In vitro differentiated HT 29-D4 clonal cell line generates leakproof and electrically active monolayers when cultured in porous-bottom culture dishes.
HT 29-D4 is a clonal cell line, derived from the human colon adenocarcinoma cell line HT 29, which can be induced to differentiate into enterocyte-like cells by replacing glucose with galactose in the culture medium (Fantini et al. [1986], J. Cell Sci. 83, 235-249). Both undifferentiated and differentiated HT 29-D4 cells have been successfully grown to confluency in Costar Transwell permeable chambers. Only HT 29-D4 cells grown in glucose-free, galactose-containing medium were able to form leakproof monolayers, as demonstrated by their ability to prevent diffusion of serum proteins. These monolayers consist of highly polarized epithelial-like cells with a well organized apical brush border. Transepithelial electrical parameters have been measured under sterile conditions for both types of monolayer. Only HT 29-D4 monolayers cultured in glucose-free, galactose-containing medium were electrically active, with a transepithelial resistance R = 172 +/- 46 omega.cm2, a potential difference PD = 0.35 +/- 0.05 mV, apical negative and a short-circuit current Isc = 2.0 +/- 0.4 microA.cm-2. Apical addition of amphotericin B induced a rapid and considerable increase in Isc and PD, which was abolished by basal ouabain. In contrast, HT 29-D4 cells grown in glucose-containing medium did not generate any potential difference (PD = 0 mV) and their resistance was very low (R = 34.1 +/- 0.9 omega.cm2). It is concluded from these studies that HT 29-D4 cells grown in glucose-free, galactose-containing medium acquire functional characteristics of epithelia, compared to HT 29-D4 cells grown in glucose-containing medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Fantini
J
J
Institut de Chimie Biologique, Université de Provence, Marseille, France.
Verrier
B
B
Marvaldi
J
J
Mauchamp
J
J
D000230:Adenocarcinoma; D000666:Amphotericin B / Q000494:pharmacology; D002454:Cell Differentiation; D002478:Cells, Cultured; D005690:Galactose / Q000494:pharmacology; D006801:Humans; D007365:Intercellular Junctions / Q000502:physiology* / Q000648:ultrastructure; D008564:Membrane Potentials / Q000187:drug effects; D008854:Microscopy, Electron; D014407:Tumor Cells, Cultured / Q000166:cytology* / Q000187:drug effects / Q000502:physiology
D000666:Amphotericin B; D005690:Galactose