Establishment and functional evaluation of a two-stage mouse hepatocyte-derived organoid culture system
Year: 2026
Type: article
Abstract: Background Primary hepatocytes rapidly lose differentiated functions during prolonged in vitro culture, limiting their use in studies of liver metabolism and zonation. This study aimed to establish a two-stage mouse hepatocyte-derived organoid system that enables serial cell propagation followed by partial recovery of hepatic identity and metabolic function in three-dimensional culture. Methods Primary mouse hepatocytes were first expanded in a two-dimensional transition and expansion medium and then transferred to low-attachment plates for three-dimensional culture in hepatocyte maturation medium. Cell growth, senescence, organoid formation, hepatic marker expression, bulk transcriptomic changes, albumin secretion, urea production, lipid and cholesterol metabolism, zonation-related responses, and post-cryopreservation recovery were evaluated. Statistical comparisons were performed using ordinary one-way or two-way analysis of variance, as appropriate. Results The expansion medium supported serial propagation, maintained albumin expression, increased the proportion of Ki67-positive cells, and reduced senescence. A seeding density of 3 × 10 4 cells per well produced compact and uniform organoids. Following transfer to 3D culture, organoids expressed albumin, E-cadherin, CYP2E1, and CK19. Bulk transcriptomic analysis showed strong cell-cycle activity in passage-2 cells, followed by partial recovery of hepatic metabolic programs during 3D culture, including cholesterol biosynthesis, the urea cycle, steroid biosynthesis, and bile-acid biosynthesis. Direct comparison with freshly isolated primary hepatocytes showed that albumin secretion remained lower than the primary hepatocytes level, whereas urea production increased during 3D culture and was not significantly different from PHCs by day 16. Exposure to 5% oxygen increased lipid-droplet accumulation. Glucose and insulin increased free and total cholesterol. Wnt/R-spondin increased the pericentral-associated marker glutamine synthetase, whereas ascorbic acid and bilirubin increased the periportal-associated marker CPS1. Early-passage cells retained organoid-forming capacity after cryopreservation and recovery. Conclusion This two-stage culture system supports serial hepatocyte propagation followed by partial and function-specific recovery of hepatic identity and function in 3D culture. Although the organoids did not fully reproduce the mature primary hepatocyte state or physiological liver zonation, the system provides an experimentally accessible in vitro platform for investigating hepatocyte expansion, partial hepatic maturation, metabolic regulation, zonation-related responses, and microenvironment-dependent modulation of hepatocyte function.
Authors: Yusi Chen, Yuting Shan, Li Tang
Institution: Academy of Military Medical Sciences
Language: English
Subfield: Hepatology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $3,801
Awards: G1425230309, G5578635483, G8800480615
OpenAlex ID: W7223185125