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A three-dimensional collagen-sponge-based culture system coated with simplified recombinant fibronectin improves the function of a hepatocyte cell line

  • Yuuki Nishida
  • Akiyoshi TaniguchiEmail author
Article

Abstract

Hepatocytes are widely used in pharmaceutical drug discovery tests, but their hepatic functions decrease rapidly during in vitro culture. Many culture systems have been devised to address this problem. We here report that a three-dimensional (3D) collagen-based scaffold coated with simplified recombinant fibronectin (FN) enhanced the function of a hepatocyte cell line. The developed culture system uses a honeycomb collagen sponge coated with collagen-binding domain (CBD)-cell attachment site (CAS), a chimeric protein comprising the CBD and CAS of FN. The function of HepG2 cells grown on honeycomb collagen sponge coated with CBD-CAS was investigated by determining the messenger RNA (mRNA) expression levels of several genes. The mRNA expression level of albumin increased 3.25 times in cells grown on CBD-CAS-coated honeycomb collagen sponge for 3 days; the expression level of CCAAT/enhancer binding protein (C/EBPα) increased 40-fold after 1 d and up to 150-fold after 3 d. These results suggested that CBD-CAS-coated honeycomb collagen sponge could improve the functions of hepatocytes by inducing C/EBPα expression. The activation of cytochrome P450 (CYP) enzymes in HepG2 cells grown on CBD-CAS-coated honeycomb collagen sponge was measured at the mRNA level and was found to increase between two and six times compared to cells grown without the CBD-CAS coating, showing that this culture system induced CYP gene expression and thus may be useful in drug metabolism assays.

