WO2014203269A2

WO2014203269A2 - Method for isolation, purification and industrial scale expansion of canine adipose tissue derived mesenchymal stem cells

Info

Publication number: WO2014203269A2
Application number: PCT/IN2013/000584
Authority: WO
Inventors: Satish Mahadeorao Totey; Manoj Kumar C REDDY; Lyle Carl FONSECA; Aarya HARI; Basavaraj CHOUGULE; Minita SODHI
Original assignee: KASIAK RESEARCH PVT Ltd
Current assignee: KASIAK RESEARCH PVT Ltd
Priority date: 2013-06-17
Filing date: 2013-09-26
Publication date: 2014-12-24
Anticipated expiration: 2015-12-17
Also published as: IN2013MU02046A; WO2014203269A3

Abstract

The invention relates to a method for isolation, purification and industrial scale expansion of canine adipose tissue derived mesenchymal stem cells (MSCs). The invention also relates to a method for treating and a therapeutic product for treating osteoarthritis, spinal cord injury, atopic dermatitis, dilated cardiomyopathy, type-1 diabetes, renal failure, hepatic disease and non healing wounds comprising MSCs.

Description

TITLE OF THE INVENTION

Method for isolation, purification and industrial scale expansion of canine adipose tissue derived mesenchymal stem cells

FIELD OF THE INVENTION

This invention relates to a method for isolation, purification and industrial scale expansion of clinical grade canine adipose tissue derived mesenchymal stem cells and to characterization of and uses for such cells.

BACKGROUND OF THE INVENTION

Stem cells are unspecialized cells that have two defining properties: the ability to differentiate into other cells and the ability to self-regenerate. The ability to differentiate is the potential to develop into other cell types.

Canine Mesenchymal Stem Cells (MSCs) are multi-potent and can be obtained from various tissues such as bone marrow, adipose tissue, umbilical cord, dental pulp etc.

Adipose tissue is an abundant source of MSCs, which have shown promise in the field of regenerative medicine. Adipose tissue is a simple and painless source of obtaining stem cells as compared to bone marrow and other tissues. Furthermore, MSCs from adipose tissue can be readily harvested in large numbers with low donor-site morbidity. During the past decade, numerous studies have provided preclinical data on the safety and efficacy of adipose-derived stem cells, supporting the use of these cells in future clinical applications.

MSCs isolated from canine adipose tissue possess different cytokines, proteins and express different genes as compared to MSCs derived from bone marrow, umbilical cord, and dental tissue. Canine adipose tissue-derived MSCs have a unique secretory profile, multi-potency, high yield, ease of availability, and self-regenerating ability. These MSCs can be maintained and propagated in culture without them losing their characteristics, thereby yielding large numbers of MSCs in fewer population doublings keeping them safe, potent and stable as appropriate for various clinical applications in canines. Since these MSCs are known to be homogeneous populations, with stable and consistent phenotypic and genotypic characteristics which aid in homing to the site of injury, these MSCs have a potential in the treatment of several clinical conditions.

Osteoarthritis (OA) is one of the most common causes of chronic pain in dogs. Studies have shown that more than 20% of dogs suffer from OA with the most common signs being pain, stiffness and loss of mobility.

Medical management with non-steroidal anti-inflammatory drugs (NSAIDs) may not provide complete pain relief in many dogs. Other sought-after modalities to augment NSAID therapy include additional pain medications, dietary supplements, and joint injections. A new and rapidly growing area of research in the treatment of OA involves the use of regenerative medicine Using this approach, MSCs are delivered to an area of damaged tissue where they stimulate regeneration and aid in repair of the damaged tissue.

Similarly, conditions like atopic dermatitis, dilated cardiomyopathy, type-I diabetes, spinal cord injury, renal failure and hepatic diseases are the most common conditions in dogs for which veterinarians have no specific treatment. Adipose derived MSCs have a promising application in these conditions to repair tissue and improve the function of damaged organs.

An important issue in stem cell therapy is to attain consistent cell numbers, with phenotypically immune privileged actively replicating cells for administration and easy isolation. It is difficult to aspirate bone marrow from an animal whereas isolation of a small piece of adipose tissue from the animal is easy and advantageous. Adipose tissue derived MSCs may also be more competent than bone marrow derived MSCs in terms of proliferative ability and they are more responsive to bFGF according to some studies. Also, canine bone marrow mesenchymal stem cells (BM-MSCs) appear to senesce much earlier than Adipose derived mesenchymal stem cells (AD-MSCs) and Umbilical cord mesenchymal stem cells (UC-MSCs). The limited passage numbers of sub-cultured BM-MSCs available for use suggests that adipose tissue and umbilical cord tissue may be preferable for therapeutic purposes. There are no reports in the literature for optimization of culture media so as to obtain a higher MSC yield. However, several nutrient media have been tried for culturing stromal vascular fraction for obtaining a homogeneous population of canine adipose MSCs. Media like DMEM- Low Glucose, DMEM/F12, DMEM - high glucose, F12, Alpha-MEM, LP02, supplemented with fetal bovine serum, knockout serum replacement, and serum replacement media result in variable and often low yields of MSCs which display variable morphology. These media have not successfully been used in industrial scale expansion of canine adipose tissue derived MSCs and are also not cost effective for producing therapeutic doses of MSCs.

In many clinical trials, efficacy has been shown to be related to the dose of MSCs administered, highlighting the need for an industrial scale process giving a high yield of MSCs. There is a high demand for canine MSCs for numerous therapeutic applications but insufficient availability in the market. There is also a need for an efficient culturing system that gives an optimum yield at an affordable cost thereby reducing the demand-supply gap.

There is also a need for an optimal method of isolating, purifying and ultimately expanding canine adipose tissue derived MSCs in the least possible passages and minimum population doublings in order to obtain highly potent and young clinical grade MSCs having multi lineage differentiation capacity, showing consistency in cell numbers and amenable to off-the-shelf clinical use.

