Advertisement

Cytotechnology

, Volume 45, Issue 3, pp 125–140 | Cite as

Three-dimensional co-culture of hepatocytes and stellate cells

  • Susan  Fugett Abu-Absi
  • Linda  K. Hansen
  • Wei-Shou Hu
Article

Abstract

Hepatocytes self-assemble in culture to form compacted spherical aggregates, or spheroids, that mimic the structure of the liver by forming tight junctions and bile canalicular channels. Hepatocyte spheroids thus resemble the liver to a great extent. However, liver tissue contains other cell types and has bile ducts and sinusoids formed by endothelial cells. Reproducing 3-D co-culture in vitro could provide a means to develop a more complex tissue-like structure. Stellate cells participate in revascularization after liver injury by excreting between hepatocytes a laminin trail that endothelial cells follow to form sinusoids. In this study we investigated co-culture of rat hepatocytes and a rat hepatic stellate cell line, HSC-T6. HSC-T6, which does not grow in serum-free spheroid medium, was able to grow under co-culture conditions. Using a three-dimensional cell tracking technique, the interactions of HSC-T6 and hepatocyte spheroids were visualized. The two cell types formed heterospheroids in culture, and HSC-T6 cell invasion into hepatocyte spheroids and subsequent retraction was observed. RT-PCR revealed that albumin and cytochrome P450 2B1/2 expression were better maintained in co-culture conditions. These three-dimensional heterospheroids provide an attractive system for in vitro studies of hepatocyte-stellate cell interactions.

