Abstract
Bioengineered livers are potential alternatives for liver transplantation in patients with end-stage liver disease. Liver scaffolds engineered through decellularization techniques have been developed for clinical applications; however, reconstruction of an integrated biliary system remains a challenge. This study aimed to structurally reconstruct a three-dimensional biliary architecture in bioengineered livers through recellularization of decellularized rat liver scaffolds with rat primary hepatocytes (PHs) and intrahepatic cholangiocyte organoids (ICOs), and to assess bile acid distribution in relation to the reconstructed hepatobiliary architecture. Decellularized rat liver scaffolds were recellularized using rat PHs and green fluorescent protein (GFP)-expressing ICOs. Dissociated ICOs were injected via the bile duct and cultured for 5 days using a perfusion device, followed by PH injection and 2 days of culture. During co-culture, biliary drainage fluid and culture medium were collected to compare total bile acid concentrations using enzyme-linked immunosorbent assay. Histological and immunofluorescence analyses were performed after 7 days of perfusion culture. Histological analyses confirmed the engraftment of GFP-expressing ICOs into bile ducts and PHs into the parenchymal space. Engrafted PHs expressed ZO-1 and MRP2, forming bile canaliculi. In specific regions, MRP2-positive PHs and KRT19-positive ICO-repopulated cells adhered to each other, resembling the native liver structure. In the samples exhibiting such structures, total bile acid concentrations in the biliary drainage fluid tended to be higher than in the culture medium. This study provides evidence supporting the structural reconstruction of a three-dimensional biliary system in bioengineered livers. These findings represent a significant step toward the development of bioengineered livers using decellularization and recellularization techniques.
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Data availability
The data that support the findings of this study are available from the corresponding author, K.F., upon reasonable request.
Abbreviations
- 3D:
-
Three-dimensional
- BD:
-
Bile duct
- ELISA:
-
Enzyme-linked immunosorbent assay
- EM:
-
Expansion medium
- ESLD:
-
End-stage liver disease
- GFP:
-
Green fluorescent protein
- ICO:
-
Intrahepatic cholangiocyte organoids
- PH:
-
Primary hepatocytes
- PV:
-
Portal vein
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Acknowledgements
We thank the Division of Electron Microscopic Study, Center for Anatomical Studies, Graduate School of Medicine, Kyoto University, for technical assistance with the analysis of electron microscopy data.
Funding
This work was supported by JSPS KAKENHI [grant numbers 22K08689, 24KJ1416].
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Conceptualization: Ken Fukumitsu. Data curation: Hiroshi Horie, Ken Fukumitsu, Yusuke Hanabata, Takuma Karasuyama, Kentaro Iwaki, Fumiaki Munekage, Kenta Makino, Takashi Ito, Katsuhiro Tomofuji, Hiroyuki Uematsu, Robert Coppo, Kunishige Onuma, Masahiro Inoue. Funding acquisition : Hiroshi Horie, Ken Fukumitsu. Investigation : Hiroshi Horie, Ken Fukumitsu, Yusuke Hanabata, Takuma Karasuyama, Kentaro Iwaki, Fumiaki Munekage, Kenta Makino, Takashi Ito, Katsuhiro Tomofuji, Hidenobu Kojima, Satoshi Ogiso, Elena Yukie Uebayashi, Hiroyuki Uematsu, Robert Coppo, Kunishige Onuma, Masahiro Inoue, Takamichi Ishii, Etsuro Hatano. Project administration : Ken Fukumitsu. Resources: Hiroshi Horie, Ken Fukumitsu, Masahiro Inoue. Supervision : Masahiro Inoue, Takamichi Ishii, Etsuro Hatano. Writing—original graft: Hiroshi Horie, Ken Fukumitsu. Writing—review and editing: Satoshi Ogiso, Masahiro Inoue, Takamichi Ishii, Etsuro Hatano.
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Horie, H., Fukumitsu, K., Hanabata, Y. et al. Three-dimensional reconstruction of a biliary system in a bioengineered liver using decellularized scaffold. Sci Rep (2026). https://doi.org/10.1038/s41598-026-39175-2
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DOI: https://doi.org/10.1038/s41598-026-39175-2