SUMMARY OF THE INVENTION

According to an embodiment of the invention there is provided a method for isolation, purification and industrial scale expansion of canine adipose tissue derived mesenchymal stem cells (MSCs) to obtain a yield of 160,000 cells per cm² pure clinical grade MSCs for allogenic use comprising over 95% cells which express positive markers CD44, CD90, and CD 105, and less than 2% cells which express negative markers CD45, CD34 and HLA-DR, the method comprising the steps of:

a) extracting 5 to 8 grams of adipose tissue from multiple canine donors; b) digesting the adipose tissue from individual canine donors with collagenase, centrifuging the digested tissue and collecting the stromal vascular fraction (SVF);

c) seeding the cells contained in the SVF into a culture medium; and d) trypsinising and washing the cells once they reach confluence at passage

0;

e) pooling the cells obtained from the adipose tissue of multiple canine donors processed through steps (a) to (d);

f) seeding the pooled cells of passage 0 into a culture medium;

g) trypsinising and washing the cells once they reach 90 % confluence at passage 1 ;

h) seeding the cells of passage 1 into a culture medium; and i) trypsinising and washing the cells once they reach confluence at passage 2,

wherein,

• step (a) is performed by biopsy assisted removal;

• cells from stromal vascular fraction are seeded in step (c) only if at least 60% cells are positive for CD 90;

• the SVF cells are seeded in to the culture medium in step (c) at a seeding density of at least 75000 cells per sq cm;

• prior to seeding in step (f), the mesenchymal stem cells are characterized based on the percentage of cells which express positive markers CD44, CD90, and CD 105, and the percentage of cells which express negative markers CD45, CD34 and HLA-DR;

• the mesenchymal stem cells are seeded in to the culture medium in step (f) and step (h) at a seeding density of 1000 to 5000 cells per sq cm and express at least 95% of the positive markers CD44, CD90, and CD105 and at most 2% of the negative markers CD45, CD34 and HLA-DR;

• the culture medium comprises 25% to 75% Dulbecco's Modified Eagle's Medium- Knockout (DMEM-KO) and 25% to 75% alpha-Minimum Essential Medium (a-MEM) not excluding 100% DMEM-KO and 100% a-MEM; and

• prior to the trypsinising at step (d), (g) and (i) 70 to 90% of the culture medium is changed at 3 to 6 day intervals after seeding until confluence is reached.

According to another embodiment of the invention there is provided a method for isolation, purification and industrial scale expansion of canine adipose tissue derived mesenchymal stem cells (MSCs) to obtain a yield of 160,000 cells per cm² pure clinical grade MSCs for autologous use comprising over 95% cells which express positive markers CD44, CD90, and CD105, and less than 2% cells which express negative markers CD45, CD34 and HLA-DR, the method comprising the steps of:

a) extracting 5 to 8 grams of adipose tissue from a canine donor;

b) digesting the adipose tissue with collagenase, centrifuging the digested tissue and collecting the stromal vascular fraction (SVF);

c) seeding the cells contained in the SVF into a culture medium; and

d) trypsinising and washing the cells once they reach confluence at passage 0;

e) seeding the washed cells of passage 0 into a culture medium;

f) trypsinising and washing the cells once they reach confluence at passage 1 ;

g) seeding the cells of passage 1 into a culture medium; and

h) trypsinising and washing the cells once they reach confluence at passage 2, wherein,

• step (a) is performed by biopsy assisted removal;

• the cells are seeded in to the culture medium in step (c) at a seeding density of at least 75000 cells per sq cm;

• prior to seeding in step (e), the mesenchymal stem cells are characterized based on the percentage of cells which express positive markers CD44, CD90, and CD105, and the percentage of cells which express negative markers CD45, CD34 and HLA-DR;

• the mesenchymal stem cells are seeded in to the culture medium in step (e) and step (g) at a seeding density of 1000 to 5000 cells per sq cm and express at least 95% of the positive markers CD44, CD90, and CD 105 and at most 2% of the negative markers CD45, CD34 and HLA-DR;

• the culture medium comprises 25% to 75% Dulbecco's Modified Eagle's Medium- Knockout (DMEM-KO) and 25% to 75% alpha-Minimum Essential Medium (a-MEM) or upto 100%^" DMEM-KO or uptol00% a-MEM; and

• prior to the trypsinising at step (d), (f) and (h) 70 to 90% of the culture medium is changed at 3 to 6 day intervals after seeding until confluence is reached.

According to another embodiment of the invention there is provided a therapeutic product for treating osteoarthritis, spinal cord injury, atopic dermatitis, dilated cardiomyopathy, type-1 diabetes, renal failure, hepatic disease and non-healing wounds comprising MSCs suspended in multiple electrolyte solution supplemented with human serum albumin and dimethyl sulfoxide (DMSO) wherein, over 95% MSCs express positive markers CD44, CD90 and CD105, and less than 2% cells express negative markers CD45, CD34 and HLA-DR, and wherein the MSCs have undergone not more than 15 population doublings in vitro and are capable of at least 30 to 35 more population doublings, the MSCs are capable of differentiating into adipocytes, osteocytes and chondrocytes, the MSCs express pluripotent markers like OCT-4, SOX2 and NANOG; and secrete growth factors like TGF-β and VEGF; and show immunomodulatory activity.

It is to be understood that both the foregoing general description and the following detailed description of the present embodiments of the invention are intended to provide an overview or framework for understanding the nature and character of the invention as it is claimed. The accompanying graphical representations are included to substantiate the invention and are incorporated into and constitute a part of this specification. BRIEF DESCRIPTION OF THE DRAWINGS

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

FIG. 1A shows the relation between yield of MSCs and seeding density of adipose derived MSCs in passage-1 (Working cell bank) and FIG. I B shows the relation between yield of MSCs and seeding density of adipose derived MSCs in and passage-2 (Final product) in various media conditions.

FIG. 2 shows morphological characteristics of adipose derived MSCs.

FIG. 3 shows immunophenotypic characterization of adipose tissues derived MSCs obtained according to an embodiment of the invention.

