Hepatocyte organoids (HOs) have superior hepatic functions to cholangiocyte-derived organoids but suffer from shorter lifespans. To counteract this, we co-cultured pig HOs with adipose-derived mesenchymal stem cells (A-MSCs) and performed transcriptome analysis. The results revealed that A-MSCs enhanced the collagen synthesis pathways, which are crucial for maintaining the three-dimensional structure and extracellular matrix synthesis of the organoids. A-MSCs also increased the expression of liver progenitor cell markers (KRT7, SPP1, LGR5⁺, and TERT). To explore HOs as a liver disease model, we exposed them to alcohol to create an alcoholic liver injury (ALI) model. The co-culture of HOs with A-MSCs inhibited the apoptosis of hepatocytes and reduced lipid accumulation of HOs. Furthermore, varying ethanol concentrations (0–400 mM) and single-versus-daily exposure to HOs showed that daily exposure significantly increased the level of PLIN2, a lipid storage marker, while decreasing CYP2E1 and increasing CYP1A2 levels, suggesting that CYP1A2 may play a critical role in alcohol detoxification during short-term exposure. Moreover, daily alcohol exposure led to excessive lipid accumulation and nuclear fragmentation in HOs cultured alone. These findings indicate that HOs mimic in vivo liver regeneration, establishing them as a valuable model for studying liver diseases, such as ALI.

In this study, we generated hepatocyte organoids (HOs) using frozen-thawed primary hepatocytes (PHs) within a three-dimensional (3D) Matrigel dome culture in a porcine model. Previously studied hepatocyte organoid analogs, spheroids, or hepatocyte aggregates created using PHs in 3D culture systems have limitations in their in vitro lifespans. By co-culturing adipose tissue-derived mesenchymal stem cells (A-MSCs) with HOs within a 3D Matrigel dome culture, we achieved a 3.5-fold increase in the in vitro lifespan and enhanced liver function compared to a conventional two-dimensional (2D) monolayer culture, i.e., more than twice that of the HO group cultured alone, reaching up to 126 d. Although PHs were used to generate HOs, we identified markers associated with cholangiocyte organoids such as cytokeratin 19 and epithelial cellular adhesion molecule (EPCAM). Co-culturing A-MSCs with HOs increased the secretion of albumin and urea and glucose consumption compared to HOs cultured alone. After more than 100 d, we observed the upregulation of tumor protein P53 (TP53)-P21 and downregulation of EPCAM, albumin (ALB), and cytochrome P450 family 3 subfamily A member 29 (CYP3A29). Therefore, HOs with function and longevity improved through co-culturing with A-MSCs can be used to create large-scale human hepatotoxicity testing models and precise livestock nutrition assessment tools.