Skip to main content
PMC home page
Cytotechnology logoLink to Cytotechnology
. 2004 Jul;45(3):125–140. doi: 10.1007/s10616-004-7996-6

Three-dimensional co-culture of hepatocytes and stellate cells

Susan Fugett Abu-Absi 1, Linda K Hansen 2, Wei-Shou Hu 1,✉
PMCID: PMC3450167  PMID: 19003250

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

Full Text

The Full Text of this article is available as a PDF (499.7 KB).

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. [DOI] [PubMed]
  2. Agius L. Metabolic interactions of parenchymal hepatocytes and dividing epithelial cells in co-culture. Biochem. J. 1988;252:23–28. doi: 10.1042/bj2520023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ankoma-Sey V., Wang Y., Dai Z. Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells. Hepatology. 2000;31:141–148. doi: 10.1002/hep.510310122. [DOI] [PubMed] [Google Scholar]
  4. Asano K., Koide N., Tsuji T. Ultrastructure of multicellular spheroids formed in the primary culture of adult rat hepatocytes. J. Clin. Electron Microsc. 1989;22:243–252. [Google Scholar]
  5. Balis U.J., Behnia K., Dwarakanath B., Bhatia S.N. Oxygen Consumption Characteristics of Porcine Hepatocytes. Metab. Eng. 1999;1:49–62. doi: 10.1006/mben.1998.0105. [DOI] [PubMed] [Google Scholar]
  6. Begue J.M., Guguen-Guillouzo C., Pasdeloup N., Guillouzo A. Prolonged maintenance of active cytochrome P-450 in adult rat hepatocytes co-cultured with another liver cell type. Hepatology. 1984;4:839–842. doi: 10.1002/hep.1840040507. [DOI] [PubMed] [Google Scholar]
  7. Bhandari R., Riccalton L., Lewis A., Fry J., Hammond A., Tendler S., Shakesheff K. Liver tissue engineering: a role for co-culture systems in modifying hepatocyte function and viability. Tissue Eng. 2001;7:345–357. doi: 10.1089/10763270152044206. [DOI] [PubMed] [Google Scholar]
  8. Bhatia S.N., Balis U.J., Yarmush M.L., Toner M. Probing heterotypic cell interactions: hepatocyte function in microfabricated co-cultures. J. Biomat. Sci. Polym. Edition. 1998;9:1137–1160. doi: 10.1163/156856298x00695. [DOI] [PubMed] [Google Scholar]
  9. Chaney A., Marback E. Modified reagents for determination of urea and ammonia. Clin. Chem. 1962;8:130–132. [PubMed] [Google Scholar]
  10. Donato M.T., Castell J.V., Gomez- Lechon M.J. Co-cultures of hepatocytes with epithelial-like cell lines: expression of drug-biotransformation activities by hepatocytes. Cell Biol. Toxicol. 1991;7:1–14. doi: 10.1007/BF00121326. [DOI] [PubMed] [Google Scholar]
  11. Fawcett J., Scott J. A rapid and precise method for the determination of urea. J. Clin. Pathol. 1960;13:156–160. doi: 10.1136/jcp.13.2.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Friedman S.L. Hepatic stellate cells. Prog. Liver Dis. 1996;14:101–130. [PubMed] [Google Scholar]
  13. Greenwel P., Rubin J., Schwartz M., Hertzberg E.L., Rojkind M. Liver 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 43. Lab. Invest. 1993;69:210–216. [PubMed] [Google Scholar]
  14. Gressner A., Lahme B., Brenzel A. Molecular dissection of the mitogenic effect of hepatocytes on cultured hepatic stellate cells. Hepatology. 1995;22:1507–1518. doi: 10.1016/0270-9139(95)90159-0. [DOI] [PubMed] [Google Scholar]
  15. Gressner A.M., Lotfi S., Gressner G., Lahme B. Identification and partial characterization of a hepatocyte-derived factor promoting proliferation of cultured fat-storing cells. (parasinusoidal lipocytes). Hepatology. 1992;16:1250–1266. [PubMed] [Google Scholar]
  16. Griffith L., Wu B., Cima M., Powers M., Chaignaud B., Vacanti J. In vitro organogenesis of liver tissue. Ann. NY. Acad. Sci. 1997;831:382–397. doi: 10.1111/j.1749-6632.1997.tb52212.x. [DOI] [PubMed] [Google Scholar]
