Summary
Primary adult rat hepatocytes were cultured in serum-free L15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml epidermal growth factor in a 5% CO2:95% air incubator. The number of cells increased and reached about 180% of the initial value by Day 4, and after 2% dimethyl sulfoxide (DMSO) was added to the culture medium at Day 4, the cells continued to proliferate until Day 6. The number of cells reached about 210% at Day 6 and they were well maintained until Day 18. The cell number gradually decreased with time in culture, but many cells remained for more than 2 mo. On the other hand, without 2% DMSO, the cells proliferated until Day 5, but thereafter they rapidly decreased. After DMSO addition, albumin and transferrin were secreted into the medium and the production of both proteins continued for more than 2 mo. Immunocytochemically both proteins were strongly stained in the cells treated with 2% DMSO. Although the expression of G6Pase in the cells disappeared at Day 6 without DMSO, the cells treated with 2% DMSO recovered G6Pase activity at Day 16. In addition, induction of peroxisomes by 2 mM sodium clofibric acid was clearly shown in the hepatocytes at Day 14 and Day 25 using enzyme-cytochemistry. Ultrastructurally, DMSO-treated hepatocytes had many mitochondria and large peroxisomes with a crystalline nucleoid, and both gap junctions and desmosomes were well developed between the cells even at Day 40. Thus, the number of cells doubled, some differentiated functions of the primary hepatocytes were well restored by the use of 2% DMSO, and these functions were maintained for more than 2 mo.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Baribault, H.; Marceau, N. Dexamethasone and dimethylsulfoxide as distinct regulators of growth and differentiation of cultured suckling rat hepatocytes. J. Cell. Physiol. 129:77–84; 1986.
Bennett, M. V. L.; Spray, D. C., editors. Gap junctions. New York: Cold Spring Harbor Laboratories; 1985.
Bissell, D. M.; Arenson, D. M.; Maher, J. J., et al. Support of cultured hepatocytes by laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver. J. Clin. Invest. 79:801–812; 1987.
Bowen, B.; Steinberg, J.; Laemmli, U. L. K., et al. The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 8:1–20; 1980.
Bucher, N. L. R. Regulation of liver growth. Historical perspective and future directions. In: Rauckman, E. J.; Padilla, G. M., eds. The isolated hepatocyte: use in toxicology and xenobiotic biotransformations. New York: Academic Press; 1987:1–19.
Chan, K.; Kost, D. P.; Michalopoulos, G. Multiple sequential periods of DNA synthesis and quiescence in primary hepatocyte cultures maintained on the DMSO-EGF on/off protocol. J. Cell. Physiol. 141:584–590; 1989.
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.
Friend, C.; Scher, W.; Holland, J. G., et al. Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid differentiation by dimethyl sulfoxide. Proc. Natl. Acad. Sci. USA 68:378–382; 1971.
Furukawa, K.; Mochizuki, Y.; Sawada, N. Properties of peroxisomes and their induction by clofibrate in normal adult rat hepatocytes in primary culture. In Vitro Cell. Dev. Biol. 20:573–584; 1984.
Furukawa, K.; Mochizuki, Y.; Sawada, N., et al. Morphometric and cytochemical evaluation of clofibrate-induced peroxisomal proliferation in adult rat hepatocytes cultured on floating collagen gels. Virchows Arch. B Cell Pathol. 55:279–285; 1988.
Guguen-Guillouzo, C. Role of homotypic and heterotypic cell interactions in expression of specific functions by cultured hepatocytes. In: Guillouzo, A.; Guguen-Guillouzo, C., eds. Research in isolated and cultured hepatocytes. France: John Libbey Eurotext Ltd.; 1986:259–284.
Higgins, P. J.; Borenfreund, E. Enhanced albumin production by malignantly transformed hepatocytes during in vitro exposure to dimethylsulfoxide. Biochim. Biophys. Acta 610:174–180; 1980.
Higgins, P. J.; Darzynkiewicz, Z.; Melamed, M. R. Secretion of albumin and alpha-foetoprotein by dimethylsulphoxide-stimulated hepatocellular carcinoma cells. Br. J. Cancer 48:485–493; 1983.
Inoue, C.; Yamamoto, H.; Nakamura, T., et al. Nicotinamide prolongs survival of primary cultured hepatocytes without involving loss of hepatocyte-specific functions. J. Biol. Chem. 132:363–366; 1989.
Isom, H. C.; Scott, T.; Georgoff, I., et al. Maintenance of differentiated rat hepatocytes in primary culture. Proc. Natl. Acad. Sci. USA 82:3252–3256; 1985.
Isom, H. C.; Georgoff, I.; Salditt-Georgoff, M., et al. Persistence of liver specific messenger RNA in cultured hepatocytes: different regulatory events for different genes. J. Cell Biol. 105:2877–2885; 1987.
Jauregui, H. O.; McMillan, P. N.; Driscoll, J., et al. Attachment and long term survival of adult rat hepatocytes in primary cultures: comparison of different substrata and tissue culture media formulations. In Vitro Cell. Dev. Biol. 22:13–22; 1986.
