Title of Invention	"PROCESS FOR PRODUCING ANTERIOR OCULAR SEGMENT RELATED CELL SHEETS AND THREE DIMENSIONAL STRUCTURES FOR REGENERATION OF CORNEAL TISSUE"
Abstract	"An anterior ocular segment related cell sheet or three-dimensional structure obtained by cultivating corneal epithelial cells in layers that are solely composed of anterior ocular segment related cells of at least one of corneal epithelial cells, corneal endothelial cells, conjunctival epithelial cells and epithelial stem cells and which have only a few structural defects as they have been recovered retaining the intercellular desmosome structure and the basement membrane-like protein between cell and substrate and having satisfactory strength as a single sheet and process for producing the same."

Title of Invention

"PROCESS FOR PRODUCING ANTERIOR OCULAR SEGMENT RELATED CELL SHEETS AND THREE DIMENSIONAL STRUCTURES FOR REGENERATION OF CORNEAL TISSUE"

Abstract

"An anterior ocular segment related cell sheet or three-dimensional structure obtained by cultivating corneal epithelial cells in layers that are solely composed of anterior ocular segment related cells of at least one of corneal epithelial cells, corneal endothelial cells, conjunctival epithelial cells and epithelial stem cells and which have only a few structural defects as they have been recovered retaining the intercellular desmosome structure and the basement membrane-like protein between cell and substrate and having satisfactory strength as a single sheet and process for producing the same."

