Loven Anne-Dorthe Bonde, Olsen Anette Kristensen, Friis Christian, Andersen Birgitte
Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.
Chem Biol Interact. 2005 Jun 30;155(1-2):21-30. doi: 10.1016/j.cbi.2005.03.040.
Primary porcine hepatocytes were cryopreserved using freezing boxes or a programmable freezer (PF). Upon thawing and culturing in 12-well plates cryopreserved hepatocytes were compared with their fresh controls on days 1 and 2 after plating. Cryopreserved hepatocytes attached approximately as well as fresh hepatocytes and useful cultures were obtained. In cryopreserved hepatocytes, coumarin 7-hydroxylation, 6beta-testosterone hydroxylation and p-nitrophenol glucuronidation were reduced to about 10-40, 35 and 40%, respectively, compared to their fresh counterparts. Glycogen synthesis in cryopreserved hepatocytes was reduced to about 30% on day 1 of culture and about 47% on day 2 of culture compared to the synthesis in fresh hepatocytes. Both fresh and cryopreserved hepatocytes increased the synthesis by twofold in response to stimulation with insulin. Reduced basal levels of glycogen and of glycogen synthesis could be explained by an increased energy demand in cryopreserved hepatocytes needing to repair damages caused by cryopreservation. Glycogenolysis was reduced to about 50% in cryopreserved hepatocytes and gluconeogenesis to about 40% of the glucose production in fresh hepatocytes. In both fresh and cryopreserved hepatocytes the glucose production from glycogenolysis and gluconeogenesis, respectively, was increased fourfold in response to stimulation with glucagon. Overall, the hepatocytes cryopreserved in boxes had a tendency to perform better than hepatocytes cryopreserved in a programmable freezer. In conclusion, the cryopreserved hepatocytes were metabolic active; however, to a lower extent than the fresh hepatocytes, although, the cryopreserved hepatocytes responded as well as the fresh hepatocytes to insulin and glucagon.
原代猪肝细胞使用冷冻盒或程序降温冷冻仪（PF）进行冻存。解冻后，将冻存的肝细胞接种于12孔板中培养，并在接种后的第1天和第2天与新鲜对照肝细胞进行比较。冻存肝细胞的贴壁情况与新鲜肝细胞大致相同，且获得了有效的培养物。与新鲜肝细胞相比，冻存肝细胞中香豆素7-羟化、6β-睾酮羟化和对硝基苯酚葡萄糖醛酸化分别降低至约10%-40%、35%和40%。与新鲜肝细胞中的糖原合成相比，冻存肝细胞在培养第1天的糖原合成降低至约30%，在培养第2天降低至约47%。新鲜和冻存的肝细胞在胰岛素刺激下，合成均增加了两倍。糖原和糖原合成基础水平的降低可能是由于冻存肝细胞需要修复冻存造成的损伤，能量需求增加所致。冻存肝细胞中的糖原分解降低至约50%，糖异生降低至新鲜肝细胞葡萄糖生成量的约40%。在新鲜和冻存的肝细胞中，糖原分解和糖异生分别产生的葡萄糖产量在胰高血糖素刺激下均增加了四倍。总体而言，用冷冻盒冻存的肝细胞比用程序降温冷冻仪冻存的肝细胞表现出更好的趋势。总之，冻存的肝细胞具有代谢活性；然而，其活性程度低于新鲜肝细胞，尽管如此，冻存肝细胞对胰岛素和胰高血糖素的反应与新鲜肝细胞相同。