FIG. 4 demonstrates the differentiation capacity of canine adipose tissue derived MSCs obtained according to an embodiment of the invention to differentiate into osteocytes, adipocytes and chondrocytes. FIG. 5 demonstrates expression of pluripotent markers like OCT4, SOX2, and NANOG in MSCs obtained according to an embodiment of the invention. FIG. 6 shows secretion of growth factors by the canine adipose derived MSCs obtained according to an embodiment of the invention.

FIG. 7 demonstrates the therapeutic efficacy of canine adipose derived MSCs obtained according to an embodiment of the invention in treating spinal cord injury in a dog.

FIG. 8 demonstrates the therapeutic efficacy of canine adipose derived MSCs obtained according to an embodiment of the invention in treating canine atopic dermatitis.

FIG. 9A and FIG. 9B respectively demonstrate the significant reduction in creatinine to basal level (9A) and blood urea nitrogen (9B) after stem cell injection indicating therapeutic efficacy of canine adipose derived MSCs obtained according to an embodiment of the invention in treating canine chronic renal failure.

FIG. 10 demonstrates the therapeutic efficacy of canine adipose derived MSCs obtained according to an embodiment of the invention in treating canine hip dysplasia.

FIG. 1 1 shows the immunomodulatory effect of canine adipose derived MSCs obtained according to an embodiment of the invention DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

For simplicity and illustrative purposes, the present invention is described by referring mainly to exemplary embodiments thereof. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one of ordinary skill in the art that the present invention may be practiced without limitation to these specific details. In other instances, well known methods have not been described in detail so as not to unnecessarily obscure the present invention. In the context of the invention, the term "combination media" as used in the specification refers to a culture media comprising 25% to 75% Dulbecco's Modified Eagle's Medium-Knockout (DMEM-KO) and 25% to 75% alpha-Minimum Essential Medium (a-MEM). In the context of the invention, the term "clinical grade" as used in the specification refers to MSCs obtained according to an embodiment of the invention and having the same efficacy and safety after isolation, purification and expansion as their parent MSCs.

In the context of the invention, the term "confluence" as used in the specification means approximately 80% to 90% confluence of cells attained during cell culture.

In the context of the invention, the term "multiple electrolyte solution" as used in the specification includes normal saline, Plasmalyte-A and/or Ringer lactate. The adipose tissue is preferably extracted from the dorsal gluteal muscle or the omentum of the donor.

The media changes were seen to help in knocking out undesired cells and toxic wastes. Only upto 90% of the media was changed and at least 10% of the spent media was left behind for the purpose of conditioning. The media change also helped to eliminate cells which were not MSCs as cells which are not MSCs do not adhere to the culture flasks/chambers. Preferably, the culture medium comprises 50% DMEM-KO + 50% a-MEM; 75% DMEM-KO + 25% a-MEM or 25% DMEM-KO + 75% a-MEM. More preferably, the culture medium comprises 25% DMEM-KO + 75% a-MEM.

According to yet another embodiment of the invention there is provided a method for treating spinal cord injury, atopic dermatitis, dilated cardiomyopathy, renal failure, hepatic disease, type- 1 diabetes, comprising administering 2 to 3 doses each of 5 to 20 million MSCs of the therapeutic product, by intravenous route or intra-articular route.

According to still another embodiment of the invention there is provided use of the therapeutic product for treating osteoarthritis, spinal cord injury, atopic dermatitis, dilated cardiomyopathy, type-1 diabetes, renal failure, hepatic disease and non-healing wounds. Optionally, the washed cells after trypsinisation at any step can be frozen in a freezing mixture comprising a cryoprotectant and stored under liquid nitrogen for subsequent use. The MSCs obtained in passage 0 can constitute a master cell bank (MCB), the MSCs obtained in passage 1 can constitute a working cell bank (WCB) and the MSCs obtained in passage 2 can constitute the clinical grade product. The cells obtained at the end of passage 0 can also be used for autologous clinical purposes. For allogenic product multiple donors cells are to be mixed together at passage-0 and seeded for getting working cell bank and expanded to passage 1 and further to passage 2. The maximum population doublings of adipose tissue derived MSCs is approximately 30. The final product in the present invention is administered at a total population doubling of 17 i.e at a stage when the MSCs are highly potent. For allogenic use, the cells obtained at the end of passage 2 from WCB are to be used as the product.

Clinical grade ready to use product is frozen in cryo vials containing 5 to 20 million cells suspended in multiple electrolyte solution supplemented with 10% injectable dimethyl sulfoxide (DMSO) as cryoprotectant and 5% injectable canine serum as a protein supplement.

The final product should be transported in liquid nitrogen charged dry shipper or dry ice for clinical use. Before transferring the cells to the dry shippers, the dry-shipper chamber should be saturated using liquid nitrogen. After saturation of the dry shipper with liquid nitrogen, excess liquid nitrogen is removed by decanting it from the dry shipper. The product which is to be transported is placed in a canister, which is provided in the dry shipper. The lid of the dry shipper is closed, the dry shipper is locked and sealed and transported to the site of administration within 7 to 9 days of charging. The dry shipper should not be exposed to direct sunlight, rain, or X-rays. After reaching the site of administration, the dry shipper should be placed at room temperature till the day of administration to the patient.

At the time of administration, the lid of the dry shipper is slowly opened. The cryo vial or cryo bags containing the product are removed from the canister and thawed in a 37°C water bath. The cryo vial or cryo bags should be held upright and swirled continuously till the last crystals melt. Immediately 3 ml to 50 of multiple electrolyte solution should be added to the cryo vials or cryo bag using a sterile syringe. The contents of the cryo vial or cryo bag should then be mixed thoroughly by swaying the vial or bag. The cell suspension is then ready to be administered at a dose of 5 million to 20 million cells intravenously, in the joint or at the affected site. In order that those skilled in the art will be better able to practice the present disclosure, the following examples are given by way of illustration and not by way of limitation.

Example 1: Determination of optimum basal medium for culture of canine adipose derived MSCs

The seeding density standardization was done to determine the optimum number of MSCs to be seeded to get maximum cell yield and a population doubling of approximately 5 to 7 per passage. Five different nutrient media were analyzed for culture of canine adipose derived MSCs. Dulbecco's Modified Eagle's Medium-knock out (DMEM-KO), Alpha modified minimum essential medium (a-MEM), 50: 50 DMEM-KO: -MEM, 75:25 DMEM-KO: a- MEM, 25:75 DMEM-KO: a-MEM were used to see the growth rate, population doubling and Fold Expansion. Seeding density was evaluated at the rate of 1000 to 5000 cells cm so as to identify optimal medium composition that gives higher yield. The process was performed for autologous clinical use and the yield of cells obtained at the end of passage 1 and passage 2 was determined. Results showed that seeding density of 5000 cells per cm² in 25: 75 DMEM-KO: a-MEM is the optimum seeding density and optimum basal medium that gives significantly higher yield i.e. 166,000 cells per cm² and found to be most optimum medium tested and significantly better than any other media and selected for large scale production as shown in Table 1 .

Table 1

5000 1 21 1,000/cm² 246,000/cm² 233,520/cm² 221,000/cm² 256,000/cm²

2 133,000/cm² 160,000/cm² 158,000/cm² 137,000/cm² 166,000/cm²

FIG. 1A (passage 1) and IB (passage 2) shows that a seeding density of 5000 cells per cm is optimum in basal media consisting of 75% alpha MEM: 25% DMEM-KO as this gave the highest cell count of 160,000 cells per cm that is equivalent to 1000 million cells in a 10-ceIl stack which will give 50 doses of 20 million cells for canine therapy.

Example 2: Isolation of Canine adipose derived MSCs

Four healthy and pedigreed dogs were sedated and their paraxial caudo dorsal gluteal regions were shaved and the skin was aseptically prepared. An inverted L pattern of local anesthetic infiltration was used for regional desensitization. A linear incision of 4-5 cm in length was made and approximately 5-8 grams of subcutaneous adipose tissue was dissected using curved scissors. The adipose tissue specimen was placed in 50 ml polypropylene centrifuge tube containing sterile DPBS solution (without Calcium and magnesium) with antibiotics and sealed using a strip of Parafilm M®. The skin incisions made were closed with simple suture pattern. Canine adipose tissue was then transported to the lab for isolation and culturing of canine adipose derived MSCs.

The adipose tissue was transferred into a sterile tube/bottle using forceps. Approximately equal volume (w/v) of washing solution, i.e., a mixture of DPBS and antibiotic/ antimycotics was added to the tissue and washed extensively. The infranatant was removed and tissue was cleaned of visible blood clots and fibrous tissue. The washing step was repeated till the infranatant became clear. The tissue was then chopped into very small pieces using sterile scissors and was then digested using approximately equal volume of 0.1-0.2 % pre warmed Type 1 Collagenase A solution. The collagenase action was neutralized by addition of Dulbecco's Modified Eagle's Media -Knock Out (DMEM-KO) or Alpha-minimum essential medium (a-MEM) or combination of DMEM-KO and a-MEM containing 10 % Fetal Bovine Serum(FBS).

The neutralized cell suspension was centrifuged at 2200 -2500 rpm for 10 minutes to pellet out Stromal Vascular Fraction (SVF) cells. The canine-SVF (CSVF) cell pellet was made into a cell suspension and filtered using 70 micron cell strainer. The filtered cells were then seeded in T 175 cm² Tissue culture Flasks and cell stacks at the rate at least 75000cells per cm². The flasks / stacks were then transferred to humidified 5% C0₂ incubator at 37 °C. After 48 - 72 hours of seeding CSVF fraction, complete media change of the tissue culture flask /chamber was done. The cultures were maintained in 5% C0₂ incubator at 37 °C in combination media containing 10% FBS, 200 Mm L-glutamine and Antibiotic- Antimycotic w/v 10,000 U Penicillin, lOmg Streptomycin and 25 μg Amphotericin B per ml in 0.9% normal saline and bFGF 2ng/ml(Sigma Afdrich. Media changes were done once in three to six days till the culture, attained 80% to 90% confluence. The cells were then trypsinized using 0.25 % Trypsin EDTA. These cells constituted Passage 0 cells and as Master cell Bank (MCB).

The trypsinized cells were cryopreserved in cryopreservation media comprising of 90% Fetal bovine Serum andl0% Dimethyl sulphoxide (DMSO,), frozen to -80°C in programmable controlled rate freezer(PLANAR) and then stored in Vapour Phase of the Liquid Nitrogen Storage Tanks at -196°C.

The isolated nucleated cells obtained after processing of fat were counted using a heamocytometer and the obtained counts were found to be 2.5 to 3 million nucleated cells /ml of fat for 4 different samples. The nucleated cells seeded in culture flasks attached to the flask surface by day 1 and at day 3, spindle shaped morphology was seen, by day 12 -14. 80% confluent cultures were obtained, which were then harvested and the SVF counts and MSC yields obtained in passage 0 as Master Cell Bank (MCB) are summarized in Table 2.

Table 2: Total cell counts obtained from fat and Passage 0 cell counts from 4 different donor samples

Sample Weight SVF cell counts per Density of SVF Passage 0 MSC

code of fat gram of fat seeding / cm² count at 80 -85 %

confluence

KRPLT/Ad- 5-8 9-1 1 x 10⁶cells/ 0.08 - 0.1 x 10⁶ 96.25 x 10⁶ cells

Ca D-01 grams gram

KRPLT/Ad- 5-8 8-10 x 10⁶cells/ 0.08 - 0.1 x 10^b 90 x 10⁶ cells

Ca D-02 grams gram

KRPLT/Ad- 5-8 7-10 x 10⁶cells/ 0.08 - 0.1 x 10⁶ 93.5 x 10^& cells

Ca/D-03 grams gram

KRPLT/ Ad- 5-8 8-12 x 10^bcells/ 0.08 - 0.1 x 10⁶ 139 x 10⁶ cells Ca/D-04 grams gram

Example 3: Expansion of canine adipose derived MSCs to Passage land Passage 2 for autologous use:

For seeding of passage 1 cells, canine Passage 0 cells (MCB) from Example 2 were thawed and seeded at the rate of 1000 cells/cm to 5000 cells/cm , , into 10-cell chamber stack having area of 6360 cm . The flasks / stacks were then transferred to humidified 5% C0₂ incubator at 37 °C. The cultures were maintained in 5% C0₂ incubator at 37°C in growth media comprising of combination media containing 10% FBS , 200 Mm L-Glutamine and Antibiotic- Antimycotic w/v 10,000 U Penicillin, lOmg Streptomycin and 25 μg Amphotericin B per ml in 0.9% normal salineand bFGF 2ng/ml. Media changes were done once in four days till the culture attained 70-80% confluency. The cells were then trypsinized using 0.25 % Trypsin EDTA. These cells constituted Passage 1 cells. The trypsinized cells were cryopreserved in cryopreservation media comprising of 90% equine serum andl0% Dimethyl sulphoxide, frozen to -80°C in programmable controlled rate freezer (PLANAR) and then stored in Liquid Nitrogen Storage Tanks at -196°C.

For seeding and maintenance of passage 2 cells, canine passage 1 cells were thawed and seeded at the rate of 1000-5000 cells/ cm into cell stacks. The stacks were then transferred to humidified 5% C0₂ incubator at 37 °C and maintained in 5% C0₂ incubator at 37 °C in growth media comprising of combination media containing 10% FBS 200 Mm L-Glutamine and Antibiotic- Antimycotic w/v 10,000 U Penicillin, lOmg Streptomycin and 25 g Amphotericin B per ml in 0.9% normal saline ) and bFGF 2ng/mlMedia changes were done once in four days till the culture attained 80% to 90%confluence. The cells were then trypsinized using 0.25 % Trypsin EDTA. These cells constituted Passage 2 cells. The cells were cryopreserved in freezing mixture comprising of 85%) multiple electrolyte solution, 10% Inject able grade Dimethyl sulphoxide and 5% equine serum.

The spent media from all three passages were checked for sterility, endotoxin and pH and these were found to be within acceptable ranges.

MSC counts obtained at passage 1 and passage 2 are shown in Table 3 below: Table 3

As is evident from Table 3, the cell count was found to be > 215,000 cells per cm² i.e. almost 1000-1400- million cells in 6360 sq cm cells chamber. This effectively yields 140 doses of MSCs per chamber and thereby reduces the cost for production and bridges the demand- supply gap for MSCs in the market at present.

Example 4: Preparation of WCB from MCB

The cryovials of four donor MCB as given in Example 2 were taken from the vapour phase of the liquid nitrogen storage tank and immediately placed in a water bath at 37°C.The vials were held straight and swirled in water bath till the last crystal dissolved out. The contents of the cryovials were then aspirated and cells from all the four donors MCB was mixed together and re-suspended in pre-thawed neutralization media. The tube containing cell suspension was centrifuged at 1400 to 1800rpm for 10 minutes. The supernatant was discarded and the pellet was re-suspended in a desired volume of complete media, mixed well and the viable cell count was taken.

Since the cells were to be seeded at the rate of 1000 cells/cm to 5000 cell/cm the cells of the four donors from the MCBs obtained in Example 2 were pooled in appropriate equal proportions to make the required quantity of cells and then the cells were seeded into culture flasks/ chambers containing combination media.

Here, the first media change was done after 3 days from seeding. 70 to 90% of the spent media was aspirated and freshly prepared combination media was then added to cell culture flask or chamber. The second media change was similarly done after 4 days of the first media change ie at around the 7^th day from seeding. Once the culture attained 80%-90% confluence, the flasks or chambers were harvested.

The spent media was then removed from the flask or chamber and two aliquots were given for checking of sterility, endotoxin, mycoplasma and pH and these were found to be within acceptable ranges. The flasks or chambers were given two washes with DPBS. The wash was removed and 0.25% Trypsin EDTA was added and kept in 5% C0₂ incubator at 37°C for 2 to 3 minutes and then the flask was observed for detachment of cells. Trypsin activity was stopped by addition of Neutralization media and the neutralized cells were collected in centrifuge tubes. The tissue culture flask or chamber was given one more wash with neutralization media and the same was collected. The neutralized cells were then centrifuged at 1400-1800rpm for 5-10 minutes. The supernatant was then discarded, and the pellet was resuspended in complete media. The cell count was taken and 1 aliquot of cells was given for FACS Flow analysis, and Differentiation. The remaining cell suspension was centrifuged at 1000-1400rpm for 5-10 minutes. The supernatant was discarded and the pellet was resuspended in the desired volume of freezing mixture such that the concentration of cells in the freezing mixture was three million cells per ml. The cells in the freezing mixture weres dispensed in to prelabelled cryovials at the rate of 1ml per cryovial. These vials constituted the working cell bank (WCB). The cryovials were then frozen in a controlled rate freezer to attain -80°C. The cryovials were then transferred to a vapour phase liquid nitrogen tank for further storage.

Example 5: Preparation of clinical grade product (Final product) from WCB

The cryovials of Example 4 were taken.from the vapour phase of the liquid nitrogen storage tank and immediately placed in a water bath 37°C. The vials were held straight and swirled in water bath till the last crystal dissolved out. The contents of the cryovials were then aspirated and resuspended in pre-thawed neutralization media. The tube containing cell suspension was centrifuged at 1400 to 1800rpm for 5 to 10 minutes. The supernatant was. discarded and the pellet was re-suspended in a desired volume of complete media, mixed well and the viable cell count was taken. The cells were seeded at the rate of 1000 cells/cm to 5000 cells/cm , into 10- cell chamber stack having area of 6360 cm .

Here, the first media change was done after 3 to 4 days from seeding. 70 to 90% of the spent media was aspirated and freshly prepared combination media was then added to cell culture flask or chamber. The second media change was similarly done after 3 to 4 days of the first media change. Once the culture attained 80%-90% confluence, the flasks or chambers were harvested.

