells and tissues.
According; to /preferred .embodiments of the present invention, the present invention is adapted for use with plant cell culture, as described above.
Preferably,: the culture, features cells that are not assembled to form a
complete.plant, such.that.at least one biological structure of a plant is not present
Optionally and preferably, the culture may feature a plurality of different types of
plant cells, but preferably the culture features a particular type of plant cell. It should
be noted that optionally plant.cultures featuring a particular type of plant cell may be
originally derived from .aiphirajity'-of different types of such plant cells. Plant cell
cultures suftable:'fbr use'Xvitlr the devices and methods of the present invention
include; ]nit ar£/ii£t 1^ cell cultures derived from plant root cells, alfalfa
cells, tobacp6;cells, ahd-tpbaoco cell Ijne cells. As used herein, tobacco cell line cells are defined ^;t©baccb cell^.that have been grown in culture as cells previous to being culturing according to the methods of the present invention. Non-limiting examples of established tobacco cell:. lines are Nicotiana tabacum L. cv Bright Yellow-2 (BYr2);and Nieotiana tabacum L. cv. Petit Havana.
The plant cell may optionally be any type of plant cell but is optionally and preferably a plant root cell (i.e. a cell derived from, obtained from, or originally based upon, a plant root), more preferably a plant root cell selected from the group consisting o£ a celery cell, a.ginger cell, a horseradish cell and a cacrot cell. As. described: Herejnaboye, and .detailed in the Examples section below, the plant root
: cell maybe an •A^pbacferiim rhizbgenes transformed root cell. Optionally and preferably;'•$&, pj'aht dells are grown in suspension. The plant cell may optionally also be a plant; feaf :cell;:or n plant shoot cell, which are respectively cells-derived from, obtdn^ftdiaa, br-originally based upon, a plant leaf or a plant shoot
. In a^prefeq^d epil^pdim^it, the plant root cell is a cairot cell. It should be noted that thetran^OTe^c^iat CCJIIS of the invention are preferably grown in suspension. 'Ah. in^ptipned[: dioye qiui described in the Examples, these cells were transformed, \yjtfr.ihe. Agrgbacterium tianefaciens cells. According to a preferred
. CTbodmif^/oTihe^-^ any suitable type of bacterial cell may
k optionally: b^;0^d. for-^jch a transformation, but preferably, an Agrobacterhan tumefaciehs cell is used fof effecting the preferred plant host cells described below.
It will:waj^redated^ by one of ordinary skill in the art, that transformation
of host cells ^QLAgrobacterium tumefadens cells can render host cells growing in
culture in the device? and by methods of the present invention capable of expressing
recombinant gtotems. • In a preferred embodiment, the recombinant proteins are
heterologbus .^rQt^ns. In yet another preferred embodiment, the recombinant
proteins are: viral- eukaryotic -and/or prokaryotic proteins. The transformed cell
cultures of the: present invention can also express chimeric polypeptides. As used
herein, cidm^ic-pblypeptides are defined as recombinant polypeptides or proteins
encoded by^pdl^uclebtides having a.fused coding sequence(s) comprising coding
sequences froifti ^ApasttWQ individual and non-identical genes. The expressed
polyp^tjde!is::^efisa:abiy a eukaryotic, non-plant protein, especially of mammalian
origin, land i^a^tbe. selected from antibody molecules, human serum albumin
(Dugaiczyk'&'&:(X9&} PNAS USA 79: 71-75(incorporated herein by reference),
erythropoietii^.OtK ther^>e\itic molecules or blood substitutes, proteins within
enhanced nutritiqjtial value? anij maybe a modified form of any of these, for instance
including pne pr more insertions, deletions, substitutions and/or additions of one or
more ainino abid|l (The cbding sequence is preferably modified to exchange codons *
ttiat are rare in the host species in accordance with principles for codon usage.).
Examples of such heterqlogpus proteins that can be expressed in host cells grown in
the devices and by tile methods of the present invention include, but are not limited
to lyso^aliozymes sucfi as glucocerebrosidase, cytokines and growth factors such
as hxmiaajiite^em^; serum^piptdns such as clotting factors, e.g. human coagulation
factor ^ ba^^M vh^ prptein^ rach as VPH. . .
* * * ** ■ • th- * * !•$ "J" " * s *"»'".■**
Acly, the deyice^ further cortq>rises a reusable harvester conqaising a flow c6nte^(&;-fofc^iabling Kary&tiiig of at least a desired portion of the medium containing qeSs iaSd/pr tissues, wheii desired, thereby enabling the device to be used
continuously for at least one fnrflieT. consecutive culturing/harvesting cycle. Optionally anil preferably, the flow controller maintains sterility of a remainder of the mediurn containing cells and/or tissue, such that the remainder of the medium remaining frorri a. previous -harvested cycle, serves as inoculant for a next culture and harvest cycle.'. /". . -'■ '->. T,
Accqrdpng:^ to-optional embodiments of the present invention, the device, system and method of- the present invention are adapted for mammalian cell culture, preferably fot:cultiirii*g miammaiian cells in suspension. One of ordinary skill in the art could easily adapt the protocols and device descriptions provided herein for mammalian cqll culture.
In QneKpreferred^. embodiment, the host cell of the invention may be a eukaiyotic,orprbteyotic celll -\ ■ .
In a prefeii;edy embodiment, the host cell of the invention is a prokaryotic cell, pTefe^Iy3.^^ac^eii^l cell:; In another embodiment, the host cell is a eukaxyotic cell, such as a pla^t-cefl-as: previously described, or a mammalian cell.
Disclosed:anddescribed, it is to be understood that this invention is not limited, to the .^particularexamples, process steps, and materials disclosed herein as such process sttps-z&d materials may vary somewhat. It is also to be understood that the terminology; us6d herein is used for the purpose of describing particular embodiments dnly and not intended to be limiting since the scope of the present invention wiU Be; limited bnly by the appended claims and equivalents thereof.
Throughout lias .specification and the claims which follow, unless the context requires iotheijvisq/ the word "coinprise**, and. variations such as "comprises" and "comprismg^^^^fllrbe un4erstoQd to imply the inclusion of a stated integer or step or group of integ^or steps J>ut not ^&e exclusion of any other integer or step or group
of integers-or;st^.V;',./v^ • " .
It mti^;bfe;^tra. ttjatt as used in this.verification and the appended claims,
the singu^f^^-."ja?i^ include plural referents unless the content
clearly dictates oiiife^isd^ -.!-' . .
The fQllo^mg 'examples are repres^itative of techniques employed by the invoators in d^xyyj^ but^ aspects of the preseait invention. It should be appreciated that while these techniques, are exemplary of preferred embodiments for the practice . of the invration) those of slsdll in.the art, in li^it of the presrat disclosure, will
. recognize that numerous mooincauons can be made without departing Scorn the spirit
;*-*.**•,- ". ** * .- . * " ' ' ■ ' :■"■■■■
and intended s^j^of the fev^itiaEL
EXAMPLE 1 TTJUSTRA TTVE DEVICE
The prnciiples. and operation of the present invention may be better . understood t^tb; reference to the drawings and the accompanying description. Figures 1-9 show! schematic illustrations of .various exemplary embodiments of the device accan|$ig-to the present invention.
It ^honld;: be noted that the device according to the present invention, as described in Jgeafer detail below, may optionally feature all components during manufacture aiid/oibefore use. Alternatively, such components may be generated at the moment of use by conveniently combining these components. For example, any one or more .pompoiients may optionally be added to the device to generate the complete device &£ title moment of use.
Refonefiigtiow to the drawings, Figures 1,2, and 3, correspond respectively to
a first, secoj4
(10), comprises.^traiispareht and/or trarislucoat container (20), having a top end (26)
and abottoni^M^ (20) comprises a side wall (22) which is
preferably ^b^atitially 'cylindrical, or at least features a rounded shape, though other shapes such as.^reetangtilar^1"or polyhedral, for example, may also be suitable. Preferably, th^ottdin,-eiid (2§) is suitably shaped to minimize sedimentation thereat For example,::&"ffie'*first embodiment, the bottom end. (28) is substantially frustro-conical pratjg^ comprises upwardly sloping walls. In the second embodiment, the bottom eiid (28) CQinprisfts. oiie upwardly sloping wall (29). In the third embodiment, the bottom ead(:(28) is.jsiibstantially cylindrical or alternatively convex. The aforemeptipnc^ cpjdSguf^tiohs.of^.the bottom end (28), in conjunction with the • location iof-m^Q^b^;'.(7.6)| .(hereinafter described) near the bottom end (28), enables air supplied vSU-pUttet (76) J6. induce a mixing motion to the container contents at the bottom end^ (^^yMm^^Bfecliydy, Tnipimizes sedimentation thereat Nevertheless,
«.»v \\*\' •. . • * y., •»_
• » ■ * * V*tf ^* * * * * *C - *• *
the bottoiiir^id>may:"ber§i^5tantially flat in othea: embodiments of the present
invoition. Tte^^tainepX2v)'cx>!iipases an internal fillable volume (30) wiuch is
ty{nc^y;;1>Blwe^5;'^d.^0^fit^ though device (10) may alternatively have an
.intemaTydfim^ :^^€f tfwjit'SO liters or less than 5 liters, Int^nal volume (30) may
be fin^^^v^l^l^l^staBle biplogLcal cell and/or tissue culture medium (65)
and/OT'-^aiteiji^^ air and/or required oth^: sterile additives
such as antibi6tjqs or fongiddes for example, as hesrednaftex described. la the aforementioned embodiments, the container (20) is substantially non-rigid, being made preferably fifem a rpinrigid plastics material chosen from the group comprising polyethylerie^^^eafbonEtte,.^ copolymer of polyethylene and nylon, PVC and EVA, for example. QpfiqiMy,' the/container (20) may be made from a laminate of more
: *'*' £v'\ '■: 'r'~~ •*!•.■' - •
than one.la.yer of materials.' . .
As sh^wn^'for the-.third embodiment in FIG. 3, the container (20) may optionally coniprise two concentric outer walls (24) to enhance mechanical strength and to minimize risk of contamination of the contents via the container walls.
In the/firs^ second aad third embodiments, device (10) is for aerobic use. Thus the.cQntgfiner. (20) further comprises at least one air inlet for introducing sterile air in the 'fbn$s>£ biibbles (70) into pulture medium (65) through at least one air inlet opening.(72).V^the.aforementioned embodiments, air inlet comprises at least one pipe (74) iW^e^tabJe to a. suitable air siipply (not shown) and extending from inlet opening (72) itoa^beatiDn inside container (20) at a distance dl from the bottom of bottom end'[($$)£Vihbxd^i dl may be typically around 1 cm, though it could be greater or st&|dler.than $'-*caL TTiepipe (74) may be made from silicon or other suitable plastic material and is" preferably flexible. The pipe (74) thus comprises an air outlet (76) ^f{$uitablp;di^oieter to produce air bubbles (70) of a required mean diameter*. These babbles not:bnly aerate the medium (65), but also serve to mix the contents of th^^xtdjaine^ thereby minimizing sedimentation at the bottom end (28) as well, aslhecfeMBejTdre described. The size of the bubbles delivered by the air inlet will vary accptdiiig to fb&.oisfr of th& device, ranging from well under 1 mm to over 10 mm jn diameter.;In spine cases, particularly relating to plant cells, small bubbles may actqaHyt^age^;tbe^ cell walls,, and a mean bubble diameter of not less than 4 mm substantial^^ Overcomes ibis potential problem. In other cases, much smaller bubbles areJb^eQpiaV ^nd a^sparger may be used at the air outlet (76) to reduce the size of the ti^Tbl^*^ 3^totiiieF".cases ?ir bubbles of diameter 10 mm or ev&i greater may ^oj^b^f^Sc^i^; q^^-(76\'m^ be restrained in posftion at bottom end ' (28) th^t^$j^^KCT|9bj ^ownXdr other means Icnown in the art
In oti^ ^bo
: :, In fourth and fifth embodiments of the present invention, and with reference to FIGS. 5 anc! 6 r^ectively,.1he device. (10) also comprises a transparent and/or translucent container (20), having a top end (26) and a bottom end (28). The container. (2d): ^mprises a side wall (22) which is preferably substantially rectangular irr cross-section, having a.large length to width aspect ratio, as shown for
the fourth embodiment qfthe present invention (FIG. 5). Thus, the container (20) of
*■*. .'-',>-' the fourth embodiment is. substantially box-like, having typical height-length-width
dimensions of 13(tcin by 70. cm by 10 cm, respectively. The height to length ratio of
the device is.typipally between, for example, about 1 and about 3, and preferably
. about 1.85. The ieight-tQrwidth ratio of the device is typically between 5 and about
30, and prefembly'aBout 13. - -
Altetnativelya and as shown in FIG. 6 with respect to the fifth embodiment of the present invention, the-.sidewall (22) may comprise a substantially accordion-shaped horizontal cross-section, having a series of parallel crests (221) intercalated with troughs (^22) "along ihe.Jength pf the container (20), thereby defining a series of adjacent, chamber^; (223) intfluid communication with each other. Optionally, the sidewaflX22)ypf}liie fiffliVeipbpdirrieiit may further comprise a plurality of vertical webs (224), each.internally'joining pairs of opposed troughs, thereby separating at least a vertical; portion, of-each chamber (223) from adjacent chambers (223). The webs (224) not only provide increased structural integrity to the container (20), but also effectively separate the.j.container (20) into smaller volumes, providing the advantage of dnhaheed ciroiilatibrL, In other words, the effectiveness of air bubbles in . promoting qeH^cidationis.far higher in smaller enclosed volumes than in a larger equivalent Vofame: In fatct,; a^proportionately higher volume flow rate for the air bubbles is rpc^fired $>r promoting.air circulation in a large volume than in a number of smaller" volumes; having the same combined volume of medium. In the fourth and.
fifth embo&riLents,. bottom end (28) is substantially semi-cylindrical or.may be
-*- *. ■-* * ••
altonatiy^^nVeXi ^4>sjantfajly flat, or any otho: suitable shape. Mthe fourth and .
fifth embodinfeqfe; Jhe; cpntaioa (20) comprises an internal fiUable volume (30)
'. ". .-.* ;-v.".r*; *. — '-■-- -z .* - * *
whidi: ist^^^b^tw^ga £0 and 100 liteas, though device (10) may alternatively
have ^.i^et^aL^oiuEQe grfet^r thmi 100 liters, and also g^eate^ than 200 liters; Inteii^^iiqiQMd)^ma^>e;filled with a suitable stacfle biological cell arid/or tissue culture medmar:(65)::^d/ox ^enic'inoculant (60) and/or sterile air and/or required
other sterile, additives sudEi as antibiotics or fimgiddes for example, as hereinafter
described. la tbe ^foremeationed fourth and fiflh embodiments, the container (20) is
substantially:p£n£-ngid9 b^ng made preferably from a non-rigid plastics material
chosen /from Ike-;^i^ ^comptidng polyethylene, polycarbonate, a copolymer of
polyethyleng.&i^ f°r example, and, optionally, the container
(20) may bei^efromalaininate 6f more than one layer of materials.