Full Text	SPECIFICATION ANTERIOR OCULAR SEGMENT RELATED CELL SHEETS, THREE-DIMENSIONAL STRUCTURES, AND PROCESSES FOR PRODUCING THE SAME TECHNICAL FIELD OF THE INVENTION This invention relates to anterior ocular segment related cell sheets and three-dimensional structures in biology, medicine and other fields, as well as processes for producing such sheets, and therapeutic methods using them. BACKGROUND ART With marked advances in medical technology, it has recently become popular to perform organ transplants, i.e., replacing a difficult-to-treat organ with another person's organ. The organs that can be transplanted are quite diverse and include the skin, cornea, kidney, liver and heart, and in addition, the postoperative progress of organ transplants has improved so remarkably that they are already becoming established as a medical procedure. Keratoplasty is one example and as early as 40 years ago, an eye bank was organized in Japan to start transplanting activities. However, as of today, the number of donors in Japan is very small and notwithstanding the fact that there are annually about 20,000 patients who need keratoplasty, only a tenth of them (ca. 2,000 in number) can actually be treated by that procedure. Although keratoplasty is a virtually established procedure, it suffers the problem of shortage in donors, giving rise to the need for the development of a next-generation medical procedure. With this background, attention has been drawn to the procedure of directly transplanting artificial substitutes or cells that were cultured into assembly. Typical examples of this approach are the artificial skin and the cultured skin. However, the artificial skin using synthetic polymers has the potential to cause rejection and other side effects that make it undesirable as skin grafts. On the other hand, the cultured skin is prepared by cultivating a portion of the normal skin of the patient until it grows to a desired size, so it can be used without the risk of causing rejection and any other side effects and may well be described as the most natural masking agent. Conventionally, such cell culture has been performed either on the surface of glass or on the surface of synthetic polymers that were subjected to a variety of treatments. For example, a variety of polystyrene vessels that were subjected to surface treatments such as y~ray irradiation and silicone coating have become popular for use in cell culture. Cells that have been cultivated to grow on those vessels for cell culture are detached and recovered from the surfaces of the vessels by treatment with proteinases such as trypsin or chemical reagents. However, it has been pointed out that the recovery of grown cells by treatment with chemical reagents involves some disadvantages such as the processing steps becoming cumbersome to increase the chance of contamination by impurities and the grown cells becoming denatured or damaged by the chemical treatment to have their inherent functions injured. In order to overcome these disadvantages, several techniques have been proposed to date . JP 2-23191 B describes a method for producing a transplantable membrane of keratin tissue which comprises the steps of cultivating human neonatal keratinized epidermic cells in a culture vessel under conditions that enable a membrane of keratin tissue to form on the surface of the vessel and detaching the membrane of keratin tissue using an enzyme. Specifically, with 3T3 cells used as a feeder layer, the epidermic cells are grown and stratified as a cell sheet which is recovered using the proteinase dispase. However, the method described in JP 2-23191 B has had the following defects. (1) Dispase is of microbial origin and the recovered cell sheet needs to be washed thoroughly. (2) The conditions for dispase treatment differ from one batch of cell culture to another and great skill is required in the treatment. (3) The cultured epidermic cells are pathologically activated by dispase treatment. (4) The extracellular matrix is decomposed by dispase treatment. (5) As the result, the diseased site to which the cell sheet has been grafted is prone to infection. However, anterior ocular segment related cells that are contemplated in the present invention, such as corneal epithelial cells, corneal endothelial cells and conjunctival epithelial cells, do not have as strong intercellular binding as dermal cells and it has been impossible to detach and recover cultivated cells as a single sheet even if the dispase is employed. In order to solve this problem, a technique has recently been devised, according to which corneal epithelial cells or conjunctival epithelial cells are cultured into assembly on an amnion deprived of the spongy layer and the epithelial layer and the assembly is used as a cell graft together with the amnion (JP 2001-161353 A). Since the amnion has adequate strength as a membrane but has no antigenicity, it is favorable as a support of cell grafts; however, the amnion is not inherently in the eye and in order to construct a more precise intraocular tissue, it has been desired that a satisfactorily strong sheet be prepared solely from the intraocular cells. The present invention has been accomplished with a view to solving the aforementioned problems of the prior art. Therefore, the present invention has as an object providing an anterior ocular segment related cell sheet or a three-dimensional structure that have only a few structural defects as they have been recovered retaining the intercellular desmosome structure and the basement membrane-like protein between cell and substrate. Another object of the present invention is to provide a process by which cultivated and grown cells can be detached and recovered from a substrate's surface easily and as a satisfactorily strong, single sheet by changing the ambient temperature without treatment with an enzyme such as dispase. SUMMARY OF THE INVENTION In order to attain the stated objects, the present inventors engaged in R&D activities taking various angles of study. As a result, the inventors found that an anterior ocular segment related cell sheet or threedimensional structure having fewer structural defects could be obtained by a process comprising the steps of cultivating anterior ocular segment related cells on a cell culture support comprising a substrate having its surface covered with a temperature responsive polymer, optionally stratifying the layer of cultured cells, thereafter adjusting the temperature of the culture solution to either above an upper critical dissolution temperature or below a lower critical dissolution temperature, bringing the cultured anterior ocular segment related cell sheet or three-dimensional structure into close contact with a polymer membrane, and detaching the sheet or threedimensional structure together with the polymer membrane. The present invention has been accomplished on the basis of this finding. Thus, the present invention first provides an anterior ocular segment related cell sheet or threedimensional structure that have only a few structural defects as they have been recovered retaining the intercellular desmosome structure and the basement membrane-like protein between cell and substrate. The present invention also provides a process for producing an anterior ocular segment related cell sheet or three-dimensional structure, comprising the steps of cultivating cells on a cell culture support comprising a substrate having its surface covered with a temperature responsive polymer having an upper or lower critical dissolution temperature of 0-80°C with respect to water, optionally stratifying the layer of cultured cells by the usual method, and thereafter, (1) adjusting the temperature of the culture solution to either above the upper critical dissolution temperature or below the lower critical dissolution temperature, (2) bringing the cultured anterior ocular segment related cell sheet or three-dimensional structure into-close contact with polymer membrane, and (3) detaching the sheet or three-dimensional structure together with the polymer membrane. In addition, the present invention provides a process for producing a three-dimensional structure by stratifying the anterior ocular segment related cell sheet or threedimensional structure in close contact with polymer membrane as obtained by the process described in the foregoing paragraph, the stratification being effected by repeating the sequence of the steps of attaching said anterior ocular segment related cell sheet or threedimensional structure to a cell culture support, with or without being covered on a surface with a temperature responsive polymer, a polymer membrane, another cell sheet, or the like, and thereafter stripping the polymer membrane out of close contact. Further in addition, the present invention provides the above-described anterior ocular segment related cell sheet or three-dimensional structure for the treatment of a tissue that has become deficient and/or wounded to a deeper area . Still further in addition, the present invention provides a method of treatment characterized in that the above-described anterior ocular segment related cell sheet or three-dimensional structure is grafted to a tissue that has become deficient and/or wounded to a deeper area. Still further, the present invention provides an anterior ocular segment related cell sheet or threedimensional structure that are useful not only in the medical field but also as cells for safety assessment of chemical substances, poisons or medicines. BEST MODES FOR CARRYING OUT THE INVENTION Cells that can suitably be used in the preparation of the anterior ocular segment related cell sheet or threedimensional structure of the present invention include corneal epithelial cells, corneal endothelial cells, conjunctival epithelial cells, and epithelial stem cells but the applicable cells are by no limited in type. In the present invention, the anterior ocular segment related cell sheet means a sheet obtained by cultivating a single layer of the above-described various kinds of anterior ocular segment forming cells in the living body on a culture support and thereafter detaching the layer from the support; the three-dimensional structure means a sheet that is obtained by stratifying the above-described sheet of various cultured epithelial cells, either on its own or in combination with a sheet or sheets of other cells. The anterior ocular segment related cell sheet or three-dimensional structure in the present invention is such that they have not been damaged during cultivation by proteinases typified by dispase and trypsin. Therefore, the anterior ocular segment related cell sheet or threedimensional structure as detached from the substrate retains the intercellular desmosome structure, has only a few structural defects, and features high strength. This means that if the obtained anterior ocular segment related cell sheet or three-dimensional structure is applied for such purposes as grafting, the anterior ocular segment related cell sheet or three-dimensional structure of the present invention having sufficient strength helps the diseased site to be completely isolated from the outside. In addition, the sheet of the present invention is characterized in that the basement membrane-like protein formed between cell and substrate during cultivation has not been destroyed by enzyme. Hence, the sheet can attach satisfactorily to the living tissue of the diseased site to which it has been grafted and this enables an efficient treatment to be performed. This is described below more specifically. If an ordinary proteinase such as trypsin is employed, the intercellular desmosome structure and the basement membrane-like protein between cell and substrate are hardly retained and, hence, the cell sheet is detached with the cells separated into discrete masses. As for the proteinase dispase, it destroys almost all of the basement membrane-like protein between cell and substrate but the cell sheet can be detached with 10-60% of the intercellular desmosome structure being retained and yet the cell sheet obtained has only low strength. In contrast, the cell sheet of the present invention keeps at least 80% of each of the desmosome structure and the basement membrane-like protein intact, thus providing the various advantages described above. As described above, the anterior ocular segment related cell sheet or three-dimensional structure in the present invention is a cell sheet that retains both the intercellular desmosome structure and the basement membrane-like protein between cell and substrate and which still features high strength; it has not been possible at all to obtain it by the prior art. The temperature responsive polymer which is used to cover the substrate of the cell culture support is characterized by having an upper or lower critical dissolution temperature of 0°C - 80°C, more preferably 20°C - 50°C, in aqueous solution. If the upper or lower critical dissolution temperature exceeds 80°C, cells may die, which is not preferred. If the upper or lower critical dissolution temperature is below 0°C, the cell growth rate will generally drop by an extreme degree or cells will die, which also is not preferred. The temperature responsive polymer to be used in the present invention may be a homopolymer or a copolymer. Examples of such polymers include the polymers described in JP 2-211865 A. Specifically, they are obtained by homo- or copolymerization of the following monomers. Monomers that can be used include, for example, (meth)acrylamide compounds, N- (or N,N-di)alkylsubstituted (meth)acrylamide derivatives, and vinyl ether derivatives; in the case of copolymers, any two or more of those monomers may be used. In addition, those monomers may be copolymerized with other monomers, or polymers may be grafted together or copolymerized, or alternatively, mixtures of polymers and copolymers may be employed. If desired, the polymers may be