Title of Invention	A PROCESS FOR GROWING MESENCHYMAL STEM CELLS [MSCS] AND A PROCESS THEREOF
Abstract	Various limitations exist in prior art towards isolating and culturing of Mesenchymal Stem Cells [MSCs] from various sources, including umbilical cord. However, the present disclosure is successful in addressing the issues associated with isolating and culturing of MSCs from umbilical cord. More vitally, the disclosure provides a step of decontaminating the umbilical cord using an antibiotic in ringer lactate solution. Accordingly, the MSCs obtained as per the present disclosure have several applications both in clinical research and also in various therapeutic areas, including the regenerative medicine. The novelty and inventive step lies in the combination of all the processing steps involved in isolating and culturing of MSCs. The success rate of the method of instant disclosure was found to be very high when compared with the existing conventional methods.

Title of Invention

A PROCESS FOR GROWING MESENCHYMAL STEM CELLS [MSCS] AND A PROCESS THEREOF

Abstract

Various limitations exist in prior art towards isolating and culturing of Mesenchymal Stem Cells [MSCs] from various sources, including umbilical cord. However, the present disclosure is successful in addressing the issues associated with isolating and culturing of MSCs from umbilical cord. More vitally, the disclosure provides a step of decontaminating the umbilical cord using an antibiotic in ringer lactate solution. Accordingly, the MSCs obtained as per the present disclosure have several applications both in clinical research and also in various therapeutic areas, including the regenerative medicine. The novelty and inventive step lies in the combination of all the processing steps involved in isolating and culturing of MSCs. The success rate of the method of instant disclosure was found to be very high when compared with the existing conventional methods.

