Abstract
A long-term primary human hepatocyte culture retraining liver-specific functions is important and essential for basic research and for the future development of hepatocyte-based applications. We established a normal hepatocyte culture system from excess normal tissues obtained from adult liver cancer patients who received partial liver resection. Hepatocytes were isolated after perfusion and enzymatic disaggregation, and were first maintained in hormonally defined media on a Matrigel matrix, and then transferred to collagen sandwich gel. The hepatocytes formed clusters on the Matrigel matrix and increased in size and numbers with time of culture and eventually grew into spheroids of variable sizes. After being transferred to collagen gel, the cells migrated outward from spheroids to form a monolayer with cuboidal or polygonal cell shapes with granular cytoplasm and continued to proliferate. Cellular functions specific for hepatocytes were analyzed using immunoblot assay for proteins specifically secreted by the liver cells on different days of culture. The cells secreted albumin, transferrin and α-fetoprotein consistently for more than 100 days, to a maximum of 150 days. Thus, we have established a long-term culture of hepatocytes from human adults, which will be useful for basic studies of liver physiology such as metabolism and morphogenesis, as well as for other applications in the study of infectious hepatitis, pharmacology, pharmacokinetics, and toxicology.
Similar content being viewed by others
References
Ballet F, Bouma ME, Wang SR, Amit N, Marais J, Infante R. Isolation culture and characterization of adult human hepatocytes from surgical liver biopsies. Hepatology 4:849–854;1984.
Berthiaume F, Moghe PV, Toner M, Yarmush ML. Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration. FASEB J 10:1471–1484;1996.
Bissell DM, Arenson DM, Maher JJ, Roll J. Support of cultured hepatocytes by a lamininrich gel. Evidence for a functionally significant subendothelial matrix in normal liver. J Clin Invest 79:801–812;1987.
Blaauboer BJ, Van Holsteijn CWM, Bleumink R. The effect of beclobric acid and clofibric acid on perixisomal oxidation and perixisome proliferation in primary cultures of rat, monkey and human hepatocytes. Biochem Pharmacol 40:521–528;1990.
Bouma ME, Pesseah M, Renaud G, Amit N, Catala D, Infante R. Synthesis and secretion of lipoproteins by human hepatocytes in culture. In Vitro Cell Dev Biol 24:85–90;1988.
Butterworth BE, Smith-Oliver T, Earle L. Use of primary cultures of human hepatocytes in toxicology studies. Cancer Res 49:1075–84;1989.
Casser-Bette M, Murray AB, Closs EI, Erfle V, Schmidt J. Bone formation by osteoblast-like cells in a three-dimensional cell culture. Calcif Tissue Int 46:46–56;1990.
Clement B, Guguen-Guillouzo C, Campion JP, Glaise D, Bourel M, Guillouzo A. Long-term co-cultures of adult human hepatocytes with rat liver epithelial cells: modulation of albumin secretion and accumulation of extracellular material. Hepatology 4:373–380;1984.
Donato MT, Castell JV, Gomez-Lechon MJ. Co-cultures of hepatocytes with epithelial-like cell lines: expression of drug-biotransformation activities by hepatocytes. Cell Biol Toxicol 7:1–14;1991.
Dunn JCY, Yarmush ML, Koebe HG, Tompkins RG. Hepatocyte function and extracellular matrix geometry: long-term cultured in a sandwich configuration. FASEB J 3:174–177;1989.
Dunn JCY, Tompkins RG, Yarmush ML. Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration. Biotechnol Prog 7:237–245;1991.
Dunn JCY, Tompkins RG, Yarmush ML. Hepatocytes in collagen sandwich configuration: evidence for transcriptional and translational control. J Cell Biol 116:1043–1053;1992.
Enat R, Jefferson DM, Ruiz-Opazo N, Gatmaitan Z, Leinwand LA, Reid M. Hepatocyte proliferation in vitro: its dependence on the use of serum-free hormonally defined medium and substrate of extracellular matrix. Proc Natl Acad Sci USA 81;1411–1415;1984.
Ezzel RM, Toner M, Hendricks K, Dunn JCY, Tompkins RG, Yarmush ML. Effect of collagen gel configuration on the cytoskeleton in cultured rat hepatocytes. Exp Cell Res 208:442–452;1993.
Fraslin JM, Kneip B, Vaulont S, Glaise D, Munnich A, Guguen-Guillouzo C. Dependence of hepatocyte-specific gene expression on cell-cell interactions in primary culture. EMBO J 4:2487–2491;1985.
Gibson-Dambrosio RE, Samuel M, Dambrosio SM. A method for isolating large numbers of viable disaggregated cells from various human tissues for cell culture establishment. In Vitro Cell Dev Biol 22:529–534;1986.
Giuket F, Normand C, Morin O. Cellular interactions promote tissue-specific function, biomatrix deposition and junctional communication of primary cultured hepatocyte. Hepatology 8:1010–1018;1988.
Gleiberman A, Kuprina-Khramkova N, Rudinskaya-Beloshapkina T, Abelev G. α-Fetoprotein synthesis in relation to structural peculiarities in postnatal and regenerating mouse liver. Int J Cancer 32:85–92;1983.
Gomez-Lechon MH, Lopez P, Donato T, Montoya A, Larrauri A, Gimenez P, Trullenque R, Fabra R, Castell JV. Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo. In Vitro Cell Dev Biol 26;67–74;1990.