. As fox;fefifs^ seqbDtfJvand third embodiments, device (10) of the fourth and fifth embodiments, is also for aerobic xise. In the fourth and fifth embodiments, the container (20)itoNher comprises at least one air inlet for introducing sterile air in the form of bubbles £70) intp. culture medium (65) through a plurality of air inlet openings (72)> -Jn^ttefourfli B®d fifth embodiments, air inlet comprises at least one air inlet pipe (74) connectable to a suitable air supply (not shown) and in communication with a plurality of secondary inlet pipes (741), each secondary inlet pipe (741) extending frjSm; iidet opening (72) to a location inside container (20) at a distance dl from the bottdmyof. bottom end (28), wherein dl may be typically around 1 cm, though itcottfd'be- greater:pr smalls than 1 cm. The plurality of inlet openings (72), are horizontally .^cedn^ from another by a suitable spacing d5, typically between about 5 cm andlayout 25 cm, and preferably about 10 cm. The at least one air inlet pipe (74) and.^pebjidary inlet pipes (741) may be made from silicon or other suitable plastic material; "ah
'■ l' ■*/*■;*-
reqtriredTne^DfjdjgmeJer; TBh^fe bubbles not only aerate the medium (65), but also s&ve to mix; &e^eotitepls of ttiQ container, th^eby minimizing sedimentation at the bottom end (28).as.well;as hereinbefore d^cribed. The size of the biibbles delivered by the air im^w^lL vary according to the use of the device, ranging from well under 1 mm to ov^lOimn^ in diameter; In some cases, particularly relating to plant cells, small butbles^^^actuallydamagethe cell walls, and a mean bubble diameter of not less than14 mm.^staatiaUy oyerroines this potential problem. In ofher cases, much smaller b^tl^^j^efei^ ^3.a sparger may be-used at least one of .air outlets (76) to rsdue^fce^size oif^&ebubbles: In yet other cases air bubbles of diameter 10 mm or pvdt^a^f-v^ay -TC^optimaL Optionally, eadh outlet (76) may be restrained in positionjrff6^ftpai^Eid (28) b^ iismg a te&^r (not shown) or by another mechanism known % j^e^rtj^^
Thefeijrflt^ adapted for processing relatively large volumes of inoculant
In all ffie aforementioned embodiments, the air inlet optionally comprises a suitable pressing.^aiige^Jor monitoxing the air pressure in the container (20). Preferably, presstifirgati^e-is operatively connected to, or alternatively comprises, a suitable shut-6fftv^ve^bich may be preset to shut off the simply of air to the container (20)if fh^pres^qre-therein exceeds a predetermined value. Such a system is useful in
The cont^^r;J(20)yfurfli€T. comprises at least one gas outlet for removing
. excess air and/or waste gatsesfrom-container (20). These gases collect at the top end (26) of.the . container (20). The gas outlet may comprise a pipe (90) having an inlet (96) at or nfcarthe top end (26), at a distance d4 from the bottom of the bottom end (28), wherein^, is-typically 90 cm for the first, second and third embodiments, for example. The:pip£ (J>0) inay b6 made from silicon or other suitable plastic material
• and is preferably ide^le.JPipie (90) ijscorinectable to a suitable exhaust (not shown) by a known :mecfiani.sm.,>Th6 exhaust means further comprises a blocker, such as a suitable onerwayfyalye or filter (typically a 0.2 micro-meter filter), for example, for substantially preventing^introduction of contaminants into container via the gas outlet At.lea^^6rti6n>af th^top'end (26) may be suitably configured to facilitate the cx>Uection^fw^te:ga§es.prior, to being removed via inlet (96), Thus, in the first and se(^nd;efe^dinien1^ J^6 jxpp^ portion of the top end (26) progressively narrows to ar.ipiMm^r^o^s.rsectioiial area near the location of the inlet (96). Altematiyely^t l^ast th? lip^er portion of the top end (26) may be correspondingly substantially fitjsiro-conii^l or^convex. In the fourth and fiflh emboc&nentsj the top
. end (26) may^e;
Tte; cpitakiCT- (20) fether comprises an additive inlet for introducing
- inoculant ahd^.j^x^ additives into container.- In the
aforementioned ^bcpd^^ifer; the additive inlet comprises a suitable pipe (80) having.m:6tt!^^^*rt^^^ly ator hesar the top raid (26), at a distance d3 from the bottom o^fe^bHattbin:&tt&'.(28)y wherein d3 for the first embodiment is typically
approximately 68 cm, for example. The pipe (80) may be made fram silicon or other suitable plastic material and is preferably flexible. Pipe (80) is connectable by a known connector to a suitable sterilized supply of inoculant and/or culture medium and/or additiye^:. The additive inlet further comprises a blocker for substantially preventing inftqopction of. ^ntemxnants into container via additive inlet, and comprise, irL^h^se enxbodimeats, a suitable one-way valve or filter (84). Typically, the level ofrcoiiteiits.(>f ife c<. remains below the level of outlet container further comprises reusable harvester for harvesting at least>
a desired first ^portion of the-medium containing cells and/or tissue when desired,
. i'» - ~., . - .* *-- ■• • * . -. • ■*■ ,■*■'*' .* * -r *."*•"-"'
thereby ^abKng^e device.to be u$ed continuously for at least one subsequent
(Staring cycle; A* T^main^ .
serves as iaoGiaant foya ii^ culture and h^vest cycle, wherein culture meditnn and/or rtpquir&3 additives provided; The harvester may also be used to introduce the original voiles of inoculant into tfie container, as well as for enabling the harvested material to floy thipreihrough ar^d out of flie container.
In the;' aforementioned embodiments, the harvester comprises a pipe (50)
having an ihl^.(52) ia c^xbmiBncation with internal voltnne (30), and an outlet (56)
outside contaijaef. (20); The pipe (50) may be made from silicon or other suitable
plastic .material .abji is;^prefearably flexible. The pipe (50) is of a relatively large .
diameter, typically abdiii ^2- cm, since the harvested cell and/or tissue flow
therethrougji! iha^isontafii^ clumps of cell particles that may clog narrower pipes.
Prefeabiy,:5S^^^sloC^^d neaf the bottom end (28) of the container (20), so that
only the contain^^jbonteots. stove inlet (52) are harvested- Thus, at the end of each
harvesting; eyclq^^ containing cells and/or tissues
automatic^at^^^aia^s: W.the bottom did (28) of the container (20), up to a level • below th£ Ibvel (5i):of tli^ &let (52), which is at a distance d2 fiom the bottom of bottom jend (28XlTs$icaO[ly .but not necessarily, d2 is about 25 cm for the first
"- V * V*-*,.". * -• "• .* .'-" -
embodiiiieiit.V:. -.'.-.. v •
;OptibM^g^.preforaibly, d2 is selectedi according to the volume of container
(20), such iB#t-J^;poiti6nbf medium and cells and/or tissue that remains is the
desii^&a$fc^;^&eyotbifie-^ Also optionally and preferably, an
additional s^pj^^A jffiay be provided (not shown) for removing a sample of the cdtm^m^^^^^iiQg'i^l^ and/pr tissue. The sampling port prefeably features
an Met.and pipe;as.for th£* harvester, and is more preferably located above the harvester! Oth§r port(s) may also optionally be provided.
Alternatively, inlet (52) may be located at the lowest point in the container = (20), wherein the operator could optionally manually ensure that a suitable portion of medium c©ntalij^g: cells- and/or tissue could remain in the container (20) after harvesting, a 4$sir£&/portion .of medium and cells and/or tissue. Alternatively, all of the medinm v^ilii4- optiopaHy t>e removed Harvester further comprises flow controUer suc|t;^:a'suitatble.valve (54) and/or an aseptic connector (55) for closing off and for pfcjmittuig the flow of material into or out of container (20) via harvester. Typically, as^tie;:connecfor (55) is made from stainless steel, and many examples thereof are^otwn in -. ths art Preferably, the harvester further comprises contamination-bteVenter' fdrVsubstantially preventing introduction of contaminants into contaiii^^-Karvesfer after haryesting.
In ^^first^vsecond," third, fourth and fifth embodiments, contamination
preventer cqifipris^s a. fluid trap (3.00). The fluid trap (300) is preferably in the form
of a substantially U-shaped hollow tube, one arm of which is mounted to the outlet
(56) of the harvester, and the other arm having an external opening (58), as shown
for the first Qmbo4niientj;for-example, in FIG. l(b). Harvested cells/tissue may flow
out of the device-(Jp) *ia: harvester,-fluid trap (300) and opening (58), to be collected
thereafter iri a^vUable.ifecdving tank as hereinafter described. After harvesting is
terminated/'aii:.cpviA possibly .be introduced into the harvester via opening (56),
accompjpu^Y'b^^^ of harvested material, thereby potentially
introduciiig cdct^^^teinfcthe device. The U-tube (300) substantially overcomes this potehtid^pro^l^^y^t^ping some harvested material, i.e.5 cells/tissues, downstrfe^ p^^fe!ppening (5f) thqreby prev^iting air, and possible contaminants, from eritering'^^^afVestferr'Once^ tiie,.harvester is closed off via valve (54), the U-tube (300) is removed and ^pically sterilized for the next use or discarded The U-tube (300) ma£'$$ inado from.staixiless steel or othra: suitable rigid plastic materials. In the aforemejitioned embddimesits, remaining second portion of medium containing . cells and/or tiskie j^caliy pomprises between 10% and 20% of the original volume of c^ture;ra«&im^d iippdant, thou^i second portion may be greater than 20%, up . to 45% or rixSj&i&less tEanlO%; down to 2.5% or less, of the original volume, if required; .
bevicfe(lb) optionally further comprises an attacher for attaching same to an overhanging support structure. In the aforementioned embodiments, support structure may comprise a bar (100) (FIGS. 1, 2, 5) or rings (not shown). In the third embodiment the. attacher may comprise a hook (25) preferably integrally attached to the top end (26);qf ihe container (20).. Alternatively, and as shown for the first and second embodm&its in FIG5- 1 and 2 respectively, the attacher may comprise a pi^ea^ly'tle^i^iind substantially cylindrical loop (27) of suitable material, typically the saifie! iaatenal 33 is used for the container (20), either integral with or suitably attatfied;(yfa foaon welding, for example) to the top end (26) of the device. Alternatively;: aik£;#s^shawn for the fourth embodiment in FIG. 5, attacher may comprise a p^ef€^bly: fle^jble; and substantially cylindrical aperture (227) made in the sidewall (22Xpf contaiaer (20), extending through the depth thereof The fifth embodiment mayoptionally be supported by a series of books (not shown) integrally or suitably arched preferably; to the top end (26) of the device (10).
C^)ti6nalljr5 the containers may be supported in a suitable support jacket. For example,,in the fourth^ anbodimetit, the device (10) may be siqpported in a support jacket consisting of a suitable outer support structure comprising an internal volume sized and shaped, to complement the datum external geometry of at least the sidewall (22) and bottpto^d.(28):of the device when nominally inflated. The outer support structure may be:rabstantidly ^continuous, with openings to allow access to the inlets and otrtlets tatH^ilevice .(^10); and further has a suitable door or opening either at the side^ top prbptlpiii to atiow .a device (10) to be inserted into the support jacket or r«noved.tli^iro^^^0{{Jatui9 geometry of the device may be defined as tiie shape' of the device ^O^henjt is^iiflatedrto its design edacity. At this point its shape is nominally is ^iesfga shq>Cj and. therefore its internal volume is nominally its design volumetriq cetpadty;. However, when such a device comprising flexible walls is actually ^^.m&^ali^dmediuin; the geometry of the device tends to deviate from the dahm^^fed^^tendSttig td bulge preferentially at the bottom the device whore the pressure i^greatest^^nd inCTeasing stresses in ftte wall material considerably. A support jacketjas ijescribedfor example and having the required structural attributes also helps in matrttaining the geome&y of the device, and reduces the wall stresses, .nrimmfTaitgjg^^j^ing^nrip of the sidewall (22), for example and th^eby ensuring a long^.wp^^l^e^r/e^ device. '- .
Alternatively; the containers may be supported in a suitable support structured
For example, in the fourth and fifth embodiments of the present invention, the device (10) maybe supported in a support structure (400) comprising a pair of opposed frames (405), (406), as illustrated, for example, in FIG. 9. Each frame (405), (406) is lypic&ily rectangular comprising substantially parallel and horizontal upper and lower load-c#rryinjg members (410) and (420) respectively, spaced by a plurality of substantially parallel vertical support members (430), at least at each longitudinal extremity of. the: load-carrying members (410), (420), and integrally or otherwise suitably joined, fp-the lipped and lower load-carrying members, (410) and (420) respectively. I'lhe? lower■supfport member (420) of each frame (405) and (406) comprises su^feLbtl^sliaped lo;wer supports adapted for receiving and supporting a corresponding.;poijion of the; bottom end (28) of the containers (20). Typically, the lower supports inay'take the form of a suitably shaped platform projecting from each of the loWer;;; support-members (420) in the direction of the opposed frame. Alternatively, the lower supports may take the form of a plurality of suitably shaped tabs (460) projecting from each of the lower support members (420) in the direction of the opposed fraxne. The frames (405), (406) are spaced from each other by strategically, located spacing bars (450), such that the container (20) may be removed relatively easily from the .support structure (400) and a new container (20) maneuvered' into place, i:e., without the need to dismantle the support frame (400). The spadiig:bars:(450) inay beintegrally connected to the frames (405), (406), as by . welding for pxaiptplQ; Preferably, though, the spacing bars (450) are releasably
connected Ito;^^;;^^^
.' ■ ■ '\* " *■-;---' .'•"•■' sq>ar^ed;oiie;frqin.ffie:ofh€^j/and also permitting the use of different sized spacing
bars to conned* the-frajnes (405), (4Q6), thereby enabling the support structure (400) to be used:;wifn;-a5tang6^ of.containers (20) having different widths. Optionally, and
preferably, th$fia^eS;(4O5), (4.06) each comprise at least one intexpartitioner (470).. .
"Vi^V'. s '••'■:*.": -"* "-. -Iht«rpmtiti6n^;(470) ihay;take the form of a vertical web projecting from each frame
(405), (406) jji the. direction of the opposed frame, and saves to push against the sidewalT(22) .at a.predetermined position, such that opposed pairs of interpartitioner (470) effectiv^yj^ace flie width of the container (20) at the predetermined position,, thereby ereafiog; }brt^€^ adjacait opposed pairs of interpartitionea: (470), for example; a parttiibiiiiia or Seritf partitioning of the internal space (30) of the container
(20). Thiis, the Ht^partifioiier (470) may typically deform the sidewall (22) of a
■';.^«:;v-/ ■ ■"■.■^»f.7"-v:-V--V'":- . ' ■ "'**■.■ "*'--,
■ - ■ ■' . .. » . - •-1 •
container (20)"accoraing to the fourth embodiment (see FIG. 5) to a shape resembling that of the sidewall (22) of the fifth embodiment (see FIG. 6). Of course, when used with a container. (20) according to the fifth embodiment of the present invention, the interpartitioneD(4?0);are located on the frames (405), (406) such as to engage with tiie troughs (222) of the sadewall (22), and thus particularly useful in maintaining the shape of fte:^fctainers (26v:Thus, adjacent partitioned (470) on each frame are spaced advarifti&feuisjly spaped a distance (d5) one from another. Preferably, int^partifioneav^^S ccfipprise suitable substantially vertical members (472) spaced from the upper aid lowersupport members, (410), (420), in a direction towards the opposed franifcrs$ksuit^le.iipper and lower struts (476), (474) respectively. The support sirttcbfo^^XSin^jQot only provides structural support for the containers (20), particula^ly^pfthe fourth and fiflh embodiments, it also provides many open spaces betwegp e^dh of -itfe-load carrying members for enabling each of the air inlet, . &e gas ouflet^this.harvester: and tiiq additive inlet to pass therethrough. Optionally, support strucitoe;: (400), may comprise rollers or castors (480) for easing transportatioifcof the ccmtainers (20) within a factory environment, for example.