Full Text	TECHNICAL FIELD The present disclosure relates to a method for isolating and culturing Mesenchymal Stem Cells [MSCs] using suitable source and that is the best for clinical and research applications. BACKGROUND Precursor cells isolated from non-embryonic tissues and capable of differentiating in to non-hematopoietic specialized cells have been generically termed Mesenchymal Stem Cells (MSCs). In other words, MSCs refer to primitive cells that are able to differentiate into bone, cartilage, adipose, nerve and muscle, for example, and are known as being contained in a large amount in bone marrow. Some of the characteristic features of MSCs include the first one that they must be plastic-adherent when maintained in standard culture conditions. Second, they must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CDllb, CD79a or CD 19 and HLA-DR surface molecules. Third, they must differentiate in to osteoblasts, adipocytes and chondroblasts in-vitro. In today's scenario, MSCs are isolated from a source called 'bone marrow' of the subjects such as human beings. However, such a source is associated with an issue of immunological rejection reaction. As an alternative source, umbilical cord is used and this is relatively easy to obtain when compared with bone marrow. During the process of child birth, it is well established in the art that the umbilical cord is one of the first structures to form following gastrulation (formation of the three embryonic germ layers). As folding is initiated, the embryonic disc becomes connected, by the primitive mid gut (embryonic origin) to the primitive yolk sac (extra embryonic origin) via the vital line and allantoic vessels which in turn develop to form the umbilical vessels. These vessels are supported in, and surrounded by, what is generally considered a primitive mesenchymal tissue of primarily extra-embryonic derivation called Wharton's Jelly (WJ). From this early stage, the umbilical cord grows, during gestation, to become the 30-50 cm cord seen at birth. It can be expected therefore, that Wharton's Jelly contains not only the fibroblast-like, or myo-fibroblast-like cells which have been described in the literature (see below), but also populations of progenitor cells which can give rise to the cells of the expanding volume of Wharton's Jelly necessary to support the growth of the cord during embryonic and fetal development. Wharton's Jelly was first described by Thomas Wharton, who published his treatise Adenographia in (1656) (Wharton T W. Adenographia, Translated by Freer (1996). Oxford, U.K.: Oxford University Press, 1656; 242-248). It has subsequently been defined as a gelatinous, loose mucous connective tissue composed of cells dispersed in an amorphous ground substance composed of proteoglycans, including hyaluronic acid and different types of collagens. In addition, usage of umbilical cord as a source for MSCs will help in employing umbilical cord stem cells that are identical with or most similar to histocompatibility genes of patients and thereby it is possible to solve problems associated with immunological rejection. The major limitations to the utilization of MSCs for therapeutic purposes arise during clinical practice, namely during cell harvesting. Culture expansion of MSCs is a prerequisite since high absolute numbers of stem cells are required to attain a clinical dose. Collection of MSCs involves invasive methodologies to the donor, such as surgical procedures that might even involve general anesthesia, like in the case of stem cells from bone marrow for example. Furthermore, because mesenchymal stem cells are generally rare, the final number of cells obtained is usually low. Furthermore, the "adult" character of most types of MSCs also presents some problems related to lack of phenotype maintenance upon expansion. This seems to be mainly caused by genetic instability derived from "aged" DNA. As an alternative, "young" foetal tissues, such as umbilical cord tissues, have been described as possible sources for MSCs. Further, the major problems in culturing cord lining derived mesenchymal stem cells is the sterility of cord, since most of the time it is got through normal vaginal delivery of the child. In this process the cord gets contaminated with vaginal flora / fauna. This leads to contamination of cultured MSCs making it unfit for clinical use. OBJECTIVES The first objective of the present disclosure is to provide a process for growing mesenchymal stem cells for various clinical and research applications, including for various therapeutic purposes. Another objective of the present disclosure is to provide a method for isolating and culturing mesenchymal stem cells from sources including umbilical cord from subjects including humans, but not limiting to it. Yet another objective of the present disclosure is to provide a simple and effective decontamination process for sources of mesenchymal stem cells including human umbilical cord. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Figure 1: Umbilical cord section showing the compartments containing mesenchymal stem cell, (A) Umbilical Cord Blood (B) Umbilical Vein Endothelium (C) Amnion (D) Wharton's jelly (E) Umbilical Cord Perivascular cells Figure 2: Cultured mesenchymal cell SUMMARY OF THE DISCLOSURE Accordingly, the present disclosure is in relation to a process for growing mesenchymal stem cells for therapeutic purposes, said process comprising steps of collecting source of mesenchymal stem cells from a subject; washing the source of mesenchymal stem cells for surface decontamination; and isolating and culturing the mesenchymal stem cells. There is also provided a method for isolating and culturing mesenchymal stem cells, said process comprising steps of collecting and washing source of mesenchymal stem cells for surface decontamination; releasing entrapped mesenchymal cells from collagen fibers by pipeting; allowing fragments of the collagen fibers to settle down followed by removing supernatant containing mesenchymal stem cells; and washing the supernatant and culturing the mesenchymal stem cells. DETAILED DESCRIPTION OF THE DISCLOSURE Before explaining any one embodiment of the present disclosure by way of drawings, experimentation, results, and pertinent procedures, it is to be understood that the disclosure is not limited in its application to the details as explained in below embodiments set forth in the following description or illustrated in the