  17. Guguen-Guillouzo C., Clement B., Baffet G., Beaumont C., Morel-Chang E., Glaise D., Guillouzo A. Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type. Exp. Cell Res. 1983;143:47–54. doi: 10.1016/0014-4827(83)90107-6. [DOI] [PubMed] [Google 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. Enhanced morphology and function in hepatocyte spheroids: A model of tissue self-assembly. Tissue Eng. 1998;4:65–74. [Google Scholar]
  19. Hsiao C.C., Friend J.R., Wu F.J., Ko W.J., Remmel R.P., Hu W.S. Receding cytochrome P450 activity in disassembling hepatocyte spheroids. Tissue Eng. 1999;5:207–221. doi: 10.1089/ten.1999.5.207. [DOI] [PubMed] [Google Scholar]
  20. Imaoka S., Enomoto K., Oda Y., Asada A., Fujimori M., Shimada T., Fujita S., Guengerich F.P., Funae Y. Lidocaine metabolism by human cytochrome P-450s purified from hepatic microsomes: comparison of those with rat hepatic cytochrome P-450s. J. Pharmacol. Exp. Ther. 1990;255:1385–1391. [PubMed] [Google Scholar]
  21. Kim S., Utsunomiya H., Koski J., Wu B., Cima M., Sohn J., Mukai K., Griffith L., Vacanti J. Survival and function of hepatocytes on a novel three-dimensional synthetic biodegradable polymer scaffold with an intrinsic network of channels. Ann. Surg. 1998;228:8–13. doi: 10.1097/00000658-199807000-00002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Koide N., Sakaguchi K., Koide Y., Asano K., Kawaguchi M., Matsushima H., Takenami T., Shinji T., Mori M., Tsuji T. Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments. Exp. Cell Res. 1990;186:227–235. doi: 10.1016/0014-4827(90)90300-Y. [DOI] [PubMed] [Google Scholar]
  23. Landry J., Bernier D., Ouellet C., Goyette R., Marceau N. Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities. J. Cell Biol. 1985;101:914–923. doi: 10.1083/jcb.101.3.914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lazar A., Mann H.J., Remmel R.P., Shatford R.A., Cerra F.B., Hu W.S. Extended liver-specific functions of porcine hepatocyte spheroids entrapped in collagen gel. In Vitro Cell. Dev. Biol.Anim. 1995;31:40–346. doi: 10.1007/BF02634282. [DOI] [PubMed] [Google Scholar]
  25. Maher J.J., Tzagarakis C. Partial cloning of the M. subunit of laminin from adult rat lipocytes: expression of the M. subunit by cells isolated from normal and injured liver. Hepatology. 1994;19:764–770. doi: 10.1002/hep.1840190332. [DOI] [PubMed] [Google Scholar]
  26. Majno G. The story of the myofibroblasts. Am. J. Surg. Pathol. 1979;6:535–542. doi: 10.1097/00000478-197912000-00006. [DOI] [PubMed] [Google Scholar]
  27. Martinez-Hernandez A., Amenta P.S. The extracellular matrix in hepatic regeneration. FASEB J. 1995;9:1401–1410. doi: 10.1096/fasebj.9.14.7589981. [DOI] [PubMed] [Google Scholar]
  28. Michalopoulos G.K., Bowen W.C., Zajac V.F., Beer-Stolz D., Watkins S., Kostrubsky V., Strom S.C. Morphogenetic events in mixed cultures of rat hepatocytes and nonparenchymal cells maintained in biological matrices in the presence of hepatocyte growth factor and epidermal growth factor. Hepatology. 1999;29:90–100. doi: 10.1002/hep.510290149. [DOI] [PubMed] [Google Scholar]
  29. Morris D.L., Davila J.C. Analysis of rat cytochrome P450 isozyme expression using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) Biochem. Pharmacol. 1996;52:781–792. doi: 10.1016/0006-2952(96)00390-5. [DOI] [PubMed] [Google Scholar]
  30. Okamoto M., Ishida Y., Keogh A., Strain A. Evaluation of the function of primary human hepatocytes co-cultured with the human hepatic stellate cell (HSC) line L.I.90. Int. J. Artif. Organs. 1998;21:353–359. [PubMed] [Google Scholar]
  31. Peshwa M.V., Wu F.J., Follstad B.D., Cerra F.B., Hu W.S. Kinetics of hepatocyte spheroid formation. Biotechnol. Prog. 1994;10:460–466. doi: 10.1021/bp00029a002. [DOI] [Google Scholar]
