Department of General, Visceral and Transplantation Surgery, University Hospital, LMU Munich, Munich, Germany.
HTCR-Services GmbH, Munich, Germany.
Biopreserv Biobank. 2023 Aug;21(4):367-377. doi: 10.1089/bio.2021.0173. Epub 2022 Nov 10.
Freshly isolated human hepatocytes are an important model for translational research, validation of experiments done in animals, and preclinical studies. Human hepatocyte isolation often cannot be carried out easily on demand in common research laboratories, and researchers often collaborate to share hepatocytes or outsource hepatocyte isolations. As a prerequisite for such a strategy, hepatocytes have to maintain their phenotypes after transport. Therefore, this study aimed to determine if overnight storage or shipment of hepatocytes affects their quality when viability, adherence, and cytochrome P450 (CYP) activities are considered. Hepatocytes were stored overnight or shipped to a collaborator in a cold storage solution on wet ice. On the next day, viability of hepatocytes was assessed before plating the cells to determine adherence. Hepatocytes were also cultured in a sandwich culture to determine CYP activities and inducibility. The results showed that although viability (79% ± 0.7% on isolation) was significantly decreased by overnight storage or shipment by 11% ( < 0.001) or 15% ( < 0.001), respectively, the viability of hepatocytes the next day at above 64% ± 2.2% remained sufficiently high for further experiments. In addition, hepatocytes stored for 18 or 24 hours were adherent the next day, and a high confluence of 81% ± 10% to 91% ± 4% was achieved after 48 hours in culture when hepatocytes were adhered on collagen-coated plates. Furthermore, CYP enzyme activities were inducible and not affected by variables such as fibrosis, age, type of operation, steatosis, and body mass index. However, our data would suggest that the type of cancer (primary/secondary), sex (male/female), hypertension, glutamic oxaloacetic transaminase activity, partial thromboplastin time, and size of perfused liver had significant effects ( < 0.05) on induction of some CYP enzymes. In conclusion, human hepatocyte isolation can be carried out at a centralized site and shared between multiple researchers, increasing flexibility and access to a representative human liver model.
新鲜分离的人肝细胞是转化研究、在动物实验中验证实验以及临床前研究的重要模型。在普通的研究实验室中，人肝细胞的分离通常不能根据需求轻松进行，研究人员经常合作共享肝细胞或外包肝细胞分离。作为这种策略的前提条件，肝细胞在运输后必须保持其表型。因此，本研究旨在确定肝细胞在运输或储存过夜后，其活力、黏附性和细胞色素 P450（CYP）活性等方面的质量是否会受到影响。肝细胞在冷藏溶液中用湿冰储存过夜或运往合作者处。第二天，在接种细胞以确定黏附性之前，评估肝细胞的活力。还将肝细胞在三明治培养中培养以确定 CYP 活性和诱导性。结果表明，尽管活力（分离时为 79%±0.7%）分别因储存过夜或运输而显著降低了 11%（ <0.001）或 15%（ <0.001），但第二天活力仍保持在 64%±2.2%以上，足以进行进一步的实验。此外，储存 18 或 24 小时的肝细胞在第二天黏附，当肝细胞在胶原涂层板上黏附时，在培养 48 小时后可达到 81%±10%至 91%±4%的高汇合度。此外，CYP 酶活性可诱导，不受纤维化、年龄、手术类型、脂肪变性和体重指数等变量的影响。然而，我们的数据表明，癌症类型（原发性/继发性）、性别（男/女）、高血压、谷氨酸草酰乙酸转氨酶活性、部分凝血活酶时间和灌注肝脏的大小对某些 CYP 酶的诱导有显著影响（ <0.05）。总之，人肝细胞的分离可以在一个集中的地点进行，并在多个研究人员之间共享，从而增加了灵活性并获得了代表性的人类肝脏模型。
