The Assessment of Cryopreservation Conditions for Human Umbilical Cord Stroma-Derived Mesenchymal Stem Cells towards a Potential Use for Stem Cell Banking

Human umbilical cord stroma-derived mesenchymal stem cells (hUCS-MSCs) are considered as a remarkable and promising stem cell source to be potentially used in cellular therapies. While no graft rejection has been reported in the recipient organism even in xeno-transplantation studies, attenuate tumor cell growth and gene transfers have been experimentally shown. In this study, we have demonstrated a reliable, reproducible and efficient cryopreservation method of hUCS-MSCs resulting in one of the highest cell survival rates reported so far. Conventional, computer-controlled multi-step slow freezing (MSSF), and vitrification methods were comparatively tested using cell permeable [dimethylsulfoxide (DMSO), ethylene glycol] and impermeable [trehalose, sucrose, hydroxyethyl starch (HES), human serum albumin] cryoprotectant agents (CPAs). After determining the ice nucleation point for each solution, latent heat evolution was suppressed during freezing, followed by a cooling process to -40 degrees C at 1 degrees C/min or 0.3 degrees C/min. The efficiency of the cryopreservation techniques used was determined by cell viability and proliferation assays, the expression of cell surface markers, cytoskeletal proteins and chromosome alignments. The cell survival rate was found to be highest (87 +/- 5%) by MSSF with sucrose (0.1 M) +DMSO (10%) at 1 degrees C/min freezing rate. In this group, no significant difference was noted before and after the cryopreservation in cell morphology, cytokeratin, vimentin, and alpha-smooth muscle actin profiles and the expressions of CD105, CD90, CD73, CD29 and HLA-DR. Second highest cell survival ratio (85 +/- 6%) was obtained in DMSO (10%) alone at 1 degrees C/min freezing rate. Interestingly, poor (18 +/- 15%) cell survival rates were obtained after vitrification. Cumulatively, results indicated that MSSF favors the other freezing protocols with an addition of sucrose or DMSO alone depending on the freezing rate used.