Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum.
Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum-free, modified L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L-15 medium differed from that in DMEM/F12 medium in that there were four peaks of 3H-thymidine incorporation in the L-15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48-72 h and 72-96 h after plating at densities between 1.5 x 10(5) and 6.0 x 10(5) cells per 35-mm dish. Even at a density of 9.0 x 10(5) cells/dish, about 40% of the cell nuclei were stained with BrdU in the periods of 48-72 h and 72-96 h. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleate with a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEM/F12 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L-15 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.
Mitaka
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McArdle Laboratory for Cancer Research, Department of Oncology, Medical School, University of Wisconsin, Madison 53706.
Sattler
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Pitot
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D000418:Albumins / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis / Q000494:pharmacology*; D000818:Animals; D001639:Bicarbonates / Q000032:analysis / Q000494:pharmacology; D001973:Bromodeoxyuridine; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media / Q000032:analysis / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000032:analysis / Q000494:pharmacology; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000032:analysis / Q000494:pharmacology; D017693:Sodium Bicarbonate; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors; D014316:Tritium
D000418:Albumins; D000596:Amino Acids; D001639:Bicarbonates; D003470:Culture Media; D014316:Tritium; D004815:Epidermal Growth Factor; D017693:Sodium Bicarbonate; D004247:DNA; D012964:Sodium; D001973:Bromodeoxyuridine; D013936:Thymidine