Optimized Cryopreservation System Of Human Oligodendrocyte Precursor Cells

Background:Oligodendrocyte Precursor Cells(OPCs),have the ability to migrate,proliferate and differentiate into mature oligodendrocytes(OLs)in the central nervous system(CNS).Demyelination related disease is a kind of nervous system disease with the absence of myelin sheath,such as spinal cord injury(SCI),multiple sclerosis(MS)and white matter injury(WMI).OPCs is considered to be the most promising cells for cerebral demyelinating disease.Cryopreservation of human OPCs was helpful for its basic and clinical transplantational researches.However,there is no more detailed systematic study on the freezing effect of human OPCs.Objective:This study aims to explore the impact of various conditions during cryopreservation on the survival rate to exploit an cryopreservation method of human OPCs.Method:We compared the cell viability of oligodendrocyte precursors harvested with or without digestion.Then we tested the impact of 4 factors during cryopreservation,cell densities on cryopreservation(.ranged from 0.5-3.5x106/mL),freezing solutions(Seven different solutions),freezing methods(the step-wised freezing or rapid freezing within liquid nitrogen)and storage durations(short-term and long-term)for the better survival rate of human OPCs.The optimized method with the best survival rate of OPCs would be implemented and the viability,OPCs specific markers:PDGFRa,A2B5,NG2,proliferation related Ki67 antigen,CCK8 proliferation and whole genome sequencing were tested and compared.Results:Harvesting with digestion contributed to higher human OPCs survival rate.Ranging from 0.5-3x106/mL on cryopreservation,the cell viability was a positive correlation with cell density.Human OPCs of rapid freezing had survival rate.less than 30%and couldn't be recultured.The step-wised freezing group shows higher recovery rate.Harvesting with digestion,preservation solution with trehalose,using 2.0 ×106/mL of cell densities,step-wised freezing,contributed to the highest OPCs survival rate,could reach to(75.73±6.66)%.Storage duration didn't affect OPCs survival rate(within 6 months).Compared to the fresh cultured group human OPCs specific marker,cell proliferation,cell migration and gene expression of human OPCs were similar,cellular differentiation ability were not affected neither.Conclusion:Harvesting with digestion,step-wised freezing,preservation solution with trehalose contributes to higher human OPCs survival rate during cryopreservation and cell-thawing,is an ideal preservation condition for OPCs derived from human fetal neural stem cells.Conditions of our experiment didn't affect phenotypes and cell function of human OPCs.