Summary
The survival of adult rat hepatocytes in monolayer culture was studied in the presence of different hormones (neurotensin, oxytocin, thyrotropin releasing hormone, luteinizing hormone releasing hormone, cholecalciferol, bradykinin, substance P, aldosterone, melanocyte stimulating hormone, 3,3′,5-triiodo-1-thyronine, corticosterone, human growth hormone, glucagon, insulin, progesterone, testosterone, estradiol, and dexamethasone phosphate) or growth factors (fetal bovine serum). For this purpose trypan blue exclusion, lactate dehydrogenase, and DNA and protein content were measured at 24 and 72 h of culture. 10−7 M Dexamethasone, a mixture of eight hormones, 10% fetal bovine serum, and a combination of the latter two supplements caused a more than 64% higher DNA content at 72 h when compared to control cultures. A striking agreement of these results with changes of lactate dehydrogenase leakage was observed, whereas trypan blue exclusion gave erratic results. Considerable changes of cell arrangement apparently specific for each supplement were ovserved by low magnification microscopy. It is concluded that glucocorticoids and fetal bovine serum have an outstanding effect on cell viability and that DNA or protein content or both are reliable indicators of cell viability in amitotic cultures.
Similar content being viewed by others
References
Asami, O.; Nakamura, T.; Mura, T. et al. Identification of trypsin inhibitor in bovine pituitary extracts as survival factor for adult rat hepatocytes in primary culture. J. Biochem. 95:299–309; 1984.
Barnes, d.; Sato, G. Methods for growth of cultured cells in serum-free medium. Anal. Biochem. 102:255–270; 1980.
Barnes, D.; Sato, G. Serum-free cell culture: an unifying approach. Cell 22:649–657; 1980.
Barth, C. A.; Hillmar, I. Taurocholate inhibits the glucocorticoid-induced rise of 3-hydroxy-3-methylglutaryl-CoA reductase in primary culture of hepatocytes. Eur. J. Biochem. 110:237–240; 1980.
Barth, C. A.; Willershausen, B.; Walther, B. et al. Morphology and metabolism of adult rat hepatocytes in primary culture. Hoppe Seylers Z. Physiol. Chem. 361:1017–1027; 1980.
Bergmeyer, H.-U.; Bernt, E. Lactat-dehydrogenase: UV-test mit pyruvat und NADH. In: Bergmeyer, H.-U., ed. Methoden der enzymatischen analyse, vol. 1. Weinheim: Verlag Chemie; 1970:533–538.
Bischoff, E.; Wilkening, J.; Tran-Thi, T.-A. et al. Differentiation of the nucleotide pyrophosphatases of rat-liver plasma membrane and endoplasmic reticulum by enzymic iodination. Eur. J. Biochem. 62:279–283; 1976.
Enat, R.; Jefferson, D. M.; Ruiz-Opazo, N. et al. Hepatocyte proliferation in vitro: Its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix. Proc. Natl. Acad. Sci. USA 81:1411–1415; 1984.
Gjessing, R.; Seglen, P. O. Adsorption, simple binding and complex binding of rat hepatocytes to various in vitro substrata. Exp. Cell Res. 129:239–249; 1980.
Gospadorowicz, d.; Moran, J. S. Growth factors in mammalian cell culture. Ann. Rev. Biochem. 45:531–558; 1976.
Hasegawa, K.; Namai, K.; Koga, M. Induction of DNA synthesis in adult rat hepatocytes cultured in a serum-free medium. Biochem. Biophys. Res. Comm. 95:243–249; 1980.
Horiuti, Y.; Nakamura, T.; Ichihara, A. Rose of serum in maintenance of functional hepatocytes in primary culture. J. Biochem. 92:1985–1994; 1982.
Jansing, R.; Samsonoff, W. A. Effect of epidermal growth factor on cultured adult rat hepatocytes. Tissue Cell 16:157–166; 1984.
