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.

肝脏由两种上皮细胞类型组成：肝细胞和肝导管细胞。先前在协议中描述了体外扩增人肝导管细胞作为类器官的培养条件；然而，直到最近，原代人肝细胞仍然难以扩张。在这个协议中，我们提供了我们如何克服这一限制的完整细节，建立了促进人类胎儿肝细胞作为类器官长期扩张的培养条件。此外，我们描述了如何在人类胎儿肝细胞和成人肝导管类器官系统中使用 CRISPR-Cas9 生成（多）基因敲除。使用 CRISPR-Cas9 和不依赖同源性的类器官转基因 (CRISPR-HOT) 方法，可以在这些系统中实现有效的基因敲入。这些基因敲入和敲除方法，以及它们的多路复用，应该可用于各种应用，例如疾病建模、研究基因功能和研究过程，例如细胞分化和细胞分裂。建立人类胎儿肝细胞类器官培养的方案需要约 1-2 个月。基因组工程人类肝导管类器官和人类胎儿肝细胞类器官的协议需要 2-3 个月。