[Optimization of cryopreservative method for human fetal brain neural stem cells-derived oligodendrocyte precursor...

Objective To explore the impact of various conditions during cryopreservation on the survival rate of oligodendrocyte precursor cells (OPCs) derived from human fetal neural stem cells. Methods We compared the cell viability of oligodendrocyte precursors harvested with or without digestion. Then we tested the impact of 3 factors during cryopreservation, freezing solutions (solution with 70 mL/L DMSO and 930 mL/L FBS; solution with 70 mL/L DMSO, 300 mL/L FBS and OPC culture medium; solution with 70 mL/L DMSO, 300 mL/L FBS, 0.2 mol/mL trehalose and OPC culture medium; solution with 70 mL/L DMSO, 300 mL/L FBS, 300 mL/L HES and OPC culture medium), freezing methods (the step-wised freezing or rapid freezing within liquid nitrogen) and storage durations for the better survival rate of OPCs. The optimized method with the best survival rate of OPCs was implemented and at day 7 after recovery, the viability, OPCs specific markers [platelet derived growth factor receptor alpha (PDGFRα), ST8 alpha-N-acetyl-neuraminide alpha-2, 8-sialyltransferase 1 (ST8SIA1/A2B5), chondroitin sulfate proteoglycan 4 (CSPG4/NG2), ki67 were tested and compared with immunofluorescent cytochemical staining. Results Harvesting with digestion contributed to higher OPCs survival rate. OPCs of rapid freezing had survival rates less than 30% and couldn't be re-cultured. The step-wised freezing group showed higher recovery rate. Harvesting with digestion, preservation solution with trehalose, using 2.0×10⁶/mL of cell number, step-wised freezing, contributed to the highest OPCs survival rate reaching (75.73±6.66)%. Compared with the fresh cultured group, cell proliferation, ki67 antigen, PDGFRα, A2B5 and NG2 expression of OPCs were similar in the recovered cells. Storage duration didn't affect OPCs survival rate. Conclusion Harvesting with digestion, step-wised freezing, preservation solution with trehalose contribute to higher OPCs survival rate during cryopreservation and cell-thawing. Storage time doesn't affect phenotypes and viability of OPCs.