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epithelia, mouse liver (5), human breast adenocarcinoma (6), pig kidney (7), and frog urinary bladder epithelium (unpublished observation).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 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 w...

Fluid and electrolyte transport by epithelial cells in vitro can be recognized by the ability of cultured cells to form domes and by the electrical properties of monolayer cultures. Pulmonary alveolar epithelial cells are thought to be partially responsible for fluid movement in the fetal lung, but their role in electrolyte transport in the adult lung is not known. We isolated alveolar type II cells from adult rat lung and maintained them on plastic culture dishes alone, on plastic culture dishes coated with an extracellular matrix, and on collagen-coated Millipore filters. Numerous large domes were formed on culture dishes coated with the extracellular matrix; smaller domes were formed on uncoated plastic culture dishes. Sodium butyrate (3 mM) stimulated dome formation. Transmission electron microscopy showed that the epithelial cells had flattened but still retained lamellar inclusions and that the cells were polarized with microvilli on the apical surface facing the culture medium. The electrical properties of the monolayers maintained on collagen-coated Millipore filters were tested in two laboratories. The transepithelial potential differences were 0.7 ± 0.1 mV (24 filters, seven experiments) and 1.3 ± 0.1 mV (13 filters, two experiments) apical side negative, and the corresponding resistances were 217 + 11 ohm.cm2 and 233 ± 12 ohm.cm2. Terbutaline (10 FM) produced a biphasic response with a transient decrease and then a sustained increase in potential difference. Amiloride (0.1 mM) completely abolished the potential difference when it was added to the apical side but not when it was added to the basal side, whereas 1 mM ouabain inhibited the potential difference more effectively from the basal side. Thus, type II cells form a polarized epithelium in culture, and these cells actively transport electrolytes in vitro.The alveolar space of adult mammalian lungs is lined by only a thin film of fluid. Although the amount offluid is not known precisely, one estimate is that 20 ml of alveolar fluid is distributed over a surface area ofabout 70 m2 (1). When lungs are fixed for electron microscopy by vascular perfusion, the only areas that appear to contain fluid are the corners ofthe alveoli, where the radius ofcurvature is short (2). At the corners, the net force of surface tension, which is a function of the radius ofcurvature and the actual surface tension, is directed to draw fluid into the alveolus (3-7). How this force is counterbalanced is not known. Although most investigators have tended to discount the possibility of active transport across alveolar epithelium (7, 8), recent observations in vivo suggest that there may be active transport of fluid from the airspace into the interstitium (9, 10).Cell culture techniques have been used to study transepithelial fluid movement. Epithelial cells that transport fluid form domes or hemicysts in culture (11,12

The pig kidney cell line LLC-PK1 cultured on a collagen coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by occluding junctions. A transepithelial electrical potential (PD) and short-circuit current (SCC) were dependent on the presence of Na and sugar in the apical bathing solution. In the presence of 5.5 mM D-glucose, a PD of 2.8 mV. apical surface negative a SCC of 13 microA cm-2 and transepithelial resistance of 211 ohm.cm2 were recorded. The SCC was promptly reduced by the addition of phlorizin to the apical bath but unaffected when placed in the basolateral bath. The effect on SCC of various sugars was compared by the concentrations required for half-maximal SCC: 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methyl-glucose. When [Na] was reduced, the concentration of D-glucose required for half-maximal SCC increase. Isotopically labeled 3H and 14C D-glucose were used to simultaneously determine bidirectional fluxes; a resultant net apical-to-basolateral transport was present and abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism of transported glucose. The pig kidney cell line, LLC-PK1, provides a cell culture model for the investigation of mechanisms of transepithelial glucose transport.

ABSTRACr The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approximately 6.6 X 105 cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 days showed that the cells accelerated at a uniform rate of 0.24 gm-hr 2 and, by extrapolation, possessed an apparent initial velocity of 2.8 gsmhr at the time of wounding. The apparent initial velocity was considered to be the result of a brief (<10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 105 cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 ;&m'hr-2) Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet. The use of epithelial cultures to study the wounding response exploits the simplicity of working with a monolayer of a single cell type. Although there are drawbacks in attempting to correlate migration on plastic or glass with an in vivo system, it is still possible to make comparisons (1). The following experiments were carried out with the dog kidney epithelial cell line (MDCK), which has the properties of a transporting epithelium (2). It will be shown that the migration of an epithelial sheet can be divided into two stages: first, a brief (<10 hr) rapid acceleration that is dependent on a threshold cell density, and second, movement at a slower constant rate of acceleration, which is considered to result from an aggregate of the individual movements of each migrating cell. MATERIALS The edge of the wound was marked by the razor at its initial placement.For the experiments begun at the maximal cell density, the times of wounding were staggered so that results at various time points could be collected at convenient hours. However, for those experiments carried out at intermediate cell densities, all of the dishes were wounded at the same time (i.e., over a period of 50 min for 45 dishes). During the experiments, the medium was changed daily. Two or three dishes were used for each time point and observations were made every 4 hr in the first 36 hr and every 6 hr up to 120 hr. Cells were fixed in 10% (vol/vol) buffered formalin and were stained with Harris-modified hematoxylin and Giemsa (both from Fischer Scientific, Fair Lawn, NJ).Distances of migration were measured with an ocular graticule by light microscopy. Fifty measurements for each ...