The container ;(20) may optionally be formed by fusion bonding two suitable sheets of suitably material, as hereinbefore exampled, along predetermined seams. Referring to-the "first and second embodiments for example, two sheets (200) of . material may bis Q^JL in;aii^approximately elongated rectangular shape and superposed one ovq:.thdJ^ti3^Pi(3::4^ 3Tie sheets are then fusion bonded together in a manner . well known iii^^^;fo fomt; seams "sflongtiie poipheries (205) and (206) of the two longer m^^M':plq^'^"pcxi^acy of one ofiho shorts ends (210), and again parallel arid'miir^dly displaced- thereto to form a seam (220) at the upper end of the container (2.Q5*; T^eVfojSon .^;eld seams (207) and (208) along the long sides and situated betwe^fbjese parallel short end seams (210) and (220) may be cut off or othCTwise vwioy^ .^ecfively leaving a loop of material (27). The bottom end (28) of die contam^ *(2(Q Is^bnned byrfuaon bonding the remaining short end of the sheets along ^v slbjnng seam lines, (230) and (240), mutually converging from the . seams (205) m& (20J5) of ftie long sides. Optionally, the two sloping seam lines (230) and (240)itoxl^ joSoed above the^pex by anoth^" fiision welded seam line (260) ^rproximat^y^Q^aipgoii^i; toe the long, side seams (205) and (206). Prior to fiision
welding the two -sheds-.:together, rigid plastic bosses (270), (290), (280) and (250) may be fusion- welded at locations corresponding to the air inlet, gas outlet, additive inlet and harvester, respectively. These bosses provide suitable mechanical attachment points for each of the corresponding input(s) and output(s). The third, fourth and.fifth embodiments of the present invention may be manufactured in a similar manner to the first and second embodiments, substantially as described above, mutatis mutandis.
In. alLenabpdiments, the device (10) is made from a material or materials that
are biologically compatible and which enable the container to be sterilized prior to
first use. .. ''•*>*:'-. - V 'v .
■;;^..;J V;'-; EXAMPLE!
[■ fi:;- ILLUSTRATIVE SYSTEM
The'.prbseiit.-invention also* relates to a battery of disposable devices for axenically cultunrig.and harvesting dells and/or tissue in cycles, wherein each of a plurality of thipse-devices, is structurally and operationally similar to device (10), hereinbefore defined and .described with reference to the first through the fifth embodiments thereof.. ;
Referring to JFIG. 10, a battery (500) comprises a plurality of devices (10), as hereinbefore described with respect, to any one of the first through the fifth emboditnents^fefchrate heft on. a frame or frames (not shown) with an attacher or support structure $00), for example* Typically, the battery (500) may be divided into a numbpr ofgrouj^eaojtf group comprising a number of devices (10).
In the j*^^ battery (500), the air inlets of the devices
(10) in .each';sp>JD$f;jffe iirterebtoected/ITius the air inlet pipes. (74) of each device (10) of the. grdup1ate conheclbd to common piping (174) having a free end (170), which is^ prpAgj^; with vsx. ^afeeptic; connector (175). Sterilized air is provided by a suitable air corn^^soi: (15&) fiaviiig a suitable st«rilizeF orblocker (110) sudi as one or more ^teas. IheVcompcesspr (130) comprises a delivery pipe (101) having an aseptic eomecjbr.(136) aWtyfr^e end which is typically connectable to the aseptic connector (175) located at tKe.free end of common p^>ing (174). This connection is made at.liebeginafing of ea6h. run of growth/harvesting cycles in a mobile sterile hood (380) .to .ensure/thai sterile conditions are .maintained durmg the connection.
The sterile hood provides a simple relatively low-cost system for connecting the various services, such as air, media, inoculant and harvested cells, to and from the group of devices (10); wider substantially sterile conditions. Similarly, at the end of each run of gr6wfli/harvesting .cycles, the connectors (175) and (176) are disconnected in the stjeril^hdod (380), and the used devices are discarded, allowing the connector* (3 75) atltKe compressor end to be connected to the connector (176) of a new group'.Qffdeyices. Sterilized /air is typically provided continuously, or alternatively att^^^fefppin^i pulses, during each culturing cycle.
In the^r^esxpd embodiment of the battery (500), excess air and/or waste
gases from eaph;£>f^be' .devices (10) is removed to the atmosphere via common-
piping (290) .^|a^;cbn&ected to each corresponding gas outlet (90). Common
piping (290) i^p^^doi^itli'a suitable contaminant preventer (210), such as one or
more filters, '$&V-pn^&ing contaminants from flowing into devices (10).
Alternatively the j^s Outlet (90) of each device (10) may be individually allowed to
vent to;ffie:dlmc$^pB«re,^ jnreYeKLbly via suitable filters which substantially prevent
a>ntamihants ifo^ (10).
M^ia^d actives-are contained in one or more holding tanks (340). For example, micro elCTnrate,'macro -elements and vitamins may be held in different tanks, while atdditives such as antibiotics and fungicides may also held in yet other separate tanks: A; pumper (345) serving each tank enable the desired relative proportions ofCeach component of the media and/or additives to be delivered at a
predeten^^;aadr^iibigllable flo\V xate. to a static mixer (350), through which
:' -• ^;:v^^v:^-v-'.. -<: :>
water—either^
(360)v|^ef6t^^^i;m^ai^';of actable pumper (365) (FIG. 10). By adjusting the flow rato pf^^S^{^5Jf^d.(365)y for exanrple, the conceatration of media as well as addi|ti^!:^^^l4fto.be deliViared into devices (10) maybe controlled. Media and/or additiy^^t^^Witliwatar may theii be delivered from the static mixer (350) under stifle^a^opsm^i filter(310) and a delivery pipe (370) having an aseptic connected (37^ a^s;fre6 €^dX39iO);:'
Li ^eT^fa^;^bodim^ the battery (500), the inlet of additive pipe (80) of pacix TOn^esponding device (10) in the grotqn of devices^ are interconnected via cx>mmou |)i|ring; (180)^wnidh ciomprises at its free ©ad a common aseptic connector Q70y^pim^p^^s^tic qbnnector (376) may then be connected, in the
sterile hood (380),to the septic connector (375) at the free end (390) of the media and additive. pipfe(37O)i: thus enabling each device (10) of the battery, or of the group, to be s^pUeid.\viai*meaia and additives. At the end of the life of the devices (10), and pridr-to-discarding the same, the aseptic connectors (375) and (376) are disconnected it the sterile h
T3ie;st^cHei:Jbi6ocl.(380) may also optionally be used for connecting the media/additi?e& tank, (350) to each one of a number of groups of devices (10) in the battery, in ti^/4]piingvthe useful lives of the devices in these groups. Thus, when one group of fleSades- has-been' serviced with media/additives, the aseptic connector (376)'ofthis;'^%pM ase^tically. sealed'temporarily in the sterile hood (380), which is then'ino^yedvtci^e neit group* of devices where their common aseptic connector {376) is cpi^e^^io;the/^q^|6 connector (375) of the pipe (370), thus enabling this group of deyice3;J9b.fe;servic^ with media/additives.
In,a diff^pteii^6dimeht of the battety (500), a mobile sterile hood (380)
may be used ite-^niiecfe together the free end (390) of a preferably flexible delivery
pipe connectedtp.static mixing/tanK (350), to the additive inlet of each device (10) in
turn. The sterile ^qpd-(3 80)^ may theii be moved from one device (10) to the next,
each time the en
(80) to enabl&toe^a* ^ each device in turn. The sterile hood (380),
together^itfr^eptife. edpneCtor^ preferably made from stainless steel, at end (390)
. and the iiilk ^fe^ipe^^^^^ device (10), respectively, enable
each devi.ee;($P3-?tbv^'e^sily.doniiefefed and subsequently disconnected to the. end
(390) and thi^iio^fe-^^ia^TO^ conditions. Many othear examples of
suitable conn^to^pr -p
Suitable;fflte6S^ (390) and at tiie pipe (80), respectively, to
. prevent or;aaJJ^^^^inffft^^ppteaatial contamination of the container contents. The sterile ]hooa^(|§Q^ro#Y tf^^W airtoiriatically or manually moved from device (10) to device (IC^2^!^.^^ ifeyicb in torn, an operator may connect the device (10) to the media §^^lj^psm§thfe sfeile liboS (380); fill the device with a suitable quantity
of medid dn^rle^kTOYes^ind.subs^entl^ disconnect ttie sterile hood (380) from
*. * ■ - ~* - *-. • - "-**.* ■• • * . • *
the device, Ttb;fti^i nioYe:Qn to.the next device. Of course, the end (390) may be
a^sqpfced to/^i^i&ti&/a^Ti3ftdifey-or comxecfedr (375)r rafter than just a sin^e sterilized connector (375)^s6 iiiatrather than one, a'similar plurality of devices (10) having con^spondiag.copi^ctor (376) iimy be.connected at a time to the media supply via thetroDey'(3§6)-- ...
. Eaqh ti^ie, prior to"; connecting end (390) to each device or set or group of devices;; fb&\
In yet-jqaoth^r embodiment of the battery (500), a single pipe or a set of pipes (not sho^yBL) (frmi&A static inixer (350), to one device (10) or to a corresponding set of de\dC^/(lt0)/.t^ec^tVely? at a time,, wherein a conveyor system transports the
'demceXl'OXbV^^dm^X^)^^ single pipe or set of pipes, respectively, or
vice yf^a:TA3^'^^i^^6\Se^ncef (1*0) or set of deuces (10), the conveyor enables a
further de^ce^i^ (10) to be connected to the static mixer
(350) fhroii^;^'§ih^ieri^^)eor set pf pipes, respectively.
-*.""" ' *••-' ■ **^L.***'• :. ~l -. -*.
hifcepreJ^^^lboiHmentrf (500), the harvesters of each of the
•"■ •5"".- .")"^v-":- •-•'T'-V'--" * .- . - -devices (10) of:thgjjgup 'are. interconnected- Thus the harvesting pipes (50) of each
device'^tQ) ^ ^isiectedto common harvesting piping (154) having a free, end
X15O)j.wM^*#;^^404>'^^-^^s9^c cqnhector (155). Preferably, each of the
harvesting pipes |50) may cqpiprise a valve (54), as hereinbefore described, to close
off or pjecqait .ffievflowof harvested cells from each corresponding device (10). Thus,
for example, i£ itisjdrtennioed that a number of devices in a particular group are
(xmtami^t^^ devices are not- then the cells in these latter devices
may be Ji&rVd$f£d;-j?wi^btt|; -fear of contamination from the former devices, so long as
the valv^?£^)::^ devices remain closed. Preferably, common.
■ m ::'7^^*&'^:: t: ;■■■' .r*-:-- - ' * ' *' . .
piping ife^^pOT shut-off valve (259) upstream of the aseptic
connector (0^Jft^^m;^ eontaniinatioii prevents: is provided for substantially pi^^^imgv3iffli^Ǥ^cm.t pt ^ntairiinanls into containear via harvester after
la wVja^abrftd* mfeoffimejii the contamination prevCTter comprises a
substantially Urshaped fluid tiap (400), having an aseptic connector (156) at one arm
C : '-'«>:>K.:-"--V.>/-'>... - ;.
th«^o^ ^^\^^^tofli';h^pig aii-bpeEning (158) in fluid communication with a.
rec^hdng.ta^5^©?^^?fe%rtic connectors (155) and (156) are then interconnected
in tiie m^fle^e^eiteN^^^ sterile conditions. Harvesting is then effected
, by,, opening the valves (54) or all the devices in the group which are not
contaminated,aswell..as- common valve (259). Cells from the group will then flow into the .receivingg tank .(590), preferably under gravity, though in some cases a
. suitable pumplinay ie.usedi -After harvesting is completed, the aseptic connectors (155) and (156) miy t>e disconnected in the sterile hood (380), which can then be moved to &e.-tteit gj^pixp of devices (10): the corresponding aseptic connector (155) of this groiip jnay;then".beinterconnected with aseptic connector (156) of theU-tube (400), and therfebjr enable the cells of this group of devices to be harvested.
In another embodiment of the battery (500), a single pipe or a set of pipes (not • shown) iriay connect qommon ireceiying tank to a device (10) or a corresponding set of devices (rO^respect^elyi at a trine, wherein a conveyor system transports the device (10) "o^ef;ofdeyices (10) to .the single pipe or set of pipes, respectively, or vice versa.; ^^^luMves^ng\the device (10) or set of devices (10), the, conveyor
. enables a :KITO!^8^ (10) to be connected to the- common
receiving iarik^ough. a: siigje pipe or set of pipes, respectively.
In mtfthete; ^^pfiigQ^nt of "the battery (500), each device (10) may be
. individually Earaesfedj\ wherein the harvester of each device comprises a contamination;^preventer/for,substantially preventing introduction of contaminants into container^{Vi^lfarvester after harvesting. In this embodiment, the contamination preventer comprises iUrshaped fluid trap (400) as hereinbefore described, having an . aseptic cpmieiipr (15.6)[$QV$ arm thereof the other arm having an opening (158) in fluid coEcmumeatipn: with, a receiving.tank (590). The harvester comprises an aseptic .
. connector (S^v^bgic^ inay be connected to the peptic connector (156) of the fluid
trap (400) in:tliei^Piobije .st^ile hood; (380) undta: sterile conditions. Harvesting is
then effect^i^y pbe^gt^h^.valve (54) of the device, wherein cells will then flow .
into the.r^eiimg^taiA; j^ierably linder gravity, though in some cases a suitable
pump may. pe2g^^;A^i^y^tiag.is completed, th^e aseptic connectors, (55) and
, (156), may:bel^isnnecfed M^flie stmle hood (380), which can then be moved to the
n^ct de^ceXlP3S3w jSM3^p6nding aseptic connector (55) of the harvester of this
device z^yih^ of the U-tube (400),
arid thefrfeby mable Jhe cdfeftf fhis next device to be harvested.
\" - *. T£- *."'. :•* *• -' "-- ^; — "
In-^^^s^^.4sS^^psa^of^^ battery (500), the harvester may also be used fo^Ai^S^^^&l^^m^iD^^X the start of a new run of growth/harvesting
cycles.thus,inoculant may be mixed Wifh sterilized medium in a suitable tanjfey
having a delivery pipe wmprising at its free end an aseptic connector which is
connected to the aseptic connector (155) of the common harvesting piping (154) in
the sterile hood £380). IiioGuiant may then he allowed to flow under gravity, or with
the aid of a statable punip, to.each of the devices (10) of the group via common
harvesting piping^(l54),vafter Which the aseptic connectors are disconnected in the
sterile hood.. . ' •/
Alternatively^ .^ he introduced into the devices via the
additive inlet, m particular; flie additive common piping (180), in a similar manner to that hereinbefore -^described regarding the harvester and the common harvesting ...
pipmg (155); iiijta& mutandis., .
* - . -i*- .• *** -• ■ • *-*v' ■*.*•■• . Ai^r^gt^pref^^embodm^^ of the present invention, the operation
of the pre^ao^l^id^c^ediindividual device and/or/battery is controlled by a computer (600)-^^^howpwnith'regard to Figure 1C. The computer is optionally and preferably able ta^ntroj ?ueh parameters of the operation of the battery and/or of a device according Ip fjiepresent invention as.one or more of temperature, amount and timing-.ofigas. br.^as: combination, entering the container, amount and timing of gas being allowed ta'tsxft the-coritainer^ amount and timing of the addition of at least one material;(suph;: as putteii^, culture medium and so forth), and/or amount of light The computer may optionally also be able to detect the amount of waste being produced. . ;~ , ;
The cOTnputer is.preferably connected to the various measuring instruments
present with regard to the .operation, of the present invention, as an example of a
system for;aufi^s^g^^ the operation of the present.invention.
For ex^pl^^^^i^^f 0QO) is pjeferably connected to a ^uge (602) or gauges
for c»ntro^g^e^qvsr.^sf■ ;a ;^as pi gas combination. Gauge (602) is preferably
connect&t&lag-^^ to a suitable air siq>ply (604), and controls the
flow of air-^r^^^s^ to;^pe(74J.