  32. Peshwa M.V., Wu F.J., Sharp H.L., Cerra F.B., Hu W-S. Mechanistics of formation and ultrastructural evaluation of hepatocyte spheroids. In Vitro Cell. Dev. Biol. 1996;32:197–203. doi: 10.1007/BF02722946. [DOI] [PubMed] [Google 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. Characterization and functional studies on rat liver fat-storing cell line and freshly isolated hepatocyte co-culture system. Am. J. Pathol. 1995;146:1508–1520. [PMC free article] [PubMed] [Google Scholar]
  34. Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring HarborNY: Cold Spring Harbor Laboratory; 1989. [Google Scholar]
  35. Schirmacher P., Geerts A., Peitrangelo A., Dienes H., Rogier C. Hepatocyte growth factor/hepatopoietin A. is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cells. Hepatology. 1992;15:5–11. doi: 10.1002/hep.1840150103. [DOI] [PubMed] [Google Scholar]
  36. Seglen P.O. Preparation of isolated rat liver cells. Method. Cell Biol. 1976;13:9–83. doi: 10.1016/s0091-679x(08)61797-5. [DOI] [PubMed] [Google Scholar]
  37. Senoo H., Imai K., Matano Y., Sato M. Molecular mechanisms in the reversible regulation of morphology, proliferation and collagen metabolism in hepatic stellate cells by the three-dimensional structure of the extracellular matrix. J. Gastroenterol. Hepatol. 1998;13:S19–32. [PubMed] [Google Scholar]
  38. Tong J.Z., Bernard O., Alvarez F. Long-term culture of rat liver cell spheroids in hormonally defined media. Exp. Cell Res. 1990;189:87–92. doi: 10.1016/0014-4827(90)90260-H. [DOI] [PubMed] [Google Scholar]
  39. Tzanakakis E.S., Hansen L.K., Hu W.S. The role of actin filaments and microtubules in hepatocyte spheroid self-assembly. Cell Motil. Cytoskel. 2001;48:175–189. doi: 10.1002/1097-0169(200103)48:3<175::AID-CM1007>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  40. Ueno K., Kobayashi A., Takegawa N., Satoh T. Rapid formation of multicellular spheroids composed of Propionibacterium acnes pretreated adult rat liver cells by rotary culture and their immunological properties. Res. Commun. Mol. Pathol. Pharmacol. 1995;90:373–387. [PubMed] [Google Scholar]
  41. Ueno K., Miyashita A., Endoh E., Takezawa T., Yamazaki M., Mori Y., Satoh T. Formation of multicellular spheroids composed of rat hepatocytes. Res. Commun. Chem. Pathol. Pharmacol. 1992;77:107–120. [PubMed] [Google Scholar]
  42. Vogel S., Piantedosi R., Frank J., Lalazar A., Rockey D.C., Friedman S.L., Blaner W.S. An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitro. J. Lipid Res. 2000;41:882–893. [PubMed] [Google Scholar]
  43. Wu F.J., Friend J.R., Hsiao C.C., Zilliox M.J., Ko W.J., Cerra F.B., Hu W.S. Efficient assembly of primary rat hepatocyte spheroids for tissue engineering applications. Biotechnol. Bioeng. 1996;50:404–415. doi: 10.1002/(SICI)1097-0290(19960520)50:4<404::AID-BIT7>3.0.CO;2-P. [DOI] [PubMed] [Google Scholar]
  44. Wu F.J., Friend J.R., Remmel R.P., Cerra F.B., Hu W.S. Enhanced cytochrome P450 lAl activity of self-assembled rat hepatocyte spheroids. Cell Transplantation. 1999;8:233–246. doi: 10.1177/096368979900800304. [DOI] [PubMed] [Google Scholar]
  45. Yagi K., Yamada C., Serada M., Sumiyoshi N., Michibayashi N., Miura Y., Mizoguchi T. Reciprocal regulation of prothrombin secretion and tyrosine aminotransferase induction in hepatocytes. Eur. J. Biochem. 1995;227:753–756. doi: 10.1111/j.1432-1033.1995.tb20198.x. [DOI] [PubMed] [Google Scholar]
  46. Zaret K.S. Liver specification and early morphogenesis. Mech. Dev. 2000;92:83–88. doi: 10.1016/S0925-4773(99)00326-3. [DOI] [PubMed] [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES