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Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets

Abstract

Hepatic tissue engineering using primary hepatocytes has been considered a valuable new therapeutic modality for several classes of liver diseases. Recent progress in the development of clinically feasible liver tissue engineering approaches, however, has been hampered mainly by insufficient cell-to-cell contact of the engrafted hepatocytes. We developed a method to engineer a uniformly continuous sheet of hepatic tissue using isolated primary hepatocytes cultured on temperature-responsive surfaces. Sheets of hepatic tissue transplanted into the subcutaneous space resulted in efficient engraftment to the surrounding cells, with the formation of two-dimensional hepatic tissues that stably persisted for longer than 200 d. The engineered hepatic tissues also showed several characteristics of liver-specific functionality. Additionally, when the hepatic tissue sheets were layered in vivo, three-dimensional miniature liver systems having persistent survivability could be also engineered. This technology for liver tissue engineering is simple, minimally invasive and free of potentially immunogenic biodegradable scaffolds.

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Figure 1: Morphological validation of the engineered hepatic tissue sheets composed of primary hepatocytes.
Figure 2: In vitro comparison of the function of isolated hepatocytes versus hepatic tissue sheets.
Figure 3: Morphological and functional characteristics of the engineered hepatic tissue sheets following transplantation into the subcutaneous space.
Figure 4: Stacking multiple monolayers of hepatic tissue for the engineering of three-dimensional hepatic tissues.

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Acknowledgements

The authors thank H. Sakai (CellSeed Inc.) for optimizing the PIPAAm culture dish conditions; G.L. Bumgardner (Ohio State University) for the hA1AT-FVB/N mouse line; and Y. Murakami (Kyoto University) for technical supervision in the development of the bFGF-releasing mesh device. This work was supported in part by Scientific Research Grants No. 15390632 (Y.N.) and No. 25691269 (K.O.), the Center of Excellence (COE) Program for the 21st Century (T.O.), and the Leading Project (K.O., M.Y., T.O.), from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, and the Terumo Life Science Foundation (K.O.).

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Authors

Contributions

K.O., M.Y., T.O. and Y.N. designed the research; K.O., T.Y., H. Kuge, H. Kanehiro and Y.N. performed the hepatic tissue sheet experiments; M.Y., J.Y. and T.O. developed the temperature-responsive culture dishes; M.T. and T.A. performed the histological analyses; H.I. manufactured and provided the bFGF-releasing devices; K.O., T.Y., H. Kuge and J.Y. interpreted and analyzed the data; and K.O., M.Y. and J.Y. wrote and drafted the manuscript.

Corresponding author

Correspondence to Kazuo Ohashi.

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Competing interests

M.Y. is a consultant for CellSeed, Inc., and T.O. is an investor in CellSeed, Inc., and an inventor/developer designated on the patent for the temperature-responsive culture surfaces (patent nos. JP1972502, US5284766, FR0382214, NL0382214, DE0382214, GB0382214, SE0382214 and CH0382214).

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Ohashi, K., Yokoyama, T., Yamato, M. et al. Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets. Nat Med 13, 880–885 (2007). https://doi.org/10.1038/nm1576

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