Gripon P, Fiot C, Theze N, Fourel I, Loreal O, Brechot C, Guguen-Guillouzo C. Hepatitis B virus infection of adult human hepatocyte culture in the presence of dimethyl sulfoxide. J Virol 62:4136–4143;1988.
Guguen-Guillouzo C, Campion JP, Brissot P, Glaise D, Launois B, Bourel M, Guillouzo A. High yield preparation of the liver. Cell Biol Int Rep 6:625–628;1982.
Guguen-Guillouzo C, Clement B, Baffet G, Beaumont C, Morel-Chany 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 143:47–54;1983.
Guillouzo A, Begue JM, Campion JP, Gascoin MN, Guguen-Guillouzo C. Human hepatocytes cultures: a model for pharmacotoxicological studies. Xenobiotica 15:635–641;1985.
Hsu IC, Lipsky MM, Cole KE, Su CH, Trump BF. Isolation and culture of hepatocytes from human liver of immediate autopsy. In Vitro Cell Dev Biol 21:154–160;1985.
Kono Y, Yang S, Letarte M, Roberts E. Establishment of a human hepatocyte line derived from primary culture in a collagen gel sandwich culture system. Exp Cell Res 221:478–485;1995.
Kono Y, Roberts EA. Modulation of the expression of liver-specific functions in novel human hepatocyte lines cultured in a collagen gel sandwich configuration. Biochem Biophys Res Commun 220:628–632;1996.
Kuhlmann WD. Immunoperoxidase labelling of α-fetoprotein (AFP) in normal and regenerating livers of a low and high AFP producing mouse strain. Histochemistry 64:67–75;1979.
Laemmli EK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685;1970.
Landry J, Bernier D, Ouellet C, Goyette R, Marceau N. Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of function activities. J Cell Biol 101;914–923;1985.
Lanford R, Carey KD, Estlack L, Smith GC, Hay RV. Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum free medium. In Vitro Cell Dev Biol 25:174–182;1989.
Lin Y, Jin DK, Vacher J, Feuerman MH. Sequence requirements for alpha-fetoprotein gene expression during liver regeneration. Cell Growth Differ 6:1549–1558;1995.
Maslanski CH, Williams GM. Primary culture and the level of cytochrome p450 in hepatocytes from mouse, rat, hamster and rabbit liver. In Vitro 18:683–639;1982.
Moghe PV, Berthiaume F, Ezzell RM, Toner M, Tompkins RG, Yarmush ML. Role of extracellular matrix composition and configuration in maintenance of hepatocyte polarity and function. Biomaterials 17:373–385;1996.
Niwa Y, Matsumura M, Shiratori Y, Imamura M, Kato N, Shina S, Okudaira T, Ikeda Y, Inoue T, Omata M. Quantitation of α-fetoprotein and albumin messenger RNAs in human hepatocellular carcinoma. Hepatology 23:1384–1392;1996.
Pasco DS, Boyum KW, Merchant SN, Chalberg SC, Fagan JB. Transcriptional and post-transcriptional regulation of the genes encoding cytochromes p-450c and p-450d in vivo and in primary hepatocyte cultures. J Biol Chem 263:8671–8676;1988.
Petropoulos C, Andrews G, Tamaoki T, Fausto N. Alpha-fetoprotein and albumin mRNA levels in liver regeneration and carcinogenesis. J Biol Chem 258:4901–4906;1983.
Reid LM, Jefferson DM. Culturing hepatocytes and other differentiated cells. Hepatology 4:548–559;1984.
Roberts EA, Letarte M, Squire J, Yang S. Characterization of human hepatocyte lines derived from normal liver tissue. Hepatology 19:1390–1399;1994.
Rojkind M, Gatmaitan Z, Mackensen S, Giambrone MA, Ponce P, Reif LM. Connective tissue biomatrix: its isolation and utilization for long-term culture of normal rat hepatocytes. J Cell Biol 87:255–263;1980.
Ruoslahti E, Seppala M. α-Fetoprotein in cancer and fetal development. Adv Cancer Res 29:275–346;1979.
Schuetz EG, Li D, Omiecinski CJ, Muller-Eberhard U, Kleinmam HK, Elswick B, Guzelian PS. Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix. J Cell Physiol 134:309–23;1988.
Seglen PO. Preparation of rat liver cells. II. Effects of ions and chelators on tissue dispersion. Exp Cell Res 76:25–30;1973.
Strom SC, Jirtle RL, Jones RS, Novicki DL, Rosenberg MR, Novotny A, Irons G. Isolation, culture and transplantation of human hepatocytes. J Natl Cancer Inst 68:771–778;1982.
Tateno C, Yoshizato K. Long-term cultivation of adult rat hepatocytes that undergo multiple cell divisions and express normal parenchymal phenotypes. Am J Pathol 148:383–392;1996.
Tong JZ, Bernard O, Alvarez F. Long-term culture of rat liver cell spheroids in hormaonally defined media. Exp Cell Res 189:87–92;1990.
Tong JZ, Sarrazin S, Cassio D, Gauthier F, Alvarez F. Application of spheroid culture to human hepatocytes and maintenance of their differentiation. Biol Cell 81:77–81;1994.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Chen, HL., Wu, HL., Fon, CC. et al. Long-term culture of hepatocytes from human adults. J Biomed Sci 5, 435–440 (1998). https://doi.org/10.1007/BF02255932
Issue Date:
DOI: https://doi.org/10.1007/BF02255932