Engineering of Implantable Liver Tissues

  • Yasuyuki SakaiEmail author
  • M. Nishikawa
  • F. Evenou
  • M. Hamon
  • H. Huang
  • K. P. Montagne
  • N. Kojima
  • T. Fujii
  • T. Niino
Part of the Methods in Molecular Biology book series (MIMB, volume 826)


In this chapter, from the engineering point of view, we introduce the results from our group and related research on three typical configurations of engineered liver tissues; cell sheet-based tissues, sheet-like macroporous scaffold-based tissues, and tissues based on special scaffolds that comprise a flow channel network. The former two do not necessitate in vitro prevascularization and are thus promising in actual human clinical trials for liver diseases that can be recovered by relatively smaller tissue mass. The third approach can implant a much larger mass but is still not yet feasible. In all cases, oxygen supply is the key engineering factor. For the first configuration, direct oxygen supply using an oxygen-permeable polydimethylsiloxane membrane enables various liver cells to exhibit distinct behaviors, complete double layers of mature hepatocytes and fibroblasts, spontaneous thick tissue formation of hepatocarcinoma cells and fetal hepatocytes. Actual oxygen concentration at the cell level can be strictly controlled in this culture system. Using this property, we found that initially low then subsequently high oxygen concentrations were favorable to growth and maturation of fetal cells. For the second configuration, combination of poly-l-lactic acid 3D scaffolds and appropriate growth factor cocktails provides a suitable microenvironment for the maturation of cells in vitro but the cell growth is limited to a certain distance from the inner surfaces of the macropores. However, implantation to the mesentery leaves of animals allows the cells again to proliferate and pack the remaining spaces of the macroporous structure, suggesting the high feasibility of 3D culture of hepatocyte progenitors for liver tissue-based therapies. For the third configuration, we proposed a design criterion concerning the dimensions of flow channels based on oxygen diffusion and consumption around the channel. Due to the current limitation in the resolution of 3D microfabrication processes, final cell densities were less than one-tenth of those of in vivo liver tissues; cells preferentially grew along the surfaces of the channels and this fact suggested the necessity of improved 3D fabrication technologies with higher resolution. In any case, suitable oxygen supply, meeting the cellular demand at physiological concentrations, was the most important factor that should be considered in engineering liver tissues. This enables cells to utilize aerobic respiration that produces almost 20 times more ATP from the same glucose consumption than anaerobic respiration (glycolysis). This also allows the cells to exhibit their maximum reorganization capability that cannot be observed in conventional anaerobic conditions.

Key words

3D culture Cellular sheet Macroporous scaffold Flow channel Oxygen Respiration 



The studies were carried out based on various scientific grants, such as Grant-in-Aids for Scientific Research from the Ministry of Health, Labor and Welfare, and those from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Some of the studies were performed in the framework of LIMMS (Laboratory for Integrated Micro-Mechatronic Systems), a joint laboratory between the CNRS (Centre National de la Recherche Scientifique) and IIS (Institute of Industrial Science), the University of Tokyo. We thank Saipaso Research Center (Tokyo, Japan) for preparing histological samples. F. Evenou and K. P. Montagne were supported by the Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science.


