Dou M, de Sousa G, Lacarelle B, Placidi M, Lechene de la Porte P, Domingo M, Lafont H, Rahmani R
INSERM U278 Faculté de Pharmacie, Marseille, France.
Cryobiology. 1992 Aug;29(4):454-69. doi: 10.1016/0011-2240(92)90048-7.
In drug metabolism studies, isolated and cultured human hepatocytes provide a useful model for overcoming the difficulty of extrapolating from animal data. In vitro studies with human hepatocytes are scarce because of the lack of livers and suitable methods of storage. After developing a new method for cryopreservation of human hepatocytes, we evaluated the effects of deep freezing storage on their viability, morphology, and functional and toxicological capabilities in classical culture conditions. Freshly isolated human hepatocytes were cryopreserved in medium containing 10% Me2SO and 20% fetal calf serum, using a Nicool ST20 programmable freezer (-1.9 degrees C/min for 18 min and -30 degrees C/min for 4 min). Cells were stored in liquid nitrogen. Viability of thawed human hepatocytes was 50-65% as assessed by erythrosin exclusion test prior to purification on a Percoll density gradient. Morphological criteria showed that thawed human hepatocytes require an adaptation period to the medium after seeding. Functional assessments showed that human hepatocytes which survive freezing and thawing preserve their protein synthesis capabilities and are able to secrete a specific protein, anionic peptidic fraction, which is involved in the hepatic uptake of bile-destined cholesterol. We then studied Midazolam biotransformation to test metabolic functions, and erythromycin toxicity by Neutral Red test (cell viability) and 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide test (cell metabolism). All of these experiments indicated that thawed human hepatocytes should be used 38 h after seeding for optimum recovery of their functions: membrane integrity, protein synthesis, and stabilization of drug metabolism enzymes.
在药物代谢研究中，分离培养的人肝细胞为克服从动物数据外推的困难提供了一个有用的模型。由于缺乏肝脏及合适的储存方法，用人肝细胞进行的体外研究较少。在开发出一种新的人肝细胞冷冻保存方法后，我们评估了深度冷冻保存对其在经典培养条件下的活力、形态以及功能和毒理学能力的影响。将新鲜分离的人肝细胞保存在含有10%二甲基亚砜和20%胎牛血清的培养基中，使用Nicool ST20可编程冷冻机（-1.9℃/分钟，持续18分钟，然后-30℃/分钟，持续4分钟）。细胞储存在液氮中。在通过Percoll密度梯度纯化之前，用赤藓红排除试验评估解冻后人肝细胞的活力为50 - 65%。形态学标准表明，解冻后人肝细胞接种后需要一段适应培养基的时间。功能评估显示，经冷冻和解冻后存活的人肝细胞保留了其蛋白质合成能力，并且能够分泌一种特定蛋白质，即阴离子肽部分，它参与肝脏对胆汁源性胆固醇的摄取。然后我们研究了咪达唑仑的生物转化以测试代谢功能，并通过中性红试验（细胞活力）和3 -（4,5 - 二甲基噻唑 - 2 - 基）- 二苯基四氮唑溴盐试验（细胞代谢）研究了红霉素毒性。所有这些实验表明，解冻后人肝细胞应在接种38小时后使用，以便最佳恢复其功能：膜完整性、蛋白质合成以及药物代谢酶的稳定性。
