Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro
Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro
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DOI：
10.3791/60507
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发表时间：
2019-12-01
影响因子：
1.2
通讯作者：
Gregor, Martin
中科院分区：
文献类型：
--
作者：
Korelova, Katerina;Jirouskova, Marketa;Gregor, Martin
肝细胞是肝脏的核心细胞，负责其代谢功能。因此，它们形成独特的极化上皮，其中两个或多个肝细胞贡献顶膜以形成胆小管网络，通过该网络分泌胆汁。肝细胞极化对于正确的小管形成至关重要，并且取决于肝细胞细胞骨架、细胞间接触和细胞外基质之间的相互作用。由于缺乏细胞培养物，肝细胞细胞骨架参与小管形成及其对病理情况的反应的体外研究受到限制，而细胞培养物与体内的小管网络结构非常相似。这里描述的是使用改良的胶原酶灌注技术从成年小鼠肝脏中分离小鼠肝细胞的方案。还描述了在 3D 胶原蛋白三明治环境中生产培养物，该培养物用于细胞骨架成分的免疫标记，以研究胆小管的形成及其对体外治疗的反应。结果表明，肝细胞 3D 胶原蛋白夹心培养物对毒素（乙醇）或肌动蛋白细胞骨架改变药物（例如，blebbistatin）的治疗有反应，并可作为体外研究胆小管形成和功能的有价值的工具。
Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.