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Abstrait
Production of Good Manufacturing Practice Grade Equine Adiposederived Mesenchymal Stem Cells for Therapeutic Use
Shashank Gowda, Aarya Hari, Basavaraj Chougule, Manoj Kumar Reddy, Abhishek Chandanan, Minita Sodhi, Nicole Koshy, Lyle Fonseca and Satish Totey
Mesenchymal stem cells (MSC) are currently being evaluated in equine clinical studies for their potential to treat various disorders and injuries. Studies have shown that both autologous and allogeneic stem cells appear to be safe. However, an efficient cell expansion method that is reliable and cost-effective is warranted to provide off-the-shelf clinical grade stem cells. Our study aimed to determine optimum culture conditions for efficient large-scale stem cell expansion. We produced cGMP standard equine adipose derived mesenchymal stem cells on a large scale. Five different medium combinations—Dulbecco’s modified Eagle’s medium-knockout (DMEM-KO), alpha modified minimum essential medium (α-MEM), 50:50 DMEM-KO/α-MEM, 75:25 DMEM-KO/α-MEM and 25:75 DMEM-KO/α- MEM—at seeding densities of 1000, 2000, 3000, 4000 and 5000 cells/cm2, were used to determine the optimum culture conditions for large-scale production. Growth kinetics, immunophenotypes, karyotypes, morphology, trilineage differentiation, T-cell proliferation, virus positivity, pre-clinical toxicity and expression of pluripotency markers were analysed. Among the medium combinations and seeding densities tested, 25:75 DMEM-KO/α-MEM at a seeding density of 5000 cells/cm2 was found to be optimum for large-scale expansion. This medium combination gave a significantly higher cell yield than the other medium combinations, while preserving their stem cell characteristics and differentiation potential. The results indicated that the adoption of an appropriate culture system significantly improved cell yield, thus enabling the production of sufficient cells for therapeutic application in a cost-effective manner. The results also showed that the method of large-scale expansion requires minimal manipulation of cells, and it could be expanded ex vivo within two passages while retaining the characteristics of true stem cells.