drawings, experimentation and/or results. The disclosure is further capable of other embodiments which can be practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary—not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. The present disclosure has been made in view of the above problems, recited under background, associated in culturing mesenchymal stem cells. Therefore it is an purpose of the present disclosure to provide a reproducible method for isolating and culturing mesenchymal stem cells, in which umbilical cord derived mesenchymal stem cells can be obtained without the loss of cells in the course of density gradient centrifugation. The present disclosure is in relation to a process for growing mesenchymal stem cells for therapeutic purposes, said process comprising steps of collecting source of mesenchymal stem cells from a subject; washing the source of mesenchymal stem cells for surface decontamination; and isolating and culturing the mesenchymal stem cells. In an illustrative embodiment the source of mesenchymal stem cells is umbilical cord from subjects including human beings but not limiting to it. In an experimental embodiment the decontamination is performed by washing the source of mesenchymal stem cells with gentamycin containing ringer lactate solution followed by dousing in alcohol for a time period ranging from about 50 seconds to 80 seconds, preferably for about 60 seconds. In an illustrative embodiment the decontamination and dousing steps are followed by air drying for a time period ranging from about 2 minutes to 4 minutes, preferably for about 3 minutes. The present disclosure is in relation to a method for isolating and culturing mesenchymal stem cells, said process comprising steps of collecting and washing source of mesenchymal stem cells for surface decontamination; releasing entrapped mesenchymal cells from collagen fibers by pipeting; allowing fragments of the collagen fibers to settle down followed by removing supernatant containing mesenchymal stem cells; and washing the supernatant and culturing the mesenchymal stem cells. In an illustrative embodiment the source of mesenchymal stem cells is umbilical cord from subjects including human beings but not limiting to it. In an experimental embodiment the said decontamination is performed by washing the source of mesenchymal stem cells with gentamycin containing ringer lactate solution followed by dousing in alcohol for a time period ranging from about 50 seconds to 80 seconds, preferably for about 60 seconds. In an illustrative embodiment the the supernatant is washed twice with ringer lactate followed by centrifuging at about 1000g at room temperature. In an illustrative embodiment the cell pellet is counted by haemocytometer and viability checked by trypan blue exclusion assay. In an illustrative embodiment the said culturing is carried out using low glucose dulbecco's modified essential medium and foetal bovine serum. Traditionally "Umbilical Cord" is discarded after division from the infant at birth, the umbilical cord is composed of a vein and two arteries surrounded by a sticky, jelly-like substance called Wharton's jelly, all encased in the surrounding tissue. The cord varies in length from inches to over three feet in length and is highly flexible. The umbilical cord is fetal tissue in a primitive state, giving it the advantage that antigenicity is lower than in adult tissue. As per figure 1, umbilical cord section showing the compartments containing mesenchymal stem cell, (A) Umbilical Cord Blood (B) Umbilical Vein Endothelium (c ) Amnion (D) Wharton's jelly (E) Umbilical Cord Perivascular cells. Umbilical cord which develops during gestation of the human embryo is the main structure which provides oxygenated blood and nutrition, while removing metabolic waste to the developing fetus; it is predominantly made up of collagen which is made by mesenchymal cells. Mesenchymal stem cells (MSCs) with their multilineage developmental plasticity comprise a promising tool for regenerative cell based therapy. Despite important biological properties which the MSCs from different sources share, the differences ii between them are poorly understood. Understanding their propensity to differentiate to different I lineages is fundamental for the development of successful cell-based therapies. Cells isolated from Wharton's jelly, referred to as umbilical cord matrix stromal (UCMS) cells, adhere to a tissue-culture plastic substrate, express mesenchymal stromal cell (MSC) surface markers, self-renew, and are multipotent (differentiate into bone, fat, cartilage, etc.) in-vitro. These properties support the notion that umbilical cord matrix stromal cells are a member of the MSC family. Here we compared the different culture conditions for long-term expansion of human MSCs isolated from the Wharton's jelly (WJ) of the umbilical cord while preserving their stem cell characteristics and differentiation potential. We find that StemPro and DMEM-F12 (Low Glucose) are superior as compared to the other media tested in supporting the in-vitro expansion of the WJ-MSCs. Hereinafter, a method of isolating and culturing mesenchymal stem cells from umbilical cord, in accordance with the present invention will be described in more detail with reference to the following examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention. Additionally, the disclosure is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope of the present invention. On the contrary, it is to be clearly understood that various other embodiments, modifications, and equivalents thereof, after reading the description herein in conjunction with the drawings and appended claims, may suggest themselves to those skilled in the art without departing from the spirit and scope of the presently disclosed and claimed invention. Example 1: Procuring and decontaminating the Umbilical Cord - a source of MSCs The umbilical cord [or any pertinent source] is usually obtained as a waste of child birth [or by any other practically feasible means]. It is one of the sources of MSCs. The sterility of the cord is of prime importance before proceeding with the isolation and culturing of MSCs. Based on the explanation provided under background and under detailed description it is evident that there are chances for the umbilical cord to get contaminated with vaginal flora/ fauna. By this contamination the culture of MSCs will be unfit for clinical and research applications. In order to