Functional characterization of primary culture cells grown in hormonally defined, serum-free medium and serum-supplemented medium

Han, Ho-jae; Kang, Ju-won; Park, Kwon-moo; Lee, Jang-hern; Yang, Il-suk

Korean Journal of Veterinary Research 1996;36(3):551-563.

Ho-jae Han¹, Ju-won Kang¹, Kwon-moo Park¹, Jang-hern Lee², Il-suk Yang²

¹College of Veterinary Medicine, Hormone Research Center, Chonnam National University
²College of Veterinary Medicine, Seoul National University

호르몬 한정배지를 이용한 세포 초대배양계의 확립

한호재¹, 강주원¹, 박권무¹, 이장헌², 양일석²

¹전남대학교 수의과대학, 호르몬연구센터
²서울대학교 수의과대학

Abstract

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 < 0.05), while the activities of brush border marker enzymes, alkaline phosphatase(AP), leucine aminopeptidase(LAP), and y-glutamyl transpeptidase(${gamma}$-GTP) were increased(p < 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 < 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 < 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 cotransportere($^{32}P$ 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 < 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.

Key Words: kidney, cell culture, serum, transporter