The je9i^a*x*(^00^;is also preferably connected to a temperature gauge (606),;wld
the computer is optionally and preferably connected to a gauge (610) for controlling* the flow of media and/or other nutrients from a nutrient/media container (612; hereinafter jefeared to * collectively as a nutrient container) to container (20)rtbrough pipe (80) of-the present invention. Computer (600) may also optionally, additionally or altanatively, control valve (84). Also optionally; only one of valve (S^qfgquige (610) is present
The* -Comfbter (600) is.preferably connected to at least one port of the ."
container, and'.inore' preferably (as shown) is connected at least to a harvest port
(shown as pipe (50)) and optionally as shown to a sample port (612). Optionally, the
sample port and the harvest port may be combined. The computer optionally may
control, an automated* sampler, and/or harvester for removing portions of the contents
of the container, for Jesting and/or harvesting (not shown). The computer may also .
optionallybe^^peted tb; an: analyze (614) for analyzing these portions of contents,
for exampte^jpr^^ for operation of the computer.
■ .; .;.* *-.. - ; ■-., •.. . EXAMPLE3
^tm-j^^'^y&^^&--S^;Tdii^'tb a method for culturing and harvesting
plant cells in \". a ^multiple-use* disposable device. The device is optionally and
preferably configured*, according to the device and/or system of Examples 1 and 2
above. La ttasTtfethpd; pliant-pells are preferably placed in a container of the device
according to \thepresentjiqyentidn. This container is preferably constructed of
plastic; wltfcfcmay; pptioniilly be traiislucent and/or transparent, and which optionally
may be.rig#;w^ have a degree of rigidity between rigid
and flexible (^g4^?^4"4^ for example)* Any other additional materials) are then
provided,^ sucfejaS; ^timje^gas or a gas combination; and/or a sterile liquid or a liquid
combmatioi%^y.^ffi^ Preferably, the device is constructed to
featured fCT^l^^i^^^'^u (plant cells and/of one of the
previously:rd^caft*d; adcfttfooaK^mat^ials) may be removed -vdiile still peacmittmg at
.- / s *.*^' * "
least on^' ad^^c^ilC^^^^tgmig^arvesting cycle to be performed. Optionally and more pr^fdrab^^fepl^pt-jCfells-.are cultured in suspoosion.
Af^^^gfe^re^S^'einbod^ of tiie present invention, the plant cells are odtin^^j^^&psidd{tii"a liqirid; medium,-with at least one staile gas or gas
combinatioa (oi^ai^jof%ases) added as required. Optionally, and preferably, the
sterile gas cpjnpBses^a sterife gas. combination which more preferably comprises
sterile, air. ~^ *s preferably added to the
container fKro^gh^ah air Mel during each cycle, dither continuously or in pulses, as
previously dese^>e
Sterile>ctj3turp /Mecliuni aiid/or sterile additives are preferably placed in the
contains flnwjj^W^ddifi^inlet as previously described.
The pl^iticells; (as: an example of an axenic inoculant) are optionally and preferably add^;l^ugfi^ fliejiarvestex* Optionally and preferably, the plant cells in the containerare^eixposed to light, for example through an external light (a source of illumination ^ej^/toVtjife container), particularly if the container is transparent and/orti^isfoc^atl ^ *v . * .
The cetf^^e Sllo^edto grow to a desired yield of cells and/or the material produced byt tM^efls, &a
Accoi^ing^to^^prefetxed mibodunents, excess air and/or waste gases are preferably 4no^^^l^e^e'.odo^iner through a gas outlet, optionally and more preferably^ cbitinuously a^jd/orihtermittejitly. •
Als^;6^1ic>rfany ah^preferably, the material in the container (such as the cell culture medipift f6r. example) is checked for one or more contaminants and/or the
quality of the cell^ and/or-ceil produces) which are produced in the container. More
v •.*A-^>/:--;*;-"1i'.*-":.1'. ••■-; preferablyif:^e|?f^ore;:C^taminants are found to be present or the cells and/or cell
* * * .* «i *r •» • ■ *• * *' , *
prodaci](^3vl3^ quality, the device and its contents are
disposedo£ X ^: .- '-U*"'*-*•
At m;a^ro|>jiato;tfirie,^partiouiarly if contaminants) and/or poor quality cells
and/or .cklptoiad^) are; fiotfound/^t least a firet portion of the material in the
contain^ is.^^^^rhgtyested^ such as medium containing cells and/or cell
produces). J mM^pTefepbl^]. a repiairung second portion of material, such as
med«m.c6i[\%^^^^ffld^: cell;product(s) is allows! to remain in&e container, .
wha^,t^^s^i^V^^pii^ii^;;-option^ as inoculant for a nert
cultura'harVQgfe *&}/§&$;.;-^at^^enle\cultur^ medium and/or sterile additives are provided ^Jfe5$&*«^^^rvest Qrcle throui^ti ttte additive inlet
The preSoOT^ d&dribed cycle is optionally performed more than once.
Also, &fe;pre«ai^^ optionally be performed with a battery
(system) .o£/$p^ces as. described with regard to Example 2.- Optionally and
preferably, th$ .method permit? cells to be cultured and/or harvested anaerobically.
. Epr.;&%^ a battery (500) of at least one group of
■ A\* ■> r>. -- /•" - * . . ' ■ . r
devices.(lO)isi^pyideq,;^gereia.'tiie devices do not comprise an air inlet For at least one. de^cfe £tO) Aibrebf the following process is performed. An axenic inoculant is iii^oSi^i tq^ deVice (10) via common harvesting piping. Next, sterile culture medium -aftd/of sterile additives is added to the device via common additive inlet piping.' Gptisially, the device is iUuminated as previously described.
The celis:ah the device are allowed to grow in medium to a desired yield of
cells and/or. pjodiiet(s) of. the cells. Optionally and preferably, excess air and/or
waste gasies ^;j?ennitted to leave the device, more preferably continuously, via
common gas outletpiping, •
As forith^previotis method,'the material in the container is monitored for the presence of 6ne^xuore ^nt3minant(s) and/or poor quality cells and/or poor quality cell product(s^;%WMct:.6a5p the container and its contents are preferably disposed of. Also' ak^f6i/^e;pre^ous^methO(^ ^e oe&s and/or cell produces) are preferably harvested a£ at>$ttitaLble^^jaes-;for exajnple when a desired amount of cell produces) has been prbduced-r;:. :.
Tbe^^ Wove.^thod^tnay/also optionally be performed aerobically in a battery of disposable/^eylGes^ ^ctjthat sterile gas and/or combination of gases, such as sterile airj: isprp^6d to dfevip^ via donamon air inlet piping.
T^ip|Ey|:a'w&t^; purification sj^tem supplies deionised and pyrogen free wafer ta a ^^i^Engri^ing^concentrated media,.and diluted media is then pumped to the device (10);$a,:addjtiyeinlet:.Filters, typically 0.2 micrometer, are installed in the feed pip^ .^idyalso^ just upstream of the additive inlet to minimize risk of . contamiiiatioavo^the^ container coiiteats in each device (10)* Alternatively or additionally,:;^^^^y ^y6;maybe also be iised to. roinimize this risk.
■ For:A&4^;
of 1hetyjK5,6?c^^hat^j| Tfsgqired to harvest in the device (10), is premixed with
. media or TCaXSjta^stSnitfs^ and is introduced into the device (10)
■. via the h^e^^ea&'i^tt&intxoduced into the device (10) via additive input For
subsequeofe b^le^^^yricieaaia and/or additives are introduced, as hereinbefore
described/
Typic^&'pa air; compressor provides substantially sterilized air to eadi device (10)v^ sbBiimbenpf filters: a coarse filter for removing particles, a dryer and humidity at^d^x^oviiig humidity, and a fine filter, typically 0.2 micro-meter, for removing ^%^^ls.;Kreferably, another filter just upstream of the air inlet further
minimizes fh&idsk Ofcontamination of the container contents.
* ■ "f • .' ..
For eadi device (iftVidt connections to the container (20), i.e., to air inlet, to
-m 'f' ~* ■"■—■.-•-.•
. * , * "•. * "'.*, i " ■ ■ - ■
additive M^t-^d^referably also to the gas outlet and to the harvester are autoclave sterilized, prior..toy^e, ?an^ 'staility is maintained during connection to periphessl eqxrfpm^t,: iiichidang; for. example, air supply and exhaust hy performing fhe connections, in &b:sterile hop4 as heareinbefore described.
Ten$^titcfe{fepnwbl for. each device (10) is prefeably provided by a suitable air conditioiidfe Optional illumination of the device may be provided by suitable
*/. V ..V- -;,-.• - -
fluorescent lights suitably, arranged around the device (10), when required for cell
growth '~'f:'&'X': V. -A" ■=':.-.. -■ '*
. 0ui^ig^%cit: (^iftiring:; cycle of each device (10), the contents--of each
* " .."■ »','
.. -A ■ ,. ■ .— . .
wirespondm^o^E^OT (^)^£u:e typically aerated and mixed for about 7 fo about 14 days, ot longgr^^tdegr conpoQed temperature and lighting conditions.
At flie'/epdirof tfee culturing cycle for each device (10), the corresponding
harvester is. ;t^pi'0^Uy connected to a presterilised environment with suitable
connectors ^K^ and during connection^ as hereinbefore
describ^;Hai^d^^^is^^:efiect^ leaving behind between about 2.5% t about
45%, thdujgh troi^lly between about 10% to about 20%, of cells and/or tissue to
sexveasmom^;jfoyAenext.c^ .
lite vl^fy^ted. cells/tissues and/or cell product(s) may then optionally be dried, orext^^Ja^xeqtar^. .
^ctor^g^foprefeed embodiments of the present invention, the process of
cell cdtti^^&^^ptfpii^ijrbe adjusted according to one or more of the following.
These adjiistiiiea^^re prefei^jty pdtfonn^i for culturing plant cells. According to a
fiist adjustp^^7^^^/t?^g gro^ suspension in culture media, the amount of •
media be^.iray^^^a^|n&ec^ on day z^o) is preferably at least
about t2^^'OTflfe^bomin€a6ded'^Qount ^nd more preferably up to about 200% of
therecomm€^^^2»^ , .
Another optional bat prefaced adjustment is the addition of media daring
growth of the.ceUs but before harvesting. More preferably, such media is added on
(lay 3 or 4 after parting the culture process. Optionally and more preferably, the
media comprises-concentrated cultur6 inedia, concentrated from about 1 to about 10
times and theteiby. providing a higher concentration of nutrients. It should be noted
that preferably >":suffioieaitvmedium is provided ihat is more preferably at a
conc^t^tiari^ Addition
of media.irie^ ^at fiestinddia is added to existing media in the container. When added as.a conc^trated solution, preferably the resultant media concentration is close to the. normal or initial concentration. Alternatively, the media in the container ■ may optionally be cdtopletely replaced with fresh media during growth, again more preferably 6ix day3Q*4 after startingthe culture process.
Another qptipnal but preferred.adjustment is the use of higher sucrose levels
-t-'" ■ •-.-* . - •
than is nbniiafly recommendedfor plant cell culture, for example by adding sucrose,
such that the 6oiicentration in the media may optionally be 40g/l rather titan 30g/l.
. One or mbrfe otibL^'sugars may optionally be added, such as glucose, fructose or other
sugars, to corfipl^qi^nt .sucrose. Sucrose (and/or one or more other sugars) is also
• -,: * ' » * : \ •■ "
, * '"'-'■•'•'.. ' - ," **•
optionally and^prefferably kidded during the cell culture process, more preferably on day 3 or 4 aftea: starting the culture process.
Anqthei- Qptibnalvadjusiment is the addition of pure oxygen during the cell culture prdctsss^ moi& prefeafely on day 3 or 4 after starting the culture process.
,Anothdr;Qprtional ^djustmegat is the use of increased aeration (gas exchange), which as sKo^ilqL greafedetail below/also results in an increased cell growth rate in the deyibe ii^ordi&g tQjihe^presentinvention.
~l-:?fr:;ii\?\ TV"'-"'."v. ■"• ■" ■ ' ■ -
■ ;•;-'!•'■.;./ -\. .' . EXAMPLE4
EXifflKJiffiNTAL EXAMPLE WITH V1NCA ROSEA CELLS
This €^pgmient was'; performed witii cells from Vinca rosea also known as
cose p^twiiMde/ -:' - ..--.-*•••••. . *
A;. g£Q>u£ o&'fO.biore^ctprs. (each a device according to the invention), each • with £ wn^i^mMe.fibm.^Jyethylerie-n copolym^:, (O.l.mm wall thickness, - 20 on &emj^i^-mh&ghi); coniplete with 30 mm ports at 5 cm (for air inlet), 25 cm (^*i^^S^^^'CQX^^-tive inl©0» and 90 an (gas outlet) from the bottom,
effective fillable vofome about 10 lifers was used. The bioreactors, together with their fittings, were sterilized by gamma irradiation (25 mRad).
Nine liters of Schenk & Hildebrandt mineral/vitamin medium, 2 mg/1 each of chlorophempiytfcetio/acad'aiid 2,4-rdichlorophenoxyacetic acid, 0.2 mg/1 ktaetin, 3% sucrose, and$)iQffip&tk6d volume initial inoculum of line V24 Catharanthus roseus (Vinca) ceUs'^dt^ mtrdSucied into each bioreactor. The yolume of air above the surfece of ih^zneSfi^x-w^ 3)L Aeration was carried out using a flow volume of 1.5 liter/minstefile WL,provided through a 4 mm orifice (air inlet), located 1 cm from the bottom of the container.
The bipreactprs were mounted in a controlled temperature room (25 ° C) and culturing y/as &n$mt$, for 10 day?, xintil the.packed volume inoreased to about 7.5 1 (75% of |he totalivpliita^-a cjpiibli%rate of % days during the logarithmic phase). At this time point, cells were harvested by withdrawing 9 liters of medium and cells through the :fc^vester and 9^ liters of fresh sterile medium together with the same additives \yar^a^ded via ihe additive inlet Cells were again harvested as above at 10-day intdcvai'sjfor .6 additional cycles, at which time the run was completed.
A totafli Veiijfcit of ]5:5 tig fresh, cells (0.5 kg dry weight) was thus collected over various* perfpdi^ of time^^ such as seveaa, ten or fourteen day intervals, from each of the I6.1 ca^ad^^ior^actors. Thesis cells had a 0.6% content of total alkaloids, the same astlxe starting line.:: Therefore, clearly the device of the present invention was able to maintain and! grow^thecells in culture in a healthy and productive state, while maintainingsimilar oridenHi^l cell characteristics as for cells from the starting line.
•" ":./-:""*r-'^"4':.: '-^""'. : EXAMPLES
Example5ai':Cloning andr Large-Scale Expression of Human Glucocerebrosidase
. >V; Vv\. ; 'in Carrot CeU Suspension
Ihis Bcamgle prpyid^ a description of experiments that wore performed with transformed plant ceJOt^ qiltored in the device of the oresent invoation, according to the method.of ^eg^CTfmvoritioEu
Materials and Experimental procedures:
Plosmid vectors: Plasmid CE-T
Plasmid (3^1* was constructed from plasmid CE obtained from Prof Galili [United States Pafent 5,367,110 November 22, (1994)].
Plasinid CE.was digested with Sail.
The/SSlI:;p0hesive\ehd was made blunt-ended using the large fragment of DNA pblymerase^;;^feitthe plasmid was digested with PstI and ligated to a DNA fragment coffingfpi' the ER targeting signal from the basic endochitinase gene: [Arabfdapsb i ;MajiqnaiATGAAGACTA ATCTTTTTCT CTTTCTCATC TTTTCACTliG JCCTATQATT ATCCTCGGCC GAATTC (SEQ ID NO: 10), and vacuolaf :.' trngdaftg-' sigpsij from Tobacco chitinase A: GATCTTTTAG TCGATACTAT^ (S;EQ;i0NO: 11) digested with Smal and PstI.