prevent contamination, the umbilical cord obtained as a waste is subjected to washing with 'Gentamycin' containing ringer lactate solution. Gentamycin can be replaced with any other similar gentamycin class of antibiotics. Practically feasible concentration of Gentamycin needs to be used for the purpose of washing/ decontamination. This step is followed by dousing the cord with absolute alcohol for a time period ranging from about 50 seconds to 80 seconds and preferably for a time period of about 60 seconds. These steps ensure a complete or total surface decontamination. Example 2: Method for isolating and culturing MSCs The surface decontaminated umbilical cord as obtained in example 1 is subjected to isolation and culturing of MSCs wherein it involves harvesting of MSCs which are present in the perivascular space (around the vein and artery) as well as Wharton' s Jelly. The steps involve slitting opening of the cord and scrapping the fragments into ringer lactate solution placed in a petridish/ vessel. It is known that the mesenchymal cells are entrapped in collagen fiber fragments, as scrapped from the cord. Hence, in order to release them, they are subjected to pipetting several times. Thereafter, the fragments of collagen fibres, are allowed to settle. Over a period of time, the fragments get settled and the supernatant containing mesenchymal cells are carefully removed. As a next step, they are washed twice in Ringer Lactate by centrifugation at around l000g, at room temperature. The pellet is counted by haemocytometer and viability checked by Trypan Blue exclusion assay followed by culturing using low glucose DMEM and 20 percent certified foetal Bovine serum. Example 3; Characterization of Cell Surface Antigen of Cultured MSCs To determine the characteristics of cell surface antigen of the MSCs isolated and cultured as described as per examples 1 and 2, the cell surface antigen was analyzed by FACS. FACS (fluorescence activated cell sorting; a flow cytometer) is used to analyze the characteristics of cells by attaching a luminescent immune antigen indicator to the surface thereof, or to separate cells containing a certain antigen indicator depending. The stem cells isolated and cultured as per the present disclosure show negative reaction in CD34, CD45 and CD 14, which are characteristic indicator of hematopoietic stem cells, positive reaction in SH2, SH3, CD29 and CD44, which are characteristic indicator of mesenchymal stem cells. Accordingly, it is confirmed that the cells isolated and cultured in accordance with the present disclosure are mesenchymal stem cells. Example 4: Comparison of instant disclosure vis-a-vis the prior art When a comparison of the culture method of instant disclosure was made with the prior art or conventional methods, it was found that the success rate of MSCs culture of the conventional method is absolutely zero percentage, while that of the present disclosure is a high success rate of about 98 percent. Example 5: Industrial utility of the present disclosure Based on examples 1 to 4 and in view of the present disclosure, it is possible to effectively isolate and culture mesenchymal stem cells from umbilical cord, and therefore, umbilical cord which is wastefully disposed may be utilized as important means for isolating and culturing MSCs for clinical and research applications. In addition, the method of instant disclosure differs from the other published methods wherein the instant method does not use enzymatic digestion, especially collagenase. It is evident from prior art and based on the experience of the skilled artesian, collagenase treatment resultsin surface modifications which does not allow the mesenchymal stem cells to adhere to culture dish, spread and divide. The method provided in the present disclosure is superior to the collagenase treatment in that no residues, however small of the collagenase remains on the cells. Since Collagenase is of bacterial origin it has adverse affect when clinically administered. Figure no.2 provides cultured mesenchymal cell. While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims. We claim: 1) A process for growing mesenchymal stem cells for therapeutic purposes, said process comprising steps of: a) collecting source of mesenchymal stem cells from a subject; b) washing the source of mesenchymal stem cells for surface decontamination; and c) isolating and culturing the mesenchymal stem cells. 2) The process as claimed in claim 1, wherein said source of mesenchymal stem cells is umbilical cord from subjects including human beings but not limiting to it. 3) The process as claimed in claim 1, wherein said decontamination is performed by washing the source of mesenchymal stem cells with gentamycin containing ringer lactate solution followed by dousing in alcohol for a time period ranging from about 50 seconds to 80 seconds, preferably for about 60 seconds. 4) The process as claimed in claim 3, wherein the decontamination and dousing steps are followed by air drying for a time period ranging from about 2 minutes to 4 minutes, preferably for about 3 minutes. 5) A method for isolating and culturing mesenchymal stem cells, said process comprising steps of: a) collecting and washing source of mesenchymal stem cells for surface decontamination; b) releasing entrapped mesenchymal ceils from collagen fibers by pipeting; c) allowing fragments of the collagen fibers to settle down followed by removing supernatant containing mesenchymal stem cells; and d) washing the supernatant and culturing the mesenchymal stem cells. 6) The method as claimed in claim 5, wherein said source of mesenchymal stem cells is umbilical cord from subjects including human beings but not limiting to it. 7) The method as claimed in claim 5, wherein said decontamination is performed by washing the source of mesenchymal stem cells with gentamycin containing ringer lactate solution followed by dousing in alcohol for a time period ranging from about 50 seconds to 80 seconds, preferably for about 60 seconds. 8) The method as claimed in claim 5, wherein the supernatant is washed twice with ringer lactate followed by centrifuging at about 1000g at room temperature. 9) The method as claimed in claim 8, wherein cell pellet is counted by haemocytometer and viability checked by trypan blue exclusion assay. 10) The method as claimed in claim 5, wherein said culturing is carried out using low glucose dulbecco's modified essential medium and foetal bovine serum.

