Pahernik S A, Thasler W E, Mueller-Hoecker J, Schildberg F W, Koebe H G
Department of Surgery, Klinikum Grosshadern, Munich, Germany.
Cryobiology. 1996 Oct;33(5):552-66. doi: 10.1006/cryo.1996.0059.
For clinical use of bioartifical liver devices a constant supply of primary liver cells has to be provided. Hypothermic storage of isolated pig hepatocytes could support large-scale stocking of cells. Freshly isolated pig hepatocytes from slaughterhouse livers were stored at 4 degrees C for 24, 48, and 72 h three different solutions: Leibovitz L-15 + 5% polyethylene glycol (PEG), University of Wisconsin (UW) solution, and a simplified UW solution. After storage, cells were cultured for 2 weeks in the collagen sandwich configuration. Viability of hepatocytes was 65, 85, and 83% after 24 h storage, 21, 74, and 70% after 48 h, and 5, 65, and 59% after 72 h in Leibovitz L-15 medium, UW, and the simplified UW, respectively. After storage in L-15 medium, cells attached poorly to collagen matrices and exhibited ultrastructural lesions. Functional performance in this group, as judged by albumin secretion and cytochrome P450-dependent activity in subsequent culture, decreased rapidly as a function of storage time, with zero values after 48 h storage. In contrast, hypothermia of hepatocytes in both UW solutions resulted in well-preserved cells with respect to ultrastructural appearance, attachment rates, and functional performance during culture. No significant differences were observed between the original and the simplified UW solution. Higher cell concentrations up to 5 x 10(7) cells/ml improved viability of hepatocytes on warmup. In terms of cell supply for hybrid artificial liver support, hypothermic storage of hepatocytes at 4 degrees C could mean an alternative to the cryopreservation of cells, which usually results in a substantial loss of cells and vital function of cells. Thus, pig hepatocytes could be stored at 4 degrees C for several days and meet the logistical need of bioartificial liver devices while avoiding the hazards of cell freezing.
对于生物人工肝装置的临床应用，必须持续供应原代肝细胞。低温保存分离的猪肝细胞有助于大规模储存细胞。从屠宰场肝脏中新鲜分离的猪肝细胞在4℃下分别在三种不同溶液中储存24、48和72小时：Leibovitz L-15 + 5%聚乙二醇（PEG）、威斯康星大学（UW）溶液和简化的UW溶液。储存后，将细胞以胶原夹心结构培养2周。在Leibovitz L-15培养基、UW溶液和简化的UW溶液中，储存24小时后肝细胞活力分别为65%、85%和83%，48小时后分别为21%、74%和70%，72小时后分别为5%、65%和59%。在L-15培养基中储存后，细胞与胶原基质的附着性较差，并出现超微结构损伤。根据后续培养中的白蛋白分泌和细胞色素P450依赖性活性判断，该组的功能性能随储存时间迅速下降，储存48小时后降至零。相比之下，两种UW溶液中肝细胞的低温保存导致细胞在超微结构外观、附着率和培养期间的功能性能方面保存良好。原始UW溶液和简化的UW溶液之间未观察到显著差异。高达5×10⁷个细胞/ml的更高细胞浓度可提高复温时肝细胞的活力。就混合人工肝支持的细胞供应而言，4℃低温保存肝细胞可能是细胞冷冻保存的一种替代方法，细胞冷冻保存通常会导致大量细胞损失和细胞重要功能丧失。因此，猪肝细胞可以在4℃下储存数天，满足生物人工肝装置的后勤需求，同时避免细胞冷冻的风险。
