Amino acid‐rich medium (Leibovitz L‐15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum

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Abstract
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 × 105 and 6.0 × 105 cells per 35‐mm dish. Even at a density of 9.0 × 105 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.

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