Full Text

TECHNICAL FIELD

The present disclosure relates to a method for isolating and culturing Mesenchymal Stem Cells [MSCs] using suitable source and that is the best for clinical and research applications.

BACKGROUND

Precursor cells isolated from non-embryonic tissues and capable of differentiating in to non-hematopoietic specialized cells have been generically termed Mesenchymal Stem Cells (MSCs). In other words, MSCs refer to primitive cells that are able to differentiate into bone, cartilage, adipose, nerve and muscle, for example, and are known as being contained in a large amount in bone marrow. Some of the characteristic features of MSCs include the first one that they must be plastic-adherent when maintained in standard culture conditions. Second, they must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CDllb, CD79a or CD 19 and HLA-DR surface molecules. Third, they must differentiate in to osteoblasts, adipocytes and chondroblasts in-vitro.

In today's scenario, MSCs are isolated from a source called 'bone marrow' of the subjects such as human beings. However, such a source is associated with an issue of immunological rejection reaction. As an alternative source, umbilical cord is used and this is relatively easy to obtain when compared with bone marrow. During the process of child birth, it is well established in the art that the umbilical cord is one of the first structures to form following gastrulation (formation of the three embryonic germ layers).

As folding is initiated, the embryonic disc becomes connected, by the primitive mid gut (embryonic origin) to the primitive yolk sac (extra embryonic origin) via the vital line and allantoic vessels which in turn develop to form the umbilical vessels. These vessels are supported in, and surrounded by, what is generally considered a primitive mesenchymal tissue of primarily extra-embryonic derivation called Wharton's Jelly (WJ). From this early stage, the umbilical cord grows, during gestation, to become the 30-50 cm cord seen at birth. It can be expected therefore, that Wharton's Jelly contains not only the fibroblast-like, or myo-fibroblast-like cells which have been described in the literature (see below), but also populations of progenitor cells which can give rise to the cells of the expanding volume of Wharton's Jelly necessary to support the growth of the cord during embryonic and fetal development. Wharton's Jelly was first described by Thomas Wharton, who published his treatise Adenographia in (1656) (Wharton T W.

Adenographia, Translated by Freer (1996). Oxford, U.K.: Oxford University Press, 1656; 242-248). It has subsequently been defined as a gelatinous, loose mucous connective tissue composed of cells dispersed in an amorphous ground substance composed of proteoglycans, including hyaluronic acid and different types of collagens. In addition, usage of umbilical cord as a source for MSCs will help in employing umbilical cord stem cells that are identical with or most similar to histocompatibility genes of patients and thereby it is possible to solve problems associated with immunological rejection.

The major limitations to the utilization of MSCs for therapeutic purposes arise during clinical practice, namely during cell harvesting. Culture expansion of MSCs is a prerequisite since high absolute numbers of stem cells are required to attain a clinical dose. Collection of MSCs involves invasive methodologies to the donor, such as surgical procedures that might even involve general anesthesia, like in the case of stem cells from bone marrow for example. Furthermore, because mesenchymal stem cells are generally rare, the final number of cells obtained is usually low. Furthermore, the "adult" character of most types of MSCs also presents some problems related to lack of phenotype maintenance upon expansion. This seems to be mainly caused by genetic instability derived from "aged" DNA. As an alternative, "young" foetal tissues, such as umbilical cord tissues, have been described as possible sources for MSCs. Further, the major problems in culturing cord lining derived mesenchymal stem cells is the sterility of cord, since most of the time it is got through normal vaginal delivery of the child. In this process the cord gets contaminated with vaginal flora / fauna. This leads to contamination of cultured MSCs making it unfit for clinical use.

OBJECTIVES

The first objective of the present disclosure is to provide a process for growing mesenchymal stem cells for various clinical and research applications, including for various therapeutic purposes.

Another objective of the present disclosure is to provide a method for isolating and culturing mesenchymal stem cells from sources including umbilical cord from subjects including humans, but not limiting to it.