crosslinked to the extent that will not impair their properties. The substrate that is to be covered with the temperature responsive polymer may be chosen from among the glass, modified glass, compounds such as polystyrene and poly(methyl methacrylate) , and all other substances that can generally be given shape, as exemplified by polymer compounds other than those compounds, and ceramics. The method of covering the support with the temperature responsive polymer is not limited in any particular way but one may follow the methods described in JP 2-211865 A. Specifically, the covering operation can be achieved by either subjecting the substrate and the abovementioned monomers or polymers to electron beam (EB) exposure, y-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment or organic polymerization reaction or by means of physical adsorption as effected by application of coating solutions or the kneading step. The support material shown in the present invention is characterized by having two regions, region A covered with the temperature responsive polymer, and the following region B on its surface: (1) a region covered with a polymer having less affinity for cells; (2) a region covered with a different amount of the temperature responsive polymer than in region A; (3) a region covered with a polymer responsive to a different temperature than in region A; or a combination of any two of regions (l)-(3) or a combination of the three. The method of preparing the support material is not limited at all as long as the final product has the abovementioned structures; to mention a few examples, they include (1) a method comprising the steps of first forming region B over the entire surface of the substrate and then superposing region A with the area masked which eventually serves as region B, or vice versa, (2) a method comprising the steps of covering the substrate with two layers of A and B and scraping either layer by an ultrasonic or scanning device, and (3) a method of offset printing the covering substances, which methods may be employed either alone or in combination. The morphology of the covered regions are not limited in any way and may include the following patterns as seen above: (1) a combination of lines and spaces, (2) polka dots, (3) a grid, or patterns made of other special shapes, or patterns of their mixtures. Considering the state of each intraocular tissue, the pattern of dots (2) is preferred. The size of the covered areas is not limited in any way but considering the size of each intraocular tissue and the possibility that a cultured anterior ocular segment related cell sheet or three-dimensional structure may shrink as they are detached from the support, the following can at least be said about a dotted pattern: if the cells within each dot are to be used, the dot diameter is generally no more than 5 cm, preferably no more than 3 cm, and more preferably 2 cm or less; if the cells outside each dot are to be used, the dot diameter is generally no more than 1 mm, preferably no more than 3 mm, and more preferably 5 mm or less. The coverage of the temperature responsive polymer is suitably in the range of 0.3-6.0 ng/cm2, preferably 0.5-3.5 (j.g/cm2, more preferably 0.8-3.0 ng/cm2. If the coverage of the temperature responsive polymer is less than 0.2 \|J,g/cm2, the cells on the polymer will not easily detach even if they are given a stimulus and the operating efficiency is considerably lowered, which is not preferred. If, on the other hand, the coverage of the temperature responsive polymer is greater than 6.0 ng/cm2, cells will not easily adhere to the covered area and adequate adhesion of the cells becomes difficult to achieve. The polymer having high affinity for cells as used in the present invention is not limited in any way as long as it is free from cell adherence; examples include hydrophilic polymers such as polyacrylamide, poly(dimethyl acrylamide), polyethylene glycol and celluloses, or highly hydrophobic polymers such as silicone polymers and fluoropolymers. In the present invention, cell cultivation is effected on the cell culture support (e.g. cell culture dish) that has been prepared in the manner described above. The temperature of the culture medium is not limited in any particular way, except that it depends on whether the aforementioned polymer the substrate's surface has been covered with has an upper critical dissolution temperature or a lower critical dissolution temperature; in the former case, the medium's temperature should not be higher than the upper critical dissolution temperature and, in the latter case, it should not be less than the lower critical dissolution temperature. It goes without saying that it is inappropriate to perform cultivation in a lower-temperature range where the cultured cells will not grow or in a higher-temperature range where the cultured cells will die. The culture conditions other than temperature may be as adopted in the usual method and are not limited in any particular way. For instance, the culture medium to be used may be one that is supplemented with serum such as known fetal calf serum (FCS); alternatively, it may be a serum-free medium. In the process of the present invention, the culture time may be set in accordance with the above-described method depending on the object of using the anterior ocular segment related cell sheet or three-dimensional structure. The cultured cells may be detached and recovered from the support material by first bringing the cultured anterior ocular segment related cell sheet or three-dimensional structure into close contact with the polymer membrane, then adjusting the temperature of the support material with adhering cells to either above the upper critical dissolution temperature of the overlying polymer on the support substrate or below its lower critical dissolution temperature, whereupon the cells can be detached together with the polymer membrane. Detachment of the anterior ocular segment related cell sheet or three-dimensional structure can be effected within the culture solution in which the cells have been cultivated or in other isotonic fluids, whichever is suitable depending on the object. The polymer membrane to be brought into close contact with the anterior ocular segment related cell sheet or three- dimensional structure may be exemplified by polyvinylidene difluoride (PVDF), polypropylene, polyethylene, celluloses, cellulose derivatives, chitin, chitosan, collagen, urethane, etc. The method of producing the three-dimensional structure in the present invention is not limited in any particular way but may be exemplified by a method in which generally known 3T3 cells are grown as a feeder layer to effect stratification, or a method in which the cultured epithelial cell sheet in close contact with the aforementioned polymer membrane is utilized to produce the three-dimensional structure. The following specific methods may be mentioned as examples. (1) The cell sheet in close contact with the polymer membrane is adhered to the cell culture support and, thereafter, the culture medium is added, whereby the polymer membrane is stripped from the cell sheet, to which another cell sheet in close contact with the polymer membrane is adhered, the process being repeated to form a stratified cell sheet. (2) The cell sheet in close contact with the polymer membrane is inverted and fixed on the cell culture support, with the polymer membrane side facing down, and another cell sheet is adhered to the first cell sheet and, thereafter, the culture medium is added, whereby the polymer membrane is stripped from the cell sheet, to which yet another cell sheet is adhered, the process being repeated to form a stratified cell sheet. (3) Two cell sheets, each in close contact with the polymer membrane, are held together in such a way that they face each other in close contact. (4) A cell sheet in close contact with the polymer membrane is pressed against the diseased site of a living body so that it is adhered to the living tissue and, thereafter, the polymer membrane is stripped away and another cell sheet is superposed on the first cell sheet. The three-dimensional structure of the present invention need not necessarily be made of corneal epithelial cells. It is also possible to overlie the cell sheet or three-dimensional structure made of corneal epithelial cells with a corneal endothelial cell sheet and/or a conjunctival epithelial cell sheet that have been prepared by following the same procedure. This procedure is extremely effective for the purpose of creating a structure closer to anterior ocular segment tissues in the living body. In order to detach and recover the anterior ocular segment related cell sheet or three-dimensional structure with high yield, the cell culture support may be lightly tapped or rocked or the culture medium may be agitated with the aid of a pipette; these and other methods may be applied either independently or in combination. In addition, the cultured cells may optionally be washed with an isotonic fluid or the like so that they are detached for recovery. The anterior ocular segment related cell sheet or three-dimensional structure obtained by the process described above far excels what are obtained by the prior art methods in terms of both easy detachment and high degree of non-invasiveness and have a great potential in clinical applications, as exemplified by corneal grafts. In particular, unlike the conventional graft sheets, the three-dimensional structure of anterior ocular segment related cells according to the present invention retains the basement membrane-like protein, so even if the diseased tissue to which it is going to be grafted is scraped by great thickness, the three-dimensional structure of the invention will take effectively. This contributes not only to improving the efficiency of treatment of the diseased site but also to reducing the burden on the patient, hence, it is anticipated to materialize as a very effective technique. Note that the cell culture support used in the process of the present invention allows for repeated use. EXAMPLES On the following pages, the present invention is described in greater detail by reference to examples which are by no means intended to limit the scope of the invention. Examples 1 and 2 To a commercial polystyrene cell culture dish (FALCON 3001 petri-dish with a diameter of 3.5 cm manufactured by Beckton Dickinson Labware), a coating solution having N- isopropylacrylamide monomer dissolved in isopropyl alcohol to give a concentration of 40% (Example 1) or 50% (Example 2) was applied in a volume of 0.10 ml. Placed on the coated surface of the Petri-dish was a metallic mask having a diameter of 3.5 cm with a center hole having a diameter of 2 cm. While being kept in that state, the surface of the culture dish was exposed to electron beams with an intensity of 0.25 MGy, whereupon an N-isopropylacrylamide polymer (PIPAAm) was immobilized in a circular form (as an island, with the area under the mask being left as the sea which was covered with nothing since it was not exposed to electron beams). Then, the metallic mask was removed and a coating solution having N-isopropylacrylamide monomer dissolved in isopropyl alcohol to give a concentration of 20% was applied in a volume of 0.10 ml. This time, a circular metallic mask having a diameter of 2 cm was placed just to cover the circular area. While being kept in that state, the culture dish was exposed to electron beams with an intensity of 0.25 MGy, whereupon an acrylamide polymer was immobilized outside the circular PIPAAm layer. After the irradiation, the metallic mask was removed and the culture dish was washed with ion-exchanged water to remove the residual monomer and the PIPAAm that did not bind to the culture dish; the culture dish was then dried in a clean bench and sterilized with an ethylene oxide gas to provide a cell culture support material. The coverage of PIPAAm in the island area was determined from a cell culture support material prepared under identical conditions to the above except that no mask was used. As the result, it was found that under the conditions employed, the substrate's surface was covered with the temperature responsive polymer in an amount of 1.6 \|J.g/cm2 (Example 1) or 2.2 \|j,g/cm2 (Example 2). On the obtained cell culture support material, normal rabbit corneal epithelial cells were cultivated by the usual method (medium used: CORNEPAK (product of KURABO INDUSTRIES, LTD.); 37°C under 5% C02) As it turned out, each of the cell culture support materials was such that the corneal epithelial cells adhered and grew normally in the central circular area. At day 14 of the culture, a 2 cm polyvinylidene difluoride (PVDF) membrane was placed over the cultivated cells and the culture medium, as gently aspirated, was subjected to incubating and cooling at 20°C for 30 minutes together with the cell culture support material, whereupon the cells on each of the cell culture support materials were detached together with the overlying membrane. The overlying membrane could be easily stripped from each of the cell sheets. The cell sheets thus detached retained the intercellular desmosome structure and the basement membrane-like protein between cell and substrate and had adequate strength as a single sheet. In each of Examples 1 and 2, "low-temperature treatment" was performed by incubating at 20°C for 30 minutes but the "low-temperature treatment" to be performed in the present invention is not limited to the aboveindicated temperature and time. The preferred temperature condition for the "low-temperature treatment" which is to be performed in the present invention is in the range of 0°C - 30°C and the preferred treatment time is in the range from two minutes to an hour. Example 3 By repeating the procedure of Example 1, normal rabbit corneal epithelial cells were cultivated on the same cell culture support, except that the medium was