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

Nat Protoc. 2021 Jan;16(1):182-217. doi: 10.1038/s41596-020-00411-2. Epub 2020 Nov 27.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Cell Culture Techniques / methods
  • Cells, Cultured
  • Fetus / cytology
  • Fetus / metabolism
  • Gene Editing / methods
  • Gene Knock-In Techniques / methods
  • Gene Knockout Techniques / methods
  • Hepatocytes / cytology*
  • Hepatocytes / metabolism
  • Humans
  • Liver / cytology*
  • Liver / metabolism
  • Organoids / cytology*
  • Organoids / metabolism