Yet another objective of the present disclosure is to provide a simple and effective
decontamination process for sources of mesenchymal stem cells including human umbilical cord.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1: Umbilical cord section showing the compartments containing mesenchymal stem cell,

(A) Umbilical Cord Blood (B) Umbilical Vein Endothelium (C) Amnion (D) Wharton's jelly (E) Umbilical Cord Perivascular cells

Figure 2: Cultured mesenchymal cell

SUMMARY OF THE DISCLOSURE

Accordingly, the present disclosure is in relation to a process for growing mesenchymal stem cells for therapeutic purposes, said process comprising steps of collecting source of mesenchymal stem cells from a subject; washing the source of mesenchymal stem cells for surface decontamination; and isolating and culturing the mesenchymal stem cells. There is also provided a method for isolating and culturing mesenchymal stem cells, said process comprising steps of collecting and washing source of mesenchymal stem cells for surface decontamination;

releasing entrapped mesenchymal cells from collagen fibers by pipeting; allowing fragments of the collagen fibers to settle down followed by removing supernatant containing mesenchymal stem cells; and washing the supernatant and culturing the mesenchymal stem cells.

DETAILED DESCRIPTION OF THE DISCLOSURE

Before explaining any one embodiment of the present disclosure by way of drawings, experimentation, results, and pertinent procedures, it is to be understood that the disclosure is not limited in its application to the details as explained in below embodiments set forth in the following description or illustrated in the drawings, experimentation and/or results. The disclosure is further capable of other embodiments which can be practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary—not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

The present disclosure has been made in view of the above problems, recited under background, associated in culturing mesenchymal stem cells. Therefore it is an purpose of the present disclosure to provide a reproducible method for isolating and culturing mesenchymal stem cells, in which umbilical cord derived mesenchymal stem cells can be obtained without the loss of cells in the course of density gradient centrifugation.

The present disclosure is in relation to a process for growing mesenchymal stem cells for therapeutic purposes, said process comprising steps of collecting source of mesenchymal stem cells from a subject; washing the source of mesenchymal stem cells for surface decontamination; and isolating and culturing the mesenchymal stem cells.

In an illustrative embodiment the source of mesenchymal stem cells is umbilical cord from subjects including human beings but not limiting to it.

In an experimental embodiment the decontamination is performed by washing the source of mesenchymal stem cells with gentamycin containing ringer lactate solution followed by dousing in alcohol for a time period ranging from about 50 seconds to 80 seconds, preferably for about 60 seconds.

In an illustrative embodiment the decontamination and dousing steps are followed by air drying for a time period ranging from about 2 minutes to 4 minutes, preferably for about 3 minutes.

The present disclosure is in relation to a method for isolating and culturing mesenchymal stem cells, said process comprising steps of collecting and washing source of mesenchymal stem cells for surface decontamination; releasing entrapped mesenchymal cells from collagen fibers by pipeting; allowing fragments of the collagen fibers to settle down followed by removing supernatant containing mesenchymal stem cells; and washing the supernatant and culturing the mesenchymal stem cells.

In an illustrative embodiment the source of mesenchymal stem cells is umbilical cord from subjects including human beings but not limiting to it.

In an experimental embodiment the said decontamination is performed by washing the source of mesenchymal stem cells with gentamycin containing ringer lactate solution followed by dousing in alcohol for a time period ranging from about 50 seconds to 80 seconds, preferably for about 60 seconds.

In an illustrative embodiment the the supernatant is washed twice with ringer lactate followed by centrifuging at about 1000g at room temperature.

In an illustrative embodiment the cell pellet is counted by haemocytometer and viability checked by trypan blue exclusion assay.

In an illustrative embodiment the said culturing is carried out using low glucose dulbecco's modified essential medium and foetal bovine serum.

Traditionally "Umbilical Cord" is discarded after division from the infant at birth, the umbilical cord is composed of a vein and two arteries surrounded by a sticky, jelly-like substance called

Wharton's jelly, all encased in the surrounding tissue. The cord varies in length from inches to over three feet in length and is highly flexible. The umbilical cord is fetal tissue in a primitive state, giving it the advantage that antigenicity is lower than in adult tissue. As per figure 1, umbilical cord section showing the compartments containing mesenchymal stem cell, (A)

Umbilical Cord Blood (B) Umbilical Vein Endothelium (c ) Amnion (D) Wharton's jelly
(E) Umbilical Cord Perivascular cells. Umbilical cord which develops during gestation of the human embryo is the main structure which provides oxygenated blood and nutrition, while removing metabolic waste to the developing fetus; it is predominantly made up of collagen which is made by mesenchymal cells. Mesenchymal stem cells (MSCs) with their multilineage developmental plasticity comprise a promising tool for regenerative cell based therapy.