changed to the ordinary medium of Green et al. containing mitomycin C (DMEM+AB (for making a feeder layer); for human neonatal keratinized epithelial cells). As the result, the corneal epithelial cells on the cell culture support material adhered and grew normally in the central circular area, and the cell layer even stratified. At day 16 of the culture, the cells were incubated and cooled at 20°C for 30 minutes together with the cell culture support material, whereupon the stratified, corneal epithelial cell sheet was detached. The stratified corneal epithelial cell sheet (threedimensional structure) as detached was circular in shape and retained the intercellular desmosome structure and the basement membrane-like protein between cell and substrate to have adequate strength as a single sheet. Comparative Examples 1 and 2 Cell culture support materials were prepared as in Example 1, except that the monomer solution for preparing the cell culture support in Example 1 was adjusted to 5% (Comparative Example 1) or 60% (Comparative Example 2). The resulting coverage on the cell culture supports was respectively 0.1 \|ig/cm2 (Comparative Example 1) and 6.2 [ig/cm2 (Comparative Example 2). Thereafter, normal rabbit corneal epithelial cells were cultivated by the same procedure as Example 1 and an attempt was made to detach them. As it turned out, the cells on the support of Comparative Example 1 were difficult to detach even if they were given the low-temperature treatment; on the other hand, cells were difficult to adhere to the support of Comparative Example 2 and, hence, it was difficult to grow them satisfactorily. Thus, neither of the comparative cell culture supports was preferred as a cell substrate. Example 4 Corneal endothelial cells were recovered from a rabbit's cornea by the usual method. The culture dish of Example 1 which had been grafted with polyisopropylamide (PIPAAm) was inoculated with those cells at a cell density of 2 x 106 cells/cm2 and cultivation was performed by the usual method (medium used: DMEM containing 10% fetal calf serum; 37°C under 5% C02) . Again, the corneal endothelial cells normally adhered and grew only in the central circular area. Ten days later, it was confirmed that the corneal endothelial cells had become confluent; thereafter, as in Example 1, a 2 cm polyvinylidene difluoride (PVDF) membrane was placed over the cultivated cells and the culture medium, as gently aspirated, was subjected to incubating and cooling at 20°C for 30 minutes together with the cell culture support material, whereupon the cells were detached together with the overlying membrane. The overlying membrane could be easily stripped from the cell sheet. The cell sheet thus detached retained the intercellular desmosome structure and the basement membrane-like protein between cell and substrate and had adequate strength as a single sheet. Example 5 The procedure of Example 2 for preparing a cell culture support material was repeated, except that a circular metallic mask having a diameter of 2 cm was put in the center of the culture dish, PIPAAm was immobilized around the mask, then a metallic mask with a center hole having a diameter of 2 cm was overlaid, thereby making a cell culture support material having the polyacrylamide immobilized in the central area (as in Example 1, except that the inner polymer layer was placed outside and the outer polymer layer, inside). The coverage of PIPAAm outside the hole was 2.1 (J,g/cm2. Subsequently, corneal endothelial cells were recovered from a rabbit's cornea by the usual method. The culture dish of Example 1 which had been grafted with polyisopropylamide (PIPAAm) was inoculated with those cells at a cell density of 2 x 106 cells/cm2 and cultivation was performed by the usual method (medium used: DMEM containing 10% fetal calf serum; 37°C under 5% 002) . Again, the corneal endothelial cells normally adhered and grew only in the central circular area. Ten days later, it was confirmed that the corneal endothelial cells had become confluent; thereafter, as in Example 1, a 2 cm polyvinylidene difluoride (PVDF) membrane was placed over the cultivated cells and the culture medium, as gently aspirated, was subjected to incubating and cooling at 20°C for 30 minutes together with the cell culture support material, whereupon the cells were detached together with the overlying membrane. The - overlying membrane could be easily stripped from the cell sheet. The cell sheet thus detached retained the intercellular desmosome structure and the basement membrane-like protein between cell and substrate and had adequate strength as a single sheet. Example 6 The corneal epithelial cell sheet on the culture dish of Example 2 from which the medium had been gently removed without cooling was immediately overlaid with the corneal epithelial cell sheet detached in Example 1. Thereafter, the culture medium used in Example 3 was gently placed to detach the polymer membrane out of close contact with the cell sheet. Kept this way, the cells were cultivated for 2 days to make a stratified sheet (three-dimensional structure) of corneal epithelial cells. The stratified sheet of corneal epithelial cells was given the same lowtemperature treatment as in Example 3, whereupon it was detached from the surface of the support. The stratified sheet (three-dimensional structure) of corneal epithelial cells as detached had satisfactory strength as a single sheet. Example 7 The corneal endothelial cell sheet on the culture dish of Example 4 from which the medium had been gently removed without cooling was immediately overlaid with the stratified sheet of corneal epithelial cells that was detached in Example 3. Thereafter, the culture medium used in Example 3 was gently placed to detach the polymer membrane out of close contact with the cell sheet. Kept this way, the cells were cultivated for 2 days to make a stratified sheet (three-dimensional structure) of corneal epithelial cells having the corneal endothelial cell layer. The stratified sheet of corneal epithelial cells was given the same low-temperature treatment as in Example 3, whereupon it was detached from the surface of the support. The detached three-dimensional structure, retaining the intercellular desmosome structure and the basement membrane-like protein between cell and substrate, had satisfactory strength as a single sheet. Example 8 The corneal endothelial cell sheet on the culture dish of Example 4 from which the medium had been gently removed without cooling was fed with a 5% IV type, dissolved collagen containing medium (the same as the medium used in Example 4, except that it contained collagen) and left to stand as such for 20 minutes. Thereafter, the medium was again gently removed without cooling. The remaining corneal endothelial cell sheet on the culture dish was immediately overlaid with the stratified sheet of corneal epithelial cells that was detached in Example 3. Thereafter, the culture medium used in Example 3 was gently placed to detach the polymer membrane out of close contact with the cell sheet. Kept this way, the cells were cultivated for 2 days to make a stratified sheet (three-dimensional structure) of corneal epithelial cells having the corneal endothelial cell layer. The stratified sheet of corneal epithelial cells was given the same low-temperature treatment as in Example 3, whereupon it was detached from the surface of the support. The detached three-dimensional structure had satisfactory strength as a single sheet. Example 9 The perforated, conjunctival epithelial cell sheet on the culture dish of Example 5 from which the medium had been gently removed without cooling was immediately overlaid partly with the stratified sheet (threedimensional structure) of corneal epithelial cells having the corneal endothelial cell layer that was detached in Example 7. Thereafter, the culture medium used in Example 3 was gently placed to detach the polymer membrane out of close contact with the cell sheet. Kept this way, the cells were cultivated for 2 days to make a stratified sheet (three-dimensional structure) of corneal epithelial cells having the corneal endothelial cell layer to which the conjunctival epithelial cell sheet had adhered. The obtained three-dimensional structure was given the same low-temperature treatment as in Example 3, whereupon it was detached from the surface of the support. The detached three-dimensional structure had satisfactory strength as a single sheet. Example 10 The stratified sheet (three-dimensional structure) of corneal epithelial cells obtained in Example 3 was grafted to a rabbit deficient of a corneal epithelial cell portion in accordance with the usual method. After grafting, the stratified sheet of corneal epithelial cells was sutured to the wound site. About 3 weeks later, the suture was removed and the stratified sheet of corneal epithelial cells had took well on the eyeball. From the foregoing results, it became clear that using the procedure of the present invention, one can fabricate satisfactorily strong sheets solely from intraocular cells. This is believed to provide a very effective technique for reducing the burden on patients by making the treatment protocol more efficient, and for constructing even more precise tissues. INDUSTRIAL APPLICABILITY The anterior ocular segment related cell sheets or three-dimensional structures of the present invention will not decompose E-cadherin or laminin 5, as opposed to the case of dispase treatment, and yet they have extremely small numbers of structural defects, thus having a great potential for use in clinical applications including skin grafting. Hence, the present invention will prove very useful in medical and biological fields such as cell engineering and medical engineering. We claim: 1. A process for producing an anterior ocular segment related cell sheet or three-dimensional structure comprising the steps of cultivating corneal epithelial cells in layers wherein said layers are solely composed of anterior ocular segment related cells of at least one of corneal epithelial cells, corneal endothelial cells, conjunctival epithelial cells and epithelial stem cells and which have only a few structural defects as they have been recovered retaining the intercellular desmosome structure and the basement membrane-like protein between cell and substrate and having satisfactory strength as a single sheet said cell culture support comprising a substrate having its surface covered with a temperature responsive polymer such as herein described having an upper or lower critical dissolution temperature of 0-80°C with respect to water, optionally stratifying the layer of cultured cells, and thereafter, (1) adjusting the temperature of the culture solution to either above the upper critical dissolution temperature or below the lower critical dissolution temperature, and further optionally (2) bringing the cultured anterior ocular segment related cell sheet or a stratified sheet thereof into close contact with a polymer membrane, and (3) detaching the sheet or stratified sheet together with the polymer membrane. 2. The process for producing the anterior ocular"segment related cell sheet or three-dimensional structure as claimed in claim 1, wherein the three-dimensional structure is a combination of at least a corneal epithelial cell sheet or a stratified product thereof with corneal endothelial cells. 3. The process for producing the anterior ocular segment related cell sheet or three-dimensional structure as claimed in claim 1, wherein the three-dimensional structure is a combination of the three-dimensional structure as claimed in claim 2 with conjunctival epithelial cells as a third component. 4. The process for producing an anterior ocular segment related cell sheet or three-dimensional structure as claimed in claim 1, wherein the cell culture support uses a material having two regions, region A covered with the temperature responsive polymer, and the following region B on its surface: (1) a region covered with a polymer having less affinity for cells; (2) a region covered with a different amount of the temperature responsive polymer than in region A; (3) a region covered with a polymer responsive to a different temperature than in region A; or a combination of any two of regions (l)-(3) or a combination of the three. 5. The process for producing a three-dimensional structure as claimed in claim 1 or 4, further comprising attaching in superposition the anterior ocular segment related cell sheet or three-dimensional structure obtained in claim 1 or 4 to a cell culture support, with or without being covered on a surface with a temperature responsive polymer, a polymer membrane or the like, or alternatively attaching in superposition, either partly or entirely, the anterior ocular segment related cell sheet or three-dimensional structure to another cell sheet or the like. 6. The process for producing an anterior ocular segment related cell sheet or three-dimensional structure as claimed in claim 1, 4 or 5, wherein the sheet or three-dimensional structure is detached without treatment with a proteinase. 7. The process for producing an anterior ocular segment related cell sheet or three-dimensional structure as claimed in claim 1, 4 or 5, wherein the temperature responsive polymer is poly(N-isopropylacrylamide). 8. The process for producing an anterior ocular segment related cell sheet or three-dimensional structure as claimed in claim 1, 4 or 5, wherein the polymer membrane is made of polyvinylidene difluoride rendered hydrophilic. 9. The process for producing a three-dimensional structure as claimed in claim 5, wherein the another cell sheet is at least one member of the group consisting of a corneal epithelial cell sheet or a stratified sheet of corneal epithelial cells, a corneal endothelial cell sheet, a conjunctival epithelial cell sheet, and the three-dimensional structure produced by the method- referred to in claim 1 or 4.

