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Dental pulp stem cells (DPSCs) from human third molar tooth germ (wisdom tooth) were isolated using a collagenase based enzymatic method, the obtained cells were analyzed as for morphology in monolayer culture, immunophenotype, proliferation and differentiation potential before and after cryopreservation. In this study, we showed that based on morphological features, surface markers profile and differentiation potential, the isolated DPSCs corresponded to multipotent mesenchymal stromal cells. DPSCs cryopreservation by slow cooling (1 °С / min) down to –80°C with subsequent immersion into liquid nitrogen in cryoprotectant-free culture medium led to cell death. Cryopreservation using the same protocol in the presence of 10% dimethyl sulfoxide (DMSO) and 20% serum ensured (82 ± 6)% cell viability; while metabolic and proliferative activity, as well as the ability to differentiate into the osteo- and adipogenic lineages of cryopreserved DPSCs were similar to their non-cryopreserved counterparts. Probl Cryobiol Cryomed 2021; 31(1): 058–069

Cryopreservation is the only established method to provide long-term storage and fast availability of cellular product for therapeutic applications. The overwhelming majority of cryopreservation media contain toxic concentrations of dimethyl sulfoxide (DMSO) limiting the possibility for the direct administration of cryopreserved cells to the patients. Here, we propose a novel approach for nontoxic xeno-free cryopreservation of human multipotent mesenchymal stromal cells (MSCs) aimed at ensuring high viability, ready-to-use availability, and localized delivery of the cell-based graft into damaged tissues. For MSC cryopreservation, we applied sucrose pretreatment procedure and xeno-free cryoprotective medium containing human platelet-poor blood plasma (PPP), sucrose, and nontoxic concentration of DMSO. Using the combination of PPP, 0.2 M sucrose, and 1% DMSO, the recovery rate of cryopreserved MSCs reached 73% of the values obtained for noncryopreserved cells. Moreover, the presence of PPP in the cryoprotective medium provided the possibility to create a ready-to-use 3D hydrogel for the localized delivery and additional support of MSCs in vivo. In a proof-of-concept study, we assessed the regenerative capacity of cryopreserved MSCs in a full-thickness wound model in mice. The positive impact of MSCs within 3D gel on wound healing rates was confirmed by morphometric and histological examinations. Our results demonstrate the possibility to apply cryopreserved cells immediately after thawing using a cryoprotective medium as the vehicle solution.

The review contains the data on scope, properties and localization of resident stem/progenitor cells of skin. The emphasis is given to populations of epithelial and dermal stem cells (SCs), as well as of melanocyte and subcutaneous fat SCs. The cells represent a prospective object for cryobiological technologies and establishing low temperature banks for use in theoretical and experimental biology, general and personalized medicine, development of bioengineered equivalents of tissues and artificial organs. Probl Cryobiol Cryomed 2014; 24(1):3–15.