Transepithelial transport in cell culture ( renal epithelium ) DAYTON

In cell culture a kidney epithelial cell line, MDCK, forms a continuous sheet of identically oriented asymmetrical cells joined by circumferential occluding junctions. The reconstructed epithelial membrane has transport and permeability qualities of in vivo transporting epithelia. The cell layer can e readily manipulated when cultured on a freely permeable membrane filter and, when placed in an Ussing chamber, electrophysiological measurements can be taken. In the absence of a chemical gradient, the cell layer generates an electrical potential of 1.42 mV, the apical surface negative. It is an effective permeability barrier and lacks significant shunting at the clamped edge, as indicated by a resistance of 84 ohms.cm2, which increased when bulk flow from basolateral to apical was induced by an osmotic gradient or electroosmosis. The MDCK cell layer is cation selective with a relative permeability ratio, PNa/PCI, of 1.7. Net water flux, apical to basolateral, was 7.3 Ml cm2 hr'1 in the absence of a chemical gradient. The morphological and functional qualities of a transporting epithelium are stable in cell culture, and the potentia use of a homogeneous cell nopulation in cell culture would enhance studies of epithelial transport at the cellular and subcellular levels. In 1969 Leighton et al. (1) and Auersperg (2) described the occurrence of domes, turgid, fluid-filled, blister-like hemicysts, in cell cultures of renal and uterine cervix epithelia, respectively. Subsequently, domes have been reported in cell cultures of normal (3) and. neoplastic (4) mouse mammary epithelia, mouse liver (5), human breast adenocarcinoma (6), pig kidney (7), and frog urinary bladder epithelium (unpublished observation). In each instance, the cells cultured were from transporting epithelia. That domes represent a transport phenomenon is suggested by the presence of morpho-logical polarity unique to transporting epithelia (8), apical microvilli extending upward into the medium and occluding junctions joining adjacent cells at the apical-basolateral membrane border. As well, time-lapse photography revealed domes to be localized regions of the cell layer that lift off the culture dish substratum, gradually expand to a maximum, and then rapidly collapse (9). The establishment and characterization of epithelial transport function in cell culture has both experimental and biological significance. The biological question relates to the stability of the differentiated state of transporting epithelia. When dissociated from in situ mesenchymal framework, can differentiated transporting epithelia establish in cell culture characteristic epithelial structure and function? Empirically, the answer rests on the comparison of features of in vivo epithelial morphology, transport, and permeability with the epithelial cell layer formed in culture. MATERIALS AND METHODS Cell Culture. Fibroblasts, a control cell population, were prepared from 16-day mouse embryos and plated at 5 X 105 cells cm-2 (10). The MDCK cell line, isolated from a whole normal adult dog kidney, was provided through the courtesy of Dr. Stewart Madin and maintained in culture by serial passage or as frozen aliquots of cells in complete medium with 10% (vol/vol) dimethylsulfoxide. The MDCK cells were plated at 5 X 105 cells cm-2. The cells were cultured in Waymouth's 752/1 medium (GIBCO) supplemented with penicillin (100 units/ml), streptomycin (100,gg/ml), insulin (26 IU/ml) (11), and 10% (vol/vol) fetal calf serum. Experiments were performed on cultures 4-21 days after plating. Membrane filters, 25 mm (Millipore HAMK 02512), were boiled for 5-10 min to remove the wetting agent and sterilize. The wet filters were affixed to plastic culture dishes by droplets of Millipore Cement Formulation no. 1 applied around the edge to hold the filter flat. The cultured cells were plated directly into the culture dish over the filter. When the cell layer and underlying filter were ready for study, the points of attachment were gently disrupted and the filter and attached cell layer removed. Light and Electron Microscopy. For light microscopy, the cells were fixed and stained with hematoxylin (12). Preparation for transmission and freeze-fracture electron microscopy was as described (13). Solutions. The salt solutions used were Hanks' (14) and Waymouth's 752/1 medium (15). The single salt solutions for dilution potential determinations were calculated as molal concentrations. The isosmotic dilutions were made by diluting the salt solutions with isotonic sucrose, assuming an osmotic coefficient of 1.0 for sucrose. Electrical Measurements. Transepithelial electrical measurements were made with a Lucite Ussing chamber (16). The electrical potential was measured by a Beckman model 76 pH meter used as a potentiometer and recorded on a Beckman model 1005 recorder. The potentiometer was connected to the bathing solutions by calomel half cells (silver/ silver chloride, saturated KCl) with 1 M NaCl-4% agar placed at opposite ends of the Lucite chambers. To measure higher currents accurately, a Simpson multimeter was inserted in series. Periodically throughout all experiments, the symmetry of the electrodes and bridges was checked by placing the agar bridges in the same container with 1 M NaCl solution and noting the absence of a potential. The potentiometer accurately measured potentials of 0.125 mV. Net Water Flux Measurements. For the measurements of net water flux, the chambers were evacuated of air and maintained watertight except for open-ended calibrated capillary tubes (Drummond Microcaps) extending horizontally from the center of each chamber. Water movement 1212 * Present address: Department of Medicine, Veterans Administration Hospital, Palo Alto, Calif. 94304; and Stanford University School of Medicine, Stanford, Calif. 94305. Author to whom requests for reprints should be sent. Proc. Nat. Acad. Sci. USA 73 (1976) 1213 :: ::: . , s . FIGS. 1-4. Fig. 1. MDCK cells grown as a monolayer on a Millipore filter. Hematoxylin stain. X156. Fig. 2. Thin section cut perpendicular to the filter (pore size 5 Mm) and monolayer. The cells send cytoplasmic processes into the pores of the filter basally and bear microvilli on their apical surfaces. Bar equals 5 ,gm. X3100. Fig. 3. A junctional complex linking two cells on the filter includes an occluding junction at the upper end, an adhering junction below this, and after a short distance, a well-developed desmosome. Bar equals 0.1 MAm. X76,000. Fig. 4. Freeze-fracture replica of a 4-gm stretch of an occluding junction linking one cell, which was removed by the fracture, to two others seen here. The fracture plane includes, from the bottom of the picture, the lateral membrane (LM) of a cell at the left, the cytoplasm (C) of a cell at the right, the ridges and grooves of the occluding junction extending across the pictures (arrows), parts of the apical membranes of the two cells (Lu), with microvilli broken off by the fracture, and finally the structureless medium (M). Circled arrow indicates shadowing direction. Bar equals 1 Am. X30,000. was measured as the progression of the meniscus along the capillary tubes. Statistical Methods. The values are expressed as the mean L standard error of the mean followed by the number of observations or samples in brackets. Statistical significance was determined by the unpaired Student's t test.