The.Sail cohesiver.end was made blunt-ended using the large fragment of DNA p^lymef^se I;-Tien the plasmid was digested with PstI and ligated to a DNA fragment coding for the ER targeting signal (SEQ ID NO: 1), a non relevant gene, and vacuolar targeting signal (SEQ ID NO: 2), digested with Smal and PstI.
pGRE^Mj^ was obtained from Dr. P. Mullineaux pioger P. Hellens et aL,
(2000) Plant "l^^$ibv:;42>819-832]. Expression from the pGREEN n vector is
controlled by ^6 5§ promoter from Cauliflower Mosaic Virus (SEQ ID NO: 9), the
TMV (TobacGO' Mdsaic:. Virus) omega translational enhancer element and the
ortopme..synthfee;tOTin^ from Agrobacterium tumefaciens.
CDNA: JiQGD^ iobtaaned from E. coli containing the human GCD cDNA
sequent .(G^B^:; AcJp^sioh^ .No: Ml 6328)(ATCC. Accession No. 65696), as -
describei:^&^ 19S5; 82:7289-7293), GC-2.2 [GCS-21cb; ; -
lambda-E^Z-g^in^S; ■; JTojito * sapiens] containing glucosidase beta acid ; [glucocerebrqsida56]v jns^*lengths (kb): 2.20; Tissue: fibroblast WI-38 cell.
Construction of expression plasmid
ThecfiNA;c^^gforhCKi>(3EQmN^ -
forward: y'C&GA^ 3*(SEQ ID NO: 3) and the reverse:
5' CTC^^^^(^qAt
PCR DNA; ]^^^/.>v^.:&gest^;witii endonudeases EcoBI and JBglll (see
ierogmfoi£&^^
vector l&;yin&^^^^ E-T digested with the same enzymes. The
expressfiwi cassette was but and eJuted from the intermediate vector and ligated into the binary vector pGREENII using restriction enzymes Smal and Xbal, forming the final expression Sector. Kanamyc&L resistance is conferred by the NPTII gene driven by the nps,p^^Qter obtained together with the pGREEN vector (Fig- 11B). The
resulting expression qassette (SJBQ'BffiNO: 13) is presented by Fig. 11 A. . ■ - : ."•'■" ^/.'. • •■'•"■■ '. ■■■ Ili€>i:e^til|Sig^yag]mdwas secfuenced to ensure correct in-frame fusion of the
• ■*'""**.. ■■ signals- using $ie-. flowing sequencing primers: 5* 35S promote: 5'
CTCAGAAGXcdAGA
Establishment of carrQi callus and cell suspension culture
Establishment of ca&ot callus (i.e« undiffer^itiated carrot cells) and cell suspension :c^tuf^5VwOTe"poformed as described previously by Torres K.C* (Tissue culturetechnkpies foritorHpujarcrops,p.p. Ill, 169).
Tratttfprniation ofcdftqt cells and isolation of transformed cells.
Transformation"^ of carrot cells was preformed using Agrobacterium
transformatioiiby:an-adaptation of amethod describedpreviotisly [Wurtele, E.S. and
Bulka, VL Plant ScL 61:253-262 (1989)]. Cells growing in liquid media were used
throughout the;pfj)cess instead of calli. Incubation and growth times were adapted for
transfomatipn^of>beUs in liquid cultufa Briefly, Agrobacteria were transformed with
the pGREEtJ.:;II|Vect6t by ^lectroppration. [den Dulk-Ra, A. and Hooykaas, PJ.
(1995) Ue&*$^ then selected using 30 mgfoal
paromomycm^ Kut«?iptic;:'Ctorof cells weare transformed with Agrobacteria and
select^ q^&^^g^^p^ in liquid media*
SjcfeeAiHglpffo cells for isolation ofcalti expressing high
JevelsofGjED: V-r,s .-• '•*'-*?* * ""■. :" . '
14 days feUoyong ^tratisformatioxi, cells from culture were plated on solid
media atdflutf^n)^3% p&^ol^cell volume for.the formation of calli fiom individual
cluster of-c^^-^yiimfiidM reached 1-2 cm in diameter, the cells wore
homogeni^:mSpS,'saEDple buffer and tho resulting protein extracts were separated
. on SDStPA0E';lI^emirfi IJ^ (1970) Nature 227:680-685] and transferred to
nitro
1^^203.0 ;Ra^|^^ as descrihed in greater det^l helow.
Westeas bIbt^fo^Td^«rfi6n ijf.GQO was preformed using polyclonal anti hGCD
antibodies (d^cnbM herein below). Calli expressing significant levels of GCD were expanded .andtr^ferredto growth in liquid media for scale up, protein purification
and analysis:
I&rge-scakcidture growth in a device according to the present invention
An ^qut-lqn: callus of genetically modified carrot cells containing the rh-GCD gene (SEQijgC); NOs: 13 "and 14) was plated onto Murashige and Skoog (MS) 9cm diam^tet? &^?me$jtin: :plate*containing 4.4gr/l MSD medium (Duchefa), 9.9mg/l Mhxmii0 (^theh% 015mg folic acid (Sigma) 0.5mg/l biotin (Duchefa), 0.8g/l Cas^jtiyctQlisaie (Duchefa), sugar 30g/l and hormones 2-4 D (Sigma). The callus was ,gfg^$br. 14 days;.at, 25 C;
SuspenMad:;C:di cdlt^re was prepared by sub-culturing the transfonned callus
in. a MSD;(M^a^fege* ^$fcp6g (1962) containing 02 mg/1 2,4-dicloroacetic acid)
liquid m^imt-a^-fs; welfjlcaown in. the art. The suspension cells were cultivated in
250ml ^le^nieyer flask (worldiig yplume starts with 25ml and after 7 days increases
to 50m}) at 2^G'^yith shaking spbed of 60rpm. Subsequently, cell culture volume
was iAcreased;to' i;L^rl€?mae;yer by addition of working volume up to 300ml under
the same cor^it^ons;'Inopulito of .the small bio-reactor (10L) [see WO 98/13469]
containing 4L>MSJ> m^liuriiy was obtaiied by addition of 400ml suspension cells
derived fro^ ^pi iL Erlerftneyer that were cultivated for seven days. After week of
cultivation: it•:25*St with 1;L pxri airflow; MSD medium was added up to 10L and the
cultivation .a)fltiiiueij flfi3er:itbe same conditions. After additional five days of
craltivafioi^]£^ and collected .by passing the cell media
through 80^fiet4pi^ex^:&^ium^as squeezed out and the packed cell cake was
store at^6dC^W^v:^^^ - /V"
■•■*?• ■ 'M J*rrr \-vW;-;:;^.; • -::: • ' -. *
.&; a mJ^Jg^^im.fenv-"growth of transfonned (Glucocarebroadase (GCD))
carrot eell ^^jq§i6n.was.m%sured in a device according to the present invention as
opposed to MMeoQieyie? fl&kJ: Growth was measured as packed cell volume (4000
ipm) and 'aa'^-yfig^^pasinng growth in the Bieraneyer flask was performed by
starting 21/ flgsSar-r^d: J^e^ing 3 flasks eveay day. The harvested flasks were
measured, for':w^t>wa^t, dry weijght and GCD content Reactor harvest was
performed bj^u^g^^lW^st'pbrt (harvests); each day 50 ml of suspension were
harvestpd"fpr^i^n3 iyK^bi^mea^ureaieiiL . -
Figu»,a2^hows tfiatlte cells grown in the flask initially show a higher rate
of grow£h,^^fy^eia the degree of aeration; however, the rates of growth for cells grown ill &£ device- and, in the flask wore ultimately found to be highly similar, and the. rapejimental results obtained in the below experiments to also be highly
similar. . ;.. . / .
The amount of protein in the transfected plant cells was then measured, GCD was exta^^^^o^phke buffer 0.5 M pH 7.2 containing 10% w/w PVPP {Poly vinyl poly pyrolidphe) ari(i1% Triton X-100. GCD content was measured in samples from flask gjowrii*^^^!^!^ and/or with samples taken from cell cultures grown in the devise of -tffelp^eDtmvfention, by using quantitative Western blot The Western blot was perf
Fwf^^.^^yj pi^tepEs frojen the obtained sample were separated in SDS
-• - - •* .* */-. t.v- - - .r---" '- ■ . •
polyacrylenmdx?;^el€sctrc5)horesis and transferred to nitrocellulose. For this purpose,
SDS pofyacryljafluHfe Vgels- were prepared as follows. The SDS gels consist of a
stackingg^l-^^r&olying^gd (inaccordance withLaemmk,UK 1970, Cleavage of
structural piptm^.di^ of the head of bacteriphage T4, Nature 227,680-
685). The coi^yposition o£Ae resolving gels was as follows: 12% acrylamide (Bio-Rad), 4 iniqcNl^-tetrametihyleihylenediamiae; Sigma catalog riupaS^ t0281) per iOrql of gel solution, 0.1% SDS, 375 mM Tris-HCl, pH 8.8 and aminpriiiim persiilfate (APS), 0.1%. TEMED and ammonium persulfate were used in -this..c^ht|x$;as free radical starters for the polymerization. About 20 minutes after the ifiitia^t gf pol^ierfeatioi^ the stacking gel (3% acrylamide, 0.1% SDS, 126 mM Ti^&;bJ&;G$£M% :^tPS and 5 microliters of TEMED per 5ml of stadkii^^^|i^pj-w%^ufpd above the resolving gel, and a 12 or 18 space comb was insegh^Ct^
The aaRdeand^eat&ode. chambers were filled with identical buffer solution: Tris glyctde.t>9^Kcdn4i^gSDS 0iorad, catalog number 161-0772), pH 8.3. The antigen-cobiaigdng: ma^i.al?\yas treated with 015 volume of sample loading buffer (30ml g^ct^f^CTpa catalog nupiber G9Q12), 9% SDS, 15 ml mercaptoethanol (Sigma-. A^^^:U6^0X lil^ mM liis-HCl, pH 6.8, 500 microliters brom9ph©aol;jHu^;aIl volranes p^ 1.00 ml sample buffer), and the mixture was then heated atdOO^e|^5 jo&oites and loaded onto the stacking gel.
Tte^e^p^"';^ performed at room temperature fi>r a suitable time period^ ists^a^^SS^ ittoiutes u^big a constant cuaent strength of 50-70 volts
fallowed by 4^0-inin at~ J8tK200 Volt for gels of 13 by 9 an in size. The antigens were then transferred to mtroceflulose (Schleicher and Schuell, Dassel).
Protdn^ransfer was performed substantially as described herein. Hie gel was located, together* ynih the adjacent nitrocellulose, between Whatmann 3 MM filter paper; conductive,0;S cm-^tbick foatiied material and wire electrodes which conduct the current bywayof platinuici electrodes* Thb filter paper, the foamed material and the nilroceUpib^Jware/jspalced thoroughly with transfer buffer (TG buffer from Biorad, catelpg ni^ber, 161-0771, diluted 10 times with methanol and water buffer (20% meflmoi)>\^6 transfer was performed at 100 volts for 90 minutes at 4°C.
'-Af^^
°C overj-ni^il^i^ Jblpd^g; buffer, containing 1% dry milk (Dairy America), and
0:1% Tvj^^ff with phosphate buffer (Riedel deHaen,
catalog.number 30435). The^blot strips were incubated with an antibody (dilution, 1:6500 mpfiio^H^febuffed cohtainihg 1% dry milk and 0.1 % Tween 20 as above, pH 7.5)at37>CMtM[ur. >: ; / ■■'f
Aft^md^atioh ^vith the aotibody, the blot was washed three times for in
each case 10 rdirftrtes wifli PBS (phosphate buffered sodium phosphate buffer (Riedel
deHaen*. catalog number 30435)), The blot strips were then incubated, at room
■temperatiire, for1 %, with > suitable secondary antibody (Goat anti rabbit (whole
molec^e)-£^:^^a:QE(t#'A-4914))> dilution 1:3000 in buffer containing 1% dry
milk '0>a^/;Jp^^)^^ukL 0.1%. Tween 20 (Sigma. Cat P1379) diluted with
. phosphate r]Si^^^€^.---d^Haai9-/catalog number 30435)). After haying been
■ washed, s^e^^timesr.^w& PBS, the blot strips were stained with ECL developer
. . reagents (^^sK^^^2'09). .
, A^^i^pa^^mg-^^^^ in the ECL reagents the blots were exposed to X-
. ray fitoVFU^5So^;BX\l&^., aiid developed with FUJI-ANATOMDC developer
and &&M&&M c^-HXRTipri out of 2). Hie bands featuring proteins that
w^ebot^b^flfeabtft^^be*^^ after this treatment
;Fi^j^i.3;^)ws^jflie^f^tdts,'indicating fliat the amount of GCD protein relative 4offie;toto|8tp6rati^ (plant cell and GCD) was highest on days 3 and 4/after winch ti^ rela^ver l^i of
Nest; ffi#'star£ point-of 7%_and 15% packed cell volume were conxpared
(again results wdre: similar for cells, grown in flasks or in the device of the present
invention). By.^aidked cell volume" it is meant the volume of cells settling within
the device of3fte p^ebt. invention after any disturbing factors have been removed,
such as aoreitibn of; the media. Figure 14 shows the growth curves, which are
parallel; Figuye 1.5 shows.the amount of GCD protein from a quantitative Western
blot, iiKKcati^&at the amount of GCD protein relative to the total protein (plant cell
and QCp);-W^ 5 and 6, after which the relative level of GCD
declined ag£dn;.^^lotild be noted that samples were taken from cells grown from
15%packi^"4Bll;^ohnrie);:; - .
..C&ow^fw^s itte^stoed over an extended period of time (14. days) to find the
stationary jwi^.^ levels of£ As shown with regard to Figure
16, this poidt?!^Relied" ;pn day 8, after which growth is reduced somewhat Therefore; jfo-:bjfde£ to; W^e to grow cells transfected with a polymicleotide expressing GGp/ptefeably cells arp grown at least until the stationary point, which in this Example is jprefeabjfy^ until day-8 (or shortly thereafter).
Figure:>47-"^^showsv&at.the maximtnn amount of GCD (relative to other proteins); is produced by transformed cells through day 8, after which the amount of GCD produce^ starts.to decline.
Adding at least some fresh media to the container was found to increase cell
growth andtfi^^^iHitbf.GCDbeing produced by the cells. As shown with regard to
Figure. |8,v^eVa^^6ii;;^ (media addition) and/or
replac^^t;oB^^a-(i^i%^^^^^ the fourth day maintains, high growth
level of cells Sfe^md4a^Sv Surthemone, fhe replacement of media with fresh media
on day fotir-ik^^^^I^^^mncKhigher amount of GCD to be produced (see
Figure. 19 for^a^qi^iritQtiv^.Westrai blot; Refreshing media" refers to replacement of
all media wffi:^ concentrated fresh media on day. four, also
results; in a i^^^p^yiiGCp b^ng produced (see Figure 20 for a quantitative
Westem]^i§.^ .
Tlieie^fei:^)fdif^e^ sugar regimes da cell growth is shown with regard to
Figure 21,;^d:^ is shown with regard to Figure 22, As
previously de^qcfl^: optionally but^preferably, hi^ber sucrose levels th$n normally reconiiri^^^f6rj)l^:c^.OTlture.are lised, for example by adding sucrose, sudi
that the. ccmtentra^bri m fh&inedia may optionally be 40g/l rather than 3OgflL One or more o&of sugars;ftiay 6ptioiially beadded, such as glucose, fructose or other sugars, to croripiemOT^se.- Sucrose (and/or one or more other sugars) is also optionally and prejfe^ly.add.ed&uijngthe cell culture process, more preferably on day 3 or 4 after starting &&culture process. The effect of these alterations to the cell culture process is desbiibed & greater detail below.
