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Three-Dimensional Culture of Fetal Mouse, Rat, and Porcine Hepatocytes

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Human Fetal Tissue Transplantation

Abstract

In this chapter, we introduce and summarize the results from our groups on typical macroporous and biodegradable poly-L-lactic acid (PLLA) scaffolds-based 3D shaking culture of fetal hepatocyte populations isolated from mice, rats, and pigs, and on some preliminary implantation of the cell-loaded scaffolds to mice and rats. In such 3D microenvironment, inoculated cells were organized into heterogenic 3D aggregates or multilayers, functional levels, and their in vitro stability was greatly enhanced when compared with those in 2D monolayer cultures. Although detoxification capacity in terms of EROD measurement did not seemed to be fully matured, other typical functions such as albumin production attained the adult level. This was enabled by the synergistic effects of 3D culture and soluble factors cocktails. Combination of nicotinamide (NA), dimethyl sulfoxide (DMSO), and oncostatin M (OSM) was very effective in fetal mice culture, but it does not support the growth and maturation of fetal rat hepatocytes, for which other cocktail composed of NA, HGF, FGF-1, FGF-4, OSM, and sodium butyrate was effective. In the case of fetal porcine hepatocytes, presumably because the obtained hepatocytes were in better matured stage than mice and rats, dependency on soluble factors was low, and 3D culture itself remarkably enhanced their spontaneous growth and maturation. The biggest problem in such 3D culture, cellular growth was limited only to the periphery of macropores of the scaffolds even with the thin disk shape of the scaffolds and with continuous shaking, resulted in about at most several times growth and one-tenth cellular density that in vivo. This indicated the insufficient mass transfer (primarily oxygen) between culture medium and inner spaces of the scaffolds. However, upon implantation to mesentery leaves of animals, almost all the remaining spaces in the scaffolds were finally filled with proliferated hepatocytes in mice and rats. These results clearly demonstrate that fetal cells that grow and mature in 3D culture with appropriate cocktails of soluble factors show promise in partly supporting the insufficient host liver functionality upon implantation.

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References

  1. Banas A, Yamamoto Y, Teratani T, Ochiya T. Stem cell plasticity: learning from hepatogenic differentiation strategies. Dev Dyn. 2007;236:3228–41.

    Article  PubMed  CAS  Google Scholar 

  2. Sakai Y, Huang H, Hanada S, Niino T. Toward engineering of vascularized three-dimensional liver tissue equivalents possessing a clinically-significant mass. Biochem Eng J. 2010;48:348–61.

    Article  CAS  Google Scholar 

  3. Landry J, Bernier D, Quellet C, Goyette R, Marceau N. Spheroidal aggregate culture of rat liver cells: hystotypic reorganization, biomatrix deposition, and maintenance of functional activities. J Cell Biol. 1985;101:914–23.

    Article  PubMed  CAS  Google Scholar 

  4. Tanimizu N, Miyajima A, Mostov KE. Liver progenitor cells develop cholangiocyte-type epithelial polarity in three-dimensional culture. Mol Biol Cell. 2007;18:1472–9.

    Article  PubMed  CAS  Google Scholar 

  5. Turner WS, Schmelzer E, McClelland R, Wauthier E, Chen W, Reid LM. Human hepatoblast phenotype maintained by hyaluronan hydrogels. J Biomed Mater Res B. 2007;82:156–68.

    Google Scholar 

  6. Xiong A, Austin TW, Lagasse E, Uchida N, Tamaki S, Bordier BB, Weissman IL, Glenn JS, Millan MT. Isolation of human fetal liver progenitors and their enhanced proliferation by three-dimensional coculture with endothelial cells. Tissue Eng Part A. 2008;14:995–1006.

    Article  PubMed  CAS  Google Scholar 

  7. Dvir-Ginzberg M, Elkayam T, Cohen S. Induced differentiation and maturation of newborn liver cells into functional hepatic tissue in macroporous alginate scaffolds. FASEB J. 2008;22:1440–9.

    Article  PubMed  CAS  Google Scholar 

  8. Ehashi T, Miyoshi H, Ohshima N. Oncostatin M stimulates proliferation and functions of mouse fetal liver cells in three-dimensional cultures. J Cell Physiol. 2005;202:698–706.

    Article  PubMed  CAS  Google Scholar 

  9. Ehashi T, Miyoshi H, Ohshima N. Three-dimensional culture of porcine fetal liver cells for a bioartificial liver. J Biomed Mater Res A. 2006;77A:90–6.

    Article  CAS  Google Scholar 

  10. Cusick RA, Lee H, Sano K, Pollok JM, Utsunomiya H, Ma PX, Langer R, Vacanti JP. The effect of donor and recipient age on engraftment of tissue-engineered liver. J Pediatr Surg. 1997;32:357–60.

    Article  PubMed  CAS  Google Scholar 

  11. Nam YS, Yoon JJ, Park TG. A novel fabrication method of macroporous biodegradable polymer scaffolds using gas foaming salt as a porogen additive. J Biomed Mater Res. 2000;53:1–7.