Despite important biological properties which the MSCs from different sources share, the differences ii between them are poorly understood. Understanding their propensity to differentiate to different I lineages is fundamental for the development of successful cell-based therapies.

Cells isolated from Wharton's jelly, referred to as umbilical cord matrix stromal (UCMS) cells, adhere to a tissue-culture plastic substrate, express mesenchymal stromal cell (MSC) surface markers, self-renew, and are multipotent (differentiate into bone, fat, cartilage, etc.) in-vitro.

These properties support the notion that umbilical cord matrix stromal cells are a member of the MSC family. Here we compared the different culture conditions for long-term expansion of human MSCs isolated from the Wharton's jelly (WJ) of the umbilical cord while preserving their stem cell characteristics and differentiation potential. We find that StemPro and DMEM-F12

(Low Glucose) are superior as compared to the other media tested in supporting the in-vitro expansion of the WJ-MSCs.

Hereinafter, a method of isolating and culturing mesenchymal stem cells from umbilical cord, in accordance with the present invention will be described in more detail with reference to the following examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.

Additionally, the disclosure is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope of the present invention. On the contrary, it is to be clearly understood that various other embodiments, modifications, and equivalents thereof, after reading the description herein in conjunction with the drawings and appended claims, may suggest themselves to those skilled in the art without departing from the spirit and scope of the presently disclosed and claimed invention.

Example 1: Procuring and decontaminating the Umbilical Cord - a source of MSCs

The umbilical cord [or any pertinent source] is usually obtained as a waste of child birth [or by any other practically feasible means]. It is one of the sources of MSCs. The sterility of the cord is of prime importance before proceeding with the isolation and culturing of MSCs. Based on the explanation provided under background and under detailed description it is evident that there are chances for the umbilical cord to get contaminated with vaginal flora/ fauna. By this contamination the culture of MSCs will be unfit for clinical and research applications. In order to prevent contamination, the umbilical cord obtained as a waste is subjected to washing with 'Gentamycin' containing ringer lactate solution. Gentamycin can be replaced with any other similar gentamycin class of antibiotics. Practically feasible concentration of Gentamycin needs to be used for the purpose of washing/ decontamination. This step is followed by dousing the cord with absolute alcohol for a time period ranging from about 50 seconds to 80 seconds and preferably for a time period of about 60 seconds. These steps ensure a complete or total surface decontamination.

Example 2: Method for isolating and culturing MSCs

The surface decontaminated umbilical cord as obtained in example 1 is subjected to isolation and culturing of MSCs wherein it involves harvesting of MSCs which are present in the perivascular space (around the vein and artery) as well as Wharton' s Jelly. The steps involve slitting opening of the cord and scrapping the fragments into ringer lactate solution placed in a petridish/ vessel. It is known that the mesenchymal cells are entrapped in collagen fiber fragments, as scrapped from the cord. Hence, in order to release them, they are subjected to pipetting several times. Thereafter, the fragments of collagen fibres, are allowed to settle. Over a period of time, the fragments get settled and the supernatant containing mesenchymal cells are carefully removed. As a next step, they are washed twice in Ringer Lactate by centrifugation at around l000g, at room temperature. The pellet is counted by haemocytometer and viability checked by Trypan Blue exclusion assay followed by culturing using low glucose DMEM and 20 percent certified foetal Bovine serum.

Example 3; Characterization of Cell Surface Antigen of Cultured MSCs

To determine the characteristics of cell surface antigen of the MSCs isolated and cultured as described as per examples 1 and 2, the cell surface antigen was analyzed by FACS. FACS (fluorescence activated cell sorting; a flow cytometer) is used to analyze the characteristics of cells by attaching a luminescent immune antigen indicator to the surface thereof, or to separate cells containing a certain antigen indicator depending. The stem cells isolated and cultured as per the present disclosure show negative reaction in CD34, CD45 and CD 14, which are characteristic indicator of hematopoietic stem cells, positive reaction in SH2, SH3, CD29 and CD44, which are characteristic indicator of mesenchymal stem cells. Accordingly, it is confirmed that the cells isolated and cultured in accordance with the present disclosure are mesenchymal stem cells.

Example 4: Comparison of instant disclosure vis-a-vis the prior art

When a comparison of the culture method of instant disclosure was made with the prior art or conventional methods, it was found that the success rate of MSCs culture of the conventional method is absolutely zero percentage, while that of the present disclosure is a high success rate of about 98 percent.

