Cryopreservation of Human Wharton's Jelly-derived Mesenchymal Stem Cells Following Controlled Rate Freezing Protocol Using Different Cryoprotectants; A Comparative Study

OBJECTIVES: To compare the effect of three different cryoprotectants on basic stem cell characteristics for the possibility of using well defined, dimethyl sulfoxide (DMSO) and serum free freezing solutions to cryopreserve human Wharton's jelly-derived mesenchymal stem cells (WJMSCs) following controlled rate freezing protocol. METHODS: The mesenchymal stem cells isolated from human Wharton's jelly were cryopreserved using 10% DMSO, 10% polyvinylpyrrolidone (PVP) and a cocktail solution comprising of 0.05 M glucose, 0.05 M sucrose and 1.5 M ethylene glycol following controlled rate freezing protocol. We investigated the post-thaw cell viability, morphology, proliferation capacity, basic stem cell characteristics, in vitro differentiation potential and apoptosis-related gene expression profile before and after cryopreservation. RESULTS: The cryoprotectant 10% DMSO has shown higher post-thaw cell viability of 81.2+/-0.58% whereas 10% PVP and cocktail solution have shown 62.87+/-0.35% and 72.2+/-0.23%, respectively at 0 h immediately thawing. The cell viability was further reduced in all the cryopreserved groups at 24 h later post-thaw culture. Further, the complete elimination of FBS in cryoprotectants has resulted in drastic reduction in cell viability. Cryopreservation did not alter the basic stem cell characteristics, plasticity and multipotency except proliferation rate. The expression of pro-apoptotic BAX and p53 genes were higher whilst p21 was lower in all the cryopreserved groups when compare to the control group of WJMSCs. CONCLUSION: Although 10% DMSO has shown higher post-thaw cell viability compare to 10% PVP and cocktail solution, the present study indicates the feasibility of developing a well-defined DMSO free cryosolution which can improve storage and future broad range applications of WJMSCs in regenerative medicine without losing their basic stem cell characteristics.

Preliminary Study of Human Ovarian Tissue Cryopreservation by Controlled-rate Freezing / 中国医科大学学报

Objective To compare the effect of different cryoprotectants and different concentrations on controlled?rate freezing of human ovarian tissues. Methods Ovarian tissues were sampled from 15 patients undergoing benign ovarian tumor surgery. Cortical slices were frozen by controlled?rate freezing using three cryoprotectants,propanediol,ethanediol,and dimethylsulphoxide,and the concentration of each cryoprotectant was 1.5 mol/L or 2.0 mol/L. Cortical slices obtained from each patient were processed with each cryopreservation procedure simultaneously. Morphology of fol?licles was studied by light and electron microscopy and the normal rate was compared with that of the fresh tissues from the patient. Results There were no significant differences in distribution of follicles of different developmental stages between each group(P0.05). However,the difference was statistically significant for 1.5 mol/L PROH,2.0 mol/L PROH, 1.5 mol/L DMSO and 2.0 mol/L DMSO groups(P0.05). Conclusion Controlled?rate freezing using 1.5 mol/L EG as the cryoprotectant can better save oocytes and granulosa cells. It is a preferable freezing procedure for ovarian tissues.