Acker Jason P, McGann Locksley E
Cell Transplant. 2002;11(6):563-71.
Extensive efforts to avoid intracellular ice formation (IF) during freezing have been central to current methods used for the preservation and long-term storage of cells and tissues. In this study, we examined the effect of intracellular ice formation on the postthaw survival of V-79W fibroblast and MDCK epithelial cells using convection cryomicroscopy and controlled-rate freezing. V-79W and MDCK cells were cultured as single attached cells or as confluent cell monolayers. Postthaw cell survival was assessed using three different indices: the presence of an intact plasma membrane, the ability to reduce alamarBlue, and the capacity to form colonies in culture. Regulating the isothermal nucleation temperature was used to control the incidence of IIF in the model systems. We report that the presence of intracellular ice in confluent monolayers at high subzero temperatures does not adversely affect postthaw cell survival. Further, we show that in the absence of chemical cryoprotectants, the formation of intracellular ice alone improves the postthaw survival of cultured V-79W fibroblast and MDCK epithelial cells. Improved long-term storage of cells and tissues will result by incorporating innocuous intracellular ice formation into current strategies for cryopreservation.
在冷冻过程中，为避免细胞内结冰（IF）所做的大量努力一直是当前用于细胞和组织保存及长期储存方法的核心。在本研究中，我们使用对流低温显微镜和程序降温法，研究了细胞内结冰对V - 79W成纤维细胞和MDCK上皮细胞解冻后存活率的影响。V - 79W和MDCK细胞以单个贴壁细胞或汇合细胞单层的形式进行培养。解冻后细胞存活率通过三种不同指标进行评估：完整质膜的存在、还原alamarBlue的能力以及在培养中形成集落的能力。调节等温成核温度用于控制模型系统中细胞内结冰（IIF）的发生率。我们报告，在零下较高温度下汇合单层细胞中存在细胞内冰并不会对解冻后细胞存活率产生不利影响。此外，我们表明，在没有化学冷冻保护剂的情况下，仅细胞内冰的形成就能提高培养的V - 79W成纤维细胞和MDCK上皮细胞解冻后的存活率。通过将无害的细胞内结冰纳入当前的冷冻保存策略，将改善细胞和组织的长期储存。
