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 cry...
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Elsevier
2025-12-01
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| Series: | Regenerative Therapy |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2352320425001361 |
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| author | Kaoruko Kikuchi Yoko Yamada Sakurako Neo Suguru Nitta Hirotaka Igarashi Akihide Kamiya Masaharu Hisasue |
| author_facet | Kaoruko Kikuchi Yoko Yamada Sakurako Neo Suguru Nitta Hirotaka Igarashi Akihide Kamiya Masaharu Hisasue |
| author_sort | Kaoruko Kikuchi |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-9433f3c8536449a285ef32cd5ebbed95 |
| institution | DOAJ |
| issn | 2352-3204 |
| language | English |
| publishDate | 2025-12-01 |
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| series | Regenerative Therapy |
| spelling | doaj-art-9433f3c8536449a285ef32cd5ebbed952025-08-20T02:56:32ZengElsevierRegenerative Therapy2352-32042025-12-013043043810.1016/j.reth.2025.06.002Development of rematuration of canine chemically induced hepatic progenitor cells using 3D rocking cultureKaoruko Kikuchi0Yoko Yamada1Sakurako Neo2Suguru Nitta3Hirotaka Igarashi4Akihide Kamiya5Masaharu Hisasue6Laboratory of Small Animal Internal Medicine, School of Veterinary Medicine, Azabu University, Sagamihara City, Kanagawa, JapanLaboratory of Small Animal Internal Medicine, School of Veterinary Medicine, Azabu University, Sagamihara City, Kanagawa, JapanLaboratory of Clinical Diagnostics, School of Veterinary Medicine, Azabu University, Sagamihara City, Kanagawa, JapanLaboratory of Small Animal Internal Medicine, School of Veterinary Medicine, Azabu University, Sagamihara City, Kanagawa, JapanLaboratory of Small Animal Internal Medicine, School of Veterinary Medicine, Azabu University, Sagamihara City, Kanagawa, JapanDepartment of Molecular Life Sciences, Tokai University School of Medicine, Kanagawa, JapanLaboratory of Small Animal Internal Medicine, School of Veterinary Medicine, Azabu University, Sagamihara City, Kanagawa, JapanIntroduction: 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.http://www.sciencedirect.com/science/article/pii/S2352320425001361Canine hepatic progenitor cellsLiver spheroids3D cultureHepatocyte maturationIn vitro liver model |
| spellingShingle | Kaoruko Kikuchi Yoko Yamada Sakurako Neo Suguru Nitta Hirotaka Igarashi Akihide Kamiya Masaharu Hisasue Development of rematuration of canine chemically induced hepatic progenitor cells using 3D rocking culture Regenerative Therapy Canine hepatic progenitor cells Liver spheroids 3D culture Hepatocyte maturation In vitro liver model |
| title | Development of rematuration of canine chemically induced hepatic progenitor cells using 3D rocking culture |
| title_full | Development of rematuration of canine chemically induced hepatic progenitor cells using 3D rocking culture |
| title_fullStr | Development of rematuration of canine chemically induced hepatic progenitor cells using 3D rocking culture |
| title_full_unstemmed | Development of rematuration of canine chemically induced hepatic progenitor cells using 3D rocking culture |
| title_short | Development of rematuration of canine chemically induced hepatic progenitor cells using 3D rocking culture |
| title_sort | development of rematuration of canine chemically induced hepatic progenitor cells using 3d rocking culture |
| topic | Canine hepatic progenitor cells Liver spheroids 3D culture Hepatocyte maturation In vitro liver model |
| url | http://www.sciencedirect.com/science/article/pii/S2352320425001361 |
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