WO2014203269A2 - Procédé d'isolement, de purification et d'expansion à l'échelle industrielle de cellules souches mésenchymateuses dérivées de tissu adipeux canin - Google Patents

Procédé d'isolement, de purification et d'expansion à l'échelle industrielle de cellules souches mésenchymateuses dérivées de tissu adipeux canin Download PDF

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WO2014203269A2
WO2014203269A2 PCT/IN2013/000584 IN2013000584W WO2014203269A2 WO 2014203269 A2 WO2014203269 A2 WO 2014203269A2 IN 2013000584 W IN2013000584 W IN 2013000584W WO 2014203269 A2 WO2014203269 A2 WO 2014203269A2
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cells
mscs
culture medium
seeding
passage
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PCT/IN2013/000584
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WO2014203269A3 (fr
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Satish Mahadeorao Totey
Manoj Kumar C REDDY
Lyle Carl FONSECA
Aarya HARI
Basavaraj CHOUGULE
Minita SODHI
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Kasiak Research Pvt. Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2511/00—Cells for large scale production

Definitions

  • 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.
  • 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 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 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.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • Other sought-after modalities to augment NSAID therapy include additional pain medications, dietary supplements, and joint injections.
  • MSCs are delivered to an area of damaged tissue where they stimulate regeneration and aid in repair of the damaged tissue.
  • Adipose derived MSCs have a promising application in these conditions to repair tissue and improve the function of damaged organs.
  • 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.
  • canine bone marrow mesenchymal stem cells appear to senesce much earlier than Adipose derived mesenchymal stem cells (AD-MSCs) and Umbilical cord mesenchymal stem cells (UC-MSCs).
  • adipose tissue and umbilical cord tissue may be preferable for therapeutic purposes.
  • 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.
  • 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 cm 2 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:
  • step (a) is performed by biopsy assisted removal
  • step (c) cells from stromal vascular fraction are seeded in step (c) only if at least 60% cells are positive for CD 90;
  • step (c) • 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 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
  • 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 cm 2 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:
  • step (a) is performed by biopsy assisted removal
  • step (c) cells from stromal vascular fraction are seeded in step (c) only if at least 60% cells are positive for CD 90;
  • step (c) • the 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 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
  • 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- ⁇ and VEGF; and show immunomodulatory activity.
  • DMSO dimethyl sulfoxide
  • 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
  • the present invention is described by referring mainly to exemplary embodiments thereof.
  • 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.
  • 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).
  • DMEM-KO Dulbecco's Modified Eagle's Medium-Knockout
  • a-MEM alpha-Minimum Essential Medium
  • 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
  • the term "confluence” as used in the specification means approximately 80% to 90% confluence of cells attained during cell culture.
  • multiple electrolyte solution 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 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.
  • 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.
  • the therapeutic product for treating osteoarthritis, spinal cord injury, atopic dermatitis, dilated cardiomyopathy, type-1 diabetes, renal failure, hepatic disease and non-healing wounds.
  • 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)
  • 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.
  • adipose tissue derived MSCs 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.
  • 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.
  • DMSO dimethyl sulfoxide
  • the final product should be transported in liquid nitrogen charged dry shipper or dry ice for clinical use.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 1A 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
  • 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.
  • washing solution i.e., a mixture of DPBS and antibiotic/ antimycotics
  • 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).
  • DMEM-KO Dulbecco's Modified Eagle's Media -Knock Out
  • a-MEM Alpha-minimum essential medium
  • FBS Fetal Bovine Serum
  • 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 cm 2 Tissue culture Flasks and cell stacks at the rate at least 75000cells per cm 2 . The flasks / stacks were then transferred to humidified 5% C0 2 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% C0 2 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 ⁇ g 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).
  • MBC Master cell Bank
  • 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.
  • 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.
  • Table 2 Total cell counts obtained from fat and Passage 0 cell counts from 4 different donor samples
  • Example 3 Expansion of canine adipose derived MSCs to Passage land Passage 2 for autologous use:
  • canine Passage 0 cells 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% C0 2 incubator at 37 °C.
  • the cultures were maintained in 5% C0 2 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 salineand bFGF 2ng/ml.
  • 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.
  • 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.
  • 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% C0 2 incubator at 37 °C and maintained in 5% C0 2 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 cell count was found to be > 215,000 cells per cm 2 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.
  • 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.
  • 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.
  • 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% C0 2 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.
  • WCB working cell bank
  • 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 .
  • 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% C0 2 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 cm 2 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.
  • 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.
  • 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
  • canine adipose tissue derived MSCs isolated, purified and culturally expanded according to an embodiment of the present invention 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.
  • FIG. 4 shows the expression levels of pluripotent markers, NANOG, SOX2 and OCT4. 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- ⁇ 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.
  • 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.
  • injectable media 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.
  • 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.
  • 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
  • canine adipose derived MSCs 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.
  • 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 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.
  • 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.
  • 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
  • immunological reactions were set up i.e. Phytoheamagglutinin (PHA) induced T-cell proliferation assay.
  • PHA Phytoheamagglutinin
  • 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 (5 ⁇ g/ml) for 15 minutes at 37°C.
  • 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%).
  • 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 10 6 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.

