Isolation, characterization, proliferation, differentiation, and freeze-thaw survival of human wharton’s jelly mesenchymal stem cells from early and late passages

Wharton’s jelly of human umbilical cord has been known to be a rich source of mesenchymal stem cells (MSCs) which have therapeutic potential for regenerative diseases. This study aimed to isolate, culture, characterize, proliferate, differentiate, and cryopreserve hWJMSCs at both early and late passages. Here hJWMSCs were isolated using explant method and propagated them up to late passage. Surface markers of hWJMSCs were analysed by flow cytometer and proliferation rate was examined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium MTS assay. Then, hWJMSCs were culture in differentiation medium and cryopreserved in different kind of freezing media. The cells at P3 and P9 were positive for CD105, CD73, CD90, and CD44 but negative for CD34, CD116, CD19, CD45, and HLA-DR. The proliferation rate of hWJMSCS at both passages increased with culture time where cell proliferation rate at P3 was higher at day 9 around 5.89±0.04 compared to P9 about 3.79±0.1 and they could be differentiated into three lineages (osteogenesis, chondrogenesis, and adipogenesis). Combination of 90% FBS and 10% DMSO showed the best cell survival rate of hWJMSCs at both passages for freeze-thawed assay. To conclude, hWJMSCs can be isolated easily using explant method, the cells retained their stem cell markers with increasing proliferation rate at both passages, showed differentiation potential and high number of live cells at cryopreservation assay.