ROCK Y-27632 Inhibitor, Ascorbic Acid, and Trehalose Increase Survival of Human Wharton Jelly Mesenchymal Stem Cells After Cryopreservation

Wharton jelly mesenchymal stem cells are good candidates for application in different aspects of regenerative medicine, and their long-time banking is important. In this study, the effects of trehalose, ascorbic acid, and Y-27632 on proliferation and survival rate of these cells after cryopreservation were investigated. Mesenchymal stem cells were isolated from human umbilical cord Wharton jelly and frozen using a slow-rate cooling process. Different concentrations of trehalose (35, 75, and 125 mM), ascorbic acid (0.06, 0.125, 0.25, and 0.5 mM), and Y-27632 (10 μM) were used to treat culture medium and/or to supplement freezing medium. Assessment of cell viability after thawing was performed using Trypan blue staining, and MTT assay was performed to measure the cell proliferation rate. We observed significantly increased postthaw viability, increased cell proliferation, and decreased doubling time of cells when 75 mM trehalose, 0.25 and 0.5 mM ascorbic acid, and 10 mM Y-27632 were used. In addition, increased viability, proliferation, and attachment were observed after 24 hours of pretreatment with these cryoprotective agents and when they were added to conventional freezing medium. The use of different cryoprotective agents in culture and freezing media could be useful for long-term storage of Wharton jelly mesenchymal stem cells.