Abstract
BackgroundVitrification is the most promising technology for successful cryopreservation of living organisms without ice crystal formation. However, high concentrations (up to ~ 6–8 M) of cryoprotective agents (CPAs) used in stem cell induce osmotic and metabolic injuries. Moreover, the application of conventional slow-freezing methods to cultures of 3-D organoids of stem cells in various studies, is limited by their size.ResultsIn this study, we evaluated the effect of high concentrations of CPAs including cytotoxicity and characterized human mesenchymal stem cell (MSC) at single cell level. The cell viability, cellular damage, and apoptotic mechanisms as well as the proliferation capacity and multipotency of cells subjected to vitrification were similar to those in the slow-freezing group. Furthermore, we identified the possibility of vitrification of size-controlled 3-D spheroids for cryopreservation of organoid with high survivability.ConclusionsOur results demonstrate successful vitrification of both single cell and spheroid using high concentration of CPAs in vitro without cytotoxicity.
Highlights
Vitrification is the most promising technology for successful cryopreservation of living organisms without ice crystal formation
Our results suggest that vitrification using a high concentration of cryoprotective agents (CPAs) facilitated successful cryopreservation of cell aggregates more efficiently than slow freezing
Several studies reported that high concentrations of CPAs such as dimethyl sulfoxide (DMSO), ethylene glycol (EG), and other compounds are used for organ storage; relatively low concentrations of CPAs are used for vitrification of single cells such as oocytes [11, 12]
Summary
Vitrification is the most promising technology for successful cryopreservation of living organisms without ice crystal formation. High concentrations (up to ~ 6–8 M) of cryoprotective agents (CPAs) used in stem cell induce osmotic and metabolic injuries. Organ cryopreservation is one of the most promising methods of storage and long-term preservation of transplantable cells and tissues. Vitrification is a novel approach to cryopreservation that facilitates freezing of living cells without crystallization. Vitrification simplifies and enhances cryopreservation by eliminating mechanical injury induced by ice crystal formation, and provides the optimal range of ‘critical cooling & rewarming rate’ for various cells and tissues [3]. The high concentration of CPAs (up to ~ 8 M) induces osmotic effects during freezing and rewarming.
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