Full Text

SPECIFICATION
ANTERIOR OCULAR SEGMENT RELATED CELL SHEETS,
THREE-DIMENSIONAL STRUCTURES, AND PROCESSES
FOR PRODUCING THE SAME
TECHNICAL FIELD OF THE INVENTION
This invention relates to anterior ocular segment
related cell sheets and three-dimensional structures in
biology, medicine and other fields, as well as processes
for producing such sheets, and therapeutic methods using
them.
BACKGROUND ART
With marked advances in medical technology, it has
recently become popular to perform organ transplants, i.e.,
replacing a difficult-to-treat organ with another person's
organ. The organs that can be transplanted are quite
diverse and include the skin, cornea, kidney, liver and
heart, and in addition, the postoperative progress of organ
transplants has improved so remarkably that they are
already becoming established as a medical procedure.
Keratoplasty is one example and as early as 40 years ago,
an eye bank was organized in Japan to start transplanting
activities. However, as of today, the number of donors in
Japan is very small and notwithstanding the fact that there
are annually about 20,000 patients who need keratoplasty,
only a tenth of them (ca. 2,000 in number) can actually be
treated by that procedure. Although keratoplasty is a
virtually established procedure, it suffers the problem of
shortage in donors, giving rise to the need for the
development of a next-generation medical procedure.
With this background, attention has been drawn to the
procedure of directly transplanting artificial substitutes
or cells that were cultured into assembly. Typical
examples of this approach are the artificial skin and the
cultured skin. However, the artificial skin using
synthetic polymers has the potential to cause rejection and
other side effects that make it undesirable as skin grafts.
On the other hand, the cultured skin is prepared by
cultivating a portion of the normal skin of the patient
until it grows to a desired size, so it can be used without
the risk of causing rejection and any other side effects
and may well be described as the most natural masking
agent.
Conventionally, such cell culture has been performed
either on the surface of glass or on the surface of
synthetic polymers that were subjected to a variety of
treatments. For example, a variety of polystyrene vessels
that were subjected to surface treatments such as y~ray
irradiation and silicone coating have become popular for
use in cell culture. Cells that have been cultivated to
grow on those vessels for cell culture are detached and
recovered from the surfaces of the vessels by treatment
with proteinases such as trypsin or chemical reagents.
However, it has been pointed out that the recovery of
grown cells by treatment with chemical reagents involves
some disadvantages such as the processing steps becoming
cumbersome to increase the chance of contamination by
impurities and the grown cells becoming denatured or
damaged by the chemical treatment to have their inherent
functions injured. In order to overcome these
disadvantages, several techniques have been proposed to
date .
JP 2-23191 B describes a method for producing a
transplantable membrane of keratin tissue which comprises
the steps of cultivating human neonatal keratinized
epidermic cells in a culture vessel under conditions that
enable a membrane of keratin tissue to form on the surface
of the vessel and detaching the membrane of keratin tissue
using an enzyme. Specifically, with 3T3 cells used as a
feeder layer, the epidermic cells are grown and stratified
as a cell sheet which is recovered using the proteinase
dispase. However, the method described in JP 2-23191 B has
had the following defects.
(1) Dispase is of microbial origin and the recovered cell
sheet needs to be washed thoroughly.
(2) The conditions for dispase treatment differ from one
batch of cell culture to another and great skill is
required in the treatment.
(3) The cultured epidermic cells are pathologically
activated by dispase treatment.
(4) The extracellular matrix is decomposed by dispase
treatment.
(5) As the result, the diseased site to which the cell
sheet has been grafted is prone to infection.
However, anterior ocular segment related cells that
are contemplated in the present invention, such as corneal
epithelial cells, corneal endothelial cells and
conjunctival epithelial cells, do not have as strong
intercellular binding as dermal cells and it has been
impossible to detach and recover cultivated cells as a
single sheet even if the dispase is employed.
In order to solve this problem, a technique has
recently been devised, according to which corneal
epithelial cells or conjunctival epithelial cells are
cultured into assembly on an amnion deprived of the spongy
layer and the epithelial layer and the assembly is used as
a cell graft together with the amnion (JP 2001-161353 A).
Since the amnion has adequate strength as a membrane but
has no antigenicity, it is favorable as a support of cell
grafts; however, the amnion is not inherently in the eye
and in order to construct a more precise intraocular
tissue, it has been desired that a satisfactorily strong
sheet be prepared solely from the intraocular cells.
The present invention has been accomplished with a
view to solving the aforementioned problems of the prior
art. Therefore, the present invention has as an object
providing an anterior ocular segment related cell sheet or
a three-dimensional structure that have only a few
structural defects as they have been recovered retaining
the intercellular desmosome structure and the basement
membrane-like protein between cell and substrate. Another
object of the present invention is to provide a process by
which cultivated and grown cells can be detached and
recovered from a substrate's surface easily and as a
satisfactorily strong, single sheet by changing the ambient
temperature without treatment with an enzyme such as
dispase.
SUMMARY OF THE INVENTION
In order to attain the stated objects, the present
inventors engaged in R&D activities taking various angles
of study. As a result, the inventors found that an
anterior ocular segment related cell sheet or threedimensional
structure having fewer structural defects could
be obtained by a process comprising the steps of
cultivating anterior ocular segment related cells on a cell
culture support comprising a substrate having its surface
covered with a temperature responsive polymer, optionally
stratifying the layer of cultured cells, thereafter
adjusting the temperature of the culture solution to either
above an upper critical dissolution temperature or below a
lower critical dissolution temperature, bringing the
cultured anterior ocular segment related cell sheet or
three-dimensional structure into close contact with a
polymer membrane, and detaching the sheet or threedimensional
structure together with the polymer membrane.
The present invention has been accomplished on the basis of
this finding.
Thus, the present invention first provides an
anterior ocular segment related cell sheet or threedimensional
structure that have only a few structural
defects as they have been recovered retaining the
intercellular desmosome structure and the basement
membrane-like protein between cell and substrate.
The present invention also provides a process for
producing an anterior ocular segment related cell sheet or
three-dimensional structure, comprising the steps of
cultivating cells on a cell culture support comprising a
substrate having its surface covered with a temperature
responsive polymer having an upper or lower critical
dissolution temperature of 0-80°C with respect to water,
optionally stratifying the layer of cultured cells by the
usual method, and thereafter,
(1) adjusting the temperature of the culture solution to
either above the upper critical dissolution temperature or
below the lower critical dissolution temperature,
(2) bringing the cultured anterior ocular segment related
cell sheet or three-dimensional structure into-close
contact with polymer membrane, and
(3) detaching the sheet or three-dimensional structure
together with the polymer membrane.
In addition, the present invention provides a process
for producing a three-dimensional structure by stratifying
the anterior ocular segment related cell sheet or threedimensional
structure in close contact with polymer
membrane as obtained by the process described in the
foregoing paragraph, the stratification being effected by
repeating the sequence of the steps of attaching said
anterior ocular segment related cell sheet or threedimensional
structure to a cell culture support, with or
without being covered on a surface with a temperature
responsive polymer, a polymer membrane, another cell sheet,
or the like, and thereafter stripping the polymer membrane
out of close contact.
Further in addition, the present invention provides
the above-described anterior ocular segment related cell
sheet or three-dimensional structure for the treatment of a
tissue that has become deficient and/or wounded to a deeper
area .
Still further in addition, the present invention
provides a method of treatment characterized in that the
above-described anterior ocular segment related cell sheet
or three-dimensional structure is grafted to a tissue that
has become deficient and/or wounded to a deeper area.
Still further, the present invention provides an
anterior ocular segment related cell sheet or threedimensional
structure that are useful not only in the
medical field but also as cells for safety assessment of
chemical substances, poisons or medicines.
BEST MODES FOR CARRYING OUT THE INVENTION
Cells that can suitably be used in the preparation of
the anterior ocular segment related cell sheet or threedimensional
structure of the present invention include
corneal epithelial cells, corneal endothelial cells,
conjunctival epithelial cells, and epithelial stem cells
but the applicable cells are by no limited in type. In the
present invention, the anterior ocular segment related cell
sheet means a sheet obtained by cultivating a single layer
of the above-described various kinds of anterior ocular
segment forming cells in the living body on a culture
support and thereafter detaching the layer from the
support; the three-dimensional structure means a sheet that
is obtained by stratifying the above-described sheet of
various cultured epithelial cells, either on its own or in
combination with a sheet or sheets of other cells.
The anterior ocular segment related cell sheet or
three-dimensional structure in the present invention is
such that they have not been damaged during cultivation by
proteinases typified by dispase and trypsin. Therefore,
the anterior ocular segment related cell sheet or threedimensional
structure as detached from the substrate
retains the intercellular desmosome structure, has only a
few structural defects, and features high strength. This
means that if the obtained anterior ocular segment related
cell sheet or three-dimensional structure is applied for
such purposes as grafting, the anterior ocular segment
related cell sheet or three-dimensional structure of the
present invention having sufficient strength helps the
diseased site to be completely isolated from the outside.
In addition, the sheet of the present invention is
characterized in that the basement membrane-like protein
formed between cell and substrate during cultivation has
not been destroyed by enzyme. Hence, the sheet can attach
satisfactorily to the living tissue of the diseased site to
which it has been grafted and this enables an efficient
treatment to be performed. This is described below more
specifically. If an ordinary proteinase such as trypsin is
employed, the intercellular desmosome structure and the
basement membrane-like protein between cell and substrate
are hardly retained and, hence, the cell sheet is detached
with the cells separated into discrete masses. As for the
proteinase dispase, it destroys almost all of the basement
membrane-like protein between cell and substrate but the
cell sheet can be detached with 10-60% of the intercellular
desmosome structure being retained and yet the cell sheet
obtained has only low strength. In contrast, the cell
sheet of the present invention keeps at least 80% of each
of the desmosome structure and the basement membrane-like
protein intact, thus providing the various advantages
described above.
As described above, the anterior ocular segment
related cell sheet or three-dimensional structure in the
present invention is a cell sheet that retains both the
intercellular desmosome structure and the basement
