Cryopreservation of human Wharton’s jelly multipotent mesenchymal stromal cells with reduced concentration of dimethyl sulfoxide

Abstract

The urgent problem of long-term storage of multipotent mesenchymal stromal cells (MMSCs) is to improve the protocol of their cryopreservation for further application maintaining the therapeutic properties and minimizing the risks of adverse effects on the health of the recipient. As a standard cryoprotectant, a mixture of 90 % fetal bovine serum (FBS) and 10 % dimethyl sulfoxide (DMSO) is used, which, however, can cause a variety of adverse reactions. Therefore, it is important to study the possibility of reducing the concentration of potentially dangerous DMSO by adding other components to the mixture for cell cryopreservation. Purpose. To determine the efficiency of cryopreservation of human Wharton's jelly MMSCs using cryoprotectants of different composition by studying the proliferative activity, phenotype and features of cell morphology in culture in vitro. Materials and methods. The cryoprotective effect of various combinations of DMSO, ethylene glycol, sucrose and trehalose was studied. The efficacy was assessed by cell viability, their adhesive properties, expansion rate and monolayer formation, as well as the expression of main MMSCs markers. Results. It is shown that the most effective combination is 4 % DMSO with 6 % trehalose which provides the highest level of preservation of cell viability, as well as their adhesive and proliferative properties during thawing. Other combinations of the cryoprotectant components showed a much slower cell division, in some cases, the monolayer was not formed at all. For all investigated variants, the main surface markers of MMSCs were preserved. Conclusions. The obtained results indicate the possibility of reducing the concentration of DMSO to 4 % in the freezing medium for MMSCs cryopreservation while maintaining their viability, proliferative activity and common surface markers.