Keywords

3-dimentional culture Co-culture Hepatocytes Stellate cell 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Abu-Absi S.F., Friend J.R., Hansen L.K. and Hu W.-S. 2002. Structural polarity and functional bile canaliculi in rat hepatocyte spheroids. Exp. Cell Res. 274: 56--67.Google Scholar
  2. Agius, L. 1988Metabolic interactions of parenchymal hepatocytes and dividing epithelial cells in co-cultureBiochem. J.2522328PubMedGoogle Scholar
  3. Ankoma-Sey, V., Wang, Y., Dai, Z. 2000Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cellsHepatology31141148CrossRefPubMedGoogle Scholar
  4. Asano, K., Koide, N., Tsuji, T. 1989Ultrastructure of multicellular spheroids formed in the primary culture of adult rat hepatocytesJ. Clin. Electron Microsc.22243252Google Scholar
  5. Balis, U.J., Behnia, K., Dwarakanath, B., Bhatia, S.N. 1999Oxygen Consumption Characteristics of Porcine HepatocytesMetab. Eng.14962CrossRefPubMedGoogle Scholar
  6. Begue, J.M., Guguen-Guillouzo, C., Pasdeloup, N., Guillouzo, A. 1984Prolonged maintenance of active cytochrome P-450 in adult rat hepatocytes co-cultured with another liver cell typeHepatology4839842PubMedCrossRefGoogle Scholar
  7. Bhandari, R., Riccalton, L., Lewis, A., Fry, J., Hammond, A., Tendler, S., Shakesheff, K. 2001Liver tissue engineering: a role for co-culture systems in modifying hepatocyte function and viabilityTissue Eng7345357CrossRefPubMedGoogle Scholar
  8. Bhatia, S.N., Balis, U.J., Yarmush, M.L., Toner, M. 1998Probing heterotypic cell interactions: hepatocyte function in microfabricated co-culturesJ. Biomat. Sci. Polym. Edition911371160Google Scholar
  9. Chaney, A., Marback, E. 1962Modified reagents for determination of urea and ammoniaClin. Chem.8130132PubMedGoogle Scholar
  10. Donato, M.T., Castell, J.V., Gomez- Lechon, M.J. 1991Co-cultures of hepatocytes with epithelial-like cell lines: expression of drug-biotransformation activities by hepatocytesCell Biol. Toxicol.7114CrossRefPubMedGoogle Scholar
  11. Fawcett, J., Scott, J. 1960A rapid and precise method for the determination of ureaJ. Clin. Pathol.13156160PubMedGoogle Scholar
  12. Friedman, S.L. 1996Hepatic stellate cellsProg. Liver Dis.14101130PubMedGoogle Scholar
  13. Greenwel, P., Rubin, J., Schwartz, M., Hertzberg, E.L., Rojkind, M. 1993Liver fat-storing cell clones obtained from a C.C.l4-cirrhotic rat are heterogeneous with regard to proliferation, expression of extracellular matrix components, interleukin-6, and connexin 43Lab. Invest.69210216PubMedGoogle Scholar
  14. Gressner, A., Lahme, B., Brenzel, A. 1995Molecular dissection of the mitogenic effect of hepatocytes on cultured hepatic stellate cellsHepatology2215071518CrossRefPubMedGoogle Scholar
  15. Gressner, A.M., Lotfi, S., Gressner, G., Lahme, B. 1992Identification and partial characterization of a hepatocyte-derived factor promoting proliferation of cultured fat-storing cells(parasinusoidal lipocytes). Hepatology1612501266PubMedGoogle Scholar
  16. Griffith, L., Wu, B., Cima, M., Powers, M., Chaignaud, B., Vacanti, J. 1997In vitro organogenesis of liver tissueAnn. NY. Acad. Sci.831382397PubMedCrossRefGoogle Scholar
  17. Guguen-Guillouzo, C., Clement, B., Baffet, G., Beaumont, C., Morel-Chang, E., Glaise, D., Guillouzo, A. 1983Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell typeExp. Cell Res.1434754CrossRefPubMedGoogle Scholar
  18. Hansen, L.K., Hsiao, C.C., Friend, J.R., Wu, F.J., Bridge, G.A., Remmel, R.P., Ceira, F.B., Hu, W.S. 1998Enhanced morphology and function in hepatocyte spheroids: A model of tissue self-assemblyTissue Eng.46574Google Scholar
  19. Hsiao, C.C., Friend, J.R., Wu, F.J., Ko, W.J., Remmel, R.P., Hu, W.S. 1999Receding cytochrome P450 activity in disassembling hepatocyte spheroidsTissue Eng.5207221PubMedGoogle Scholar
  20. Imaoka, S., Enomoto, K., Oda, Y., Asada, A., Fujimori, M., Shimada, T., Fujita, S., Guengerich, F.P., Funae, Y. 1990Lidocaine metabolism by human cytochrome P-450s purified from hepatic microsomes: comparison of those with rat hepatic cytochrome P-450sJ. Pharmacol. Exp. Ther.25513851391PubMedGoogle Scholar
  21. Kim, S., Utsunomiya, H., Koski, J., Wu, B., Cima, M., Sohn, J., Mukai, K., Griffith, L., Vacanti, J. 1998Survival and function of hepatocytes on a novel three-dimensional synthetic biodegradable polymer scaffold with an intrinsic network of channelsAnn. Surg.228813CrossRefPubMedGoogle Scholar
  22. Koide, N., Sakaguchi, K., Koide, Y., Asano, K., Kawaguchi, M., Matsushima, H., Takenami, T., Shinji, T., Mori, M., Tsuji, T. 1990Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environmentsExp. Cell Res.186227235CrossRefPubMedGoogle Scholar
  23. Landry, J., Bernier, D., Ouellet, C., Goyette, R., Marceau, N. 1985Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activitiesJ. Cell Biol.101914923CrossRefPubMedGoogle Scholar
  24. Lazar, A., Mann, H.J., Remmel, R.P., Shatford, R.A., Cerra, F.B., Hu, W.S. 1995Extended liver-specific functions of porcine hepatocyte spheroids entrapped in collagen gelIn Vitro Cell. Dev. Biol.Anim.3140346Google Scholar
