National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, PR China.
J Biotechnol. 2012 Dec 31;162(2-3):224-31. doi: 10.1016/j.jbiotec.2012.09.004. Epub 2012 Sep 16.
Cryopreservation is one of the most practical methods for the long-term storage of cell-matrix systems to ensure off-shelf availability in tissue engineering, stem cell therapy and drug testing. The aim of this study is to investigate the effects of osmotic and cold shock caused by the procedures of cryoprotectant agent addition/removal and freezing during cryopreservation on cell viability, intracellular properties, such as filamentous actin distribution, mitochondria localization and intracellular pH, and further recovery of adherent human mesenchymal stem cells. Our results shows a significant decrease in cell viability around 30% after cryopreservation at the cooling rates of 1, 5 and 10°C/min in comparison to the adherent cells and the cells in suspension, implicating that the adherent cells are more vulnerable than the suspension cells. The osmotic shock and cold shock induced by freezing lead to dramatic changes in the intracellular properties. The cooling rate of 10°C/min results in acidification of intracellular pH, distortion and accumulation of filamentous actin, and aggregation of mitochondria. Our findings also suggest that the cooling rate of 1°C/min helps to maintain cell morphology and attachment, integrity and uniformity of filamentous actin, and leads to better cell recovery after cryopreservation.
冷冻保存是细胞基质系统长期储存的最实用方法之一，可确保组织工程、干细胞治疗和药物测试中货架上的可用性。本研究旨在研究在冷冻保存过程中添加/去除冷冻保护剂和冷冻过程中渗透压和冷冲击对细胞活力、细胞内特性（如丝状肌动蛋白分布、线粒体定位和细胞内 pH）的影响，以及进一步恢复贴壁人骨髓间充质干细胞。我们的结果表明，与贴壁细胞和悬浮细胞相比，在 1°C/min、5°C/min 和 10°C/min 的冷却速率下冷冻保存后，细胞活力显著下降约 30%，这表明贴壁细胞比悬浮细胞更脆弱。冷冻引起的渗透压冲击和冷冲击导致细胞内特性发生剧烈变化。10°C/min 的冷却速率导致细胞内 pH 酸化、丝状肌动蛋白变形和聚集、线粒体聚集。我们的研究结果还表明，1°C/min 的冷却速率有助于保持细胞形态和附着、丝状肌动蛋白的完整性和均匀性，并在冷冻保存后实现更好的细胞恢复。
