The DOI matches its Crossref record; 30 of the 30 references it cites were found in a registry or an index.
Establishment of human fetal hepatocyte organoids and CRISPR–Cas9-based gene knockin and knockout in organoid cultures from human liver
Open accessAbstract
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. Culture conditions are described for long-term expansion of human fetal hepatocytes as 3D organoids. Gene knockin and knockout approaches are also described for organoids derived from human fetal hepatocytes and human adult liver ductal cells.
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Cites
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Self-citationOrganoids: The current status and biomedical applications
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Self-citationOrganoids in gastrointestinal diseases: From experimental models to clinical translation
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Urciuolo, A., Giobbe, G. G., Dong, Y., Michielin, F., Brandolino, L., Magnussen, M., Gagliano, O., Selmin, G., Scattolini, V., Raffa, P., et al. (2023) · Nature Communications · doi:10.1038/s41467-023-37953-4 · Cited by 121
Self-citationFetal liver development and implications for liver disease pathogenesis
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Metadata
- Authors
- Hendriks, D., Artegiani, B., Hu, H., Chuva de Sousa Lopes, S., & Clevers, H.
- Venue
- Nature Protocols
- Published
- 2020-11-27
- Volume
- 16
- Issue
- 1
- Pages
- 182-217
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W3110547538
- Cited by
- 159
- References
- 30
- FWCI
- 7.75
- Type
- journal article
- Language
- en
- Open access
- green
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- Hendriks, D., Artegiani, B., Hu, H., Chuva de Sousa Lopes, S., & Clevers, H. (2021). Establishment of human fetal hepatocyte organoids and CRISPR–Cas9-based gene knockin and knockout in organoid cultures from human liver. Nature Protocols. https://doi.org/10.1038/s41596-020-00411-2
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