Chen Bowen, Dou Xiaoning, Zhang Dan, Liu Tiaoguo, Yang Bohui, Lu Zengkui
Key Laboratory of Animal Genetics and Breeding on Tibetan Plateau, Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou 730050, China.
Sheep Breeding Engineering Technology Research Center of Chinese Academy of Agricultural Sciences, Lanzhou 730050, China.
Curr Issues Mol Biol. 2022 Aug 12;44(8):3621-3631. doi: 10.3390/cimb44080248.
The liver plays a crucial role in metabolism, synthesis, biotransformation, secretion, and excretion. Hepatocytes are the main cells of the liver and can be used as a cell model to study liver function. The classic method of collagenase perfusion to isolate hepatocytes is a two-step technique that is time-consuming, labor-intensive, and has high technical requirements. Therefore, in this study, we compared different methods for isolating and culturing primary hepatocytes. We found that the 0.25% trypsin and 0.1 mg/mL type IV collagenase mixture at a 1:1 ratio showed the most efficient cell digestion, and William’s Medium E complete medium showed the best growth and proliferation. The isolated cells showed the typical irregular polygonal morphology of hepatocytes. Periodic acid−Schiff staining and immunofluorescence confirmed that the isolated cells were positive for glycogen and hepatocyte-specific markers cytokeratin 18, AFP, and albumin. On subculturing, stable cell lines were obtained. Therefore, we optimized the isolation and in vitro culture method to obtain highly pure (>95%) sheep primary hepatocytes from newborn sheep liver tissue.
肝脏在代谢、合成、生物转化、分泌和排泄过程中发挥着关键作用。肝细胞是肝脏的主要细胞，可作为研究肝功能的细胞模型。传统的胶原酶灌注法分离肝细胞是一种两步技术，耗时、费力且技术要求高。因此，在本研究中，我们比较了分离和培养原代肝细胞的不同方法。我们发现，0.25%胰蛋白酶和0.1 mg/mL IV型胶原酶按1:1比例混合显示出最有效的细胞消化效果，而William’s Medium E完全培养基显示出最佳的生长和增殖效果。分离出的细胞呈现出肝细胞典型的不规则多边形形态。过碘酸-希夫染色和免疫荧光证实，分离出的细胞糖原及肝细胞特异性标志物细胞角蛋白18、甲胎蛋白和白蛋白呈阳性。传代培养时，获得了稳定的细胞系。因此，我们优化了分离和体外培养方法，从新生羊肝脏组织中获得了高纯度（>95%）的羊原代肝细胞。