The spent media was then removed from the flask or chamber and two aliquots were given for checking of sterility, endotoxin, mycoplasma and pH and these were found to be within acceptable ranges. The flasks or chambers were given two washes with DPBS. The wash was removed and 0.25% Trypsin EDTA was added and kept in 5% C0₂ incubator at 37°C for 2 to 3 minutes and then the flask is observed for detachment of cells. Trypsin activity was stopped by addition of Neutralization media and the neutralized cells were collected in centrifuge tubes. The tissue culture flask or chamber was given one more wash with neutralization media and the same was collected. The neutralized cells were then centrifuged at 1400-1800rpm for 5-10 minutes. The supernatant was then discarded, and the pellet was resuspended in multiple electrolyte solution. The cell count was taken and 1 aliquot of cells was given for FACS Flow analysis, and Differentiation. The cell count was found to be > 160,000 cells per cm² i.e. almost 1000 million cells in 6360 sq cm cells chamber. This effectively yields 50 doses of 20 million each per chamber and thereby reduces the cost for production and bridges the demand- supply gap for MSCs in the market at present.

The remaining cell suspension was centrifuged at 1000-1400rpm for 5-10 minutes. The supernatant was discarded and the pellet was resuspended in multiple electrolyte solution. The cell suspension was then filtered through a 20-40micron strainer and then centrifuged at 1000- 1400rpm for 5-10minutes. The washing with multiple electrolyte solution was repeated twice and the supemantant was discarded and the pellet was resuspended in the desired volume of multiple electrolyte solution. The cell suspension was then filtered through a 40micron strainer and then centrifuged at 1400rpm for l Ominutes. The washing with multiple electrolyte solution was repeated twice and the supemantant was discarded and the pellet was resuspended in the desired volume of multiple electrolyte solution containing 5% canine serum and 10%dimethyl sulfoxide (DMSO) which serves as a cryoprotectant such that the concentration of cells in the freezing mixture was 5 to 20 million cells per ml. The cells in freezing mixture were dispensed in to pre-labelled cryovials at the rate of 1ml per cryovial. These vial constituted the final Product. The cryovial were then frozen in a controlled rate freezer to attain -80°C. The cryovial were then transferred to a vapour phase liquid nitrogen tank for further storage. Example 6: Morphological analysis of adipose derived MSCs

Adipose derived MSCs when cultured in basal medium comprising 75% a-MEM: 25% KO- DMEM maintained the typical fibroblastic spindle-shaped morphology as shown in FIG. 2. Population doubling (PD) time of MSC on an average in nutrient media was 33± 1.12 hours. Example 7: Analysis of Cell Surface Markers

The surface markers of adipose derived MSCs are analyzed by FACS antibodies after dissociation. Cells are stained with fluorescein or phycoerythrin coupled antibodies, including Cluster of Differentiation, CD34, CD44, CD45, CD90, CD105 (all antibodies purchased from Becton-Dickinson, San Jose, CA, USA). Stained samples and unstained control cells are analyzed with BD FACS Calibure. Immunophenotyping of the adipose derived MSCs shows high expression of stromal specific markers specific for MSCs and negligible expression of endothelial and hematopoietic markers as shown in FIG. 3, which is a characteristic of a pure MSCs population. Example 8: Pluripotentent markers, Differentiation and Secretome analysis

The ability of canine adipose tissue derived MSCs isolated, purified and culturally expanded according to an embodiment of the present invention to differentiate into the various lineages was investigated. Cells obtained as per Example 2, 3, 4 and 5 were plated and cultured in the specific differentiation media for adipogenic, chondrogenic and osteogenic differentiation, and an undifferentiated unstained control of canine adipose tissue derived MSCs was also maintained. Differentiation into adipocytes was confirmed by observing the lipid droplets after Oil red O staining as seen in (FIG. 4), mineralization of the matrix / calcium deposition as assessed by Alizarin red S staining demonstrated the osteogenic differentiation potential of AD- MSCs as seen in (FIG. 4) and a micromass culture, characteristic to chondrocytes was observed on staining with Alacian blue as seen in (FIG. 4). Notably, differentiation into adipocytes, osteocytes and chondrocytes was more than 90%. In order to determine the pluripotent capacity of the MSCs, the expression levels of pluripotent markers, NANOG, SOX2 and OCT4 were determined. The expression levels of these pluripotent markers were found to be significantly high as shown in FIG. 5. Spent media was collected at the end of passage 1 as per Example 4 and passage 2 as per Example 5. FIG. 6 shows the levels of VEGF and TGF-β in the spent media. Levels of growth factors were more or less similar in MCB, WCB and Final product. This shows that culture method was consistent and uniform yielding uniform MSCs at the end of each passage up to passage 2.

Example 9: Preparation of Off-The-Shelf-Product for clinical use

As mentioned in Example 5, industrial scale production of adipose derived MSCs was done in cell stacks so as to get multiple dosages. MSCs were washed and trypsinized. Cells were washed minimum 3 to 4 times to remove all the trypsin. Cells were re-suspended in injectable media (freezing mixture) comprising multiple electrolyte solution supplemented with 10% DMSO, 5% canine serum. 5 to 20 million cells were packed in cryo vial in 1 ml of freezing mixture and gradually frozen in a control rate freezer at the rate of 1°C per minute until -80°C. The cryo vial was then stored in vapour phase liquid nitrogen container. For clinical use, the cryo vial was thawed at 37°C in a water bath for 2 minutes. 0.5 to 50 ml of normal saline was added to make volume 2 to 50 ml. This dilution made DMSO 1.5 to 3.5% and canine serum from 0.75 to 1.25%o which was within the allowable range for canine use without causing any toxicity, side effects or shock. The diluted solution could then be safely and efficaciously injected intravenously at the rate of 1 ml per minute.

