用于生物人工肝的大量猪肝细胞低温冻存技术

目的：比较大量猪肝细胞程序冻存与常规冻存的效果。方法采用二步法胶原酶经肝静脉逆行灌注的方法分离猪肝细胞，分离的肝细胞根据冻存方法与冻存袋的不同分5组：a 组，50 mL 冻存袋，程序冻存；b 组，100 mL 冻存袋，程序冻存；c 组，50 mL 冻存袋，常规冻存；d 组，100 mL 冻存袋，常规冻存；e 组，新鲜分离的肝细胞。各冻存组解冻后，比较各组的肝细胞活性、贴壁率、LDH 漏出量及白蛋白合成能力。结果新鲜分离的肝细胞产量为（1．8＆#177;0．4）1010/肝，活性高达（90．5＆#177;1．7）％，培养后贴壁率为（70．5＆#177;8．5）％。采用程序冻存的 a、b 组在肝细胞活性、贴壁率、LDH 漏出量及白蛋白合成能力方面明显优于常规冻存的 c、d 组（P＜0．05），但与 e 组新鲜分离的肝细胞相比，其解冻后肝细胞的活性、贴壁率及蛋白合成能力均降低（P＜0．05），LDH 漏出量增加（P＜0．05）。相同冻存方法条件下，50 mL 袋装与100 mL 袋装冻存效果差异无统计学意义（P＞0．05）。结论采用袋装程序冻存法大量冻存猪肝细胞可满足生物人工肝对肝细胞活性及数量的要求。

Cryopreservation for large quantity of porcine hepatocytes used in bioartificial liver support systems

Objective To study the effects of cryopreserving porcine hepatocytes in large quatities using the controlled-rate freezer program and the standard cryopreservation procedure.Methods Primary porcine hepatocytes were harvested by a modified two-step collagenase digestion method via hepatic vein perfusion,then stored in the Williams E medium solution .They were cryopreserved in large quantities （50 or 100 mL/bag）with the controlled-rate freezer program or the standard cryopreservation procedure.Once they were frozen,they were stored in the liquid nitrogen for 7 d.The effects on thawed cell viability and attachment,lactate dehydrogenase （LDH）release,and albumin synthesis were deter-mined.Results The hepatocytes yield was （1 .8 ＆#177;0.4）1010/liver with a viability of （90.5 ＆#177;1 .7）% and a plating rate of （70.5 ＆#177;8.5 ）%.Viability,attachment,LDH release and albumin synthesis of the thawed hepatocytes were significantly better in the group a and b with the controlled-rate freezer program compared with the group c and d with the standard cryopreservation procedure （P＆lt;0.05）,but they were all inferior to the group d,freshly isolated hepatocytes （P＆lt;0.05）. There was no significance between 50 mL bags and 100 mL bags if using the same freezing mehods（P＆gt;0.05）.Conclusion Successful cryopreservation of isolated porcine hepatocytes in large quantities using the controlled-rate freezer program could meet with the need of hepatocyte to the bioartificial liver support systems.