Study On Culture Process Of Embryonic Stem Cells

Embryonic stem cells(ESCs)have great potentials in cell therapy and regenerative medicine due to their unlimited self-renewal capabilities and pluripotency.However,successful applications of ESCs require large numbers of cells with good quality.Ex vivo expansion of ESCs is a feasible strategy which could overcome this problem.However,these cells tend to differentiate and become senescent during expansion.The objective of this study was to investigate the culture process of ESCs.First,effects of cryopreservation on the quality of feeder layer generally from mouse embryonic fibroblasts(MEF)were investigated.It was shown that cell density of the feeder layers made by freshly isolated MEF remained constant on day 6 and cells began to show senescent afterwards.In contrast,the feeder layers prepared from cryopreserved MEF became senescent as early as on day 4.Based on this finding,in order to eliminate any effect of feeder layer on the growth of mouse ESCs(mESCs),the passaging of mESCs was performed every 3 days instead of 5 days.Secondly,the feeder-free culture process of mESCs was explored.After continuous cultivation for 20 passages,an expansion fold of 1.3×1017 was achieved,the percentage of SSEA-1+cells was maintained above 75%for all passages and the cells showed too lows senescence.Finally,the growth characteristics of mESCs in dynamic culture was studied.It was demosntrated that with the addition of 50 ?g/ml asorbic acid,senescence of mESCs was delayed,and total expansion fold of mESCs and the percentage of SSEA-1+cells were 53.9±1.5 and 74.9%±6.0%,respectively,both of which were significantly higher than those in control group without asorbic acid,respectively(38.5±3.9 and 62.1%±3.3%).These data prove that asorbic acid has anti-aging effect,thus promoting ex vivo expansion of mESCs.In conclusion,our work provided solid base for further optimization on ex vivo expansion of ESCs.