In Hgdxe&l; the label 40g sucrose indicates that 40g of sucrose was added at the start of cell growth;, the label "30g sucrose + lOg glucose" indicates that this combination ^f':.sugars; .was ^present at the start of cell growth; the label "extra . sucrose" irid5c^tt^,^iat^ 30^JbiPsucrose was present at day zero (start of cell growth) and tiiat 30g/I;^^Q"se ^|a.^ded to the medium on day 4; the label "extra M3D" indicates t^at^^rififedrum^was added; and the label "control" indicates that 30g/l sucrose; was gresf&j-jit day ierp (start of cell growth). As shown, the presence of extra MSI5 ]hs$ th&j^eatfest effect by day 7, followed by the use of a higher amount of sucrose (40:g#); followed by the addition of sucrose mid-way through the growth
cycle/ ■ V .; '"'■■ ■ /,' '..".*
Figure. 22-shows that both the use of a higher amount of sucrose (40g/l) in Figure 22A and. th-e addition of sucrose on day four (Figure 22B) incre&sed the amount of GCt) pfoduc^dj. however, the latter condition produced a spike of GCD
. production o^day:5, while the former.condition provided overall higher amounts of GCDpr6dncEpnfc>r'sev&al.days. *. . ' '
]bcxfea|^C;9^JiQi 'generally (ie. - the presence of. a more-rapid gas .
•. exel^ge)c^^inSt€iksed::Q^gen.spedfic^ hoQx inoreased the rate of growth of GCD tran^pim^-^laht^Ils,.. For these experiments, the cultures were initially aexated'^Va!^^^.]:*tii^]6j^afr per^minute. Increased aeration was performed by increasing rthe^Stof'air.floW.tQ 1^5 or 2 liters per minute, as shown with regard to Figure 23;* Q^yg^^^/^ibi starting on the fourth day, with up to 300% oxygen added te ^o^^^fti.-f^^.to Figure 24 (solid line without symbols shows the oxygCTi pressra£)V .^dfli^wise t&e conditions were idmtical.
• • ' \" ' - ~ ■ -'r
FigurQ;23:.^idws-llie effect of aeration rate on cell growth in a 10 L device accordinjg fo/^ipi^^eat'iayeatioiL As shown, increased aeration (greater than the base of IX' aqr^c&aage $&^ nnnute); provided as 13 L p^: minute (Figure 23A) or 2 L per minute ^igi^;23B)tesiilted in= an increased level of cell growth.
Figure• l^showsiKe .effect of adding more oxygen to the device according to the preseant invention; Oxygen was added starting on day 4; fhe pressure of the additional oxygen^ shown aS ;a solid black line without symbols. It should be noted that because tfie cell^ultttfs'inediBm becomes increasingly viscous as the cells grow and mul^ly/^b^tneasuf^nmt.of oxygen pressure can be somewhat variable, even thou^;the'fldW;of oxygen was maintained at a constant level. As shown, cells . recriving extr^oxyieii clearly showed a higher growth rate, particularly after day 7, when the: gip^fli; rate typically starts to level offj as shown for cells which did not
* * * • •#*- *
receive oxygeitV * : . - . . *
■••"-* T ***-'•' - . \ ..."
-. )*.? .; : . • t Example 5b:
■ .'•.'-•■'1': :/'-v->* "•'* -K-- - '
. Cloning and E>qptession of Biologically Active Human Coagulation Factor
- « -r."f •"•-;' :5jv^-/ X-in Carrot Calti
M^n&ls'^dz^^0nntentalFrocedures
Plasmid^ieciors:'^ V.:f -
^k"^l^mi4:^6;^kd^yonp of the -CE-K plasmid is a Bluescript SK+ plasnrid (Strat^^e, La JollaCA)(SEQ ID NO:15) with an additional cassette in the polycloning site ccfhtaining fall the necessary elements for high level expression and retention^in tfe eadoplasniio reticuhmd of the plant cells. This cassette includes (see sequence (SECg.ID NO;1
of the recpipJMg&J: gepK LKDEL, ER retention signal, and the transoiption
.•-•-^'*:^?*^^*->-^ .T,=:'.'V'. • - - * " • -■/".--
f .teniiiniatibix-ai^'t^JX^^lahbn signal of the Agrobactacium tiimefaciens bctopine
"• : *i ■'•- Vv -H*»^9:'?v\/*-'^v. •"^•* -■'.■". - . * -' -
synth^YO^^g^eJv;;^ , "
pGHen^yJkm^r: .'Bjhasi^ plasmid vectors are designed to integrate manipulated
DNA into^e^OT^e.^ipj^tsl pGREEN, is a second generation binary vector for
plant trans&i^fii^ a .^^ . .
JnJ^fM^BtS^'yp^rj^\cmcfft of seperating functions which can act in trans w^e.iat^o%T&$ &$&&- -The RepA gme is not. present on the cloning vector, but is pioyid^i. m -a^c0raMi3)l^ plasmid, which is co-resident within transformed Agrobacteriu^. 'j£B&i &X^ rranovfig the RqpA function and other unnecssary conjugation ^fiiuciiohs, .fii^/tiyprall plasmid size has beai dramaticaly reduced. . (HeHens^alfpi^^
Cfoq&pctfjfre Buman Factor Xgene: The cDNA for human coagulation, factor X (k^AG^Gen^^nkAccession No: M57285)(SEQ EDNOs:17 and 18XXX was pr^ared^iiditf Hie..plasmid Sig-€EXGLY-FX-HDEL, which includes the complete cDNA for Factor XL 'The coding region was amplified and restriction sites for EcoRI and:SM-added'for sub-cloning according to art recognized protocols. Briefly, ihevic^ing-sequence of mature Human Factor X was amplified using the forward priin^ci :\ ; •,-■"- -..*
Fx start^ :^bM:5^-CCGAATTCCGCGTAAGCTCTGCAGCC 3* (SEQ ID
NO:19) • ;X '":'-■'■ .\ »'■'.
A&dy%*$h&:. rteyetse primer Fx end Sail kdel:
5'GCOTCC^^^GmGGprTG:3' (SEQ IDNO:20);
dspyOTab|^g:i^dfi*o^signals, at the N- and C- terminals of the gene via the
*•':-::■---•-*■/■""" •■ ^iz ':
incorporated; rfs©%ipn'^f^^^
Tbfe a^p^jf^^on je^ctions wbre carried out using the Expand High Fidelity
PCR System^^ ■•^^wheTAppKa3-S.cience catalogue numbenl-732650), according to
manufacta^jii^v^^^^T^i^ PCR products were separated on a 1% agarose gel
for identificaiip^fo^; the; fector X: sequence. Figure 25 shows the predominant
amplified HS^AlJXsbtodparked.by arrow). The band was eluted, cut with the -
restriction/eh^^^>EeoM& ligated into a purified CE-K expression
cassette, acc^r^g.toiroanufacturer's instructions-
• '""*■"'*'
T^xc ligatioii ipi^}xiret was iased to transform E-Coli DHSct and transformed '. *. *** ■ ***** **■." .-.-■' *'- ■ •> '. bacteda were^elefet^d pn agarVplates with lOO^ml ampicilline. Positive clones
were sel^t^^^E0K;^^ysis. using FX forward and reverse primers,, and further verifiedlyTe^c^bkfliial^fe^uangSmal + Xbal,HiridllL andNotL
7fae*^eXp^^hr:^Sette was cut from the CEK-FX-ER plasmid using
restriction ^n^^ls^As^iS.^id XKaL The binary vector pGREEN nos-kana was cut
with the. S^E£ ei^^ eluted from 1% agarose gel. The
Unai7
E. coSrJ>^tt^^^%ter transformation, growth and plasmid extraction, ^sitiveiclo»^w^uverifie4iy PCR.and restriction analysis with HindEff and BglTT, The sd^^.^i^^R^l^sfc^'FX--ER (Figaro 28,) was furthta: verified by sequencing:^
plant transformation :transformation of carrot cells was performed using
Agrobacterp^^^mfoj^adttjqn .by an adaptation of a method described previously [Wurtele,-E^^a^^Q^-li^PJbQf&l 61:253-262 (1989)]. Cells growing in liquid media were xeqdjQi^^bBtffie process instead of callL Incubation and growth times were ad^ted^rfrirk^c^mation of cells in liquid culture. Briefly, Agrobacteria LB4404 \yes0? ^msfonn^d; witii. the pGREEN noskana EX-ER vector by electroporatipir[dea Dulk-Ra, A, and Hooykaas, VJ. (1995) Methods Mol Bioh
■ " -'.V C *-. «* "" ■ • ■
'•3 .'..._ ' ^
55:63-72].M3 tH^^lec^ uang 30 mg'ml parornomycine antibiotic. Carrot cells . {J)aucuscarQt
". —■/*;-> *..'***»A> •- ■ -
Expre^Q^p^^^dMecpntbincmit Human Factor X in Cultured Carrot Cells
Ej^r£^on~:tind^hMys^ in carrot cells; Transformed carrot cells were
g/xrnn'mys^S^^m\J^itt^i^ &.Sk6og medium (PhysioL Plant, 15, 473, 1962)
*suppleri^1^*y^ acetic acid, as descaribed for GCD
hereiimbove;l £bII^Wdre.,^wi-fof seven.days after which the cells were harvested.
Excess Kqittd;^^^ara^1on 100: mesh filter. The cell contents were extracted
for the evaliiati6fiof.prot^n"'content as described in detail hereinabove. Carrot cells
- transformed*;^^.^^;FX-bp^A were analysed for FX expression by Western blot
analysis Ti^gR^bbife X purified IgG From Affinity Biologicals
(Hamilton Gutari^-CJa^Si); A-number of diffearent cell lines were analysed (Figure
.-," .* * "sl >//.-■■ ' •*• *'.-
30). Fi^e.3^Xlia^S;l .^di) demonstrate the strong expression of Human factor X
V:*:*'- -*v-"■£"* r-:*- ";"•. *. '* ': • -in tiie oatrot ^Hs^33ie difeent sizes observed are due to partial proccessing of the
reromfeinibifc^^ -
^Tolpoilmp&fai^^ protein, it's ability to be cleaved
■" - • .»Vi"S ■ n?,T * • -" -* ■••. *" • . •* * '
by furfn wa^^^^UJ^R'3^ a caldtim dependent serine protease, and a major processmgiao^EQeiof tK^^c^lory piihway. Fiirin cleaves Factor X as well as other
clottiBg facto^^.^S^fi^^ . Furin was purchased from New England Biolabs
. \*'*l2Z*'jg/"-pi!;*: .
and fb£--*de&i^^*fc$^^ according to the manufacturer's
recomendatip^;^j[^TO.3i;s^ws the accurate digestion of the recombinant fector X by the finm^ JsJndS coriipared to lane 6). .
A
cteonwgcpicrp^^o substrate for factor Xa* Figure 32 (see soKd lines as compared, to the broicen iines) clearly show accurate Factor X activity in the extracts from carrot coils', ex^ressujig;'fheiecombinant FX grown in large scale culture.
Larg&segie, culture growth in a device according to the present invention .Anabotrt"tUaii. callus of genetically modified carrot cells containing, the recombinant h^^ttEX g6ne (SEQTD NOs;l*6 and 21) are plated onto Murashige and Skoog (MS) ftcm diameter, agar medium plate containing 4.4grA MSD medium (Duchefa);, V^0^2c^U^^u^Qf?), 0.5mg folic acid (Sigma) 0.5mg/l biotin (Duchefa), Otffgfl Gaseia tiydrolisate (Duchefa), sugar 30g/l and hormones 2-4 D (SigmajSt.Lo^ IvIO); The callus is grown for 14 days at 25°C.
Susp^ogLdtfcbli cqltdre is prepared by sub-culturing the transformed callus in
a MSD (N&it^Mge-^^&.Skobg (196^) containing 0.2 ingfl' 2,4-dicloroacetic acid)
liquid mediiiBTi^ ;4§J-js i^elfl^b\vn ih.the art. The suspension cells are cultivated in
25Qml^epJ9i^^ with 25ml and after 7 days increases
to 5Qnd)\at2-S%Ib^^ speed of 60rpm. Subsequently, cell culture volume is
increased -tb> 113 ^Ienmeyer by addition of working volume up to 300ml oinder the
same conditibns^.^ bio-reactor (10L) [see WO 98/13469]
containHg;4L/:lviS£) medium, is obtained by addition of 400ml suspension cells derived.from ftvp/1-L Brlenmpyer flasks that, was cultivated for seven days. After a week of cultivation at 25°C ^vith lliter per minute airflow, MSD medium is added up to 10L ^^^^cdltivatibn continued under the same conditions. After additional five days of .^fiyaJion,.mbst of the cells are harvested and collected by passing the cell mQai'a.tfi^qugIj.*§P{i net.ITie extra medium is squeezed out and the packed cell cakesboT&a€£z$Q; . -.:■'■::. -V ' .
• ■.. Exampfe>Si;: Cloning and. Expression of Human Interferon ft in Carrot
'. ■ .\ ■;••■■.:v:-4^'::..'■■■■•.■ ^.v ■■;- ■ • ' '
..- a-^-i:/?:•■'■.■■■•:-.: .. . - Cam
MaierittWaHdT^eriMental Procedures
(^K&ti^&:r^%k(±bone of the CE-K plasmid is a Bluescript SK+ plasmid (S^^^^i^IoTlaLMSAXSEQ ID NO: 15) witb EIA additional cassette in the polyclooing sfe wiita^triSg afl the necessary elements for high level expression and r^entiotLiir i&^&ddplasmdreiiculuDa of tiie plant cells. This cassette includes (see sequence. " NOSTand map, Pigure 37): CaMV35S promoter, omega enhancer? -:PT^;;fe^CTt.io^g:for the ER targeting signal from the basic
endocMtiii^ j^ ihaliana], EcoRI and Sail restriction sites for fusion
of the mxa^S^^;^0ae>-:-'^^--JSR retention signal, and the transcription termination anjd ^lya^enyiation signal of the Agrobacterium tumefaciens octopine synthase (OCS) gepeT ... .
p$ZPllI:^i&^y^or are designed to integrate manipulated DNA into the genome of plaqt&>T% bin^Sy Ti vector pPZPlll (Hajdukiewicz, et aL Plant Mol Biol 1994; \2&9j59^94) carries the gene fo* kanamycdn resistance, adjacent to the left border. 0P); of ^^ithe teinsfetred region. A lacZ alpha-peptide, with the pUC18 multiple clon&g'site(M£S), lies between the plant marker gene and the right border (RB). Tbusi, since the RIHs transferred first, drug resistance is obtained only if the passenger gengispresfcntin the transgenic plants.