Example 5: Industrial utility of the present disclosure

Based on examples 1 to 4 and in view of the present disclosure, it is possible to effectively isolate and culture mesenchymal stem cells from umbilical cord, and therefore, umbilical cord which is wastefully disposed may be utilized as important means for isolating and culturing MSCs for clinical and research applications.

In addition, the method of instant disclosure differs from the other published methods wherein the instant method does not use enzymatic digestion, especially collagenase. It is evident from prior art and based on the experience of the skilled artesian, collagenase treatment resultsin surface modifications which does not allow the mesenchymal stem cells to adhere to culture dish, spread and divide. The method provided in the present disclosure is superior to the collagenase treatment in that no residues, however small of the collagenase remains on the cells.

Since Collagenase is of bacterial origin it has adverse affect when clinically administered. Figure no.2 provides cultured mesenchymal cell.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

We claim:

1) A process for growing mesenchymal stem cells for therapeutic purposes, said process comprising steps of:

a) collecting source of mesenchymal stem cells from a subject;

b) washing the source of mesenchymal stem cells for surface decontamination; and

c) isolating and culturing the mesenchymal stem cells.

2) The process as claimed in claim 1, wherein said source of mesenchymal stem cells is umbilical cord from subjects including human beings but not limiting to it.

3) The process as claimed in claim 1, wherein said decontamination is performed by washing the source of mesenchymal stem cells with gentamycin containing ringer lactate solution followed by dousing in alcohol for a time period ranging from about 50 seconds to 80 seconds, preferably for about 60 seconds.

4) The process as claimed in claim 3, wherein the decontamination and dousing steps are followed by air drying for a time period ranging from about 2 minutes to 4 minutes, preferably for about 3 minutes.

5) A method for isolating and culturing mesenchymal stem cells, said process comprising steps of:

a) collecting and washing source of mesenchymal stem cells for surface decontamination;

b) releasing entrapped mesenchymal ceils from collagen fibers by pipeting;

c) allowing fragments of the collagen fibers to settle down followed by removing supernatant containing mesenchymal stem cells; and
d) washing the supernatant and culturing the mesenchymal stem cells.

6) The method as claimed in claim 5, wherein said source of mesenchymal stem cells is umbilical cord from subjects including human beings but not limiting to it.

7) The method as claimed in claim 5, wherein said decontamination is performed by washing the source of mesenchymal stem cells with gentamycin containing ringer lactate solution followed by dousing in alcohol for a time period ranging from about 50 seconds to 80 seconds, preferably for about 60 seconds.

8) The method as claimed in claim 5, wherein the supernatant is washed twice with ringer lactate followed by centrifuging at about 1000g at room temperature.

9) The method as claimed in claim 8, wherein cell pellet is counted by haemocytometer and viability checked by trypan blue exclusion assay.

10) The method as claimed in claim 5, wherein said culturing is carried out using low glucose dulbecco's modified essential medium and foetal bovine serum.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=nxg9y+QiIqzXOKK5gSru8w==&loc=egcICQiyoj82NGgGrC5ChA==

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Patent Number

272353

Indian Patent Application Number

132/CHE/2010

PG Journal Number

14/2016

Publication Date

01-Apr-2016

Grant Date

30-Mar-2016

Date of Filing

20-Jan-2010

Name of Patentee

SRI RAGHAVENDRA BIOTECHNOLOGIES PVT. LIMITED

Applicant Address

NO: 117, VENKATESHPURA, NEAR SAMPIGE HALLI, JAKKUR POST, BANGALORE-560 064

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. SRIMUSHNAM GOPALAKRISHNA ANAND RAO	NO: 117, VENKATESHPURA, NEAR SAMPIGE HALLI, JAKKUR POST, BANGALORE-560 064
2	DR. JYOTHSNA ANAND RAO	NO: 117, VENKATESHPURA, NEAR SAMPIGE HALLI, JAKKUR POST, BANGALORE-560 064
3	PURUSHOTHAMA HANUMANTHAIAH	NO: 18, KARAGADA MAHALAKSHMI NILAYA, CHIKKANAYA LAYOUT, HENNUR, KALYAN NAGAR POST, BANGALORE-560 043.
4	DR. GURURAJ ANAND RAO	NO: 117, VENKATESHPURA, NEAR SAMPIGE HALLI, JAKKUR POST, BANGALORE-560 064

PCT International Classification Number

C12N

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA