Hubel A, Toner M, Cravalho E G, Yarmush M L, Tompkins R G
Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge 02139.
Biotechnol Prog. 1991 Nov-Dec;7(6):554-9. doi: 10.1021/bp00012a011.
During freezing, intracellular ice formation (IIF) has been correlated with loss in viability for a wide variety of biological systems. Hence, determination of IIF characteristics is essential in the development of an efficient methodology for cryopreservation. In this study, IIF characteristics of hepatocytes cultured in a collagen matrix were determined using cryomicroscopy. Four factors influenced the IIF behavior of the hepatocytes in the matrix: cooling rate, final cooling temperature, concentration of Me2SO, and time in culture prior to freezing. The maximum cumulative fraction of cells with IIF increased with increasing cooling rate. For cultured cells frozen in Dulbecco's modified Eagle's medium (DMEM), the cooling rate for which 50% of the cells formed ice (B50) was 70 degrees C/min for cells frozen after 1 day in culture and decreased to 15 degrees C/min for cells frozen after 7 days in culture. When cells were frozen in a 0.5 M Me2SO + DMEM solution, the value of B50 decreased from 70 to 50 degrees C/min for cells in culture for 1 day and from 15 to 10 degrees C/min for cells in culture for 7 days. The value of the average temperature for IIF (TIIF) for cultured cells was only slightly depressed by the addition of Me2SO when compared to the IIF behavior of other cell types. The results of this study indicate that the presence of the collagen matrix alters significantly the IIF characteristics of hepatocytes. Thus freezing studies using hepatocytes in suspension are not useful in predicting the freezing behavior of hepatocytes cultured in a collagen matrix. Furthermore, the weak effect of Me2SO on IIF characteristics implies that lower concentrations of Me2SO (0.5 M) may be just as effective in preserving viability. Finally, the value of B50 measured in this study indicates that cooling rates nearly an order of magnitude faster than those previously investigated could be used for cryopreservation of the hepatocytes in a collagen gel.
在冷冻过程中，细胞内冰晶形成（IIF）与多种生物系统的活力丧失相关。因此，确定IIF特征对于开发有效的冷冻保存方法至关重要。在本研究中，使用低温显微镜确定了在胶原基质中培养的肝细胞的IIF特征。有四个因素影响基质中肝细胞的IIF行为：冷却速率、最终冷却温度、Me2SO浓度以及冷冻前的培养时间。具有IIF的细胞的最大累积分数随冷却速率的增加而增加。对于在杜氏改良 Eagle 培养基（DMEM）中冷冻的培养细胞，培养1天后冷冻的细胞形成冰的细胞比例达到50%（B50）时的冷却速率为70℃/分钟，而培养7天后冷冻的细胞该冷却速率降至15℃/分钟。当细胞在0.5 M Me2SO + DMEM溶液中冷冻时，培养1天的细胞B50值从70℃/分钟降至50℃/分钟，培养7天的细胞从15℃/分钟降至10℃/分钟。与其他细胞类型的IIF行为相比，添加Me2SO时培养细胞的IIF平均温度（TIIF）值仅略有降低。本研究结果表明，胶原基质的存在显著改变了肝细胞的IIF特征。因此，使用悬浮肝细胞进行的冷冻研究对于预测在胶原基质中培养的肝细胞的冷冻行为并无帮助。此外，Me2SO对IIF特征的微弱影响意味着较低浓度的Me2SO（0.5 M）在保存活力方面可能同样有效。最后，本研究中测得的B50值表明，比先前研究快近一个数量级的冷却速率可用于胶原凝胶中肝细胞的冷冻保存。