  25. Maher, J.J., Tzagarakis, C. 1994Partial cloning of the M. subunit of laminin from adult rat lipocytes: expression of the M. subunit by cells isolated from normal and injured liverHepatology19764770PubMedGoogle Scholar
  26. Majno, G. 1979The story of the myofibroblastsAm. J. Surg. Pathol.6535542CrossRefGoogle Scholar
  27. Martinez-Hernandez, A., Amenta, P.S. 1995The extracellular matrix in hepatic regenerationFASEB J.914011410PubMedGoogle Scholar
  28. Michalopoulos, G.K., Bowen, W.C., Zajac, V.F., Beer-Stolz, D., Watkins, S., Kostrubsky, V., Strom, S.C. 1999Morphogenetic events in mixed cultures of rat hepatocytes and nonparenchymal cells maintained in biological matrices in the presence of hepatocyte growth factor and epidermal growth factorHepatology2990100CrossRefPubMedGoogle Scholar
  29. Morris, D.L., Davila, J.C. 1996Analysis of rat cytochrome P450 isozyme expression using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)Biochem. Pharmacol.52781792CrossRefPubMedGoogle Scholar
  30. Okamoto, M., Ishida, Y., Keogh, A., Strain, A. 1998Evaluation of the function of primary human hepatocytes co-cultured with the human hepatic stellate cell (HSC) line L.I.90Int. J. Artif. Organs21353359PubMedGoogle Scholar
  31. Peshwa, M.V., Wu, F.J., Follstad, B.D., Cerra, F.B., Hu, W.S. 1994Kinetics of hepatocyte spheroid formationBiotechnol. Prog.10460466CrossRefGoogle Scholar
  32. Peshwa, M.V., Wu, F.J., Sharp, H.L., Cerra, F.B., Hu, W-S 1996Mechanistics of formation and ultrastructural evaluation of hepatocyte spheroidsIn Vitro Cell. Dev. Biol.32197203Google Scholar
  33. Rojkind, M., Novikoff, P.M., Greenwel, P., Rubin, J., Rojas-Valencia, L., de Carvalho, A.C., Stockert, R., Spray, D., Hertzberg, E.L., Wolkoff, A.W. 1995Characterization and functional studies on rat liver fat-storing cell line and freshly isolated hepatocyte co-culture systemAm. J. Pathol.14615081520PubMedGoogle Scholar
  34. Sambrook, J., Fritsch, E.F., Maniatis, T. 1989Molecular Cloning: A Laboratory Manual, 2nd edCold Spring Harbor LaboratoryCold Spring HarborNYGoogle Scholar
  35. Schirmacher, P., Geerts, A., Peitrangelo, A., Dienes, H., Rogier, C. 1992Hepatocyte growth factor/hepatopoietin A. is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cellsHepatology15511PubMedGoogle Scholar
  36. Seglen, P.O. 1976Preparation of isolated rat liver cellsMethod. Cell Biol.13983Google Scholar
  37. Senoo, H., Imai, K., Matano, Y., Sato, M. 1998Molecular mechanisms in the reversible regulation of morphology, proliferation and collagen metabolism in hepatic stellate cells by the three-dimensional structure of the extracellular matrixJ. Gastroenterol. Hepatol.13S1932PubMedGoogle Scholar
  38. Tong, J.Z., Bernard, O., Alvarez, F. 1990Long-term culture of rat liver cell spheroids in hormonally defined mediaExp. Cell Res.1898792CrossRefPubMedGoogle Scholar
  39. Tzanakakis, E.S., Hansen, L.K., Hu, W.S. 2001The role of actin filaments and microtubules in hepatocyte spheroid self-assemblyCell Motil. Cytoskel.48175189CrossRefGoogle Scholar
  40. Ueno, K., Kobayashi, A., Takegawa, N., Satoh, T. 1995Rapid formation of multicellular spheroids composed of Propionibacterium acnes pretreated adult rat liver cells by rotary culture and their immunological propertiesRes. Commun. Mol. Pathol. Pharmacol.90373387PubMedGoogle Scholar
  41. Ueno, K., Miyashita, A., Endoh, E., Takezawa, T., Yamazaki, M., Mori, Y., Satoh, T. 1992Formation of multicellular spheroids composed of rat hepatocytesRes. Commun. Chem. Pathol. Pharmacol.77107120PubMedGoogle Scholar
  42. Vogel, S., Piantedosi, R., Frank, J., Lalazar, A., Rockey, D.C., Friedman, S.L., Blaner, W.S. 2000An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitroJ. Lipid Res.41882893PubMedGoogle Scholar
  43. Wu, F.J., Friend, J.R., Hsiao, C.C., Zilliox, M.J., Ko, W.J., Cerra, F.B., Hu, W.S. 1996Efficient assembly of primary rat hepatocyte spheroids for tissue engineering applicationsBiotechnol. Bioeng.50404415CrossRefPubMedGoogle Scholar
  44. Wu, F.J., Friend, J.R., Remmel, R.P., Cerra, F.B., Hu, W.S. 1999Enhanced cytochrome P450 lAl activity of self-assembled rat hepatocyte spheroidsCell Transplantation8233246PubMedGoogle Scholar
  45. Yagi, K., Yamada, C., Serada, M., Sumiyoshi, N., Michibayashi, N., Miura, Y., Mizoguchi, T. 1995Reciprocal regulation of prothrombin secretion and tyrosine aminotransferase induction in hepatocytesEur. J. Biochem.227753756CrossRefPubMedGoogle Scholar
  46. Zaret, K.S. 2000Liver specification and early morphogenesisMech. Dev.928388CrossRefPubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Susan  Fugett Abu-Absi
    • 1
  • Linda  K. Hansen
    • 2
  • Wei-Shou Hu
    • 1
  1. 1.Departments of Chemical Engineering and Materials ScienceUniversity of MinnesotaMinneapolisUSA
  2. 2.Laboratory Medicine and PathologyUniversity of MinnesotaMinneapolisUSA

Personalised recommendations