Development of rematuration of canine chemically induced hepatic progenitor cells using 3D rocking culture

Abstract

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 AggreWell400^TM 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.