de Sousa G, Nicolas F, Placidi M, Rahmani R, Benicourt M, Vannier B, Lorenzon G, Mertens K, Coecke S, Callaerts A, Rogiers V, Khan S, Roberts P, Skett P, Fautrel A, Chesne C, Guillouzo A
INSERM/Centre de Recherche Agronomique, Antibes, France.
Chem Biol Interact. 1999 Jun 1;121(1):77-97. doi: 10.1016/s0009-2797(99)00092-7.
Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.
伦理、经济和技术方面的原因阻碍了从猴子等高等级哺乳动物获取新鲜分离的肝细胞用于药物临床前评估的常规供应。因此，我们旨在开发优化且可重复的方案，用于冷冻保存和解冻来自这一主要毒理学研究物种的肝实质细胞。在常规使用这些方案之前，我们通过一项多实验室研究对其进行了验证。对整个动物肝脏进行解离后，获得了10亿至50亿个肝实质细胞，活力约为86%。将刚分离出的细胞的适当部分（约20%）立即进行原代培养，并进行各种肝特异性测试，以检查其代谢、生化和毒理学功能以及超微结构特征。将大部分肝细胞冷冻，并在解冻后使用相同参数检查其功能。对新鲜和解冻后的猴子肝细胞的表征证明了各种肝特异性功能得以维持。事实上，冷冻保存的肝细胞能够在培养中存活并发挥功能，与新鲜肝细胞相当。在深度冷冻保存后，合成（蛋白质、ATP、谷胱甘肽）以及胆汁酸结合和分泌的能力得以保留。在猴子身上观察到药物代谢活性的稳定性比啮齿动物肝细胞更好。解冻后，I相和II相活性（细胞色素P450、乙氧基香豆素-O-脱乙基酶、艾氏剂环氧化酶、环氧化物水解酶、谷胱甘肽转移酶、谷胱甘肽还原酶和谷胱甘肽过氧化物酶）得到了很好的保留。几种药物的代谢模式在冷冻保存前后在定性和定量方面相似。最后，细胞毒性测试表明，冷冻/解冻步骤并未改变细胞对有毒化合物的敏感性。