    Article  PubMed  CAS  Google Scholar 

  12. Kinoshita T, Sekiguchi T, Xu MJ, Ito Y, Kamiya A, Tsuji K, Nakamura T, et al. Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis. Proc Natl Acad Sci USA. 1999;96:7265–70.

    Article  PubMed  CAS  Google Scholar 

  13. Seglen PO. Preparation of isolated liver cells. In: Prescott DM, editor. Methods in cell biology, vol. 13. New York: Academic; 1976. p. 29–83.

    Google Scholar 

  14. Hammond DK, Strobel HW. Ethoxy resorufin O-deethylase activity in intact human cells. Toxicol In Vitro. 1992;6:41–6.

    Article  PubMed  CAS  Google Scholar 

  15. Sakai Y, Naruse K, Nagashima I, Muto T, Suzuki M. A new bioartificial liver using porcine hepatocyte spheroids in high-cell-density suspension perfusion culture: in vitro performance in synthesized culture medium and 100 % human plasma. Cell Transplant. 1999;8:531–41.

    PubMed  CAS  Google Scholar 

  16. Brunk CK, Jones KC, James TW. Assay for nanogram quantities of DNA in cellular homogenates. Anal Biochem. 1979;92:497–500.

    Article  PubMed  CAS  Google Scholar 

  17. Okayasu T, Tochimaru H, Hyuga T, Takahashi T, Takekoshi Y, Li Y, Togashi Y, Takeichi N, Kasai N, Arashima S. Inherited copper toxicity in Long-Evans cinnamon rats exhibiting spontaneous hepatitis: a model of Wilson’s disease. Pediatr Res. 1992;31:253–7.

    Article  PubMed  CAS  Google Scholar 

  18. Mitaka T, Sattler GL, Pitot HC, Mochizuki Y. Characteristics of small cell colonies developing in primary cultures of adult rat hepatocytes. Virchows Arch B Cell Pathol. 1992;62:329–35.

    Article  CAS  Google Scholar 

  19. Sakai Y, Jiang J, Kojima N, Kinoshita T, Miyajima A. Enhanced in vitro maturation of fetal liver cells with oncostatin M, nicotinamide and dimethylsulfoxide. Cell Transplant. 2002;11:435–41.

    PubMed  CAS  Google Scholar 

  20. Jiang J, Kojima N, Kinoshita T, Miyajima A, Yan W, Sakai Y. Cultivation of fetal mouse liver cells in a three-dimensional poly-L-lactic acid scaffold in the presence of oncostatin M. Cell Transplant. 2002;11:403–6.

    PubMed  Google Scholar 

  21. Jiang J, Kojima N, Guo L, Naruse K, Makuuchi M, Miyajima A, Yan W, Sakai Y. 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. 2004;10:1577–86.

    PubMed  CAS  Google Scholar 

  22. Inoue S, Tahara K, Shimizu H, Yoshino H, Suzuki C, Kaneko T, Hakamata Y, Takahashi M, Murakami T, Kaneko M, Kobayashi E. Rat liver transplantation for total vascular reconstruction, using a suture method. Microsurgery. 2003;23:470–5.

    Article  PubMed  Google Scholar 

  23. Hanada S, Kojima N, Sakai Y. Soluble factor-dependent in vitro growth and maturation of rat fetal liver cells in a three-dimensional culture system. Tissue Eng. 2008;14:149–60.

    Article  CAS  Google Scholar 

  24. Teratani T, Yamamoto H, Aoyagi K, Sasaki H, Asari A, Quinn G, Sasaki H, Terada M, Ochiya T. Direct hepatic fate specification from mouse embryonic stem cells. Hepatology. 2005;41:836–46.

    Article  PubMed  CAS  Google Scholar 

  25. Brill S, Zvibel I, Reid LM. Expansion conditions for early hepatic progenitor cells from embryonal and neonatal rat livers. Dig Dis Sci. 1999;44:364–71.

    Article  PubMed  CAS  Google Scholar 

  26. Teratani T, Quinn G, Yamamoto Y, Sato T, Yamanokuchi H, Asari A, Ochiya T. Long-term maintenance of liver-specific functions in cultured ES cell-derived hepatocytes with hyaluronan sponge. Cell Transplant. 2005;14:629–35.

    Article  PubMed  Google Scholar 

  27. Katsuda T, Teratani T, Ochiya T, Sakai Y. Transplantation of a fetal liver cell-loaded hyaluronic acid sponge onto the mesentery recovers a Wilson’s disease model rat. J Biochem. 2010;148(3):281–8.

    Article  PubMed  CAS  Google Scholar 

  28. Bacon BR, Schilsky ML. New knowledge of genetic pathogenesis of hemochromatosis and Wilson’s disease. Adv Intern Med. 1999;44:91–116.

    PubMed  CAS  Google Scholar 

  29. Wu J, Forbes JR, Chen HS, Cox DW. The LEC rat has a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene. Nat Genet. 1994;7:541–5.

