Zhang Kun, Yuan Xiang, Lu Shuang, Shu Yajing, Wang Chenhua, Cen Jin, Wu Baihua, Hui Lijian
Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China.
Department of Histoembryology, Genetics and Developmental Biology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Nat Protoc. 2025 Jul 25. doi: 10.1038/s41596-025-01211-2.
Hepatocytes are one of the most important cell types in the liver, carrying out key functions. They are essential for hepatocyte-based therapy, disease modeling and drug development. However, the availability of primary human hepatocytes (PHHs) is limited by a shortage of donors. It is therefore of great value to expand PHHs in large quantities. Here we provide a detailed protocol for the large-scale expansion of PHHs (proliferating human hepatocytes, ProliHHs) derived from healthy donors and patients with inherited liver diseases, which can be rematured in a three-dimensional culture system. Moreover, we provide a protocol for the genetic manipulation of ProliHHs, including lentivirus transduction and CRISPR-Cas9-mediated knockout and knock-in. The protocol described here will help to realize the full potential of ProliHH-based therapy, organoid-based liver disease modeling and drug screening. The protocol to expand PHHs takes ~1-2 months, the protocol to establish the 3D-cultured ProliHHs takes ~8 d and the protocol to perform gene editing takes ~3 d. Personnel with basic scientific training can conduct these protocols.
肝细胞是肝脏中最重要的细胞类型之一，执行着关键功能。它们对于基于肝细胞的治疗、疾病建模和药物开发至关重要。然而，原代人肝细胞（PHH）的可获得性受到供体短缺的限制。因此，大量扩增PHH具有重要价值。在此，我们提供了一个详细方案，用于大规模扩增源自健康供体和遗传性肝病患者的PHH（增殖性人肝细胞，ProliHHs），这些细胞可在三维培养系统中重新成熟。此外，我们还提供了ProliHHs基因操作的方案，包括慢病毒转导以及CRISPR-Cas9介导的基因敲除和敲入。本文所述方案将有助于充分发挥基于ProliHHs的治疗、基于类器官的肝病建模和药物筛选的潜力。扩增PHH的方案耗时约1至2个月，建立三维培养的ProliHHs的方案耗时约8天，进行基因编辑的方案耗时约3天。具备基础科学培训的人员即可开展这些方案。
