Establishment and functional evaluation of a two-stage mouse hepatocyte-derived organoid culture system
- 1State Key Laboratory of Medical Proteomics, National Center for Protein Sciences (Beijing), Academy of Military Medical Sciences, Beijing, China
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 × 104 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.
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BibTeX
@article{chen2026establishment,
title = {{Establishment and functional evaluation of a two-stage mouse hepatocyte-derived organoid culture system}},
author = {Yusi Chen and Yuting Shan and Li Tang},
journal = {Frontiers in Cell and Developmental Biology},
year = {2026},
month = oct,
doi = {10.3389/fcell.2026.1974273},
url = {https://doi.org/10.3389/fcell.2026.1974273},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Establishment and functional evaluation of a two-stage mouse hepatocyte-derived organoid culture system
AU - Yusi Chen
AU - Yuting Shan
AU - Li Tang
PY - 2026
DA - 2026/10/22
JO - Frontiers in Cell and Developmental Biology
DO - 10.3389/fcell.2026.1974273
UR - https://doi.org/10.3389/fcell.2026.1974273
AB - 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 × 104 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.
SN - 2296-634X
ER -