Organoids and Spheroids as Models for Studying Cholestatic Liver Injury and Cholangiocarcinoma

Abstract

Cholangiopathies, such as primary sclerosing cholangitis, biliary atresia, and cholangiocarcinoma, have limited experimental models. Not only cholangiocytes but also other hepatic cells including hepatic stellate cells and macrophages are involved in the pathophysiology of cholangiopathies, and these hepatic cells orchestrate the coordinated response against diseased conditions. Classic two-dimensional monolayer cell cultures do not resemble intercellular cell-to-cell interaction and communication; however, three-dimensional cell culture systems, such as organoids and spheroids, can mimic cellular interaction and architecture between hepatic cells. Previous studies have demonstrated the generation of hepatic or biliary organoids/spheroids using various cell sources including pluripotent stem cells, hepatic progenitor cells, primary cells from liver biopsies, and immortalized cell lines. Gene manipulation, such as transfection and transduction can be performed in organoids, and established organoids have functional characteristics which can be suitable for drug screening. This review summarizes current methodologies for organoid/spheroid formation and a potential for three-dimensional hepatic cell cultures as in vitro models of cholangiopathies.

FIG. 1.

Generation of hepatic organoids and spheroids. Liver organoids are generally established from ESCs, iPSCs, HPCs, or primary cells isolated from liver or bile duct tissue samples. Cells are embedded with ECM components, such as Matrigel or collagen type I, to support organoid formation. Cells differentiate into hepatic cells depending on culture media. Mature organoids are functional and consist of multiple cell types, such as hepatocytes and cholangiocytes. Hepatocytes in organoids express HNF4α and albumin, as well as show tight junction, bile canaliculi, and CYP3A4 activity. Populating cholangiocytes can form cyst-like structures and have primary cilia and GGT activity expressing biliary markers, such as CK-7 and CK-19. Cell aggregates established from a single cell type, especially immortalized or cancer cells, are referred to as “spheroids.” Spheroid formation can be achieved using scaffold or scaffold-free techniques. Scaffold-free methods inhibit cells to adhere on the bottom surface by hanging drop culture media, constant rotation of flasks, or culture plates with an ultralow attachment surface. Although spheroids do not have multiple cell types, they are suitable for gene manipulation and drug screening tests.

FIG. 2.

Generation of liver organoids using isolated human hepatic cells and scaffold-free methods. (A) Left: Representative phase contrast microscopic image showing the isolated human intrahepatic cholangiocyte in culture. Right: Immunofluorescent staining showing CK-19 expression in isolated cholangiocytes. (B) Representative microscopic image showing the formation of liver organoids by coculturing of primary human hepatocytes, cholangiocytes, and LSECs for 14 days at low adhesive plates. (C) Immunofluorescent staining shows the expression of albumin and CYP3A4 in hepatocytes populating the generated organoid. (D) Immunofluorescence shows the expression of AQP1 in LSECs and CK-19 in cholangiocytes populating the organoid. Abbreviation: CHO, cholangiocyte.

FIG. 3.

Experimental potentials of hepatic organoids. Generated liver organoids/spheroids are suitable for drug screening and gene manipulation, such as transfection and transduction. Effects can be obtained by RNA sequencing for gene expression profiling, immunofluorescence for protein expression and localization, dose–response curve analysis for drug sensitivity tests, and enzyme-linked immunosorbent assay for protein secretion into culture media. Patient-derived organoids can be used to observe morphology as well as mutation screening to identify gene aberrations. Abbreviation: ELISA, enzyme-linked immunosorbent assay.