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KMID : 0359319960360030551
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Korean Journal of Veterinary Research
1996 Volume.36 No. 3 p.551 ~ p.563
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Functional characterization of primary culture cells grown in hormonally defined , serum - free medium and serum - supplemented medium
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Abstract
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This study investigated the properties of primary cultured proximal tubule cells in hormonally defined(insulin, transferrin, and hydrocortisone), serum-free medium or 10% serum-supplemented medium.
The growth rate of the primary cultured proximal tubule cells was lower in the hormonally defined, serum-free medium than in the 10% serum-supplemented medium(p$lt; 0.05), while the activities of brush border marker enzymes, alkaline phosphatase(AP), leucine aminopeptidase(LAP), and γ-glutamyl transpeptidase(γ-GTP) were increased(p$lt; 0.05). The activities of these enzymes, however, decreased with the lapse of incubation time to 50-70% after 6 days culture compared to those of the freshly-prepared proximal tubules. The enzymatic activities of the primary cultured proximal tubul cells on 6, 9, 12, and 15 days of culture were significantly increased in the hormonally defined, serum-free medium compared to the 10% serum-supplemented medium(p$lt; 0.05). The functional differentiation of the primary culture was examined by observing multicellular domes of the confluent monolayer, which is indicative of transepithelial solute transport. The dome formation by the proximal tubule cultures occurred at a higher frequency in the hormonally defined, serum-free medium than in the 10% serum-supplemented medium(p$lt; 0.05). Upon electron microscopic examination, an increased density of the brush border was observed in the hormonally defined, serum-free medium compared to the cells grown in 10% serum supplemented medium.
The activities of Na^+/glucose cotransporter(^(14)C-a-MG uptake), Na^+/phosphate cotransporter(^(32)P uptake) and Na^+ transporter(^(22)Na^+ uptake) in the brush border membrane, and of Na^+/K^+ -ATPase(^(86)Rb uptake) in the basolateral membrane were significantly stimulated in the hormonally defined, serum-free medium than in 10% serum-supplemented medium(p$lt; 0.05).
In conclusion, the primary cultured proximal tubule cells grown in the hormonally defined, serum-free medium demonstrated a slower growth rate, but the functions of cell were enhanced.
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