Optimized 3D Culture of Hepatic Cells for Liver Organoid Metabolic Assays

Abstract

The liver is among the principal organs for glucose homeostasis and metabolism. Studies of liver metabolism are limited by the inability to expand primary hepatocytes in vitro while maintaining their metabolic functions. Human hepatic three-dimensional (3D) organoids have been established using defined factors, yet hepatic organoids from adult donors showed impaired expansion. We examined conditions to facilitate the expansion of adult donor-derived hepatic organoids (HepAOs) and HepG2 cells in organoid cultures (HepGOs) using combinations of growth factors and small molecules. The expansion dynamics, gluconeogenic and HNF4α expression, and albumin secretion are assessed. The conditions tested allow the generation of HepAOs and HepGOs in 3D cultures. Nevertheless, gluconeogenic gene expression varies greatly between conditions. The organoid expansion rates are limited when including the TGFβ inhibitor A8301, while are relatively higher with Forskolin (FSK) and Oncostatin M (OSM). Notably, expanded HepGOs grown in the optimized condition maintain detectable gluconeogenic expression in a spatiotemporal distribution at 8 weeks. We present optimized conditions by limiting A8301 and incorporating FSK and OSM to allow the expansion of HepAOs from adult donors and HepGOs with gluconeogenic competence. These models increase the repertoire of human hepatic cellular tools available for use in liver metabolic assays.

Evaluation of different media compositions promoting hepatocyte differentiation in the canine liver organoid model

“…Six hepatocyte differentiation protocols that had previously been used for hepatic organoid cultures in four different species (human [ [8] , [24] , [25] ], rodent [ 12 ], dog [ 13 ], and cat [ 14 ]) were identified. The expansion and differentiation media composition was analyzed and compared to previously established protocols for the culture of canine organoids [ 15 ] ( Table 1 A).…”

In vitro functional models for human liver diseases and drug screening: beyond animal testing

“…62,63 Parallelly, ascertained medium glucose levels have to be utilized to control cellular proliferation, investigate drug, and hepatic metabolism outcomes, as well as glucose production. [64][65][66] Moreover, specific interactions among cell populations and biophysical environment should also be strictly controlled to recapitulate heterogeneous structures like the liver tissue. 67,68 Organoids can be formed via 3D suspension culture systems (i.e., using ultra-low attachment plates), spinning bioreactors, air-liquid interface methods, and extracellular matrix (ECM)-based embedding matrices (e.g., Matrigel).…”

Oncostatin M: From Intracellular Signaling to Therapeutic Targets in Liver Cancer

“…Accordingly, treatment with OSM of EpCAM + HCC cells, but not CD133 + HepG2 cells, induced colony formation and cell proliferation [ 75 , 76 ]. OSM-supplemented media also led to an expansion rate of 3D organoids populated by adult-donor-derived hepatocytes or by HepG2 cells [ 77 ]. Moreover, OSM, through the activation of the STAT3, MEK, and JAK pathways, induced the expression of glucose-regulated protein 78 (Grp78) in HepG2 and Huh7 cells, a factor involved in proliferation, cancer progression, and the survival of tumor cells [ 78 ].…”