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.

Ultrastructural changes in the spinal cord of rats with experimental allergic encephalomyelitis under the influence of human umbilical cord-derived multipotent mesenchymal stromal cells cryopreserved according to different protocols

“…The obtained data are fully consistent and are explained with the use of previously obtained data on the maximum preservation of the proliferative activity of UC-MMSCs after the cryopreservation in the cryoprotective media with the reduced concentration of DMSO from 10 % to 4 % with the addition of 6 % trehalose (cryo-2), as well as high proliferative activity of cells after the cryopreservation in a standard medium (cryo-3), in contrast to the multicomponent (cryo-1), which is obviously associated with the changes in cell metabolism during cryopreservation in this medium [19].…”

“…The number and viability of the obtained cells was determined by counting in a Goryaev's chamber after staining with 0.2 % trypan blue solution (AppliChem, Germany). The accordance of UC-MMSCs to the multipotent mesenchymal stromal cells was confirmed by the ability to differentiate in the osteogenic, chondrogenic and adipogenic directions and persisted after freezing and thawing [19]. The variants of the composition of cryopreservation media did not affect the expression of specific surface markers CD73 + , CD90 + , CD105 + , determined by flow cytometry [19].…”

“…Previous in vitro studies have shown the possibility of using different modes of cryopreservation of UC-MMSCs while preserving their phenotypic characteristics and proliferative activity [19]. The next task is to evaluate the possibility of in vivo application of UC-MMSCs, cryopreserved under different protocols.…”

“…UC-MMSCs were obtained in accordance with the principles of bioethics from the umbilical cord during normal childbirth from a healthy mother by the informed consent. Cultivation, cryopreservation, thawing and washing were performed according to the previously described protocol [19]. The number and viability of the obtained cells was determined by counting in a Goryaev's chamber after staining with 0.2 % trypan blue solution (AppliChem, Germany).…”