Koide, N.; Shinji, T.; Tanabe, T., et al. Continued high albumin production by multicellular spheroids of adult rat hepatocytes formed in the presence of liver-derived proteoglycans. Biochem. Biophys. Res. Commun. 161:385–391; 1989.
Kreamer, B. L.; Staecker, J. L.; Sawada, N., et al. Use of a low-speed, isodensity Percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell. Dev. Biol. 22:201–211; 1986.
Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685; 1970.
McGowan, J. A. Hepatocyte proliferation in culture. In: Guillouzo, A.; Guguen-Guillouzo, C., eds. Research in isolated and cultured hepatocytes. France: John Libbey Eurotext Ltd.; 1986:14–38.
McGowan, J. A. Reciprocal regulation of adult rat hepatocyte growth and functional activities in culture by dimethyl sulfoxide. J. Cell. Physiol. 137:497–504; 1988.
Michalopoulos, G. K.; Pitot, H. C. Primary cultures of parenchymal liver cells on collagen membranes: morphological and biological observations. Exp. Cell Res. 94:70–78; 1975.
Michalopoulos, G. K.; Sattler, G. L.; Pitot, H. C. Maintenance of microsomal cytochromes b5 and p-450 in primary cultures of parenchymal liver cells on collagen membranes. Life Sci. 18:1139–1144; 1976.
Michalopoulos, G. K. Liver regeneration: molecular mechanisms of growth control. FASEB J. 4:176–187; 1990.
Michalopoulos, G. K.; Ciancilulli, H. D.; Novotny, A. R., et al. Liver regeneration studies with rat hepatocytes in primary culture. Cancer Res. 42:4673–4682; 1982.
Mitaka, T.; Sattler, C. A.; Sattler, G. L., et al. Multiple cell cycles occur in rat hepatocytes cultured in the presence of nicotinamide and epidermal growth factor. Hepatology 13:21–30; 1991.
Mitaka, T.; Sattler, G. L.; Pitot, H. C. Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum. J. Cell Physiol. 147:495–504; 1991.
Mitaka, T.; Sattler, G. L.; Pitot, H. C. The bicarbonate ion is essential for efficient DNA synthesis by primary cultured rat hepatocytes. In Vitro Cell. Dev. Biol. 27A:549–556; 1991.
Miyazaki, M.; Handa, Y.; Oda, M., et al. Long-term survival of functional hepatocytes from adult rats in the presence of phenobarbital in primary culture. Exp. Cell Res. 159:176–190; 1985.
Nakamura, T.; Yoshimoto, K.; Nakayama, Y., et al. Reciprocal modulation of growth and differentiated functions of mature rat hepatocytes in primary culture by cell-cell contact and cell membrane. Proc. Natl. Acad. Sci. USA 80:7229–7233; 1983.
Paine, A. J.; Hockin, L. J.; Legg, R. F. Relationship between the ability of nicotinamide to maintain nicotinamide adenine dinucleotide in rat liver cell culture and its effect on cytochrome P-450. Biochem. J. 184:461–463; 1979.
Rojkind, M.; Gatmaintan, Z.; Mackensen, S., et al. Connective tissue biomatrix: its isolation and utilization for long-term cultures of normal rat hepatocytes. J. Cell Biol. 87:255–263; 1980.
Seglen, P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 13:29–83; 1976.
Spray, D. C.; Bennett, M. V. L. Physiology and pharmacology of gap junctions. Annu. Rev. Physiol. 47:281–302; 1985.
Spray, D. C.; Fujita, M.; Saez, J. C., et al. Proteoglycans and glycosaminoglycans induce gap junction synthesis and function in primary liver cultures. J. Cell Biol. 105:541–551; 1987.
Staecker, J. L.; Sattler, C. A.; Pitot, H. C. Sodium butyrate preserves aspects of the differentiated phenotype of normal rat hepatocytes in culture. J. Cell Physiol. 135:367–376; 1988.
Towbin, H.; Stachelim, T.; Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350–4354; 1979.
Traub, O.; Druge, P. M.; Willecke, K. Degradation and resynthesis of gap junction protein in plasma membranes of regenerating liver after partial hepatectomy or cholestasis. Proc. Natl. Acad. Sci. USA 80:255–259; 1983.
Wachstein, M.; Meisel, E. On the histochemical demonstration of glucose-6-phosphatase. J. Histochem. Cytochem. 4:592; 1956.
Yancy, S. B.; Easter, D.; Revel, J. P. Cytological changes in gap junctions during liver regeneration. J. Ultrastruct. Res. 67:229–242; 1979.
Yee, A. G.; Revel, J. P. Loss and reappearance of gap junctions in regenerating liver. J. Cell Biol. 78:554–564; 1978.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Mitaka, T., Norioka, KI. & Mochizuki, Y. Redifferentiation of proliferated rat hepatocytes cultured in L15 medium supplemented with EGF and DMSO. In Vitro Cell Dev Biol - Animal 29, 714–722 (1993). https://doi.org/10.1007/BF02631428
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02631428