Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes

  • Maria Shulman
  • Yaakov NahmiasEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 945)


The liver is the largest internal organ in mammals, serving a wide spectrum of vital functions. Loss of liver function due to drug toxicity or viral infection is a major cause of death in the United States. The development of Bioartificial Liver (BAL) devices and the demand for pharmaceutical and cosmetic toxicity screening require the development of long-term hepatocyte culture techniques. However, primary hepatocytes rapidly lose their cuboidal morphology and liver-specific functions over a few days in culture. Accumulation of stress fibers, loss of metabolic function, and cell death are known phenomena. In recent years, several techniques were developed that can support high levels of liver-specific gene expression, metabolic and synthetic function for several weeks in culture. These include the collagen double-gel configuration, hepatocyte spheroids, coculture with endothelial cells, and micropatterned cocultures with 3T3-J2 fibroblasts. This chapter covers the current status of hepatocyte culture techniques, including: hepatocyte isolation, media formulation, oxygen supply, heterotypic cell–cell interactions, and basic functional assays.

Key words

Liver Hepatocytes Metabolism Oxygen Coculture Culture medium Non-parenchymal cells 



The authors wish to thank Ms. Candice Calhoun for technical advice. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (K01DK080241) and the European Research Council Starting Grant (TMIHCV 242699). Resources were provided by the Silberman Institute of Life Sciences and the Center for Bioengineering in the Service of Humanity.