membrane-like protein between cell and substrate and which
still features high strength; it has not been possible at
all to obtain it by the prior art.
The temperature responsive polymer which is used to
cover the substrate of the cell culture support is
characterized by having an upper or lower critical
dissolution temperature of 0°C - 80°C, more preferably 20°C
- 50°C, in aqueous solution. If the upper or lower
critical dissolution temperature exceeds 80°C, cells may
die, which is not preferred. If the upper or lower
critical dissolution temperature is below 0°C, the cell
growth rate will generally drop by an extreme degree or
cells will die, which also is not preferred.
The temperature responsive polymer to be used in the
present invention may be a homopolymer or a copolymer.
Examples of such polymers include the polymers described in
JP 2-211865 A. Specifically, they are obtained by homo- or
copolymerization of the following monomers. Monomers that
can be used include, for example, (meth)acrylamide
compounds, N- (or N,N-di)alkylsubstituted (meth)acrylamide
derivatives, and vinyl ether derivatives; in the case of
copolymers, any two or more of those monomers may be used.
In addition, those monomers may be copolymerized with other
monomers, or polymers may be grafted together or
copolymerized, or alternatively, mixtures of polymers and
copolymers may be employed. If desired, the polymers may
be crosslinked to the extent that will not impair their
properties.
The substrate that is to be covered with the
temperature responsive polymer may be chosen from among the
glass, modified glass, compounds such as polystyrene and
poly(methyl methacrylate) , and all other substances that
can generally be given shape, as exemplified by polymer
compounds other than those compounds, and ceramics.
The method of covering the support with the
temperature responsive polymer is not limited in any
particular way but one may follow the methods described in
JP 2-211865 A. Specifically, the covering operation can be
achieved by either subjecting the substrate and the abovementioned
monomers or polymers to electron beam (EB)
exposure, y-ray irradiation, ultraviolet irradiation,
plasma treatment, corona treatment or organic
polymerization reaction or by means of physical adsorption
as effected by application of coating solutions or the
kneading step.
The support material shown in the present invention
is characterized by having two regions, region A covered
with the temperature responsive polymer, and the following
region B on its surface:
(1) a region covered with a polymer having less affinity
for cells;
(2) a region covered with a different amount of the
temperature responsive polymer than in region A;
(3) a region covered with a polymer responsive to a
different temperature than in region A; or a combination of
any two of regions (l)-(3) or a combination of the three.
The method of preparing the support material is not
limited at all as long as the final product has the abovementioned
structures; to mention a few examples, they
include (1) a method comprising the steps of first forming
region B over the entire surface of the substrate and then
superposing region A with the area masked which eventually
serves as region B, or vice versa, (2) a method comprising
the steps of covering the substrate with two layers of A
and B and scraping either layer by an ultrasonic or
scanning device, and (3) a method of offset printing the
covering substances, which methods may be employed either
alone or in combination.
The morphology of the covered regions are not limited
in any way and may include the following patterns as seen
above: (1) a combination of lines and spaces, (2) polka
dots, (3) a grid, or patterns made of other special shapes,
or patterns of their mixtures. Considering the state of
each intraocular tissue, the pattern of dots (2) is
preferred.
The size of the covered areas is not limited in any
way but considering the size of each intraocular tissue and
the possibility that a cultured anterior ocular segment
related cell sheet or three-dimensional structure may
shrink as they are detached from the support, the following
can at least be said about a dotted pattern: if the cells
within each dot are to be used, the dot diameter is
generally no more than 5 cm, preferably no more than 3 cm,
and more preferably 2 cm or less; if the cells outside each
dot are to be used, the dot diameter is generally no more
than 1 mm, preferably no more than 3 mm, and more
preferably 5 mm or less.
The coverage of the temperature responsive polymer is
suitably in the range of 0.3-6.0 ng/cm2, preferably 0.5-3.5
(j.g/cm2, more preferably 0.8-3.0 ng/cm2. If the coverage of
the temperature responsive polymer is less than 0.2 |J,g/cm2,
the cells on the polymer will not easily detach even if
they are given a stimulus and the operating efficiency is
considerably lowered, which is not preferred. If, on the
other hand, the coverage of the temperature responsive
polymer is greater than 6.0 ng/cm2, cells will not easily
adhere to the covered area and adequate adhesion of the
cells becomes difficult to achieve.
The polymer having high affinity for cells as
used in the present invention is not limited in any way as
long as it is free from cell adherence; examples include
hydrophilic polymers such as polyacrylamide, poly(dimethyl
acrylamide), polyethylene glycol and celluloses, or highly
hydrophobic polymers such as silicone polymers and
fluoropolymers.
In the present invention, cell cultivation is
effected on the cell culture support (e.g. cell culture
dish) that has been prepared in the manner described above.
The temperature of the culture medium is not limited in any
particular way, except that it depends on whether the
aforementioned polymer the substrate's surface has been
covered with has an upper critical dissolution temperature
or a lower critical dissolution temperature; in the former
case, the medium's temperature should not be higher than
the upper critical dissolution temperature and, in the
latter case, it should not be less than the lower critical
dissolution temperature. It goes without saying that it is
inappropriate to perform cultivation in a lower-temperature
range where the cultured cells will not grow or in a
higher-temperature range where the cultured cells will die.
The culture conditions other than temperature may be as
adopted in the usual method and are not limited in any
particular way. For instance, the culture medium to be
used may be one that is supplemented with serum such as
known fetal calf serum (FCS); alternatively, it may be a
serum-free medium.
In the process of the present invention, the culture
time may be set in accordance with the above-described
method depending on the object of using the anterior ocular
segment related cell sheet or three-dimensional structure.
The cultured cells may be detached and recovered from the
support material by first bringing the cultured anterior
ocular segment related cell sheet or three-dimensional
structure into close contact with the polymer membrane,
then adjusting the temperature of the support material with
adhering cells to either above the upper critical
dissolution temperature of the overlying polymer on the
support substrate or below its lower critical dissolution
temperature, whereupon the cells can be detached together
with the polymer membrane. Detachment of the anterior
ocular segment related cell sheet or three-dimensional
structure can be effected within the culture solution in
which the cells have been cultivated or in other isotonic
fluids, whichever is suitable depending on the object. The
polymer membrane to be brought into close contact with the
anterior ocular segment related cell sheet or three-
dimensional structure may be exemplified by polyvinylidene
difluoride (PVDF), polypropylene, polyethylene, celluloses,
cellulose derivatives, chitin, chitosan, collagen,
urethane, etc.
The method of producing the three-dimensional
structure in the present invention is not limited in any
particular way but may be exemplified by a method in which
generally known 3T3 cells are grown as a feeder layer to
effect stratification, or a method in which the cultured
epithelial cell sheet in close contact with the
aforementioned polymer membrane is utilized to produce the
three-dimensional structure. The following specific
methods may be mentioned as examples.
(1) The cell sheet in close contact with the polymer
membrane is adhered to the cell culture support and,
thereafter, the culture medium is added, whereby the
polymer membrane is stripped from the cell sheet, to which
another cell sheet in close contact with the polymer
membrane is adhered, the process being repeated to form a
stratified cell sheet.
(2) The cell sheet in close contact with the polymer
membrane is inverted and fixed on the cell culture support,
with the polymer membrane side facing down, and another
cell sheet is adhered to the first cell sheet and,
thereafter, the culture medium is added, whereby the
polymer membrane is stripped from the cell sheet, to which
yet another cell sheet is adhered, the process being
repeated to form a stratified cell sheet.
(3) Two cell sheets, each in close contact with the polymer
membrane, are held together in such a way that they face
each other in close contact.
(4) A cell sheet in close contact with the polymer membrane
is pressed against the diseased site of a living body so
that it is adhered to the living tissue and, thereafter,
the polymer membrane is stripped away and another cell
sheet is superposed on the first cell sheet.
The three-dimensional structure of the present
invention need not necessarily be made of corneal
epithelial cells. It is also possible to overlie the cell
sheet or three-dimensional structure made of corneal
epithelial cells with a corneal endothelial cell sheet
and/or a conjunctival epithelial cell sheet that have been
prepared by following the same procedure. This procedure
is extremely effective for the purpose of creating a
structure closer to anterior ocular segment tissues in the
living body.
In order to detach and recover the anterior ocular
segment related cell sheet or three-dimensional structure
with high yield, the cell culture support may be lightly
tapped or rocked or the culture medium may be agitated with
the aid of a pipette; these and other methods may be
applied either independently or in combination. In
addition, the cultured cells may optionally be washed with
an isotonic fluid or the like so that they are detached for
recovery.
The anterior ocular segment related cell sheet or
three-dimensional structure obtained by the process
described above far excels what are obtained by the prior
art methods in terms of both easy detachment and high
degree of non-invasiveness and have a great potential in
clinical applications, as exemplified by corneal grafts.
In particular, unlike the conventional graft sheets, the
three-dimensional structure of anterior ocular segment
related cells according to the present invention retains
the basement membrane-like protein, so even if the diseased
tissue to which it is going to be grafted is scraped by
great thickness, the three-dimensional structure of the
invention will take effectively. This contributes not only
to improving the efficiency of treatment of the diseased
site but also to reducing the burden on the patient, hence,
it is anticipated to materialize as a very effective
technique. Note that the cell culture support used in the
process of the present invention allows for repeated use.
EXAMPLES
On the following pages, the present invention is
described in greater detail by reference to examples which
are by no means intended to limit the scope of the
invention.
Examples 1 and 2
To a commercial polystyrene cell culture dish (FALCON
3001 petri-dish with a diameter of 3.5 cm manufactured by
Beckton Dickinson Labware), a coating solution having N-
isopropylacrylamide monomer dissolved in isopropyl alcohol
to give a concentration of 40% (Example 1) or 50% (Example
2) was applied in a volume of 0.10 ml. Placed on the
coated surface of the Petri-dish was a metallic mask having
a diameter of 3.5 cm with a center hole having a diameter
of 2 cm. While being kept in that state, the surface of
the culture dish was exposed to electron beams with an
intensity of 0.25 MGy, whereupon an N-isopropylacrylamide
polymer (PIPAAm) was immobilized in a circular form (as an
island, with the area under the mask being left as the sea
which was covered with nothing since it was not exposed to
electron beams). Then, the metallic mask was removed and a
