Summary
High yields of human hepatocytes (up to 23×106 viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham’s F12 containing 0.2% bovine serum albumin, 10−8 M insulin, and 10−8 M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250±177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50±0.17 nmol glucose·mg−1·min−1) similar to that reported for human liver. Insulin at 10−8 M activated glycolysis (×1.40) and glycogenesis (×1.34), and glucagon at 10−9 M stimulated gluconeogenesis (×1.35) and glycogenolysis (×2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, α1-antitrypsin, α1-antichymotrypsin, α1-acid glycoprotein, haptoglobin, α2-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10−9 M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol·mg−1 cell protein·min−1, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol·mg−1).
The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.
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Guguen-Guillouzo, C.; Campion, J. P.; Brissot, D., et al.: High yield preparation of isolated human adult hepatocytes by enzymatic perfusion of the liver. Cell. Biol. Int. Rep. 6: 625–628; 1982.
Strom, S. C.; Jirtle, R. L.; Jones, R. S., et al. Isolation, culture, and transplantation of human hepatocytes. J. Natl. Cancer Inst. 68: 771–778; 1982.
Ballet, F.; Bouma, M. E.; Wang, S. R., et al. Isolation, culture and characterization of adult human hepatocytes from surgical liver biopsies. Hepatology 4: 849–854; 1984.
Guguen-Guillouzo, C.; Bourel, M.; Guillouzo, A.. Human hepatocyte cultures. In: Popper, H.; Schaffner, F., eds. Progress in liver diseases, vol. 8. New York: Grune and Stratton; 1986: 33–49.
Feliu, J. E.; Coloma, J.; Gómez-Lechón, M. J., et al. Effect of dexamethasone on isozyme patterns of adult rat liver parenchymal cells in primary culture. Mol. Cell. Biochem. 45: 73–81; 1984.
Gómez-Lechón, M. J.; García, M. D.; Castell, J. V. Effect of glucocorticoids on the expression of GGT and TAT in serum-free cultured hepatocytes. Hoppe-Seyler’s Z. Physiol. Chem. 364: 501–508; 1983.
López, P.; Gómez-Lechón, M. J.; Castell, J. V. Glycogen synthesis in serum-free cultured hepatocytes in response to insulin and dexamethasone. In Vitro 20: 923–931; 1984.
Castell, J. V.; Gómez-Lechón, M. J.; Coloma, J., et al. Preservation of the adult functionality of hepatocytes in serum-free cultures. In: Fisher, G.; Wieser, R., eds. Hormonally defined medium. A tool in cell biology. Berlin: Springer Verlag; 1983: 333–336.
Ichiara, A.; Nakamura, T.; Tanaka, H. Use of hepatocytes in primary culture for biochemical studies on liver functions. Mol. Cell. Biol. Chem. 43: 145–160; 1982.
Guillouzo, A.; Beaune, P.; Gascoin, M. N. Maintenance of cytochrome P-450 in cultured human hepatocytes. Biochem. Pharmacol. 34: 2995–2997; 1985.
Grant, M. G.; Burke, M. D.; Haswksworth, G. M., et al. Human hepatocytes in primary monolayer cultures. Maintenance of mixed function oxidase and conjugation pathways of drug metabolism. Biochem. Pharmacol. 36: 2311–2316; 1986.
Bouma, M. E.; Pessah, G.; Renaud, G., et al. Synthesis and secretion of lipoproteins by human hepatocytes in culture. In Vitro Cel. Dev. Biol. 24: 850–89; 1989.
Gómez-Lechón, M. J.; López, P.; Castell, J. V. Biochemical functionality and recovery of hepatocytes after deep freezing storage. In Vitro. 20: 826–832; 1984.
Andus, T.; Gross, V.; Tran-Thi, T. A., et al. The biosynthesis of acute-phase proteins in primary cultures of rat hepatocytes. Eur. J. Biochem. 133: 561–571; 1983.
Hames, B. D. Detection of radioactive proteins. In: Hames, B. D.; Rickwood, D., eds. Gel electrophoresis of proteins: a practical approach. London: IRL Press Limited. 1981: 50–59.
Castell, J. V.; Montoya, A.; Larrauri, A., et al. Effects of benorylate and impacina on the metabolism of cultured hepatocytes. Xenobiotica 15: 743–749; 1985.
Gutmann, I.; Wahlefeld, A. W.; Determination ofl(+)-lactate with lactate dehydrogenase and NAD. In: Bergmeyer, H., ed. Methods of enzymatic analysis, vol 4. New York: Academic Press. 1974: 1464–1468.
Good, C. A.; Kramer, H.; Somogyi, M. The determination of glycogen. J. Biol. Chem. 100: 485–491; 1933.