  1. 1.
    Nahmias Y, Berthiaume F, Yarmush ML (2007) Integration of technologies for hepatic tissue engineering. Adv Biochem Eng Biotechnol 103:309–329PubMedGoogle Scholar
  2. 2.
    Heron M, Hoyert DL, Murphy SL, Xu J, Kochanek KD, Tejada-Vera B (2009) Deaths: final data for 2006. Natl Vital Stat Rep 57:1–134PubMedGoogle Scholar
  3. 3.
    Block GD, Locker J, Bowen WC, Petersen BE, Katyal S, Strom SC, Riley T, Howard TA, Michalopoulos GK (1996) Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium. J Cell Biol 132:1133–1149PubMedCrossRefGoogle Scholar
  4. 4.
    Allen JW, Hassanein T, Bhatia SN (2001) Advances in bioartificial liver devices. Hepatology 34:447–455PubMedCrossRefGoogle Scholar
  5. 5.
    DiMasi JA, Hansen RW, Grabowski HG (2003) The price of innovation: new estimates of drug development costs. J Health Econ 22:151–185PubMedCrossRefGoogle Scholar
  6. 6.
    Pritchard JF, Jurima-Romet M, Reimer ML, Mortimer E, Rolfe B, Cayen MN (2003) Making better drugs: decision gates in non-clinical drug development. Nat Rev Drug Discov 2:542–553PubMedCrossRefGoogle Scholar
  7. 7.
    Rodrigues MA, Gomes DA, Andrade VA, Leite MF, Nathanson MH (2008) Insulin induces calcium signals in the nucleus of rat hepatocytes. Hepatology 48:1621–1631PubMedCrossRefGoogle Scholar
  8. 8.
    Hewitt NJ, Lechon MJ, Houston JB, Hallifax D, Brown HS, Maurel P, Kenna JG, Gustavsson L, Lohmann C, Skonberg C, Guillouzo A, Tuschl G, Li AP, LeCluyse E, Groothuis GM, Hengstler JG (2007) Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev 39:159–234PubMedCrossRefGoogle Scholar
  9. 9.
    Kamiya A, Kinoshita T, Ito Y, Matsui T, Morikawa Y, Senba E, Nakashima K, Taga T, Yoshida K, Kishimoto T, Miyajima A (1999) Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer. EMBO J 18:2127–2136PubMedCrossRefGoogle Scholar
  10. 10.
    Michalopoulos GK, Khan Z (2005) Liver regeneration, growth factors, and amphiregulin. Gastroenterology 128:503–506PubMedCrossRefGoogle Scholar
  11. 11.
    Nahmias Y, Casali M, Barbe L, Berthiaume F, Yarmush ML (2006) Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes. Hepatology 43:257–265PubMedCrossRefGoogle Scholar
  12. 12.
    Richards CD, Brown TJ, Shoyab M, Baumann H, Gauldie J (1992) Recombinant oncostatin M stimulates the production of acute phase proteins in HepG2 cells and rat primary hepatocytes in vitro. J Immunol 148:1731–1736PubMedGoogle Scholar
  13. 13.
    Dunn JC, Tompkins RG, Yarmush ML (1991) Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration. Biotechnol Prog 7:237–245PubMedCrossRefGoogle Scholar
  14. 14.
    Koide N, Shinji T, Tanabe T, Asano K, Kawaguchi M, Sakaguchi K, Koide Y, Mori M, Tsuji T (1989) Continued high albumin production by multicellular spheroids of adult rat hepatocytes formed in the presence of liver-derived proteoglycans. Biochem Biophys Res Commun 161:385–391PubMedCrossRefGoogle Scholar
  15. 15.
    Bhatia SN, Balis UJ, Yarmush ML, Toner M (1999) Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. FASEB J 13:1883–1900PubMedGoogle Scholar
  16. 16.
    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 133:253–260PubMedCrossRefGoogle Scholar
  17. 17.
    Seglen PO (1976) Preparation of isolated rat liver cells. Methods Cell Biol 13:29–83PubMedCrossRefGoogle Scholar
  18. 18.
    Berry MN, Grivell AR, Grivell MB, Phillips JW (1997) Isolated hepatocytes—past, present and future. Cell Biol Toxicol 13:223–233PubMedCrossRefGoogle Scholar
  19. 19.
    Kidambi S, Yarmush RS, Novik E, Chao P, Yarmush ML, Nahmias Y (2009) Oxygen-mediated enhancement of primary hepatocyte metabolism, functional polarization, gene expression, and drug clearance. Proc Natl Acad Sci U S A 106:15714–15719PubMedCrossRefGoogle Scholar
  20. 20.
    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
  21. 21.
    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
  22. 22.
    Taub R (2004) Liver regeneration: from myth to mechanism. Nat Rev Mol Cell Biol 5:836–847PubMedCrossRefGoogle Scholar
  23. 23.
    Michalopoulos GK, DeFrances MC (1997) Liver regeneration. Science 276:60–66PubMedCrossRefGoogle Scholar
  24. 24.
    Yarmush ML, Toner M, Dunn JC, Rotem A, Hubel A, Tompkins RG (1992) Hepatic tissue engineering. Development of critical technologies. Ann N Y Acad Sci 665:238–252PubMedCrossRefGoogle Scholar
  25. 25.
    Bhatia SN, Yarmush ML, Toner M (1997) Controlling cell interactions by micropatterning in co-cultures: hepatocytes and 3 T3 fibroblasts. J Biomed Mater Res 34:189–199PubMedCrossRefGoogle Scholar
  26. 26.
    Kmiec Z (2001) Cooperation of liver cells in health and disease. Adv Anat Embryol Cell Biol 161:III–XIII, 1–151PubMedGoogle Scholar
  27. 27.
    Strain AJ (1999) Ex vivo liver cell morphogenesis: one step nearer to the bioartificial liver? Hepatology 29:288–290PubMedCrossRefGoogle Scholar
  28. 28.