Abstract

La présente invention concerne un procédé d'isolement, de purification et d'expansion à l'échelle industrielle de cellules souches mésenchymateuses (MSC) dérivées de tissu adipeux canin. L'invention concerne également un procédé de traitement et un produit thérapeutique pour traiter l'arthrose, une lésion de la moelle épinière, la dermatite atopique, une cardiomyopathie dilatée, le diabète de type 1, l'insuffisance rénale, une maladie hépatique et des plaies ne cicatrisant pas, comprenant des MSC.
PCT/IN2013/000584 2013-06-17 2013-09-26 Procédé d'isolement, de purification et d'expansion à l'échelle industrielle de cellules souches mésenchymateuses dérivées de tissu adipeux canin WO2014203269A2 (fr)

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CN105685015A (zh) * 2016-03-10 2016-06-22 广州赛莱拉干细胞科技股份有限公司 一种细胞冻存液
EP3100738A1 (fr) * 2015-05-08 2016-12-07 DeltaBio 2000 Kft Procédé et préparation pour le traitement de maladies orthopédiques, c'est à dire une lésion du cartilage articulaire, en particulier des maladies articulaires et des traumatismes orthopédiques impliquant une arthrose et un détachement du cartilage
CN107746829A (zh) * 2017-10-19 2018-03-02 佛山科学技术学院 一种分离与原代培养犬胎盘来源的间充质干细胞的方法
WO2019175773A1 (fr) 2018-03-12 2019-09-19 Universidade Do Porto Compositions destinées à être utilisées dans le traitement d'affections musculo-squelettiques et méthodes de production de celles-ci tirant profit de l'activité synergique de deux types différents de cellules souches/stromales mésenchymateuses
CN111621469A (zh) * 2020-06-01 2020-09-04 重庆医科大学 一种改良酶消化法分离骨细胞的技术及其应用
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CN113774020A (zh) * 2021-08-24 2021-12-10 南通美韦德生命科学有限公司 一种脂肪间充质干细胞库的构建方法
WO2023280834A1 (fr) * 2021-07-08 2023-01-12 Boehringer Ingelheim Veterinary Medicine Belgium Cellules souches mésenchymateuses destinées à être utilisées dans le traitement de la dermatite atopique
WO2024036387A1 (fr) * 2022-08-16 2024-02-22 Omics Biotecnologia Animal Ltda. Concentré de protéines et de peptides dérivés de cellules stromales mésenchymateuses, procédé d'obtention et utilisation du concentré

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CN105238748A (zh) * 2015-10-27 2016-01-13 上海百众源生物科技有限公司 一种胎盘源壁蜕膜间充质干细胞分离冷冻的制备方法
CN105685015B (zh) * 2016-03-10 2018-11-27 广州赛莱拉干细胞科技股份有限公司 一种细胞冻存液
CN105685015A (zh) * 2016-03-10 2016-06-22 广州赛莱拉干细胞科技股份有限公司 一种细胞冻存液
CN107746829B (zh) * 2017-10-19 2022-03-22 佛山科学技术学院 一种分离与原代培养犬胎盘来源的间充质干细胞的方法
CN107746829A (zh) * 2017-10-19 2018-03-02 佛山科学技术学院 一种分离与原代培养犬胎盘来源的间充质干细胞的方法
WO2019175773A1 (fr) 2018-03-12 2019-09-19 Universidade Do Porto Compositions destinées à être utilisées dans le traitement d'affections musculo-squelettiques et méthodes de production de celles-ci tirant profit de l'activité synergique de deux types différents de cellules souches/stromales mésenchymateuses
CN112584846A (zh) * 2018-06-15 2021-03-30 路易斯·马里纳斯·帕尔多 用于皮肤病的药物组合物及其用途
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WO2023280834A1 (fr) * 2021-07-08 2023-01-12 Boehringer Ingelheim Veterinary Medicine Belgium Cellules souches mésenchymateuses destinées à être utilisées dans le traitement de la dermatite atopique
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CN113774020B (zh) * 2021-08-24 2023-04-14 河北朋和生物科技有限公司 一种脂肪间充质干细胞库的构建方法
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