Abstract
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.
Key points
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This Protocol is for the large-scale expansion of proliferating human hepatocytes derived from human donor livers. Proliferating human hepatocytes can be rematured in a three-dimensional culture system and are amenable to genetic manipulation.
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This overcomes limitations in using primary human hepatocytes, for which there is a shortage of donors and which display a low efficiency of gene manipulation.
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Data availability
All data generated or analyzed during this study are included in either this paper or in our original research study15,18,19,20,21. The RNA-seq data were reanalyzed from the public repository Gene Expression Omnibus database: GSE112866 (related to Extended Data Fig. 1f and Supplementary Code 1). All raw data are provided as source data files. Any additional data required for research purposes are available from the corresponding authors upon request. Source data are provided with this paper.
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Acknowledgements
We thank the core facilities for molecular biology, cell biology and animal care at the Center for Excellence in Molecular Cell Science (CEMCS) (Shanghai Institute of Biochemistry and Cell Biology, China). This project was supported by the National Natural Science Foundation of China (NSFC) (grant nos. 92168202, 32370793 and 32221002), the Shanghai Science and Technology Committee (grant no. 22JC1403001), the Shanghai Municipal Science and Technology Major Project and the National Center of Technology Innovation for Biopharmaceuticals (grant no. NCTIB2023XB01019). The schematic view was adapted from BioRender.com.
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Contributions
The data in the paper are derived from independent experiments conducted by multiple individuals (K.Z., X.Y., S.L., Y.S. and C.W.). K.Z. performed most of the experiments; X.Y. and C.W. performed PHHs and 3D-cultured ProliHHs experiments; S.L. and Y.S. assisted with the cell culture and the manuscript preparation; J.C. performed the HTx; K.Z., X.Y. and L.H. analyzed the data and wrote the manuscript and L.H. supervised and coordinated the project.
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L.H., L.Z. and K.Z. declare financial interests via patents filed by Center for Excellence in Molecular Cell Science on the production of ProliHHs.
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Nature Protocols thanks Dongho Choi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references
Zhang, K. et al. Cell Stem Cell 23, 806–819 e804 (2018): https://doi.org/10.1016/j.stem.2018.10.018
Wang, C. et al. Hepatology 76, 1690–1705 (2022): https://doi.org/10.1002/hep.32436
Zhang, K. et al. Cell Prolif. 56, e13467, (2023): https://doi.org/10.1111/cpr.13467
Yuan, X. et al. Cell Stem Cell 31, 484–498 e485 (2024): https://doi.org/10.1016/j.stem.2024.02.005
Zhang, K. et al. Cell Stem Cell 31, 1187–1202 e8 (2024): https://doi.org/10.1016/j.stem.2024.04.022
Extended data
Extended Data Fig. 1 ProliHHs culture in modified HM.
a. Time-lapse images of PHHs (healthy donor: MRW) cultivated in HM and modified HM. b. Relative cell proliferation (fold change) of ProliHHs (P1) in HM and modified HM (n = 3). The data are shown as mean ± SD. ****p < 0.0001; Student’s t test. c. Expression of hepatocyte genes and progenitor-associated genes determined by qPCR in HM and modified HM. Data are normalized to PHH (n = 3). The data were shown as the mean ± SD. d. Representative cell images of cultured hepatocytes (donor: MRW). e. Growth curves of cultured PHHs from healthy donor (LTV and MRW) were analyzed at indicated passages. f. Pearson correlation coefficient-based heat map analysis was performed to compare global gene expression profiles in ProliHHs (P2, P4, P6 and P8 under hypoxia), PHH and hiPSC-derived liver progenitor-like cells (LPCs). The RNA-seq data have been published previously and the accession number of the RNA-seq data in the GEO database is GSE112866. g. Representative images of fibroblast-like ProliHHs.
Extended Data Fig. 2 Functional identification of Liver Organoid.
a. Expression of hepatocyte genes was determined by qPCR after ProliHHs maturation induction. Data are normalized to freshly thawed PHH (n =5). The data were shown as the mean ± SD. b. Co-immunofluorescent staining of CYP1A2 and CYP3A4 in ProliHH-derived liver organoids. Scale bars, 100 mm. c. Co-immunofluorescent staining of E-Cadherin and multidrug resistance-associated protein 2 in ProliHH-derived liver organoids. Scale bars, 100 mm. d. Immunofluorescent staining of Ki67 in ProliHH-derived liver organoids. Scale bars, 100 mm. e. Expression of progenitor-associated genes was determined by qPCR after ProliHHs maturation induction (n = 5). Data are normalized to freshly thawed PHH and the data were shown as the mean ± SD. f. Immunofluorescent staining of EPCAM in ProliHH-derived liver organoids. Scale bars, 100 mm. g. Expression of AFP was determined by qPCR after ProliHHs maturation induction (n = 3), the data were shown as the mean ± SD. Data provide the amplification cycles of the AFP gene, using PHH and HepG2 cells as negative and positive controls for AFP expression, respectively. h. Representative time-lapse images of ProliHH-derived liver organoids from a healthy donor demonstrating the uptake of pHrodo™ Green-LDL.
Extended Data Fig. 3 ProliHH cryopreservation and resuscitation.
a. Representative images of Trypan Blue staining and live/dead staining of resuscitated ProliHH after cryopreservation b. The percentage of live cells of resuscitated ProliHH as determined by Trypan Blue exclusion assay (n = 3), the data were shown as the mean ± SD. c. Time-lapse images of resuscitated ProliHH cultivation. d. Relative cell proliferation (fold change) of ProliHHs in normal (n = 3) and resuscitated ProliHH (n = 5), the data were shown as the mean ± SD. e. Expression of hepatocyte genes and progenitor-associated genes in normal and resuscitated ProliHH cells was determined by qPCR (n = 4), the data were shown as the mean ± SD.
Supplementary information
Supplementary Code 1
The code for the Pearson correlation coefficient.
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Zhang, K., Yuan, X., Lu, S. et al. Expansion of human hepatocytes and their application in three-dimensional culture and genetic manipulation. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01211-2
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DOI: https://doi.org/10.1038/s41596-025-01211-2