    Khetani SR, Bhatia SN (2008) Microscale culture of human liver cells for drug development. Nat Biotechnol 26:120–126PubMedCrossRefGoogle Scholar
  29. 29.
    Mitaka T, Sato F, Mizuguchi T, Yokono T, Mochizuki Y (1999) Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells. Hepatology 29:111–125PubMedCrossRefGoogle Scholar
  30. 30.
    Mitaka T (2002) Reconstruction of hepatic organoid by hepatic stem cells. J Hepatobiliary Pancreat Surg 9:697–703PubMedCrossRefGoogle Scholar
  31. 31.
    Harada K, Mitaka T, Miyamoto S, Sugimoto S, Ikeda S, Takeda H, Mochizuki Y, Hirata K (2003) Rapid formation of hepatic organoid in collagen sponge by rat small hepatocytes and hepatic nonparenchymal cells. J Hepatol 39:716–723PubMedCrossRefGoogle Scholar
  32. 32.
    Michalopoulos GK, Bowen WC, Mule K, Stolz DB (2001) Histological organization in hepatocyte organoid cultures. Am J Pathol 159:1877–1887PubMedCrossRefGoogle Scholar
  33. 33.
    Goulet F, Normand C, Morin O (1988) Cellular interactions promote tissue-specific function, biomatrix deposition and junctional communication of primary cultured hepatocytes. Hepatology 8:1010–1018PubMedCrossRefGoogle Scholar
  34. 34.
    Morin O, Normand C (1986) Long-term maintenance of hepatocyte functional activity in co-culture: requirements for sinusoidal endothelial cells and dexamethasone. J Cell Physiol 129:103–110PubMedCrossRefGoogle Scholar
  35. 35.
    Nahmias Y, Schwartz RE, Verfaillie CM, Odde DJ (2005) Laser-guided direct writing for three-dimensional tissue engineering. Biotechnol Bioeng 92:129–136PubMedCrossRefGoogle Scholar
  36. 36.
    Milosevic N, Schawalder H, Maier P (1999) Kupffer cell-mediated differential down-regulation of cytochrome P450 metabolism in rat hepatocytes. Eur J Pharmacol 368:75–87PubMedCrossRefGoogle Scholar
  37. 37.
    Allen JW, Khetani SR, Bhatia SN (2005) In vitro zonation and toxicity in a hepatocyte bioreactor. Toxicol Sci 84:110–119PubMedCrossRefGoogle Scholar
  38. 38.
    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 73:379–389PubMedCrossRefGoogle Scholar
  39. 39.
    Arno WT, Harihara B, Partha R, Martin LY, Mehmet T (2001) Effects of oxygenation and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor. Biotechnol Bioeng 73:379–389CrossRefGoogle Scholar
  40. 40.
    Fisher R, Peattie R (2007) Controlling tissue microenvironments: biomimetics, transport phenomena, and reacting systems. Adv Biochem Eng Biotechnol 103:1–73PubMedGoogle Scholar
  41. 41.
    Fariss MW (1990) Oxygen toxicity: unique cytoprotective properties of vitamin E succinate in hepatocytes. Free Radic Biol Med 9:333–343PubMedCrossRefGoogle Scholar
  42. 42.
    Martin H, Sarsat JP, Lerche-Langrand C, Housset C, Balladur P, Toutain H, Albaladejo V (2002) Morphological and biochemical integrity of human liver slices in long-term culture: effects of oxygen tension. Cell Biol Toxicol 18:73–85PubMedCrossRefGoogle Scholar
  43. 43.
    Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS (1986) Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc Natl Acad Sci U S A 83:2496–2500PubMedCrossRefGoogle Scholar
  44. 44.
    Smedsrod B, Pertoft H (1985) Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence. J Leukoc Biol 38:213–230PubMedGoogle Scholar
  45. 45.
    Moghe PV, Berthiaume F, Ezzell RM, Toner M, Tompkins RG, Yarmush ML (1996) Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function. Biomaterials 17:373–385PubMedCrossRefGoogle Scholar
  46. 46.
    Abu-Absi SF, Friend JR, Hansen LK, Hu W-S (2002) Structural polarity and functional bile canaliculi in rat hepatocyte spheroids. Exp Cell Res 274:56–67PubMedCrossRefGoogle Scholar
  47. 47.
    Davidson AJ, Zon LI (2003) Love, honor, and protect (your liver). Science 299:835–837PubMedCrossRefGoogle Scholar
  48. 48.
    LeCouter J, Moritz DR, Li B, Phillips GL, Liang XH, Gerber H-P, Hillan KJ, Ferrara N (2003) Angiogenesis—independent endothelial protection of liver: role of VEGFR-1. Science 299:890–893PubMedCrossRefGoogle Scholar
  49. 49.
    Sugimachi K, Sosef MN, Baust JM, Fowler A, Tompkins RG, Toner M (2004) Long-term function of cryopreserved rat hepatocytes in a coculture system. Cell Transplant 13:187–195PubMedGoogle Scholar
  50. 50.
    Bhandari RN, Riccalton LA, Lewis AL, Fry JR, Hammond AH, Tendler SJ, Shakesheff KM (2001) Liver tissue engineering: a role for co-culture systems in modifying hepatocyte function and viability. Tissue Eng 7:345–357PubMedCrossRefGoogle Scholar
  51. 51.
    Khetani SR, Szulgit G, Rio JAD, Barlow C, Bhatia SN (2004) Exploring interactions between rat hepatocytes and nonparenchymal cells using gene expression profiling. Hepatology 40:545–554PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.The Selim and Rachel Benin School of EngineeringThe Hebrew University of JerusalemJerusalemIsrael

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