Hassid, W. Z.; Abraham, S. Chemical procedures for analysis of polysaccharides. In: Colowick, S. P.; Kaplan, N. O., eds. Methods in enzymology, vol III. New York: Academic Press. 1957: 37–54.
Holme, J. A.; Soderlund, E.; Dybing, E. Drug metabolism activities in isolated rat hepatocytes in monolayer culture. Acta Pharmacol. Toxicol. 52: 348–356; 1983.
Omura, T.; Sato, R. The carbon monoxide-binding pigment of liver microsomes, J. Biol. Chem. 239: 2370–2378; 1964.
Nebert, D. W.; Gelboin, H. V.; Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. J. Biol. Chem. 243: 6242–6249; 1968.
Greenlee, W. F.; Poland, A.. An improved assay of 7-ethoxycoumarin-O-deethylase activity: induction of hepatic enzyme activity in C57BL/GJ and BDA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachorodibenzo-p-dioxin. J. Pharmacol. Exp. Ther. 205: 596–605; 1978.
Hissin, P. J.; Hilf, R. A fluorimetric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 74: 214–226; 1976.
Lowry, O. H.; Rosebrough, N. J.; Farr, A. L., et al. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275; 1951.
Topping, D. L.; Trimble, R. P.; Storer, G. B. O2 dependence of insulin stimulation of glucose uptake by perfused rat liver: effects of carboxyhemoglobin and haematocrit. Horm. Metab. Res. 17: 281–284; 1985.
Sharma, R. J.; Rodrigues, L. M.; Whitton, P. O., et al. Control mechanism in the acceleration of hepatic glycogen degradation during hypoxia. Biochim. Biophys. Acta 630: 414–424; 1980.
Bonney, J. R.; Becker, J. E.; Walker, P. R. et al. Primary monolayer culture of adult rat liver parenchymal cells suitable for study of the regulation of the enzyme synthesis. In Vitro 9: 399–413; 1974.
Seifter, S.; Englard, S. Energy metabolism. In: Arias, I. M.; Popper, H.; Schachter, D., et al., eds. The Liver: Biology and Pathobiology. New York: Raven Press. 1982: 219–231.
Krebs, H. A.; Woods, H. F.; Alberti, K. G. M. Hyperlactemia and lactic acidosis. Essays Med. Biochem. 1: 81–92; 1975.
Munck Petersen, C.; Christiansen, B. S.; Heickendorff, L., et al. Synthesis and secretion of 2-macroglobulin by human hepatocytes in culture. Eur. J. Clin. Invest. 18: 543–548; 1988.
Christiansen, B. S.; Ingerslev, J.; Heickendorff, L., et al. Human hepatocytes in culture synthesize and secrete fibronectin. Scand. J. Clin. Lab. Invest. 48: 685–690; 1988.
Gordon, A. H.; Koj, A., eds. The acute phase response to injury and infection. Elsevier. Amsterdam. New York; 1985.
Meier, K. P.; Talke, H.; Gerok, W., Harnstoffzyklusenzyme und Harnstoffsynthese bei chronischen Lebererkrankungen. Intern Ammoniak Symposium. Wien, May 1977. Aminosauren, Ammoniak und hepatische Encephalopathie. Vol 3. Stiittgart-New York: Gustav Fisher Verlag; 1977: 33–45.
Butterworth, B.; Smith-Oliver, T.; Earle, L., et al. Use of primary cultures of human hepatocytes in toxicological studies. Cancer Res. 49: 1075–1084; 1989.
Le Bot, M. A.; Bégué, J. M.; Kernaleguen, J., et al. Different cytotoxicity and metabolism of doxorubicin, daunorubicin, epirubicin, esorubicin and idarubicin in cultured human and rat hepatocytes. Biochem. Pharmacol. 37: 3877–3887; 1988.
Berthou, F.; Ratanasavanh, D.; Alix, D., et al. Caffeine and theophylline metabolism in newborn and adult human hepatocytes; comparison with adult rat hepatocytes. Biochem. Pharmacol. 37: 3691–3700; 1988.
Bégué, J. M.; Baffet, G.; Campion, J. P., et al. Differential response of primary cultures of human and rat hepatocytes to aflatoxin B1-induced cytotoxicity and protection by the hepatoprotective agent (+)-cyanidanol-3. Biol. Cell 63: 327–333; 1988.
Ratanasavanh, D.; Beaune, P.; Baffet, G., et al. Immunocytochemical evidence for the maintenance of cytochrome P-450 isozymes. NADPH cytochrome C reductase, epoxide hydrolase in pure and mixed primary cultures of adult human hepatocytes. J. Histochem. Cytochem. 4: 527–533; 1986.
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Gómez-Lechón, M.J., López, P., Donato, T. et al. 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). https://doi.org/10.1007/BF02624157
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DOI: https://doi.org/10.1007/BF02624157