coating solution having N-isopropylacrylamide monomer
dissolved in isopropyl alcohol to give a concentration of
20% was applied in a volume of 0.10 ml. This time, a
circular metallic mask having a diameter of 2 cm was placed
just to cover the circular area. While being kept in that
state, the culture dish was exposed to electron beams with
an intensity of 0.25 MGy, whereupon an acrylamide polymer
was immobilized outside the circular PIPAAm layer. After
the irradiation, the metallic mask was removed and the
culture dish was washed with ion-exchanged water to remove
the residual monomer and the PIPAAm that did not bind to
the culture dish; the culture dish was then dried in a
clean bench and sterilized with an ethylene oxide gas to
provide a cell culture support material. The coverage of
PIPAAm in the island area was determined from a cell
culture support material prepared under identical
conditions to the above except that no mask was used. As
the result, it was found that under the conditions
employed, the substrate's surface was covered with the
temperature responsive polymer in an amount of 1.6 |J.g/cm2
(Example 1) or 2.2 |j,g/cm2 (Example 2). On the obtained
cell culture support material, normal rabbit corneal
epithelial cells were cultivated by the usual method
(medium used: CORNEPAK (product of KURABO INDUSTRIES,
LTD.); 37°C under 5% C02) As it turned out, each of the
cell culture support materials was such that the corneal
epithelial cells adhered and grew normally in the central
circular area. At day 14 of the culture, a 2 cm
polyvinylidene difluoride (PVDF) membrane was placed over
the cultivated cells and the culture medium, as gently
aspirated, was subjected to incubating and cooling at 20°C
for 30 minutes together with the cell culture support
material, whereupon the cells on each of the cell culture
support materials were detached together with the overlying
membrane. The overlying membrane could be easily stripped
from each of the cell sheets. The cell sheets thus
detached retained the intercellular desmosome structure and
the basement membrane-like protein between cell and
substrate and had adequate strength as a single sheet.
In each of Examples 1 and 2, "low-temperature
treatment" was performed by incubating at 20°C for 30
minutes but the "low-temperature treatment" to be performed
in the present invention is not limited to the aboveindicated
temperature and time. The preferred temperature
condition for the "low-temperature treatment" which is to
be performed in the present invention is in the range of
0°C - 30°C and the preferred treatment time is in the range
from two minutes to an hour.
Example 3
By repeating the procedure of Example 1, normal
rabbit corneal epithelial cells were cultivated on the same
cell culture support, except that the medium was changed to
the ordinary medium of Green et al. containing mitomycin C
(DMEM+AB (for making a feeder layer); for human neonatal
keratinized epithelial cells). As the result, the corneal
epithelial cells on the cell culture support material
adhered and grew normally in the central circular area, and
the cell layer even stratified. At day 16 of the culture,
the cells were incubated and cooled at 20°C for 30 minutes
together with the cell culture support material, whereupon
the stratified, corneal epithelial cell sheet was detached.
The stratified corneal epithelial cell sheet (threedimensional
structure) as detached was circular in shape
and retained the intercellular desmosome structure and the
basement membrane-like protein between cell and substrate
to have adequate strength as a single sheet.
Comparative Examples 1 and 2
Cell culture support materials were prepared as in
Example 1, except that the monomer solution for preparing
the cell culture support in Example 1 was adjusted to 5%
(Comparative Example 1) or 60% (Comparative Example 2).
The resulting coverage on the cell culture supports was
respectively 0.1 |ig/cm2 (Comparative Example 1) and 6.2
[ig/cm2 (Comparative Example 2). Thereafter, normal rabbit
corneal epithelial cells were cultivated by the same
procedure as Example 1 and an attempt was made to detach
them. As it turned out, the cells on the support of
Comparative Example 1 were difficult to detach even if they
were given the low-temperature treatment; on the other
hand, cells were difficult to adhere to the support of
Comparative Example 2 and, hence, it was difficult to grow
them satisfactorily. Thus, neither of the comparative cell
culture supports was preferred as a cell substrate.
Example 4
Corneal endothelial cells were recovered from a
rabbit's cornea by the usual method. The culture dish of
Example 1 which had been grafted with polyisopropylamide
(PIPAAm) was inoculated with those cells at a cell density
of 2 x 106 cells/cm2 and cultivation was performed by the
usual method (medium used: DMEM containing 10% fetal calf
serum; 37°C under 5% C02) . Again, the corneal endothelial
cells normally adhered and grew only in the central
circular area. Ten days later, it was confirmed that the
corneal endothelial cells had become confluent; thereafter,
as in Example 1, a 2 cm polyvinylidene difluoride (PVDF)
membrane was placed over the cultivated cells and the
culture medium, as gently aspirated, was subjected to
incubating and cooling at 20°C for 30 minutes together with
the cell culture support material, whereupon the cells were
detached together with the overlying membrane. The
overlying membrane could be easily stripped from the cell
sheet. The cell sheet thus detached retained the
intercellular desmosome structure and the basement
membrane-like protein between cell and substrate and had
adequate strength as a single sheet.
Example 5
The procedure of Example 2 for preparing a cell
culture support material was repeated, except that a
circular metallic mask having a diameter of 2 cm was put in
the center of the culture dish, PIPAAm was immobilized
around the mask, then a metallic mask with a center hole
having a diameter of 2 cm was overlaid, thereby making a
cell culture support material having the polyacrylamide
immobilized in the central area (as in Example 1, except
that the inner polymer layer was placed outside and the
outer polymer layer, inside). The coverage of PIPAAm
outside the hole was 2.1 (J,g/cm2. Subsequently, corneal
endothelial cells were recovered from a rabbit's cornea by
the usual method. The culture dish of Example 1 which had
been grafted with polyisopropylamide (PIPAAm) was
inoculated with those cells at a cell density of 2 x 106
cells/cm2 and cultivation was performed by the usual method
(medium used: DMEM containing 10% fetal calf serum; 37°C
under 5% 002) . Again, the corneal endothelial cells
normally adhered and grew only in the central circular
area. Ten days later, it was confirmed that the corneal
endothelial cells had become confluent; thereafter, as in
Example 1, a 2 cm polyvinylidene difluoride (PVDF)
membrane was placed over the cultivated cells and the
culture medium, as gently aspirated, was subjected to
incubating and cooling at 20°C for 30 minutes together with
the cell culture support material, whereupon the cells were
detached together with the overlying membrane. The -
overlying membrane could be easily stripped from the cell
sheet. The cell sheet thus detached retained the
intercellular desmosome structure and the basement
membrane-like protein between cell and substrate and had
adequate strength as a single sheet.
Example 6
The corneal epithelial cell sheet on the culture dish
of Example 2 from which the medium had been gently removed
without cooling was immediately overlaid with the corneal
epithelial cell sheet detached in Example 1. Thereafter,
the culture medium used in Example 3 was gently placed to
detach the polymer membrane out of close contact with the
cell sheet. Kept this way, the cells were cultivated for
2 days to make a stratified sheet (three-dimensional
structure) of corneal epithelial cells. The stratified
sheet of corneal epithelial cells was given the same lowtemperature
treatment as in Example 3, whereupon it was
detached from the surface of the support. The stratified
sheet (three-dimensional structure) of corneal epithelial
cells as detached had satisfactory strength as a single
sheet.
Example 7
The corneal endothelial cell sheet on the culture
dish of Example 4 from which the medium had been gently
removed without cooling was immediately overlaid with the
stratified sheet of corneal epithelial cells that was
detached in Example 3. Thereafter, the culture medium used
in Example 3 was gently placed to detach the polymer
membrane out of close contact with the cell sheet. Kept
this way, the cells were cultivated for 2 days to make a
stratified sheet (three-dimensional structure) of corneal
epithelial cells having the corneal endothelial cell layer.
The stratified sheet of corneal epithelial cells was given
the same low-temperature treatment as in Example 3,
whereupon it was detached from the surface of the support.
The detached three-dimensional structure, retaining the
intercellular desmosome structure and the basement
membrane-like protein between cell and substrate, had
satisfactory strength as a single sheet.
Example 8
The corneal endothelial cell sheet on the culture
dish of Example 4 from which the medium had been gently
removed without cooling was fed with a 5% IV type,
dissolved collagen containing medium (the same as the
medium used in Example 4, except that it contained
collagen) and left to stand as such for 20 minutes.
Thereafter, the medium was again gently removed without
cooling. The remaining corneal endothelial cell sheet on
the culture dish was immediately overlaid with the
stratified sheet of corneal epithelial cells that was
detached in Example 3. Thereafter, the culture medium used
in Example 3 was gently placed to detach the polymer
membrane out of close contact with the cell sheet. Kept
this way, the cells were cultivated for 2 days to make a
stratified sheet (three-dimensional structure) of corneal
epithelial cells having the corneal endothelial cell layer.
The stratified sheet of corneal epithelial cells was given
the same low-temperature treatment as in Example 3,
whereupon it was detached from the surface of the support.
The detached three-dimensional structure had satisfactory
strength as a single sheet.
Example 9
The perforated, conjunctival epithelial cell sheet on
the culture dish of Example 5 from which the medium had
been gently removed without cooling was immediately
overlaid partly with the stratified sheet (threedimensional
structure) of corneal epithelial cells having
the corneal endothelial cell layer that was detached in
Example 7. Thereafter, the culture medium used in Example
3 was gently placed to detach the polymer membrane out of
close contact with the cell sheet. Kept this way, the
cells were cultivated for 2 days to make a stratified sheet
(three-dimensional structure) of corneal epithelial cells
having the corneal endothelial cell layer to which the
conjunctival epithelial cell sheet had adhered. The
obtained three-dimensional structure was given the same
low-temperature treatment as in Example 3, whereupon it was
detached from the surface of the support. The detached
three-dimensional structure had satisfactory strength as a
single sheet.
Example 10
The stratified sheet (three-dimensional structure) of
corneal epithelial cells obtained in Example 3 was grafted
to a rabbit deficient of a corneal epithelial cell portion
in accordance with the usual method. After grafting, the
stratified sheet of corneal epithelial cells was sutured to
the wound site. About 3 weeks later, the suture was
removed and the stratified sheet of corneal epithelial
cells had took well on the eyeball.
From the foregoing results, it became clear that
using the procedure of the present invention, one can
fabricate satisfactorily strong sheets solely from
intraocular cells. This is believed to provide a very
effective technique for reducing the burden on patients by
making the treatment protocol more efficient, and for
constructing even more precise tissues.
INDUSTRIAL APPLICABILITY
The anterior ocular segment related cell sheets or
three-dimensional structures of the present invention will
not decompose E-cadherin or laminin 5, as opposed to the
case of dispase treatment, and yet they have extremely
small numbers of structural defects, thus having a great
potential for use in clinical applications including skin
grafting. Hence, the present invention will prove very
useful in medical and biological fields such as cell
engineering and medical engineering.