Keywords

Hepatocyte 3D culture Biomaterials Recombinant protein 

References

  1. Askari JA, Tynan CJ, Webb SED, Fernandez ML, Ballestrem C, Humphries MJ (2010) Focal adhesions are sites of integrin extension. J Cell Biol 188:891–903CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bader A, Rinkes IH, Closs EI, Ryan CM, Toner M, Cunningham JM, Tompkins RG, Yarmush ML (1992) A stable long-term hepatocyte culture system for studies of physiologic processes: cytokine stimulation of the acute phase response in rat and human hepatocytes. Biotechnol Prog 8:219–225CrossRefPubMedGoogle Scholar
  3. Berthiaume F, Moghe PV, Toner M, Yarmush ML (1996) Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration. FASEB J 10:1471–1484PubMedGoogle Scholar
  4. Bi Y, Kazolias D, Duignan DB (2006) Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport. Drug Metab Dispos 34:1658–1665CrossRefPubMedGoogle Scholar
  5. Bokhari M, Carnachan RJ, Cameron NR, Przyborski SA (2007) Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge. J Anat 211:567–576PubMedPubMedCentralGoogle Scholar
  6. de Graaf IA, Draaisma AL, Schoeman O, Fahy GM, Groothuis GM, Koster HJ (2007) Cryopreservation of rat precision-cut liver and kidney slices by rapid freezing and vitrification. Cryobiology 54:1–12CrossRefPubMedGoogle Scholar
  7. Dunn JC, Yarmush ML, Koebe HG, Tompkins RG (1989) Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. FASEB J 3:174–177PubMedGoogle Scholar
  8. Friedman AD, Landschulz WH, McKnight SL (1989) CCAAT/enhancer binding protein activates the promoter of the serum albumin gene in cultured hepatoma cells. Genes Dev 3:1314–1322CrossRefPubMedGoogle Scholar
  9. Harunaga JS, Yamada KM (2011) Cell-matrix adhesions in 3D. Matrix Biol 30:363–368CrossRefPubMedPubMedCentralGoogle Scholar
  10. Hewitt NJ, Hewitt P (2004) Phase I and II enzyme characterization of two sources of HepG2 cell lines. Xenobiotica 34:243–256CrossRefPubMedGoogle Scholar
  11. Horiuchi S, Ishida S, Hongo T, Ishikawa Y, Miyajima A, Sawada J, Ohno Y, Nakazawa K, Ozawa S (2009) Global gene expression changes including drug metabolism and disposition induced by three-dimensional culture of HepG2 cells—involvement of microtubules. Biochem Biophys Res Commun 378:558–562CrossRefPubMedGoogle Scholar
  12. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110:673–687CrossRefPubMedGoogle Scholar
  13. Itho H, Aso Y, Furuse M, Noishiki Y, Miyata T (2001) A honeycomb collagen carrier for cell culture as a tissue engineering scaffold. Artif Organs 25:213–217CrossRefGoogle Scholar
  14. Kleinman HK, McGarvey ML, Liotta LA, Robey PG, Tryggvason K, Martin GR (1982) Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21:6188–6193CrossRefPubMedGoogle Scholar
  15. Kono Y, Roverts EV (1996) Modulation of the expression of liver-specific functions in novel human hepatocyte lines cultured in a collagen gel sandwich configuration. Biochem Biophys Res Commun 220:628–632CrossRefPubMedGoogle Scholar
  16. Kowles BB, Howe CC, Aden DP (1980) Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209:497–499CrossRefGoogle Scholar
  17. Kurosawa Y, Taniguchi A, Okano T (2005) A novel method to examine hepatocyte specific gene expression in a functional co-culture system. Tissue Eng 11:1650–1657CrossRefPubMedGoogle Scholar
  18. Landry J, Bernier D, Ouellet R, Goyette R, Marceau N (1985) Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities. J Cell Biol 101:914–923CrossRefPubMedGoogle Scholar
  19. LeCluyse EL (2001) Human hepatocyte culture systems for the in vitro evaluation of cytochrome P450 expression and regulation. Eur Pharm Sci 13:343–368CrossRefGoogle Scholar
  20. Li JW, Wang GP, Fan JY, Chang CF, Xu CS (2011) Eight path of ERK1/2 signaling pathway regulating hepatocyte proliferation in rat liver regeneration. J Genet 90:435–442CrossRefPubMedGoogle Scholar
  21. Meli L, Jordan ET, Clark DS, Linhardt RJ, Dordick JS (2012) Influence of a three-dimensional, microarray environment on human cell culture in drug screening systems. Biomaterials 33:9087–9096CrossRefPubMedPubMedCentralGoogle Scholar
  22. Nakamura K, Kato N, Aizawa K, Mizutani R, Yamauchi J, Tanoue A (2011) Expression of albumin and cytochrome P450 enzymes in HepG2 cells cultured with a nanotechnology-based culture plate with micro fabricated scaffold. J Toxicol Sci 36:625–633CrossRefPubMedGoogle Scholar
  23. Nishida Y, Taniguchi A (2013) Induction of albumin expression in Hepg2 cells using immobilized simplified recombinant fibronectin protein. In Vitro Cell Dev Biol Anim 49:400–407CrossRefPubMedGoogle Scholar
  24. Nishimura M, Hag M, Ejiri Y, Kishimoto S, Horie T, Narimatsu S, Nito S (2010) Secretion of albumin and induction of CYP1A2 and CYP3A4 in novel three-dimensional culture system for human hepatocytes using micro-space plate. Drug Metab Pharmacokinet 25:236–242CrossRefPubMedGoogle Scholar
  25. Page JL, Johnson MC, Olsavsky KM, Strom SC, Zarbl H, Omiecinski CJ (2007) Gene expression profiling of extracellular matrix as an effector of human hepatocyte phenotype in primary cell culture. Toxicol Sci 97:384–397CrossRefPubMedPubMedCentralGoogle Scholar
  26. Pankov R, Cukierman E, Katz BZ, Matsumoto K, Lin DC, Lin S, Hahn C, Yamada KM (2000) Integrin dynamics and matrix assembly: tensin-dependent translocation of α5β1 integrins promotes early fibronectin fibrillogenesis. J Cell Biol 148:1075–1090CrossRefPubMedPubMedCentralGoogle Scholar
  27. Paszek MJ, Boettiger D, Weaver VM, Hammer DA (2009) Integrin clustering is driven by mechanical resistance from the glycocalyx and the substrate. PLoS Comput Biol 5:e1000604CrossRefPubMedPubMedCentralGoogle Scholar
  28. Pereira-Rodrigues N, Poleni PE, Guimard D, Arakawa Y, Sakai Y, Fujii T (2010) Modulation of hepatocarcinoma cell morphology and activity by parylene-c coating on PDMS. PLoS One 5:e9667CrossRefPubMedPubMedCentralGoogle Scholar
  29. Rodriguez-Antona C, Donato MT, Boobis A, Edwards RJ, Watts PS, Castell JV, Gomez-Lechon MJ (2002) Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells. Xenobiotica 32:505–520CrossRefPubMedGoogle Scholar
  30. Takagi J, Springer TA (2002) Integrin activation and structural rearrangement. Immunol Rev 186:141–163CrossRefPubMedGoogle Scholar
  31. Takayama G, Taniguchi A, Okano T (2007) Identification of differentially expressed genes in hepatocyte endothelial cell co-culture system. Tissue Eng 13:159–166CrossRefPubMedGoogle Scholar
  32. Tan EH, Ma FJ, Gopinadhan S, Sakban RB, Wang ND (2007) C/EBPα knock-in hepatocytes exhibit increased albumin secretion and urea production. Cell Tissue Res 330:427–435CrossRefPubMedGoogle Scholar
  33. Westerink WM, Schoonen WG (2007) Cytochrome P450 enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicol In Vitro 21:1581–1591CrossRefPubMedGoogle Scholar
  34. Wilkening S, Stahl F, Bader A (2003) Comparison of primary human hepatocytes and hepatoma cell line HepG2 with regard to their biotransformation properties. Drug Metab Dispos 3:1035–1042CrossRefGoogle Scholar
  35. Wu J, Buye’ NM, Muino’s TF, Borro’s S, Favia P, Semino CE (2010) Nanometric self-assembling peptide layers maintain adult hepatocyte phenotype in sandwich cultures. J Nanobiotechnol 8:29CrossRefGoogle Scholar
  36. Xu C, Yang Y, Yang J, Chen X, Wang G (2011) Analysis of the role of the integrin pathway in hepatocyte during rat liver regeneration. Cell Mol Biol Lett 17:274–288Google Scholar
  37. Yamada KM, Cukierman E (2007) Modeling tissue morphogenesis and cancer in 3D. Cell 130:601–610CrossRefPubMedGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2015

Authors and Affiliations

  1. 1.Cell-Materials Interaction Group, Biomaterials Unit, Nano-Life Field, International Center for Materials Nanoarchitectonics (MANA)National Institute for Materials Science (NIMS)TsukubaJapan
  2. 2.Graduate School of Advanced Science and EngineeringWaseda UniversityShinjukuJapan

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