Related Experiment Video
Updated: Sep 11, 2025
Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research
Published on: January 31, 2022
Development of rematuration of canine chemically induced hepatic progenitor cells using 3D rocking culture
Kaoruko Kikuchi1, Yoko Yamada1, Sakurako Neo2
1Laboratory of Small Animal Internal Medicine, School of Veterinary Medicine, Azabu University, Sagamihara City, Kanagawa, Japan.
Introduction:
Developing canine hepatocyte culture systems is critical for liver transplantation, toxicity evaluation, and drug metabolism studies. However, maintaining viable and functional hepatocytes in long-term cultures remains challenging. Our prior research demonstrated differentiation of cryopreserved canine hepatocytes into hepatic progenitor cells (cHPCs) using three small-molecule compounds: Y-27632 (ROCK inhibitor), A-83-01 (TGFβ inhibitor), and CHIR99021 (GSK3 inhibitor). Nevertheless, rematuration into functional hepatocytes was not achieved. This study aimed to evaluate the differentiation of progenitor cells into mature hepatocytes, compare two-dimensional (2D) and three-dimensional (3D) culture systems, and determine the advantages of 3D culture.
Methods:
cHPCs were cultured in 2D cultures with HGF and oncostatin M or in 3D cultures using AggreWell400TM plates to form spheroids, transferred to low-adherent plates, and cultured with shaking. Cells were analyzed for morphology, gene expression, and protein markers using immunocytochemistry.
Results:
In 2D cultures, rematuration produced cells with wider cytoplasm, multiple nuclei, and a paving stone-like morphology. Spheroids in 3D cultures reached 150 μm in diameter with irregular edges by day 5. Quantitative real-time polymerase chain reaction analysis revealed significant upregulation of liver-specific genes. In 2D cultures, KRT19 expression increased 1.7-fold compared with cHPCs(p < 0.01). In 3D cultures, ALB (63-fold), TAT (9-fold), MRP2 (34-fold), EpCAM (1.6-fold), CYP2E1 (10-fold), and CYP3A12 (56-fold) were all significantly upregulated compared with cHPCs (p < 0.05). Immunohistochemistry showed robust AFP, ALB, and CYP2E1 expression in 3D cultures, with 87.6 % of cells AFP-positive and 100 % CYP2E1-positive compared to 11.4 % and 7.9 % in 2D cultures, respectively.
Conclusions:
3D rocking culture markedly enhanced liver-specific gene and protein expression, producing functional liver spheroids. These findings underscore the potential of 3D rocking cultures to create reliable, in vivo-like liver models for research and therapeutic applications.