We claim:
1. A process for producing an anterior ocular segment related cell sheet or three-dimensional structure comprising the steps of
cultivating corneal epithelial cells in layers wherein said layers are solely composed of anterior ocular segment related cells of at least one of corneal epithelial cells, corneal endothelial cells, conjunctival epithelial cells and epithelial stem cells and which have only a few structural defects as they have been recovered retaining the intercellular desmosome structure and the basement membrane-like protein between cell and substrate and having satisfactory strength as a single sheet
said cell culture support comprising a substrate having its surface covered with a temperature responsive polymer such as herein described having an upper or lower critical dissolution temperature of 0-80°C with respect to water, optionally stratifying the layer of cultured cells, and thereafter,
(1) adjusting the temperature of the culture solution
to either above the upper critical dissolution temperature
or below the lower critical dissolution temperature, and
further optionally
(2) bringing the cultured anterior ocular segment
related cell sheet or a stratified sheet thereof into close
contact with a polymer membrane, and
(3) detaching the sheet or stratified sheet together

with the polymer membrane.
2. The process for producing the anterior ocular"segment related cell sheet or three-dimensional structure as claimed in claim 1, wherein the three-dimensional structure is a combination of at least a corneal epithelial cell sheet or a stratified product thereof with corneal endothelial cells.
3. The process for producing the anterior ocular segment related cell sheet or three-dimensional structure as claimed in claim 1, wherein the three-dimensional structure is a combination of the three-dimensional structure as claimed in claim 2 with conjunctival epithelial cells as a third component.
4. The process for producing an anterior ocular segment related cell sheet or three-dimensional structure as claimed in claim 1, wherein the cell culture support uses a material having two regions, region A covered with the temperature responsive polymer, and the following region B on its surface:
(1) a region covered with a polymer having less
affinity for cells;
(2) a region covered with a different amount of the temperature responsive polymer than in region A;
(3) a region covered with a polymer responsive to a different temperature than in region A; or a combination of

any two of regions (l)-(3) or a combination of the three.
5. The process for producing a three-dimensional structure as claimed in claim 1 or 4, further comprising attaching in superposition the anterior ocular segment related cell sheet or three-dimensional structure obtained in claim 1 or 4 to a cell culture support, with or without being covered on a surface with a temperature responsive polymer, a polymer membrane or the like, or alternatively attaching in superposition, either partly or entirely, the anterior ocular segment related cell sheet or three-dimensional structure to another cell sheet or the like.
6. The process for producing an anterior ocular segment related cell sheet or three-dimensional structure as claimed in claim 1, 4 or 5, wherein the sheet or three-dimensional structure is detached without treatment with a proteinase.
7. The process for producing an anterior ocular segment related cell sheet or three-dimensional structure as claimed in claim 1, 4 or 5, wherein the temperature responsive polymer is poly(N-isopropylacrylamide).
8. The process for producing an anterior ocular segment related cell sheet or three-dimensional structure as claimed in claim 1, 4 or 5, wherein the polymer membrane is made of polyvinylidene difluoride rendered hydrophilic.

9. The process for producing a three-dimensional structure as claimed in claim 5, wherein the another cell sheet is at least one member of the group consisting of a corneal epithelial cell sheet or a stratified sheet of corneal epithelial cells, a corneal endothelial cell sheet, a conjunctival epithelial cell sheet, and the three-dimensional structure produced by the method- referred to in claim 1 or 4.

Documents:

3967-DELNP-2005-Abstract-(20-04-2009).pdf

3967-delnp-2005-abstract.pdf

3967-DELNP-2005-Claims-(16-06-2009).pdf

3967-DELNP-2005-Claims-(20-04-2009).pdf

3967-delnp-2005-claims.pdf

3967-DELNP-2005-Correspondence-Others-(16-06-2009).pdf

3967-DELNP-2005-Correspondence-Others-(20-04-2009).pdf

3967-DELNP-2005-Correspondence-Others-(22-04-2009).pdf

3967-delnp-2005-correspondence-others.pdf

3967-delnp-2005-description (complete).pdf

3967-DELNP-2005-Form-1-(20-04-2009).pdf

3967-delnp-2005-form-1.pdf

3967-delnp-2005-form-13.pdf

3967-delnp-2005-form-18.pdf

3967-DELNP-2005-Form-2-(20-04-2009).pdf

3967-delnp-2005-form-2.pdf

3967-DELNP-2005-Form-3-(22-04-2009).pdf

3967-delnp-2005-form-3.pdf

3967-delnp-2005-form-5.pdf

3967-delnp-2005-gpa.pdf

3967-DELNP-2005-Others-Document-(22-04-2009).pdf

3967-delnp-2005-pct-210.pdf

3967-DELNP-2005-Petition-137-(22-04-2009).pdf

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Patent Number

234916

Indian Patent Application Number

3967/DELNP/2005

PG Journal Number

31/2009

Publication Date

31-Jul-2009

Grant Date

19-Jun-2009

Date of Filing

05-Sep-2005

Name of Patentee

CELLSEED INC.

Applicant Address

33-8, WAKAMATSU-CHO, SHINJUKU-KU, TOKYO 158-0097, JAPAN

Inventors:

#	Inventor's Name	Inventor's Address
1	YAMATO MASAYUKI	2-28-16, YOUGA, SETAGAYA-KU, TOKYO 158-0097, JAPAN
2	OKANO TERUO	6-12-12, KOUNODAI, ICHIKAWA-SHI, CHIBA 272-0827, JAPAN.

PCT International Classification Number

C12N 5/06

PCT International Application Number

PCT/JP2003/001248

PCT International Filing date

2003-02-06

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	PCT/JP03/001248	2003-02-06	Japan