Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide

Abstract

BackgroundMesenchymal stem cells (MSCs) are increasingly used as therapeutic agents as well as research tools in regenerative medicine. Development of technologies which allow storing and banking of MSC with minimal loss of cell viability, differentiation capacity, and function is required for clinical and research applications. Cryopreservation is the most effective way to preserve cells long term, but it involves potentially cytotoxic compounds and processing steps. Here, we investigate the effect of decreasing dimethyl sulfoxide (DMSO) concentrations in cryosolution by substituting with hydroxyethyl starch (HES) of different molecular weights using different freezing rates. Post-thaw viability, phenotype and osteogenic differentiation capacity of MSCs were analysed.ResultsThe study confirms that, for rat MSC, cryopreservation effects need to be assessed some time after, rather than immediately after thawing. MSCs cryopreserved with HES maintain their characteristic cell surface marker expression as well as the osteogenic, adipogenic and chondrogenic differentiation potential. HES alone does not provide sufficient cryoprotection for rat MSCs, but provides good cryoprotection in combination with DMSO, permitting the DMSO content to be reduced to 5%. There are indications that such a combination would seem useful not just for the clinical disadvantages of DMSO but also based on a tendency for reduced osteogenic differentiation capacity of rat MSC cryopreserved with high DMSO concentration. HES molecular weight appears to play only a minor role in its capacity to act as a cryopreservation solution for MSC. The use of a ‘straight freeze’ protocol is no less effective in maintaining post-thaw viability of MSC compared to controlled rate freezing methods.ConclusionA 5% DMSO / 5% HES solution cryopreservation solution using a ‘straight freeze’ approach can be recommended for rat MSC.

Meta-analysis of preclinical studies of mesenchymal stromal cells for ischemic stroke

“…Optimal translation of MSCs to humans may therefore require additional studies focused on issues such as effects of storage, transportation, and thawing. [37][38][39] Second, which MSC administration route(s) should be pursued as a priority? The intracerebral route showed larger effects than IV, but a neurosurgical procedure may not be trivial for some patients with recent stroke, and the IV route did provide very large behavioral benefits ( figure 1A).…”

Enhanced viability and neural differential potential in poor post-thaw hADSCs by agarose multi-well dishes and spheroid culture

“…Therefore, effective cellular cryopreservation offers an opportunity to advance the potential use and implementation of these cells into clinical applications. Sometimes, storage of hADSCs could be seen as an intermediary GMP product to be needed in the future for many differentiation protocols to be developed [54]. Yet, the processes of multi-passage or cryopreservation invariably leaded these cells into adverse growth status, which affected their viability, morphology and differentiation potential [21][22][23][24][25].…”

Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena)

“…In spite of using same protocol and careful cell management in all independent experiments, subtle difference in the initial condition before being frozen might provoke high variation at the end point. Suboptimal culture condition for O. dancena embryonic cells can be indirectly proven by relatively low post-thaw cell viability (73.5 ± 10.2 % in maximum) in comparison with those from higher vertebrates largely reaching more than 80 % (Ock and Rho 2011;Naaldijk et al 2012;Lee et al 2013c). Different sets of experiments focusing on freezing duration and employing short-term cultured cells or another batch of O. dancena cell lines will provide more clear evidence.…”