Stability of MSCs was checked in normal saline for frozen canine MSCs as shown in Table 4

Table: 4:

Stability (Hours) for frozen sample Normal Saline (% viability)

0 95.65%

24 93.26%

48 87.41% It is clear from Table 4 that injectable normal saline gives more than 85% viability and stability up to 48 h during transport and after final dilution of the Final product.

Example 10: Efficacy of canine adipose derived MSCs in treating spinal cord injury by using a therapeutic product prepared according to the method of preparing the product for allogenic use according to an embodiment of the invention

The efficacy of canine adipose derived MSCs was studied by injecting these MSCs into a study subject dog paralysed due to spinal cord injury by distemper. Distemper is a viral infection that destroys nerve and muscle.

Three injections of 20 million each were given every month in three doses by intra-thecal and intra-muscular route. MRI was observed and injection was given near site of injury. The condition of the study subject was recorded and videographed before and after injection. Three months after injection the study subject showed signs of recovery and was able to stand and walk on its own as is evident from FIG. 7. i.e. after giving 3 injection of 20 million each at monthly interval, the study subject showed significant improvement in sensory and motor neuron improvement and increase in muscle tonicity.

Example 11: Clinical efficacy study in canine atopic dermatitis by using a therapeutic product prepared according to the method of preparing the product for allogenic use according to an embodiment of the invention

Canine atopic dermatitis (CAD) is the second most common skin disease in dogs; it is estimated to affect 10% of the canine population. Neither the incidence nor the prevalence of atopic dermatitis in the general canine population has been studied.

Ten study subject dogs with atopic dermatitis were recruited in this study. Two doses of 20 million cells were given intravenously at weekly intervals. The base line symptoms and results after 31 days post-injection of MSCs to 10 dogs having CAD is shown in Table 5. Table 5

It is clear from Table 5 that the CAD condition in the ten study subjects improved significantly after 31 days. Also, FIG. 8 graphically represents the results after giving 1 injection of 20 million each showing significant improvement in dermatitis, reduction in pruritus and hair growth.

Example 12: Efficacy in canine chronic renal failure by using a therapeutic product prepared according to the method of preparing the product for allogenic use according to an embodiment of the invention

A dog with a history of chronic renal failure and continuous elevated level of blood urea nitrogen (BUN) and creatinine were used as study subjects. The study subject's medical history showed that it had elevated BUN and creatinine levels for the last 7 years and was on dialysis and several other standard medications. The study subject was given two doses of adipose derived MSCs at the rate of 20 million cells one month apart. After such treatment, BUN and creatinine levels significantly reduced to normal levels and continued to remain normal until last follow-up of one year after treatment ended. FIG. 9A and 9B respectively show reduction in creatinine and blood urea nitrogen in the study subject after two doses of injections of the MSC Final product.

Example 13: Efficacy in hip dysplasia by using a therapeutic product prepared according to the method of preparing the product for autologous/allogenic? use according to an embodiment of the invention

The study subject was a dog with broken acetabula margin and extensive lytic area of hip bone. This condition had persisted for 2 years and was continuously deteriorating. ^{^}Stem cell injections (Final product) were given at the rate of 5 million cells per dose one month apart. After 3 months of follow-up, the hip bone showed well defined cortical margin and addition of sclerosis suggesting increase in calcium and reduced osteopenia, as is evident from FIG.1 OA and 10B. In FIG. 10A, the dog's X-ray film shows broken acetabula margin with extensive lytic area. But after intra-articular stem cell injection, the study subject canine showed well defined cortical margin, and sclerosis suggestive of increased calcium and reduced osteopenia as shown in FIG 10B. Adipose derived MSCs obtained according to an embodiment of this invention were clearly effective in treating hip dysplasia.

Example 14: Immunomodulatory activity of canine adipose derived MSCs

To assess the immunomodulatory activity of the MSCs, immunological reactions were set up i.e. Phytoheamagglutinin (PHA) induced T-cell proliferation assay.

MSCs obtained at the end of Example 5 were harvested and seeded into 96 well plates at 1000 or 5000 cells/well densities and allowed to adhere for 24 hours. The next day, they were inactivated by incubation with Actinomycin-D (5μg/ml) for 15 minutes at 37°C.

To set up the PHA stimulation assay, individual populations of 3 native peripheral blood mononuclear cells (PBMCs) were added to wells containing inactivated MSCs. The positive control wells contained no MSCs. PHA was added to each test and positive control well at a concentration of 10 μg/ml. The negative controls included inactivated MSCs and native PBMCs plated individually in the same concentrations, as in the test wells. No PHA was added to the negative control wells. The assay was allowed to proceed for 3 days before estimation of cell proliferation using the BrdU cell proliferation kit. The endpoint was calculated in terms of percentage T-cell proliferation relative to the positive control, which is assumed to show 100% proliferation. FIG.1 1 shows the shows the dose dependent suppression of PHA induced T-cell proliferation by 1000 and 5000 Canine MSCs (T-cell proliferation induced by PHA, in the absence of MSCs, serves as the positive control and is assumed to be 100%).

Example 15: Safety profile of canine MSCs

In vivo acute toxicity study was conducted to evaluate the effect of a single dose of intravenous (IV) infusion of canine adipose derived MSCs on male and female Swiss Albino mice and^' Wistar rats. The mice and rats were into six groups of 10 animals (5 males and 5 females) each. The first three groups were the control groups. The first group received only Plasmalyte A, the second control group received Plasmalyte A + canine serum while the third control group received the vehicle i.e. Plasmalyte A + canine serum (5%) + DMSO (3%). The other three test groups received IP investigational product IP i.e mesenchymal stem cells at concentrations of 2, 10 and 20 x 10⁶ cells/kg body weight by intravenous route. Following single dose of IV injection, the animals were observed for a minimum period of 14 days. Following single dose of intravenous injection, the animals were observed for a minimum period of 14 days. All animals were sacrificed at termination of the study and subjected to a complete necropsy. No clinical signs, mortality or morbidity was observed in the injected animals. The body weight gain by treated animals was not adversely affected during the 14 day observation period post-dosing. No gross pathological alterations were encountered at terminal necropsy in tissues /organs of any of the animals in this study. No adverse effects were observed in vehicle control group animals treated with the vehicle, with respect to survival, clinical signs, body weight gain and necropsy findings. No mortality or morbidity was encountered in the study groups. Based on the findings of this acute intravenous toxicity study, in absence of any incidence of deaths and adverse effects among treated animals, the maximum tolerated dose (MTD) of canine adipose derived MSCs in Swiss albino mice and Wistar rats was estimated to be greater than 20 X 10⁶ cells/ kg body weight when administered intravenously. Canine adipose derived MSCs obtained according to an embodiment of the invention also showed a safety margin of greater than ten times (1 OX) the maximum therapeutic dose anticipated for use with canine subjects.

