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

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 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, 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,