Support for animal cell culture
Surface study of polycarbonate membranes used as a substratum for animal cell culture
Investigated were the relations between the chem. compn. of a polymer surface and its physicochem. properties. The study of track-etched membranes was done to understand and control better the attachment of epithelial cells for reconstituting a highly differentiated cell tissue. The bisphenol A polycarbonate membrane surface properties were modified by various procedures: sulfochromic acid, nitration, oxygen and ammonia radio frequency (rf) glow discharge, corona discharge. The surface chem. compn. was detd. by XPS and the surface free energy was computed from the contact angle of various liqs. The presence of S- and N-contg. groups bound to the surface was demonstrated after application of the corresponding wet surface treatments. All surface treatments lead to an increase of the O surface concn., the highest value being obtained after nitration. As a result, the polar contribution to the surface free energy also is increased; this seems to be correlated with the surface concn. of O bearing a high electron d. The cell adhesion and growth on the membranes was improved by the surface treatments applied, in particular by O rf glow discharge. However, stream sterilization tends to remove the effects of earlier surface modifications; this seems to be more pronounced when the sample is in direct contact with liq. water.
Iron absorption by intestinal epithelial cells: 1. CaCo2 cells cultivated in serum-free medium, on polyethyleneterephthalate microporous membranes, as an in vitro model
Iron absorption by intestinal epithelial cells, passage onto plasmatic apotransferrin, and regulation of the process remain largely misunderstood. To investigate this problem, we have set up an in vitro model, consisting in CaCo2 cells (a human colon adenocarcinoma line, which upon cultivation displays numerous differentiation criteria of small intestine epithelial cells). Cells are cultivated in a serum-free medium, containing 1 microgram/ml insulin, 1 ng/ml epidermal growth factor, 10 micrograms/ml albumin-linoleic acid, 100 nM hydrocortisone, and 2 nM T3 on new, transparent, Cyclopore polyethyleneterephthalate microporous membranes coated with type I collagen. Cells rapidly adhere, grow, and form confluent monolayers; after 15 days, scanning electron microscopy reveals numerous uniform microvilli. Domes, which develop on nonporous substrata, are absent on high porosity membranes. Culture medium from upper and lower compartments of microplate inserts and cell lysates were immunoprecipitated after labeling with [3H]glucosamine and leucine; analysis was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by autoradiography. [3H]transferrin is found mainly in the lower compartment and in cells; [3H]apolipoprotein B is released in both compartments, and fibronectin almost entirely recovered in the lower compartment; [3H]transferrin receptors and ferritin are only present in cell lysates. Binding experiments also show that transferrin receptors are accessible from the lower compartment. These results suggest that CaCo2 cells, cultivated in synthetic medium on membranes of appropriate porosity, could provide an in vitro model of the intestinal barrier, with the upper compartment of the culture insert corresponding to the apical pole facing the intestinal lumen and the lower one to the basal pole in contact with blood.
Phase I and II biotransformations in living CaCo 2 cells cultivated under serum-free conditions. Selective apical excretion of reaction products
CaCo 2 cells, cultivated in a synthetic, serum-free nutritive medium on poly (ethylene terephthalate) membranes, form a confluent monolayer of differentiated cells, with the apical and basolateral poles exposed to the upper and lower compartments, respectively, of bicameral culture inserts (Halleux and Schneider, In Vitro Cell Dev Biol, 27A: 293-302, 1991). This cell culture system allows the passage of intact mannitol by the paracellular route and the transcellular diffusion of testosterone which appears mainly as a biotransformed unconjugated metabolite. When ethoxyresorufin is added to either the apical or basolateral poles of living CaCo 2 cells, resorufin is formed, and more than 80% is excreted at the apical pole. Under our experimental conditions, no detectable amounts of glucurono- or sulfconjugates are found. Methylcholanthrene and phenobarbital increase the biotransformation of ethoxyresorufin 50 and 3 times, respectively, and induce that of benzoxyresorufin, but not of pentoxyresorufin which remains absent under all conditions. These substances do not affect the proportion of resorufin recovered at the apical role. Verapamil inhibits by 25% the release of resorufin but does not affect its distribution. Chlorodinitrobenzene is conjugated with glutathione and at least two-thirds of the product is excreted at the apical pole; methylcholanthrene and phenobarbital do not increase this activity. These results demonstrate that differentiated CaCo 2 cells, under serum-free conditions, perform phase I and II reactions and that the biotransformation products are selectively excreted at the apical pole.
Iron absorption by CaCo 2 cells cultivated in serum-free medium as in vitro model of the human intestinal epithelial barrier
A cell culture system consisting of confluent monolayer of human enterocyte-like CaCo 2 cells, cultivated in a serum-free nutritive medium, on microporous synthetic membranes has been used as an in vitro model of the intestinal epithelial barrier. The uptake of 55ferric citrate, as well as the transepithelial passage from the apical to the basolateral pole, have been studied. CaCo 2 cells accumulate iron in a time- and concentration-dependent process, largely specific from the apical pole. When 55ferric citrate is added at the apical pole, radioiron appears at the basal pole and the clearance rate is approximately four times higher than in the opposite direction; the amounts of 55Fe increase with the concentration in iron citrate and the duration of incubation. At least two concurrent mechanisms could be involved in iron absorption across monolayers of CaCo 2 cells. A first route would correspond to a paracellular passage of the metal from the apical to the basal pole. The second route would involve a selective intake of iron at the apical pole and could require a reduction of ferric iron, prior to the entry. Iron accumulated by the cells would, for a minor part, be stored within ferritin, whereas the major part would be excreted at the basolateral pole, either as low molecular weight material of undetermined chemical composition but from which iron is easily mobilized by apotransferrin or associated with neosynthesized apotransferrin. Vesicular transport and protein synthesis seem to be required.