Cryopreservation Of Neural Stem Cells Encapsulated In Rat Tail Collagen Type I

The cryopreservation of neural stem cells(NSCs)for clinical medicine transplantation and the establishment of stem cell bank are of important significance.This study followed the slow-cooling method with the NSCs encapsulated in rat tail collagen typeⅠto develop a new cryopreservation protocol.DMSO is used as the cryoprotectants.The concentration of rat tail collagen typeⅠis 0.5mg/ml,1.0mg/ml和1.5mg/ml respectively,and the concentration of DMSO is 10%(v/v),15%(v/v),20%(v/v)and 25%(v/v).From the variation of the concentration of DMSO at the external system after loading,the average concentration of DMSO solution within the collagen system can be estimated.And then the average internal concentrations of 8%to 15%were selected for the cryopreservation experiments and the effect of cooling rate on NSCs survival rate at the same time.The survival rate of NSCs at 0h, 24h after freeze-thawing was assayed,and NSCs were assayed by flow cytometry for the apoptosis and the ATP enzyme activity after freeze-thawing as well.The NSCs stem cells after freeze-thawing were assayed by immunofluorescence(Nestin-positive,βtubulinⅢ-positive,GFAP-positive and RIP-positive).The results show that neural stem cells in the collagen keep their good shape,groups of experimental conditions were tested for the slow-cooling cryopreservition of NSCs.The equilibrium time of encapsulated NSCs system was longer than that of cell suspension system, which slows osmosis of DMSO solution.The survival rates of the encapsulated NSCs after freezing and thawing operations with cooling rates of 1℃/min and 3.4℃/min are higher than that of cryopreservation of cell suspension,and could be about 88.5%for the conditon of 1.5mg/ml+20%DMSO.The results of apoptosis and ATP enzyme activity are also better than that of the traditional method,with the healthy cells of 86.7%.The NSCs are assayed by immunofluorescence after freeze-thawing,the Nestin-positive cells were found.So the NSCs kept the good function of stem cells.The cultured NSCs could be differentiated into neuron (βtubulinⅢ-positive),astrocyte(GFAP-positive)and oligodendrocyte(RIP-positive).