Vitrification 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. In 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. Our results demonstrate successful vitrification of both single cell and spheroid using high concentration of CPAs in vitro without cytotoxicity.

中文翻译：

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使用高浓度的冷冻保护剂进行2D和3D干细胞培养的玻璃化冷冻保存。

玻璃化是成功冷冻生物而不形成冰晶的最有前途的技术。但是，干细胞中使用的高浓度（最多约6-8 M）冷冻保护剂（CPA）会引起渗透和代谢损伤。此外，在各种研究中，将常规的慢速冷冻方法应用于干细胞的3-D类器官的培养受到其大小的限制。在这项研究中，我们评估了高浓度CPA的作用，包括细胞毒性，并在单细胞水平上表征了人间充质干细胞（MSC）。玻璃化后的细胞活力，细胞损伤和凋亡机制以及细胞的增殖能力和多能性与慢速冷冻组相似。此外，我们确定了玻璃化大小控制的3D球体的玻璃化可能性，以使其具有较高的存活率。我们的结果表明，使用高浓度的CPA在体外对单细胞和球体均能成功进行玻璃化，而无细胞毒性。