Clonfn%rp^iheHjimanJnterferon pgene The cDNA for Human loterferon j3
(Ifin^/HU^Dfff©^^ No. M28622, SEQ ED NOs: 22 and 23).
g«ae was Qbt^^frd^jH^^eprot^h Inc. Princeton, NJ). The coding region was . amplified md^^pfioripites EcoRI .and Sail addition for sub-cloning. Two portions of the cx^in^re^n-of-^ahire Human Interferon j3 sequences were-amplified, alteniatiyely tBtgetedtothe: endoplasmic reticulum (using primers 1 and 2) or to the apoplast (using ppiriers 1 ,and;3):
L vpoi^Vard priiaersr Ifnfi start EcoRI:
,2; ,.._Rey$£s6- primpien , Ifii^ end Sail kdel
5'GGATGTGOA^Ab.GCAGGTAG 3' (SEQ ID NO: 25)
3. ' :Rex^.r\|)rimer , H: IfiajS end Sail STOP
Aiso: ^i^5^(^;:fc^^i 6f ^sigaals at the N- and C- terminals of the gene via the mcomoratedxes^t^^^
•\T[6e! a^p^ifjaipif Reactions were carried out using the Expand High Fidelity
PCR System-|Rda^^plied-Scietice catalogue number 1732650), according to the
. manufactoe^^insthiGti6ii&. 'Tfee PCR products weace separated on a 1% agarose gel
for id^itificafigtf^^i^ kmifeb^IntCTferon iS sequ^ice. The PCR product band was
. eluted &g desc^l^^eremabove, and 10% of the elated DNA was separated again on
. a 1% agaro^liffcl- J5jr vefiftpation .-anfl purification. Figure 33diows the purified
* "■ - -."*-■*-*.*-." - *-.:;.-_ *..-■•
cloned Hum^Bjt^d^nrjSs^aic^ (arrow marks the PCR product).
the pck product was eluted, cut with the restriction enzymes EcoRI and
Sail, and lifted into 3(JB-K expression cassette according to manufacturer's
instructions^ V. j
TBe Motion-inixture.was -.used to transform E-Coli DH5o; transformed bacteria w^e sel^pted on agar plat^ with lOOftg/ml ampiciline. Positive clones were selected by PGRliialysis using 35S forward (SEQ ID NO: 5) and Terminator reverse (SEQ H>N0; ;6X^rahers (Figtires.34 arid 35). The cloning was further verified by restriction analysis using EcoRI + $£11, and Kpnl + Xbal (Figure 36).
the expression cassettes were cut from the CEK-ifd-ER (Figure 37) and
CEK-ifh-STOP. plasmids using restriction enzymes Kpnl and Xbal. The binary
vector pPZPl IT (Kgure 38) was also cut with Kpnl and Xbal, dephosphorylated and
elated frbm.is?o: agaros'e gel. Hie binary vector and the interferon expression cassettes
were ligated'3^^ coli DH5ot and plasmid extraction, jpositive
clones wd:4 ve^^d .by^C^: and restriction analysis.
'Plant$tyi$>rjhc$ of carrot cells was performed using
Agrobacteriuni, frsoisfoTi^ation by an adaptation of a method described previously
I^urtele/E^^ 61:253-262 (1989)]. CeUs growing in liquid
media;were.usgd:£^^ instead of calli. Incubation and growth times
were adaptedvfot.^ansformation of cells in liquid culture. Briefly, Agrobacteria
LB44(H. were ;^ and pzp-ifii-STOP" vectors by
electrop'Qrati()U £&^ Hooykaas, PJ. (1995) Methods Mol. Biol.
55:63-72] ^n^ttlien, jselected itising 30 mg/rol paromomycine antibiotic. Carrot cells
(Z)aMC!wcorofe).WCTe-tnin^f6m and selected using 60 mg'inl of
paromomycm^mt^ioti^inU^uid .
■.-'-, i>v^:!. -: :. >':"... - ' - . - Results
Expreptionof^^ Interferon ft in Cultured Carrot Cells.
. .Expr$$&0^ Initial.analysis: Transformed
carot;'c«l]^;^ & SkoogmecKinn (PhysioL Plant,
15, 473y-l^|y;^^l^^t^.w&0^ mg/1 2,4 didbloromethoxy acetic acid, as descdbed^for^G3E)^€ar^bqiS>6ve» Cellwere grown for seven days after which fee cells were haryesfed>^^^ss fiquid was separated on a 100 mesh filter. Two weeks foUowiig the'ffan^rmatiOiitell samples were collected for preliminary analysis of interfa^m expfesyoa usffig-a.fdot.blot assay using monoclonal mouse anti human
interffenm B^tfa@tK^eslk&VafB^ty;iRnified' rabbit and inierferon beta antibodies (C^Ibio^^;La:; Jolla,CA)/Both antibodies gave a strong and specific signal in interferon # t^03^mned:QelIs, and no signal in nontransfbrmed cells.
Selection; of best expressing calli: Two weeks after transformation, human interferon ^^presang -cells'were^poured over solid agar with selection antibiotics (Kanamycfctod;0eifQta?&ie) to isolate calli representing individual transformation - events. \&ft%^e£cajQj[ )yere,-f6rmed.they were transferred to individual plates and grown for tiir^^ioriflis.-.^EnOugh oiaterial was recovered from the resultant calli to analyze "the"expression levels.:in' individual calli, and identify the calli having strongest expf^ssipii.. Figure 40 shows a sample Western blot for screening fhe transformed- ^lli;for:thesta)ngest expression of human interferon j8 (see> for example, lan^-l^aitid 2)., . :.
Acttt&Qridfysis in carrot cells: In order to assess the biological activity of the recomb^i^tsbumah linteafeon ^ produced in carrot cells, the reeombinant expressed p^t$^i^as%*&s^£yed"fi>r the viral cytopathic inhibition effect (Rubinstein, et al J YiroI?J^l;37t75>758)l Briefly, reeombinant human interferon & samples were pre-dihit^i.-^id ^jied to a pre-forded monolay^ of WISH cells (a human
."£; .. --■ w . .'"•"£ .';■. . • ' ■
amnionic epithelial-; cellv line): The WISH cells were challenged with vesicular stomatitis virus (^SAQ and cell viability monitored. The titer (expressed in U/ml) is • determined relafivie to an itEH. standard human interferon /?. Table 1 shows the results of flie-sjiial cytopaihic iehibition assay using protdai extracts prepared from different trans eeriic oarrotjlines. -
-• ■.■•■.": .-»-•.. *•*•_.. ._••* ....-'
. Ttiu^iii,$itiSr ofraesb'rescats, reeombinant human iAteferon /S expressed in
carrot calfi i$\i^c^ identity wifli nat5"^
human iiteifetefif pi
large scale culture growth in a device according to the present invention
An about ;lcm callus of genetically modified carrot cells containing the. recombinant tmniangene interferon £ (SEQ ID NOs: 27 and 28) are plated onto Murashige ari^ ,$cpog ;(MS)T9cm diameter agar medium plate containing 4.4gr/l MSD m^imr(^chefa);^.^g/l thiaroin HC1 (Duchefa), 0.5mg folic acid (Sigma) 0,5mg/l biotiav^uctiefa)^ 6/8g/l "Casein hydrolysate (Duchefa), sugar 30g/l and hormones 2-4 jE) (Sigtiia, $t~$Xyms> Mp). The callus is grown for 14 days at 25°C.
Suspension cell culture.is prepared by sub-culturing the transformed callus in a MSD (Mxi^lSge ^ Sfcoog, (19)52) containing 0.2 mg/1 2,4-dicloroacetic acid) liquid mediunjy as is:well.kQown in the art. The suspension cells are cultivated in 250ml ErliMMey^^.agk\(^o^drig volume starts with 25ml and after 7 days increases to 50ml) at 25?C wiJb^haMng'.speed of 6Grpm. Subsequently, cell culture volinne is increased to it Erl^imeyer by addition of working volume up to 300ml under the same a>ndjtiohsl;vliioculuiri of^ the small bio-reactor (10L) [see WO 98/13469] containing-■4L^S0;*iiiii^uai,'4is obtained by addition of 400ml suspension cells derived froxi^twdviL_ ^rj'^imeyer flasks that was cultivated for seven days. After a week of'^tx^^^^i^C/^^-liiteai per minute airflow, MSD medium is added up to 10L and.the:lcoltivation continued "under the same conditions. After additional five days of cUlUyatioii,,^p^ bf the cells are harvested and collected by passing the cell media. thro|u^ 80|j: iietr^e extfa medium is squeezed out and the packed cell cake stored atf^G^fe,'. ■'-£';'•-*) \ '/ ■
Jfcampl&5ik-]&fanmgjin& bursal disease virus viral
; • vif ?; r prdteihZfKPII) in Carrot Catti
Matertfflti dpd Experimental Procedures
GE P^^^;.;^e,tackborie. of the GB plasmid is a Bluescript SK+ plasmid
(Stxatagene, E^^ 15) with an additional cassette in the
polyclonin&sii^c^tqiBiiQg a|l the. necessary elements for high level expression and retention itit th^i e^d^a^a^cdhtti of the plant cells; This cassette includes (see sequence'^($BQ;.p>^^^^d tn^>, Figure XXX): CaMV35S promoter, omega enhance, •J03^^fej^^rcoaii]§'-for the ER targeting signal from fee basic . endo(M^^^g^6p^fdMq^is thaliana\ EcoRI and Sail restriction sites for fusion of the recombinant -g^i^^^I>EL "ER retention signal, and the transcriptior
termination and polyadeijylation signal of die Agrobacterium tumefaciens octopine
synthase(OGS)gai4- -V f .
j^JW&r^inaay vecfor are designed to integrate manipulated DNA inta the
gencmie ofxplapt^. ^^ ^e. b|nkfyr 11 vector pGA492 (An, Methods in Enzymol 1987;
•:*?l"7^-;..--"'-:*:;%?.'.-> * ';-153:292-3O5);ba|ji^ih0;geiie for kaaamycin resistance.
■ • • * • ■ p . * ™ • ■•
Cloning of Meinfectious bursal disease virus viral protein 2 (VPII) gene: The CDNAJ. sequence fpiv^ectious bursal disease virus viral protein 2 (VPII) gene (GenBank: A^&essloii TS6^L/$2284) (SEQ ID NO: 29) was obtained from DR. J. Pitkovski, MKAL^Kiryai^JSheiaoiia Israel). The virus genome is formed by two segments of^(toiafcl^shrod^/RNA^ Segment A (3.2 kb) contains two open reading frames (Oia?^ Al'^d^; bj^ Al des for a polyprotdn of 108 kDa that, after proteolytic proBes§jiig^^.yields 'three mature polypeptides: VP2 (VPE^ (37 to 40 kDa), VP3 (3Q; to 3? ke^kX:an4 YP4 (22 k£>a). VPII and VP3 form the virus capsid, and VP4 is Te^flsible fer,.tii6 pl^yage of the polyprotein.
The'cE)J^.a^m|ffof *yPH was amplified with primers to facilitate cloning and signal fu^Qni^Bn^flyJ thef coding sequence of VPII was amplified using the -forwardpnmen-v- o \ .:>> •■.»-...
VPIt(SEQ]BN0:iO);vVvv; ..""..-
5> GCCTTCTdATCGCGfeATGCAAATGGCAAACCTGCAAGATCAAACCS'
And the feYer^e grim^n "v -. ;•->;." . -
VPH-(SEQI^lfefBj): v; - '/■.-:
5' GCCQGT(3Gf &f
v •..-■-■i..^?-f-V:?-/.'-%"^x-- ■■
Also; ^A^s^\^mp^^f ^^ais at the N- terminal of the gene via the ;
mcorpoiated/ri^^ . .
The amplificgfion- reactions were carried out using tiie Expand High Fidelity PCR
. System; ; (Rodi^^jgpi5ed-:Seience, catalogue number 1732650), according to
manufacturer's1 iistrublioti?. Hie PGR.products were separated on a 1% agarose gel
■ r '-i"fe -, -• ;'£• ..' •*.-/■:-•-••••.'*. - * ■ .
for idenUfic^ohWftej^lt.^eqaence. figure 40 shows the predominant VPH band
(marked by-tii^ jca^w)C. The band ^a? duted, dit with the restriction enzymes EcoRI
and. Spli^ v^i JSrateafiftt6 .purified CB expression cassette according to the
manufectyrer^^jBsttvcttQiis: *|K' - .- *%■.■- * .
The BgaHtfa-inixture was used to transform E-Coli DHSa, and transformed bacteria were selected on. agar plates with 100/ig/ml ampiciline. Positive clones were selected by PCJR. analysis "Using 3 5^ forward and Terminator reverse primers: Forward pr^ne* if&ntke 35S promoter. 5* CTCAGAAGACCAGAGGGCT 3' (SEQ
ID NO? 5)-" :>-[i^':- :-;:' ■':■.,.
BackwardfcrMie^^^ 5' CAAAGCGGCCATCGTGC3' (SEQID
Iiiev©xpr€ssi6ii. cassettes', were cut from the CE-VPII plasroids using restrictioid en^^os^ainJn arid XbaL The pGA492 vector was cut with Bgin and Xbal (Bglll agii B^aHI i^ye compatible sticky ends), and ehrted from 1% agarose. gel. The binary y^torapd. the VPII expression cassettes were ligated and used to transfoi^."EAw|£_.©H5ar;hqst cells: After transformation, growth and plasmid extraction, poSitivie cl6ries:were verified by PCR and restriction analysis.
PtaMvir^^ormafioh: Traiisformation of carrot cells was performed using
Agrobaeieritiitt.traasforaiaLtion. by.an adaptation of a method described'previously
[Wratde; ^SvS^^ 61:253^262 (1989)]. CeUs gxwing in liquid -
-*■*■.■*— ^**"-" ■ . ■■
media we^ep$^i^u^D^A^ instead of calli. Incubation and growth times
were adapfed^tV^ in liquid culture. Briefly, Agrobacteria .
LB4404 w^;tx^fotm^[^&i tibe. "pGA492-CE-VPIIn vector by electroporation
[den DiifeRaf^^^ Tf'J: (1995) Methods MoL BioL 55:63-72] and then
selected usmg:3d^gi^ antibiotic. Cairot cells (Daucus carota) were
... -ftc- -v^'-*' ■ .-•*."'V".-
transfoinieil ^m^Agrobcicieiia and. selected using 60 mg/ml of paromomycine
antibioti&^^l^d^^^.; .. ..
■ .*.«"■ '-'-:;.-^v";}:\;-' .c-y ;\\*'-' ] ■ . ■•"'..
/ ' $&p?e?}sion o/RecombinantVPIIin Cultured Carrot Cells
Expression and analysis in carrot cells: Initial analysis: Transformed
carrot cells.w%^;gp6Wn;iii^ccdtures^^inMurashige & Skoog medium (Physiol. Plant,
15, 473, 196i),si^lfei^^t^.^ 2,4 dichloromethoxy acetic add, as .
descaib^foppC^^j^a^Vel Cettwere grown for seven days after which the cells weire Haiyos!^;^&ce»*lk5nd W^s separated on a 100 mesh filter. Two weeks followibg-ftg;fi^Q^prQjito.c^U samples^ were collected for preliminary analysis of -,
:-: '~.;^^:^.'f':':!?\>f- UN/ >""*{;/-■ - " - " "
VPII exp^^iii^fil* -^atrtlot^^ assay using chicken antHBDV and rabbit anti-
IBDV antibodies, /.Both.antibodies gave a strong and specific signal ia VBII transformed cells, aid no signal in nontransfonned cells.
Selection oj best^expressing caltt: Two weeks after transformation, human interfeon $ expi^arig cells were poured over solid agar with selection antibiotics ^anamyciAVaii4^fota5u^e).to isolate calli representing individual transformation events. AftetW^-caaii^er^formed they were transferred to individual plates and grown for ili^ inbntti^^ Bnou^i material was recovered from the resultant calli to analyze AoV^^ceasion' levelsin individual calli by Western blot analysis, and identify fhe c^ paying itpngest expression. Figure 44 shows a sample Western blot for screenihgihe transformed calli for the strongest, expression of VP11 (see, for -. example, lanes^ 2:and 11:;); Following the screening the best expressing callus-(vp2R21) wgsjsel^ed arid transferred to. liquid media for expansion.
Recombinant VPJ'l- Chicken vaccination assay:
Rtcombinant^yPll was. assayed for effectiveness as a vaccine against
infectioiis btttisal^disQaise M chickens. Total protein extract was prepared from calh
from line vp2jR2i/ and administeared (to 10 4 weeks old chickens in each group) by
injection (Img X-OT^.otalljr (3 X 100{ig). Oral administration was. performed by
feedmg-2;giiti^^£ffl^ chicken on three successive days. The
. protective eflfects;:
'^•'^s^^^^B^a^^\SW)^ vpII w«:e administeared orally, resulting in
immiiriia#6nS^fe?|i'of^h^diidcGn^ (resuts not shown). Thus, recombinant Vpll
expressed in caaOT^feisS^ffective as &n injected vaccine. . .
