Department of Pediatrics, Johns Hopkins University School of Medicine;
Department of Pediatrics, Seattle Children's Hospital.
J Vis Exp. 2021 Oct 25(176). doi: 10.3791/61812.
Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.
原代肝细胞广泛用于肝脏体外研究，特别是在葡萄糖代谢研究中。根据时间、劳动力、成本和原代肝细胞使用等不同需求，已经适应了一种基本技术，从而产生了各种原代肝细胞分离方案。然而，原代肝细胞分离过程中的众多步骤和耗时的试剂制备是效率的主要障碍。在比较了不同方案的优缺点后，我们结合了每种方案的优点，并制定了一种快速有效的原代肝细胞分离方案。仅需约 35 分钟，该方案就可以获得与其他方案一样多（如果不是更多）的健康原代肝细胞。此外，使用分离的原代肝细胞进行的葡萄糖代谢实验验证了该方案在体外肝脏代谢研究中的有用性。我们还广泛回顾和分析了本研究中每个步骤的意义和目的，以便未来的研究人员可以根据需要进一步优化该方案。