  1. 1.
    Demetrious AA, Jr. Brown RS, Busuttil RW, Fair J, McGuire BM, Rosenthal P, II Am Esch JS, Lerut J, Nyberg SL, Salizzoni M, Fagan EA, de Hamptinne B, Broelsch CE, Muraca M, Salmeron JM, Rablin JM, Metselaar HJ, Pratt D, DE La Mata M, McChesney LP, Everson GT, Lavin PT, Stevens AC, Pitkin Z, Solomon BA (2004) Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure, Ann Surg 239, 660–667.Google Scholar
  2. 2.
    Fox IJ, Chowdhury JR, Kaufman SS, Goertzen TC, Chowdhury NR, Warkentin PI, Dorko K, Sauter BV, Strom SC (1998) Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation, New Eng J Med., 338, 1422–1426.PubMedCrossRefGoogle Scholar
  3. 3.
    Ohashi K (2008) Liver tissue engineering: The future of liver therapeutics, Hepatol Res, 38, S1, S76-87.Google Scholar
  4. 4.
    Fiegel HC, Kaufmann PM, Bruns H, Kluth D, Horch RE, Vacanti JP, Kneser U (2008) Hepatic tissue engineering: from transplantation to customized cell-based liver directed therapies from the laboratory. J Cell Mol Med, 12, 56–66.PubMedCrossRefGoogle Scholar
  5. 5.
    Lovett M, Lee K, Edwards A, Kaplan DL (2009) Vascularization strategies for tissue engineering, Tissue Eng B, 15, 353–370.CrossRefGoogle Scholar
  6. 6.
    Hoganson DM, Pryor II HI., Vacanti JP (2008) Tissue engineering and organ structure: a vascularized approach to liver and lung, Pediat Res, 63, 520–526.PubMedCrossRefGoogle Scholar
  7. 7.
    Ohashi K, Yokoyama T, Yamato M, Kuge H, Kanehiro H, Tsutsumi M, Amanuma T, Iwata H, Yang J, Okano T, Nakajima Y (2007) Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets, Nat Med, 13, 880–885.PubMedCrossRefGoogle Scholar
  8. 8.
    Mooney DJ, Kaufmann PM, Sano K, McNamara KM, Vacanti JP, Langer R (1994), Transplantation of hepatocytes using porous biodegradable sponge, Transplant Proc, 26, 3425–3426.PubMedGoogle Scholar
  9. 9.
    Smith MK, Riddle KW, Mooney DJ (2006), Delivery of heterotrophic factors fails to enhance longer-term survival of subcutaneously transplanted hepatocytes, Tissue Eng, 12, 235–244.PubMedCrossRefGoogle Scholar
  10. 10.
    Sakai Y, Huang H, Hanada S, Niino T (2010) Toward engineering of vascularized three-dimensional liver tissue equivalents possessing a clinically-significant mass, Biochem Eng J, 48, 348–361.CrossRefGoogle Scholar
  11. 11.
    Seglen PO (1976) Preparation of isolated liver cells. In Methods in Cell Biology, vol. 13 (Etd. by Prescott, D. M.), pp. 29–83, Academic Press, New York.Google Scholar
  12. 12.
    Nishikawa M, Kojima N, Komori K, Yamamoto T, Fujii T, Sakai Y (2008) Enhanced maintenance and functions of rat hepatocytes induced by combination of on-site oxygenation and coculture with fibroblasts, J Biotechnol, 133, 253–260.PubMedCrossRefGoogle Scholar
  13. 13.
    Hanada S, Kojima N, Sakai Y (2008) Soluble factor-dependent in vitro growth and maturation of rat fetal liver cells in a three-dimensional culture system, Tissue Eng A, 14, 149–160.CrossRefGoogle Scholar
  14. 14.
    Hamon M, Hanada S, Fujii T, Sakai Y (2011) Direct oxygen supply with polydimethylsiloxane (PDMS) membranes induces a spontaneous organization of thick heterogeneous liver tissues from rat fetal liver cells in vitro, Cell Transplant, in press.Google Scholar
  15. 15.
    Huang H, Hanada S, Kojima N, Sakai Y. (2006) Enhanced functional maturation of fetal porcine hepatocytes in three-dimensional poly-L-lactic acid scaffolds: a culture condition suitable for engineered liver tissues in large-scale animal studies, Cell Transplant, 15, 799–809.PubMedCrossRefGoogle Scholar
  16. 16.
    Jiang J, Kojima N, Guo L, Naruse K, Makuuchi M, Miyajima A, Yan W, Sakai Y (2004) Efficacy of engineered liver tissue based on poly-L-lactic acid scaffolds and fetal mouse liver cells cultured with oncostatin M, nicotinamide and dimethyl sulfoxide, Tissue Eng, 10, 1577–1586.PubMedGoogle Scholar
  17. 17.