Kissane, J. M.; Ribons, E. J. The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system. J. Biol. Chem. 233:184–188; 1958.
Kleinmann, H. K.; Klebe, R. J.; Martin, G. R. Role of collagenous matrices iin the adhesion and growth of cells. J. Cell Biol. 88:473–485; 1981.
Koch, K. S.; Leffert, H. L. Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation. Cell 18:153–163; 1979.
Laishes, B. A.; Williams, G. M. Conditions affecting primary cell cultures of functional adult rat hepatocytes. 1. The effect of insulin. In Vitro 12:521–532; 1976.
Laishes, B. A.; Williams, G. M. Conditions affecting primary cell cultures of functional adult rat hepatocytes. 2. Dexamethasone enhanced longevity and maintenance of morphology. In Vitro 12:821–832; 1976.
McGowan, J. a.; Strain, A. J.; Bucher, N. L. R. DNA synthesis in primary culturess of adult rat hepatocytes in a defined medium: Effects of epidermal growth factor, insulin, glucagon, and cyclic AMP. J. Cell. Physiol. 108:353–363; 1981.
McGowan, J. A.; Bucher, N. L. R. Pyruvate promotion of DNA synthesis in serum-free primary cultures of adult rat hepatocytes. In vitro 19:159–166; 1983.
Michalopoulos, G.; Pitot, H. C. Primary culture of parenchymal liver cells on collagen membranes. Exp. Cell Res. 94:70–78; 1975.
Nakamura, T.; Asami, O.; Tanaka, K. et al. Increased survival of rat hepatocytes in serum-free medium by inhibition of a trypsin-like protease associated with their plasma membrane. Exp. Cell Res. 154:81–91; 1984.
Reid, L. M.; Roijkind, M. New techniques for culturing differentiated cells: reconstituted basement membrane rafts. Methods Enzymol. 58:263–278; 1979.
Phillips, H. J. Dye exclusion tests for cell viability. In: Kruse, P. F., Patterson, M. K. Jr., eds. Tissue culture methods and application. New York: Academic Press; 1973:406–408.
Richman, R. A.; Claus, T. H.; Pilkis, S. J. et al. Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes. Proc. Natl. Acad. Sci. USA 73:3589–3593; 1976.
Roijkind, M.; Gatmaitan, Z.; Mackensen, S. et al. Connective biomatrix: its isolation and utilization for long term cultures of normal rat hepatocytes. J. Cell Biol. 87:255–263; 1980.
Sato, G.; Reid, L. The replacement of serum in cell culture by hormones. In: Rickenberg, H. V., ed. Biochemistry and mode of action of hormones II. International Review of Biochemistry, vol. 20. Baltimore: University Park Press; 1978:219–251.
Schudt, C. Influence of insulin, glucocorticoids and glucose on glycogen synthase activity in hepatocyte cultures. Biochem. Biophys. Acta 629:499–509; 1980.
Schwarze, P. E.; Seglen, P. O. Protein metabolism and survival of rat hepatocytes in carly culture. Exp. Cell Res. 130:185–190; 1980.
Wanson, J. C.; Bernaert, D.; May, C. Morphology and functional properties of isolated cultured hepatocytes. In: Popper, H.; Schaffner, F., eds. Progress in liver disease, vol. 6. New York: Grune and Stratton: 1979:1–22.
Williams, G. M.; Bermudez, E.; Scaramuzzino, D. Rat hepatocyte primary cell cultures III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium. In Vitro 13:809–817; 1977.
Wilson, E. J.; McMurray, W. C. Effect of hormones on the maintenance and mitochondrial functions of rat hepatocytes cultured in serum-free medium. Can. J. Biochem. Cell Biol. 61:636–643; 1983.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Wirthensohn, K., Barth, C.A. Influence of hormones and growth factors on viability, DNA, and protein content of adult hepatocytes in primary culture. In Vitro Cell Dev Biol 21, 546–552 (1985). https://doi.org/10.1007/BF02620884
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02620884