Kikuchi Kaoruko, Yamada Yoko, Neo Sakurako, Nitta Suguru, Igarashi Hirotaka, Kamiya Akihide, Hisasue Masaharu
Laboratory of Small Animal Internal Medicine, School of Veterinary Medicine, Azabu University, Sagamihara City, Kanagawa, Japan.
Laboratory of Clinical Diagnostics, School of Veterinary Medicine, Azabu University, Sagamihara City, Kanagawa, Japan.
Regen Ther. 2025 Aug 5;30:430-438. doi: 10.1016/j.reth.2025.06.002. eCollection 2025 Dec.
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.
cHPCs were cultured in 2D cultures with HGF and oncostatin M or in 3D cultures using AggreWell400 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.
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, expression increased 1.7-fold compared with cHPCs( < 0.01). In 3D cultures, (63-fold), (9-fold), (34-fold), (1.6-fold), (10-fold), and (56-fold) were all significantly upregulated compared with cHPCs ( < 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.
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, -like liver models for research and therapeutic applications.
开发犬肝细胞培养系统对于肝移植、毒性评估和药物代谢研究至关重要。然而，在长期培养中维持有活力且功能正常的肝细胞仍然具有挑战性。我们之前的研究表明，使用三种小分子化合物：Y-27632（ROCK抑制剂）、A-83-01（TGFβ抑制剂）和CHIR99021（GSK3抑制剂），可将冷冻保存的犬肝细胞分化为肝祖细胞（cHPCs）。然而，未能使其再成熟为功能正常的肝细胞。本研究旨在评估祖细胞向成熟肝细胞的分化情况，比较二维（2D）和三维（3D）培养系统，并确定3D培养的优势。
将cHPCs在含HGF和抑瘤素M的2D培养体系中培养，或使用AggreWell400平板在3D培养体系中培养以形成球体，转移至低附着平板并振荡培养。使用免疫细胞化学分析细胞的形态、基因表达和蛋白质标志物。
在2D培养中，再成熟产生的细胞具有更宽的细胞质、多个细胞核以及铺路石样形态。3D培养中的球体在第5天时直径达到150μm，边缘不规则。定量实时聚合酶链反应分析显示肝特异性基因显著上调。在2D培养中，与cHPCs相比， 表达增加了1.7倍（<0.01）。在3D培养中，与cHPCs相比， （63倍）、 （9倍）、 （34倍）、 （1.6倍）、 （10倍）和 （56倍）均显著上调（<0.05）。免疫组织化学显示3D培养中有强大的AFP、ALB和CYP2E1表达，3D培养中87.6%的细胞AFP阳性，100%CYP2E1阳性，而2D培养中分别为11.4%和7.9%。
3D振荡培养显著增强了肝特异性基因和蛋白质表达，产生了功能性肝球体。这些发现强调了3D振荡培养在创建用于研究和治疗应用的可靠的类肝模型方面的潜力。
