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Establishment of human fetal hepatocyte organoids and CRISPR–Cas9-based gene knockin and knockout in organoid cultures from human liver


The liver is composed of two epithelial cell types: hepatocytes and liver ductal cells. Culture conditions for expansion of human liver ductal cells in vitro as organoids were previously described in a protocol; however, primary human hepatocytes remained hard to expand, until recently. In this protocol, we provide full details of how we overcame this limitation, establishing culture conditions that facilitate long-term expansion of human fetal hepatocytes as organoids. In addition, we describe how to generate (multi) gene knockouts using CRISPR–Cas9 in both human fetal hepatocyte and adult liver ductal organoid systems. Using a CRISPR–Cas9 and homology-independent organoid transgenesis (CRISPR-HOT) approach, efficient gene knockin can be achieved in these systems. These gene knockin and knockout approaches, and their multiplexing, should be useful for a variety of applications, such as disease modeling, investigating gene functions and studying processes, such as cellular differentiation and cell division. The protocol to establish human fetal hepatocyte organoid cultures takes ~1–2 months. The protocols to genome engineer human liver ductal organoids and human fetal hepatocyte organoids take 2–3 months.

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Fig. 1: Schematic overview of this protocol.
Fig. 2: Human fetal hepatocyte organoid line establishment.
Fig. 3: Genome engineering of human fetal hepatocyte organoids.
Fig. 4: Genome engineering of human liver ductal organoids.
Fig. 5: Representative follow-up and characterization of human fetal hepatocyte organoids and genome-engineered human liver ductal organoids.


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We thank H. Gehart for providing fluorescent images of human liver ductal organoids; S. van der Brink for RSPO1-conditioned medium production; and L. Huang and L. Luciana for critically reading the manuscript. We acknowledge all of the anonymous tissue donors. This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society. D.H. is supported by an SSMF postdoctoral fellowship (P19-0074).

Author information




Conceptualization: D.H. and B.A.; methodology: D.H., B.A. and H.H; formal analysis: D.H. and B.A.; investigation: D.H. and B.A.; resources: S.C.v.S.L. and H.C.; data curation: D.H. and B.A.; writing—original draft: D.H., B.A. and S.C.v.S.L.; writing—review and editing: D.H., B.A. and H.C.; visualization: D.H. and B.A.; project administration: D.H. and B.A.; and funding acquisition: H.C.

Corresponding authors

Correspondence to Delilah Hendriks or Benedetta Artegiani or Hans Clevers.

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Competing interests

H.C. holds several patents on organoid technology.

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Peer review information Nature Protocols thanks Ömer Yilmaz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Hu, H. et al. Cell 175, 1591–1606 (2019):

Artegiani., B. et al. Cell Stem Cell 24, 927–943 (2019):

Artegiani, B. et al. Nat Cell Biol. 22, 321–331 (2020):

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Hendriks, D., Artegiani, B., Hu, H. et al. Establishment of human fetal hepatocyte organoids and CRISPR–Cas9-based gene knockin and knockout in organoid cultures from human liver. Nat Protoc 16, 182–217 (2021).

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