Preparation of master cell bank (MCB), working cell bank (WCB) and investigational product (IP)

Clinical grade MCB, WCB and IP was prepared as per the method described earlier (21). Briefly, SVF from each donor was seeded separately on to 2-chamber cell stack (corning plastic wares supplied by Sigma Aldrich, Bangalore, India) in growth medium at a density of 85,000~100,000 cells/cm² and incubated at 37°C in 5% humidified CO₂. The medium was changed every third day, and cells were harvested once they became confluent ~70% with 0.05% trypsin plus 0.2% Ethylenediaminetetraacetic acid (EDTA), washed with DPBS and counted with haemocytometer. The cells from the individual donors were tested for quality parameters. The cells were aliquoted and frozen at the concentration of 1 million cells/ml and stored in vapour phased liquid nitrogen at passage-0.

For WCB, frozen vials of MCB from five different donors were thawed and cells were pooled together. Mixed donor cells were then seeded onto 5-chamber cells stack (Corning) in growth medium at the seeding density of 3000 cells/cm² and cultured as described above. Cells were harvested once they reached ~70% confluence. Cells of the WCB were tested for quality parameters and were frozen at the concentration of 3 million cells/vial and stored in vapour phase liquid nitrogen at passage-1.

The IP was prepared by seeding frozen-thawed WCB at a seeding density of 3000 cells/cm² in a 10-chamber cells stack (Corning) containing growth medium and cultured as described above. The cells were harvested once they reached ~70% confluent. IP was then tested for quality parameters and used for pre-clinical study. Remaining batch was frozen at the concentration of 100 million cells/cryocyte bag and stored in vapour phased liquid nitrogen at passage-2.