Xarse^scaJf^dfW^e gwwth in,a device according to the present invention :Aa.^M^i^"wiD^V6f;g^ij^ modified carrot cells containing the recombinaa^^^C^m%Os: '32=and 33) are plated onto Morashige and Skoog
(MS) 9cm dia^et^r.agar inedium plate containing 4.4gr/l MSD medium (Duchefa), 9.9mg/l thiamin HC1 (Duchefa), 0.5mg folic acid (Sigma) 0.5mg/l biotin (Duchefa), O.Sg/l Casein .hydrolisate (Duchefa), sugar 30g/l and hormones 2-4 D (Sigma, St Louis, MO). The cjalius is grown for 14 days at 25°C.
Suspension; cell culture is prepared by sub-culturing the transformed callus in a MSD.(Miu&^ge/&-^oojg; (1962) containing 0.2 mg/1 2,4-dicloroacetic acid) liquid mediin^i:^7fe wefl;kiip^;iii"-^ art The suspension cells are cultivated in 250ml Erltiw\eyp?:"flask(woiimg volume starts with 25ml and after 7 days increases to 5Qml).at25.C with.shakingv,speed of 60rpm. Subsequently, cell culture volume is
. increased to it Erlenmeypr by addition of working volume up to 300ml under the
-■***-..» ■
sane conditiQ^.^oculinn ;6f the 'small bio-reactor (10L) [see WO 98/13469] containing; 4PJ^D medium, is obtained by addition of 400ml suspension cells derived irom two. It, Erlenmeyer flasks that was cultivated for seven days. After a week ofptiltiyation at;25°C with ILiter per minute airflow, MSD medium is added up to 10L and'theicioltivation continued under the same conditions. After additional five days of cultivation, most of the cells are harvested and collected by passing the cell media;tteou^'80^ iiet The extra medium is squeezed out and the packed cell cake.storedat^7^fe:' '■.'>•' V • /-'■
It is ajxjprefciated..Qiat certain features of the invention, which are, for clarity, described in[;jijiechcontext bf separate embodiments, may also be provided in combination:£$.a;SaiMe*i^VJ6olineaiL Conversely, various features of the invention, which are, fo^r^]fy,: diesprftied iqt tie context of a single embodiment, may also be provided.S^^telyjt*: in;^apy.^taolesubcombination.
*-*■-..*."* M^pvghl-|he - urvehtion has-been described in conjunction with specific
embodiments^iei^o^.it i$;evidGnt that many alternatives, modifications and variations
will be. apparfe^t to-thpse skilled in tbe art. Accordingly, it is intended to embrace all
-%*■ •*-"•***..*"*.--■**" * . *
such alt^atiV^'-ino^ificationS and Variations that fell within the spirit and broad
sa>pe.of..th^^j^aai^;cl^Eiis. ., All publications, patents and patent applications
mentioned &^n£spe^ incorporated in ttidr ditirefy by referoice.
into t£esp£i^
patent i^lfc^^^^ individually indicated to be incorporated
herein by reference.in.addition, citation or identification of any reference in this application shall '-rotfe construed as an admission that such reference is available as prior art to the present itiyeatioiL .
WHAT IS CLAIMED IS:
1. A system for expressing a recombinant protein in a plant cell culture,
the system comprising:
(a) at least one disposable device for axenically culturing and harvesting cells
and/or tissue in at least one cycle, said device comprising a sterilisable disposable
container comprising a reusable harvester comprising a flow controller for enabling
harvesting of at least a desired portion of culture medium containing cells and/or
tissues when desired, thereby enabling said device to be used continuously for at least
one further consecutive culturing/harvesting cycle, wherein a remainder of said
medium containing cells and/or tissue, remaining from a previous harvested cycle,
may serve as inoculant for a next culture and harvest cycle; and
(b) plant cells genetically modified to express a recombinant protein.
2. A method for axenically culturing and harvesting plant cells capable of
expressing a recombinant protein in at least one disposable device comprising :
providing said at least one device which comprises a sterilisable transparent and/or translucent disposable container having a reusable harvester comprising a flow controller for enabling harvesting of at least a portion of culture medium containing cells when desired, thereby enabling said device to be used continuously for at least one further consecutive cycle, wherein a remainder of said medium containing cells and/or tissue, remaining from a previously harvested cycle may serve as inoculant for a next culture and harvest cycle; and;
providing axenic inoculant via said harvester;
providing sterile said culture medium and/or, sterile additives;
optionally illuminating said container with external light; and
allowing said cells and/or tissue to grow in said medium to a desired yield, wherein said plant cells are selected from the group consisting of celery cells, ginger cells, horseradish cells, carrot cells, Agrobacterium rhizogenes transformed root cells, alfalfa cells, tobacco cells, and tobacco cell line cells.
3. A method for producing a recombinant protein in plant cells axenically
cultured in at least one disposable device, the method comprising:
providing said at least one device which comprises a sterilisable transparent and/or translucent disposable container having a reusable harvester comprising a flow controller for enabling harvesting of at least a portion of culture medium containing cells when desired, thereby enabling said device to be used continuously for at least one further consecutive cycle, wherein a remainder of said medium containing cells, remaining from a previously harvested cycle may serve as inoculant for a next culture and harvest cycle; and;
providing axenic inoculant via said harvester; providing sterile said culture medium and/or, sterile additives; optionally illuminating said container with external light; and allowing said cells to grow in said medium to a desired yield; and harvesting said cells expressing said recombinant protein from said cells or medium, wherein said recombinant protein is selected from the group consisting of a prokaryotic protein, a viral protein a eukaryotic protein and a chimeric protein.
4. An axenic culture of plant cells genetically modified to express a
recombinant protein, the culture comprising:
plant cells expressing a recombinant protein cultured in at least one disposable device, said device comprising a sterilisable transparent and/or translucent disposable container having a reusable harvester comprising a flow controller for enabling harvesting of at least a portion of culture medium containing cells when desired, thereby enabling said device to be used continuously for at least one further consecutive cycle, wherein a remainder of said medium containing cells, remaining from a previously harvested cycle may serve as inoculant for a next culture and harvest cycle, wherein said plant cells are selected from the group consisting of celery cells, ginger cells, horseradish cells, carrot cells, Agrobacterium rhizogenes transformed root cells, alfalfa cells, tobacco cells, and tobacco cell line cells.
5. An axenic culture of plant cells genetically modified to express a
recombinant protein, the culture comprising:
plant cells expressing a recombinant protein cultured in at least one disposable device, said device comprising a sterilisable transparent and/or translucent disposable container having a reusable harvester comprising a flow controller for enabling harvesting of at least a portion of culture medium containing cells when desired,
thereby enabling said device to be used continuously for at least one further consecutive cycle, wherein a remainder of said medium containing cells, remaining from a previously harvested cycle may serve as inoculant for a next culture and harvest cycle, wherein said recombinant protein is selected from the group consisting of a prokaryotic protein, a viral protein a eukaryotic protein and a chimeric protein.
6. The system, method or culture of claims 1, 3 or 5, wherein said plant
cells are selected from the group consisting of celery cells, ginger cells, horseradish
cells, carrot cells, Agrobacterium rhizogenes transformed root cells, alfalfa cells,
tobacco cells, and tobacco cell line cells.
7. The system, method or culture of claims 1, 2 or 4 wherein said
recombinant protein is selected from the group consisting of a prokaryotic protein, a
viral protein, a eukaryotic protein and a chimeric protein.
8. The system, method or culture of claim 7 wherein said viral protein is
the infectious bursal disease virus viral protein VPII.
9. The system, method or culture of claim 7, wherein said eukaryotic
protein is Human interferon p.
10. The system, method or culture of claim 7, wherein said eukaryotic
protein is a Human clotting factor.
11. The system, method or culture of claim 10, wherein said clotting factor
is Human Factor X.
12. The system, method or culture of claim 7, wherein said eukaryotic
protein is a Human lysosomal enzyme.
13. The system, method or culture of claim 12, wherein said lysosomal
enzyme is Human glucocerebrosidase.
14. The system, method or culture of claim 12, wherein said lysosomal
enzyme is Human alpha-galactosidase.
15. The system, method or culture of claims 1-5, wherein said device
further comprises at least one air inlet for introducing sterile gas in the form of
bubbles into said culture medium through a first inlet opening, and wherein said air
inlet is connectable to a suitable gas supply.
16. The system, method or culture of claim 15, wherein a plurality of
different gases are introduced at different times and/or concentrations through said at
least one air inlet.
17. The system, method or culture of claims 1-5, said harvester comprising
a contamination preventer for substantially preventing introduction of contaminants
into said container via said harvester.
18. The system, method or culture of claims 1-5, wherein said container is
made from a material selected from the group comprising polyethylene,
polycarbonate, a copolymer of polyethylene and nylon, PVC and EVA.
19. The system, method or culture of claim 18, wherein said container is
made from a laminate of more than one layer of said materials.
20. The system, method or culture of claim 15, wherein said at least one
air inlet comprises an air inlet pipe extending from said inlet opening to a location
inside said container at or near said bottom end thereof.
21. The system, method or culture of claim 20, wherein said at least one
air inlet comprises a least one air inlet pipe connectable to a suitable air supply and in
communication with a plurality of secondary inlet pipes, each said secondary inlet
pipe extending to a location inside said container, via a suitable inlet opening therein,
for introducing sterile air in the form of bubbles into said culture medium.
22. The system, method or culture of claims 1-5, wherein said device
comprises a substantially box-like geometrical configuration, having an overall
length, height and width, and having a height-to-length ratio between about 1 and
about 3, and preferably about 1.85, and a height to width ratio between about 5 and
about 30, and preferably about 13.
23. The system, method or culture of claims 1-5, wherein said device
comprises a substantially cylindrical-like geometrical configuration, having a height
to width ratio between about 2.5 and about 5, and preferably about 2.7.
24. The system, method or culture of claims 1-5, wherein said device
comprises a support aperture substantially spanning the depth of said device, said
aperture adapted to enable said device to be supported on a suitable pole support.
25. The system, method or culture of claim 15, wherein at least some of
said air bubbles comprise a mean diameter of between about 1 mm and about 10 mm.
26. The system, method or culture of claim 15, wherein at least some of
said air bubbles comprise a mean diameter of about 4 mm.
27. The system, method or culture of claims 1-5, wherein said container
comprises a suitable filter mounted on a gas outlet for substantially preventing
introduction of contaminants into said container via said gas outlet.
28. The system, method or culture of claims l-5? wherein said container
further comprises a suitable filter mounted on an additive inlet for substantially
preventing introduction of contaminants into said container via said additive inlet.
29. The system, method or culture of claims 1-5, said device further
comprises a contamination preventer comprising a U-shaped fluid trap, wherein one
arm thereof is aseptically mounted to an external outlet of said harvester by suitable
aseptic connector.
30. The system, method or culture of claims 1-5, wherein said harvester is
located at the bottom of a bottom end of said container.
31. The system, method or culture of claims 1-5, wherein said harvester is
located near the bottom of a bottom end of said container, such that at the end of each
harvesting cycle said remainder of said medium containing cells and/or tissue
automatically remains at said bottom end of said container up to a level below the
level of said harvester.
32. The system, method or culture of claims 1-5, wherein said remainder
of said medium containing cells and/or tissue comprises from about 2.5% to about
45% of the original volume of said culture medium and said inoculant.
33. The system, method or culture* of claims 1-5, wherein said remainder
of said medium containing cells and/or tissue comprises from about 5% to about 30%
of the original volume of said culture medium and said inoculant
34. The system, method or culture of claim 33, wherein said remainder of
said medium containing cells and/or tissue comprises from about 10% to about 20%
of the original volume of said culture medium and said inoculant.
35. The system, method or culture of claims 1-5, wherein the bottom end
of said container is substantially convex.
36. The system, method or culture of claims 1-5, wherein the bottom end
of said container is substantially frusta-conical.
37. The system, method or culture of claims 1-5, wherein said container
comprises an internal fillable volume of between about 5 liters and about 1000 liters.
38. The system, method or culture of claim 37, wherein said internal
fillable volume is between about 20 liters and 800 liters.
39. The system, method or culture of claim 38, wherein said internal
fillable volume is between about 50 liters and 200 liters, and most preferably about
100 liters.
40. The system, method or culture of claims 1-5, wherein said device
further comprises suitable attacher for attaching said device to a suitable support
structure.
41. The system, method or culture of claim 39, wherein said attacher
comprises a loop of suitable material preferably integrally attached to said top end of
said container.
42. The system, method or culture of claims 1-5, comprising a battery of at
least two said disposable devices.
43. The system, method or culture of claim 41, wherein said devices are
supported by a suitable support structure via an attacher of each said device.
44. The system, method or culture of claim 41, wherein said gas outlet of
each said device is suitably connected to a common gas outlet piping which optionally
comprises a blocker for preventing contaminants from flowing into said devices.
45. The system, method or culture of claim 43, wherein said blocker
comprises a suitable filter.
46. The system, method or culture of claim 41, wherein an additive inlet of
each said device is suitably connected to a common additive inlet piping having a free
end optionally comprising suitable aseptic connector thereat.
47. The system, method or culture of claim 45, wherein said free end is
connectable to a witable supply of medium and/or additives.
48. The system, method or culture of claim 41, wherein said harvester of
each said device is suitably connected to a common harvesting piping having a free
end optionally comprising suitable aseptic connector thereat.
49. The system, method or culture of claim 47, further comprising a
contamination preventer for substantially preventing introduction of contaminants into
said container via said common harvesting piping.
50. The system, method or culture of claim 48, wherein said contamination
preventer comprises a U-shaped fluid trap, wherein one arm thereof is free having an
opening and wherein the other end thereof is aseptically mountable to said free end of
said common harvesting piping via suitable aseptic connector.
51. The system, method or culture of claim 49, wherein said free end of
said U-tube is connectable to a suitable receiving tank.
52. The system, method or culture of claim 41, wherein said air inlet of
each said device is suitably connected to a common air inlet piping having a free end
optionally comprising suitable aseptic connector thereat.
53. The system, method or culture of claim 51, wherein said free end is
connectable to a suitable air supply.
54. The method of claims 2 and 3, further comprising:
allowing excess air and/or waste gases to leave said container continuously via a gas outlet.
54. The method of claims 2 and 3, further comprising:
checking for contaminants and/or the quality of the cells which are produced in said container: if contaminants are found or the cells which are produced are of poor quality, the device and its contents are disposed of;
if contaminants are not found, harvesting said desired portion of said medium containing cells.
55. The method of claim 54, wherein while harvesting said desired portion,
leaving a remainder of medium containing cells in said container, wherein said
remainder of medium serves as inoculant for a next culture/harvest cycle.
56. The method of claim 55, further comprising:
providing sterile said culture medium and/or sterile said additives for the next culture/harvest cycle via an additive inlet; and
repeating the growth cycle until said contaminants are found or the cells which are produced are of poor quality, whereupon the device and its contents are disposed of.
57. The method of claims 2 and 3, wherein said device further comprises
an air inlet for introducing sterile air in the form of bubbles into said culture medium
through a first inlet opening connectable to a suitable sterile air supply, said method
further comprising the step of providing sterile air to said air inlet during the first and
each subsequent cycle.
5 8. The method of claim 5 7 9 wherein said sterile air is supplied continuously throughout at least one culturing cycle.
59. The method of claim 57, wherein said sterile air is supplied in pulses during at least one culturing cycle.
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