What has been described and illustrated herein are preferred embodiments of the invention along with some of their variations. The terms, descriptions and figures used herein are set forth by way of illustration only and are not meant as limitations. Those skilled in the art will recognize that many variations are possible within the spirit and scope of the invention, which is intended to be defined by the claims in the complete specification— and their equivalents— in which all terms are meant in their broadest reasonable sense unless otherwise indicated.

Claims

Claims:

1. A method for isolation, purification and industrial scale expansion of canine adipose tissue derived mesenchymal stem cells (MSCs) to obtain a yield of 160,000 cells per cm² pure clinical grade MSCs for allogenic use comprising over 95% cells which express positive markers CD44, CD90, and CD 105, and less than 2% cells which express negative markers CD45, CD34 and HLA-DR, the method comprising the steps of:

c) seeding the cells contained in the SVF into a culture medium; and d) trypsinising and washing the cells once they reach confluence at passage 0;

f) seeding the pooled cells of passage 0 into a culture medium;

wherein,

• step (a) is performed by biopsy assisted removal;

• cells from stromal vascular fraction are seeded in step (c) only if at least 60% cells are positive for CD 90; • the SVF cells are seeded in to the culture medium in step (c) at a seeding density of at least 75000 cells per sq cm;

• the mesenchymal stem cells are seeded in to the culture medium in step (f) and step (h) at a seeding density of 1000 to 5000 cells per sq cm and express at least 95% of the positive markers CD44, CD90, and CD 105 and at most 2% of the negative markers CD45, CD34 and HLA-DR;

• the culture medium comprises 25% to 75% Dulbecco's Modified Eagle's Medium- Knockout (DMEM-KO) and 25% to 75% alpha-Minimum Essential Medium (a-MEM), or upto 100% DMEM-KO or uptol00% a-MEM; and

2. A method for isolation, purification and industrial scale expansion of canine adipose tissue derived mesenchymal stem cells (MSCs) to obtain a yield of 160,000 cells per cm² pure clinical grade MSCs for autologous use comprising over 95% cells which express positive markers CD44, CD90, and CD 105, and less than 2% cells which express negative markers CD45, CD34 and HLA-DR, the method comprising the steps of:

a) extracting 5 to 8 grams of adipose tissue from a canine donor;

c) seeding the cells contained in the SVF into a culture medium; and

d) trypsinising and washing the cells once they reach confluence at passage 0;

e) seeding the washed cells of passage 0 into a culture medium;

f) trypsinising and washing the cells once they reach confluence at passage 1 ;

g) seeding the cells of passage 1 into a culture medium; and

• step (a) is performed by biopsy assisted removal;

• cells from stromal vascular fraction are seeded in step (c) only if at least 60% cells are positive for CD 90; • the ^'cells are seeded in to the culture medium in step (c) at a seeding density of at least 75000 cells per sq cm;

• prior to seeding in step (e), the mesenchymal stem cells are characterized based on the percentage of cells which express positive markers CD44, CD90, and CD 105, and the percentage of cells which express negative markers CD45, CD34 and HLA-DR;

• the culture medium comprises 25% to 75% Dulbecco's Modified Eagle's Medium- Knockout (DMEM-KO) and 25% to 75% alpha-Minimum Essential Medium (a-MEM) or upto 100% DMEM-KO or upto 100% a-MEM; and

3. The method as claimed in claim 1 or 2, wherein the adipose tissue is extracted from the dorsal gluteal muscle or the omentum of the donor.

4. The method as claimed in claim 1 or 2, wherein the washed cells after trypsinisation are frozen in a freezing mixture comprising multiple electrolyte solution supplemented with 5% canine serum and 10% dimethyl sulfoxide (DMSO).

5. The method as claimed in claim 1 or 2, wherein the culture medium comprises 25% DMEM-KO and 75% a-MEM.

6. A therapeutic product prepared according to the method of claim 1 or 2 for clinical use.

7. A therapeutic product for treating osteoarthritis, spinal cord injury, atopic dermatitis, dilated cardiomyopathy, type-1 diabetes, renal failure, hepatic disease and non-healing wounds comprising MSCs suspended in multiple electrolyte solution supplemented with human serum albumin and dimethyl sulfoxide (DMSO) wherein, over 95% MSCs express positive markers CD44, CD90 and CD 105, and less than 2% cells express negative markers CD45, CD34 and HLA-DR, and wherein the MSCs have undergone not more than 15 population doublings in vitro and are capable of at least 30 to 35 more population doublings, the MSCs are capable of differentiating into adipocytes, osteocytes and chondrocytes, the MSCs express pluripotent markers like OCT4, SOX2 and NANOG; and secrete growth factors like TGF-β and VEGF; and show immunomodulatory activity.

8. A method for treating spinal cord injury, atopic dermatitis, dilated cardiomyopathy, renal failure, hepatic disease, type-1 diabetes, comprising administering 2 to 3 doses each of 5 to 20 million MSCs of the therapeutic product as claimed in claim 7, by intravenous route or intra-articular route.

9. Use of the therapeutic product as claimed in claim 7 for treating osteoarthritis, spinal cord injury, atopic dermatitis, dilated cardiomyopathy, type-1 diabetes, renal failure, hepatic disease and non-healing wounds.