    Article  PubMed  CAS  Google Scholar 

  30. Chen Q, Kon J, Ooe H, Sasaki K, Mitaka T. Selective proliferation of rat hepatocyte progenitor cells in serum-free culture. Nat Protoc. 2007;2:1197–205.

    Article  PubMed  CAS  Google Scholar 

  31. Gregory PG, Connolly CK, Toner M, Sullivan SJ. In vitro characterization of porcine hepatocyte function. Cell Transplant. 2000;9:1–10.

    PubMed  CAS  Google Scholar 

  32. Elcin YM, Dixit V, Gitnick G. Hepatocyte attachment on biodegradable modified chitosan membranes: in vitro evaluation for the development of liver organoids. Artif Organs. 1998;22:1525–94.

    Google Scholar 

  33. Elcin YM, Dixit V, Lewin K, Gitnick G. Xenotransplantation of fetal porcine hepatocytes in rats using a tissue engineering approach. Artif Organs. 1999;23:146–52.

    Article  PubMed  CAS  Google Scholar 

  34. Huang H, Hanada S, Kojima N, Sakai Y. 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. 2006;15:799–809.

    Article  PubMed  Google Scholar 

  35. Mitaka T. The current status of primary hepatocyte culture. Int J Exp Pathol. 1998;79:393–409.

    Article  PubMed  CAS  Google Scholar 

  36. Nishikawa M, Kojima N, Komori K, Yamamoto T, Fujii T, Sakai Y. Enhanced maintenance and functions of rat hepatocytes induced by combination of on-site oxygenation and coculture with fibroblasts. J Biotechnol. 2008;133:253–60.

    Article  PubMed  CAS  Google Scholar 

  37. Evenou F, Fujii T, Sakai Y. Spontaneous formation of highly functional three-dimensional multilayer from human hepatoma Hep G2 cells cultured on an oxygen-permeable polydimethylsiloxane membrane. Tissue Eng Part C Methods. 2010;16:311–8.

    Article  PubMed  CAS  Google Scholar 

  38. Hamon M, Hanada S, Fujii T, Sakai Y. 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. 2012;21(2–3):401–10.

    Article  PubMed  Google Scholar 

  39. Ohashi K. Liver tissue engineering: the future of liver therapeutics. Hepatol Res. 2008;38(S1):S76–87.

    Article  PubMed  CAS  Google Scholar 

  40. Sudo S, Kohara H, Mitaka T, Ikeda M, Tanishita K. Coordination of bile canalicular contraction in hepatic organoid reconstructed by rat small hepatocytes and nonparenchymal cells. Ann Biomed Eng. 2005;33:696–708.

    Article  PubMed  Google Scholar 

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Acknowledgments

The works were done 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. J. Jiang and T. Katsuda were supported by the Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science.

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Authors and Affiliations

  1. Organs and Biosystems Engineering Laboratory, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan

    Yasuyuki Sakai Ph.D., Jinlan Jiang, Sanshiro Hanada, Hongyug Huang & Takeshi Katsuda

  2. Cellular Dynamics International, Madison, WI, USA

    Jinlan Jiang

  3. International Clinical Research Center, Research Institute, International Medical Center of Japan, Tokyo, Japan

    Sanshiro Hanada

  4. Okami Chemical Industry Co., Ltd, Tokyo, Japan

    Hongyug Huang

  5. Section for Studies on Metastasis, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

    Takeshi Katsuda, Takumi Teratani & Takahiro Ochiya

  6. BEANS Project, Ministry of Economy, Trade and Industry, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan

    Nobuhiko Kojima

  7. Center for Development of Advanced Medical Technology, Jichi Medical University, Tochigi, Japan

    Takumi Teratani

  8. Laboratory for Cell Growth and Differentiation, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan

    Atsushi Miyajima

Authors
  1. Yasuyuki Sakai Ph.D.
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  2. Jinlan Jiang
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  3. Sanshiro Hanada
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  6. Nobuhiko Kojima
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  7. Takumi Teratani
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  8. Atsushi Miyajima
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  9. Takahiro Ochiya
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Correspondence to Yasuyuki Sakai Ph.D. .

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Editors and Affiliations

  1. Southend Park 55, Kolkata, 700029, India

    Niranjan Bhattacharya

  2. Dept. Obstetrics & Gynaecology, Boston University Medical Centre, E. Concord Street 85, Boston, 02118, Massachusetts, USA

    Phillip Stubblefield

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Sakai, Y. et al. (2013). Three-Dimensional Culture of Fetal Mouse, Rat, and Porcine Hepatocytes. In: Bhattacharya, N., Stubblefield, P. (eds) Human Fetal Tissue Transplantation. Springer, London. https://doi.org/10.1007/978-1-4471-4171-6_4

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  • DOI: https://doi.org/10.1007/978-1-4471-4171-6_4

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  • Publisher Name: Springer, London

  • Print ISBN: 978-1-4471-4170-9

  • Online ISBN: 978-1-4471-4171-6

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