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


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.


  1. Strom, S. & Fisher, R. Hepatocyte transplantation. New possibilities for therapy. Gastroenterology 124, 568–571 (2003).

    Article  Google Scholar 

  2. Ohashi, K., Park, F. & Kay, M.A. Hepatocyte transplantation: clinical and experimental application. J. Mol. Med. 79, 617–630 (2001).

    Article  CAS  Google Scholar 

  3. Griffith, L.G. & Naughton, G. Tissue engineering—current challenges and expanding opportunities. Science 295, 1009–1014 (2002).

    Article  CAS  Google Scholar 

  4. Gouon-Evans, V. et al. BMP-4 is required for hepatic specification of mouse embryonic stem cell–derived definitive endoderm. Nat. Biotechnol. 24, 1402–1411 (2006).

    Article  CAS  Google Scholar 

  5. Yokoyama, T. et al. In vivo engineering of metabolically active hepatic tissues in a neovascularized subcutaneous cavity. Am. J. Transplant. 6, 50–59 (2006).

    Article  CAS  Google Scholar 

  6. Ohashi, K. et al. Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases. Hepatology 41, 132–140 (2005).

    Article  Google Scholar 

  7. Ohashi, K. et al. Stability and repeat regeneration potential of the engineered liver tissues under the kidney capsule in mice. Cell Transplant. 14, 621–627 (2005).

    Article  Google Scholar 

  8. Ohashi, K. et al. Sustained survival of human hepatocytes in mice: a model for in vivo infection with human hepatitis B and hepatitis delta viruses. Nat. Med. 6, 327–331 (2000).

    Article  CAS  Google Scholar 

  9. Kuge, H. et al. Genetic modification of hepatocytes towards hepatocyte transplantation and liver tissue engineering. Cell Transplant. 15, 1–12 (2006).

    Article  Google Scholar 

  10. Lee, H. et al. Effect of implantation site on hepatocytes heterotopically transplanted on biodegradable polymer scaffolds. Tissue Eng. 9, 1227–1232 (2003).

    Article  CAS  Google Scholar 

  11. Yang, J. et al. Cell sheet engineering: recreating tissues without biodegradable scaffolds. Biomaterials 26, 6415–6422 (2005).

    Article  CAS  Google Scholar 

  12. Yamada, N. et al. Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells. Makromol. Chem. Rapid Commun. 11, 571–576 (1990).

    Article  CAS  Google Scholar 

  13. Okano, T., Yamada, N., Sakai, H. & Sakurai, Y. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide). J. Biomed. Mater. Res. 27, 1243–1251 (1993).

    Article  CAS  Google Scholar 

  14. Nishida, K. et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N. Engl. J. Med. 351, 1187–1196 (2004).

    Article  CAS  Google Scholar 

  15. Hirose, M., Kwon, O.H., Yamato, M., Kikuchi, A. & Okano, T. Creation of designed shape cell sheets that are noninvasively harvested and moved onto another surface. Biomacromolecules 1, 377–381 (2000).

    Article  CAS  Google Scholar 

  16. Shimizu, T. et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circ. Res. 90, e40–e48 (2002).

    Article  CAS  Google Scholar 

  17. Miyahara, Y. et al. Monolayered mesenchymal stems cells repair scarred myocardium after myocardial infarction. Nat. Med. 12, 459–465 (2006).

    Article  CAS  Google Scholar 

  18. Harimoto, M. et al. Novel approach for achieving double-layered cell sheets co-culture: overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature-responsive culture dishes. J. Biomed. Mater. Res. 62, 464–470 (2002).

    Article  CAS  Google Scholar 

  19. Stephenne, X. et al. Sustained engraftment and tissue enzyme activity after liver cell transplantation for argininosuccinate lyase deficiency. Gastroenterology 130, 1317–1323 (2006).

    Article  Google Scholar 

  20. Nahmias, Y., Casali, M., Barbe, L., Berthiaume, F. & Yarmush, M.L. Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes. Hepatology 43, 257–265 (2006).

    Article  CAS  Google Scholar 

  21. Michalopoulos, G.K. & DeFrances, M.C. Liver regeneration. Science 276, 60–66 (1997).

    Article  CAS  Google Scholar 

  22. Fox, I.J. et al. Treatment of the Cirgler-Najjar syndrome type I with hepatocyte transplanation. N. Engl. J. Med. 338, 1422–1426 (1998).

    Article  CAS  Google Scholar 

  23. Fox, I.J., Schafer, D.F. & Yannam, G.R. Finding a home for cell transplants: location, location, location. Am. J. Transplant. 6, 5–6 (2006).

    Article  CAS  Google Scholar 

  24. Dhawan, A., Mitry, R.R. & Hughes, R.D. Hepatocyte transplantation for liver-based metabolic disorders. J. Inherit. Metab. Dis. 29, 431–435 (2006).

    Article  Google Scholar 

  25. Muraca, M. et al. Hepatocyte transplantation as a treatment for glycogen storage disease type Ia. Lancet 359, 317–318 (2002).

    Article  Google Scholar 

  26. Dhawan, A. et al. Hepatocyte transplantation for inherited factor VII deficiency. Transplantation 78, 1812–1814 (2004).

    Article  Google Scholar 

  27. Ohashi, K., Park, F. & Kay, M.A. Efficient gene transduction to cultured hepatocytes by HIV-1 derived lentiviral vector. Transplant. Proc. 34, 1431–1433 (2002).

    Article  CAS  Google Scholar 

  28. Bumgardner, G.L., Li, J., Heininger, M., Ferguson, R.M. & Orosz, C.G. In vivo immunogenicity of purified allogeneic hepatocytes in a murine hepatocyte transplant model. Transplantation 65, 47–52 (1998).

    Article  CAS  Google Scholar 

  29. Jolley, M.E. et al. Fluorescence polarization immunoassay. I. Monitoring aminoglycoside antibiotics in serum and plasma. Clin. Chem. 27, 1190–1197 (1981).

    CAS  PubMed  Google Scholar 

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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 and Affiliations



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.

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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).

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