Huang Haishui, Zhao Gang, Zhang Yuntian, Xu Jiangsheng, Toth Thomas L, He Xiaoming
Department of Biomedical Engineering, The Ohio State University, 1080 Carmack Road, Columbus, Ohio 43210, United States.
Department of Mechanical Engineering, The Ohio State University, 201 W 19th Avenue, Columbus, Ohio 43210, United States.
ACS Biomater Sci Eng. 2017 Aug 14;3(8):1758-1768. doi: 10.1021/acsbiomaterials.7b00201. Epub 2017 Jun 12.
Conventional approaches for cell cryopreservation require the use of toxic membrane-penetrating cryoprotective agents (pCPA), which limits the clinical application of cryopreserved cells. Here, we show intentionally induced ice formation at a high subzero temperature (> -10 °C) during cryopreservation, which is often referred to as ice seeding, could result in significant cell injury in the absence of any pCPA. This issue can be mitigated by predehydrating cells using extracellular trehalose to their minimal volume with minimized osmotically active water before ice seeding. We further observe that ice seeding can minimize the interfacial free energy that drives the devastating ice recrystallization-induced cell injury during warming cryopreserved samples. Indeed, by combining predehydration using extracellular trehalose with ice seeding at high subzero temperatures, high cell viability or recovery is achieved for fibroblasts, adult stem cells, and red blood cells after cryopreservation without using any pCPA. The pCPA-free technology developed in this study may greatly facilitate the long-term storage and ready availability of living cells, tissues, and organs that are of high demand by modern cell-based medicine.
传统的细胞冷冻保存方法需要使用有毒的膜穿透性冷冻保护剂（pCPA），这限制了冷冻保存细胞的临床应用。在此，我们表明，在冷冻保存过程中，在较高的零下温度（> -10°C）下有意诱导冰形成（通常称为冰种形成），在没有任何pCPA的情况下可能会导致显著的细胞损伤。通过在冰种形成之前使用细胞外海藻糖将细胞预脱水至最小体积并使具有渗透活性的水最小化，可以缓解这个问题。我们进一步观察到，冰种形成可以最小化在解冻冷冻保存样品期间驱动破坏性的冰重结晶诱导的细胞损伤的界面自由能。事实上，通过将使用细胞外海藻糖的预脱水与在较高零下温度下的冰种形成相结合，在不使用任何pCPA的情况下，冷冻保存后的成纤维细胞、成体干细胞和红细胞可实现高细胞活力或回收率。本研究中开发的无pCPA技术可能会极大地促进现代细胞医学高度需求的活细胞、组织和器官的长期储存和随时可用。
