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 similar to 1-2 months. The protocols to genome engineer human liver ductal organoids and human fetal hepatocyte organoids take 2-3 months.

Automated summary

This protocol provides detailed culture conditions for long-term expansion of human fetal hepatocytes as organoids and introduces methods for gene editing using CRISPR-Cas9. These approaches are valuable for disease modeling, gene function studies, as well as research on cellular differentiation and cell division processes.