    Nishikawa M, Yamamoto T, Kojima N, Komori K, Fujii T, Sakai Y (2008) Stable immobilization of rat hepatocytes as hemispheroids onto collagen-conjugated poly-dimethylsiloxane (PDMS) surfaces: importance of direct oxygenation through PDMS for both formation and function, Biotechnol Bioeng, 99, 1472–1481.PubMedCrossRefGoogle Scholar
  18. 18.
    Huang H, Oizumi S, Kojima N, Niino T, Sakai Y (2007) Avidin-biotin binding–based cell seeding and perfusion culture of liver-derived cells in a porous scaffold with a three-dimensional interconnected flow-channel network, Biomat., 28, 3815–3823.CrossRefGoogle Scholar
  19. 19.
    Stevens KM (1965) Oxygen requirement for liver cells in vitro, Nature, 206, 199.PubMedCrossRefGoogle Scholar
  20. 20.
    Smith MD, Smirthwaite AD, Cairns DE, Cousins RB, Gaylor JD (1996) Techniques for measurement of oxygen consumption rates of hepatocytes during attachment and post-attachment. Int J Artif Organs, 19, 36–44.PubMedGoogle Scholar
  21. 21.
    Tilles AW, Baskaran H, Roy P, Yarmush ML, Toner M (2001) Effects of oxygenation and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor. Biotechnol Bioeng, 73, 379–389.PubMedCrossRefGoogle Scholar
  22. 22.
    de Bartolo L, Salerno S, Morelli S, Giorno L, Rende M, Memoli B, Procino A, Andreucci VE, Bader A, Drioli E (2006) Long-term maintenance of human hepatocytes in oxygen-permeable membrane bioreactor. Biomat, 27, 4794–4803.CrossRefGoogle Scholar
  23. 23.
    Wang N, Ostuni E, Whitesides GM, Ingber DE (2002) Micropatterning tractional forces in living cells. Cell Motil Cytoskeleton, 52, 97–106.PubMedCrossRefGoogle Scholar
  24. 24.
    Matsui H, Osada T, Moroshita Y, Sekijima M, Fujii T, Takeuchi S, Sakai Y (2010) Rapid and enhanced repolarization in sandwich-cultured hepatocytes on an oxygen-permeable membrane, Biochem Eng. J, 52, 255–262.Google Scholar
  25. 25.
    Evenou F, Fujii T, Sakai Y (2010), Spontaneous formation of highly functional three-dimensional multilayer from human hepatoma Hep G2 cells cultured on an oxygen-permeable polydimethylsiloxane membrane, Tissue Eng C, 16, 311–318.CrossRefGoogle Scholar
  26. 26.
    Alberti KG (1977) The biochemical consequences of hypoxia, J Clin Pathol, Suppl (R Coll Pathol) 11, 14–20.Google Scholar
  27. 27.
    Sakai Y, Jiang J, Hanada S, Huang H, Katsuda T, Kojima N, Teratani T, Ochiya T (2011) Three-dimensional culture of fetal mouse, rat and porcine hepatocytes, in “Fetal Tissue Transplantation”, etd. by N. Bhattacharya and P. Stubblefield, Springer-Verlag UK, in press.Google Scholar
  28. 28.
    Nam YS, Yoon JJ, Park TG (2000) A novel fabrication method of macroporous biodegradable polymer scaffolds using gas foaming salt as a porogen additive. J Biomed Mater Res, 53, 1–7.PubMedCrossRefGoogle Scholar
  29. 29.
    Teratani T, Yamamoto H, Aoyagi K, Sasaki H, Asari A, Quinn G, Sasaki H, Terada M, Ochiya T (2005) Direct hepatic fate specification from mouse embryonic stem cells, Hepatol, 41, 836–846.CrossRefGoogle Scholar
  30. 30.
    Katsuda T, Teratani T, Ochiya T, Sakai Y (2010) Transplantation of a fetal liver cell-loaded hyaluronic acid sponge onto the mesentery recovers a Wilson’s disease model rat, J Biochem, 148, 281–288.PubMedCrossRefGoogle Scholar
  31. 31.
    Park J, Li Y, Berthiaume F, Toner M, Yarmush ML, Tilles AW (2008) Radial flow hepatocyte bioreactor using stacked microfabricated grooved substrates, Biotechnol Bioeng, 99, 455–467.PubMedCrossRefGoogle Scholar
  32. 32.
    Carraro A, Hsu WM, Kulig KM, Cheung WS, Miller ML, Weinberg EJ, Swart EF, Kaazempur-Mofrad M, Borenstein JT, Vacanti JP, Neville C (2008) In vitro analysis of a hepatic device with intrinsic microvascular-based channels, Biomed Microdevices, 10, 795–805.PubMedCrossRefGoogle Scholar
  33. 33.
    Hoganson DM, Pryor II HI, Spool ID, Burns OH, Gilmore JR, Vacanti JP (2010) Principles of biomimetic vascular network design applied to a tissue-engineered liver scaffold, Tissue Eng A, 16, 1469–1477.CrossRefGoogle Scholar
  34. 34.
    McGuigan AP, Sefton MY (2006) Vascularized organoid engineering by modular assembly enables blood perfusion., Proc Nat Acad Sci, 103, 11461–11466.Google Scholar
  35. 35.
    Inamori M, Mizumoto H, Kajiwara T (2009) An approach for formation of vascularized liver tissue by endothelial cell–covered hepatocyte spheroid integration, Tissue Eng A, 15, 2029–2037.CrossRefGoogle Scholar
  36. 36.
    Krogh A (1919) The number and the distribution of capillaries in muscle with the calculation of the oxygen pressure necessary for supplying the tissue, J Physiol. (Lond) 52, 409–515.Google Scholar
  37. 37.
    Niino T, Sakai Y, Huang H, Naruke H (2006) SLS fabrication of highly porous model including fine flow channel network aiming at regeneration of highly metabolic organs, Solid Freeform Fab. Proc., 160–170.Google Scholar
  38. 38.
    Kojima N, Matsuo T, Sakai Y (2006) Rapid hepatic cell attachment onto biodegradable polymer surfaces without toxicity using an avidin-biotin binding system, Biomat, 27, 4904–4910.CrossRefGoogle Scholar
  39. 39.
    Vozzi G, Previti A, De Rossi D, Ahluwalia A (2002) Microsyringe-based deposition of tow-dimensional and three-dimensional polymer scaffolds with a well-defined geometry for application to tissue engineering, Tissue Eng, 8, 1089–1098.PubMedCrossRefGoogle Scholar
  40. 40.
    Sudo R, Mitaka T, Ikeda S, Sugimoto S, Harada K, Hirata K, Tanishita K, Mochizuki Y (2004) Bile canalicular formation in hepatic organoid reconstructed by rat small hepatocytes and hepatic nonparenchymal cells, J Cell Physiol, 199 252–261.PubMedCrossRefGoogle Scholar
  41. 41.
    Sudo R, Kohara H, Mitaka T, Ikeda M, Tanishita K (2005) Coordination of bile canalicular contraction in hepatic organoid reconstructed by rat small hepatocytes and nonparenchymal cells. Ann Biomed Eng, 33, 696–708.PubMedCrossRefGoogle Scholar
  42. 42.
    Lokmic Z, Mitchell GM (2008) Engineering the microcirculation, Tissue Eng B, 14, 87–103.CrossRefGoogle Scholar
  43. 43.
    Wang Y, Yao HL, Cui CB, Wauthier E, Barbier C, Costello MJ, Moss N, Yamauchi M, Sricholpech M, Gerber D, Loboa EG, Reid LM (2010) Paracrine signals from mesenchymal cell populations govern the expansion and differentiation of human hepatic stem cells to adult liver fates, Hepatol, 52, 1443–1454.CrossRefGoogle Scholar
  44. 44.
    Matsumoto K, Yoshitomi H, Rossant J, Zaret KS (2001) Liver organogenesis promoted by endothelial cells prior to vascular function, Science, 294, 559–563.PubMedCrossRefGoogle Scholar
  45. 45.
    Lammert E, Cleaver O, Melton D (2001) Induction of pancreatic differentiation by signals from blood vessels, Science, 294, 564–567.PubMedCrossRefGoogle Scholar
  46. 46.
    Takahashi A (1995) Characterization of Neo Red Cell (NRCs), their function and safety; in vitro test, Artif. Cells Blood Subst Immob Biotechnol, 23, 347–354.CrossRefGoogle Scholar
  47. 47.
    Naruto H, Huang H, Nishikawa M, Kojima N, Mizuno A, Ohta K, Sakai Y (2007) Feasibility of direct oxygenation of primary-cultured rat hepatocytes using polyethylene glycol decorated liposome-encapsulated hemoglobin (LEH), J Biosci Bioeng, 104, 343–346.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Yasuyuki Sakai
    • 1
    Email author
  • M. Nishikawa
    • 2
  • F. Evenou
    • 3
  • M. Hamon
    • 4
  • H. Huang
    • 5
  • K. P. Montagne
    • 1
  • N. Kojima
    • 1
  • T. Fujii
    • 1
  • T. Niino
    • 1
  1. 1.Institute of Industrial ScienceUniversity of TokyoTokyoJapan
  2. 2.Renal Regeneration LaboratoryVAGLAHS at Sepulveda & UCLA David Geffen School of MedicineLos AngelsUSA
  3. 3.Laboratoire Matière et Systèmes Complexes (MSC), Bâtiment CondorcetUniversité Paris DiderotParis 7France
  4. 4.Department of Mechanical EngineeringAuburn UniversityAuburnUSA
  5. 5.Okami Chemical Industry Co. LtdKyotoJapan

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