Proc. Nat. Acad. Sci. USAVol. 73, No. 4, pp. 1212-1216, April 1976Cell Biology

Transepithelial transport in cell culture(renal epithelium)

DAYTON S. MISFELDT*, SUSAN T. HAMAMOTO, AND DOROTHY R. PITELKADepartment of Zoology and Cancer Research Laboratory, University of California, Berkeley, Calif. 94720

Communicated by Howard A. Bern, December 18,1975

ABSTRACT In cell culture a kidney epithelial cell line,MDCK, forms a continuous sheet of identically orientedasymmetrical cells joined by circumferential occluding junc-tions. The reconstructed epithelial membrane has transportand permeability qualities of in vivo transporting epithelia.The cell layer can e readily manipulated when cultured ona freely permeable membrane filter and, when placed in anUssing chamber, electrophysiological measurements can betaken. In the absence of a chemical gradient, the cell layergenerates an electrical potential of 1.42 mV, the apical sur-face negative. It is an effective permeability barrier andlacks significant shunting at the clamped edge, as indicatedby a resistance of 84 ohms.cm2, which increased when bulkflow from basolateral to apical was induced by an osmoticgradient or electroosmosis. The MDCK cell layer is cation se-lective with a relative permeability ratio, PNa/PCI, of 1.7. Netwater flux, apical to basolateral, was 7.3 Ml cm2 hr'1 in theabsence of a chemical gradient. The morphological and func-tional qualities of a transporting epithelium are stable in cellculture, and the potentia use of a homogeneous cell nopula-tion in cell culture would enhance studies of epithelial trans-port at the cellular and subcellular levels.

In 1969 Leighton et al. (1) and Auersperg (2) described theoccurrence of domes, turgid, fluid-filled, blister-like hemi-cysts, in cell cultures of renal and uterine cervix epithelia,respectively. Subsequently, domes have been reported in cellcultures of normal (3) and. neoplastic (4) mouse mammaryepithelia, mouse liver (5), human breast adenocarcinoma (6),pig kidney (7), and frog urinary bladder epithelium (unpub-lished observation). In each instance, the cells cultured werefrom transporting epithelia. That domes represent a trans-port phenomenon is suggested by the presence of morpho--logical polarity unique to transporting epithelia (8), apicalmicrovilli extending upward into the medium and occludingjunctions joining adjacent cells at the apical-basolateralmembrane border. As well, time-lapse photography revealeddomes to be localized regions of the cell layer that lift off theculture dish substratum, gradually expand to a maximum,and then rapidly collapse (9). The establishment and charac-terization of epithelial transport function in cell culture hasboth experimental and biological significance.The biological question relates to the stability of the dif-

ferentiated state of transporting epithelia. When dissociatedfrom in situ mesenchymal framework, can differentiatedtransporting epithelia establish in cell culture characteristicepithelial structure and function? Empirically, the answerrests on the comparison of features of in vivo epithelial mor-phology, transport, and permeability with the epithelial celllayer formed in culture.

MATERIALS AND METHODSCell Culture. Fibroblasts, a control cell population, were

prepared from 16-day mouse embryos and plated at 5 X 105cells cm-2 (10). The MDCK cell line, isolated from a wholenormal adult dog kidney, was provided through the courtesyof Dr. Stewart Madin and maintained in culture by serialpassage or as frozen aliquots of cells in complete mediumwith 10% (vol/vol) dimethylsulfoxide. The MDCK cellswere 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 oncultures 4-21 days after plating. Membrane filters, 25 mm(Millipore HAMK 02512), were boiled for 5-10 min to re-move the wetting agent and sterilize. The wet filters wereaffixed to plastic culture dishes by droplets of Millipore Ce-ment Formulation no. 1 applied around the edge to hold thefilter flat. The cultured cells were plated directly into theculture dish over the filter. When the cell layer and underly-ing filter were ready for study, the points of attachmentwere gently disrupted and the filter and attached cell layerremoved.

Light and Electron Microscopy. For light microscopy,the cells were fixed and stained with hematoxylin (12).Preparation for transmission and freeze-fracture electronmicroscopy was as described (13).

Solutions. The salt solutions used were Hanks' (14) andWaymouth's 752/1 medium (15). The single salt solutionsfor dilution potential determinations were calculated asmolal concentrations. The isosmotic dilutions were made bydiluting the salt solutions with isotonic sucrose, assuming anosmotic coefficient of 1.0 for sucrose.

Electrical Measurements. Transepithelial electrical mea-surements were made with a Lucite Ussing chamber (16).The electrical potential was measured by a Beckman model76 pH meter used as a potentiometer and recorded on aBeckman model 1005 recorder. The potentiometer was con-nected to the bathing solutions by calomel half cells (silver/silver chloride, saturated KCl) with 1 M NaCl-4% agarplaced at opposite ends of the Lucite chambers. To measurehigher currents accurately, a Simpson multimeter was in-serted in series. Periodically throughout all experiments, thesymmetry of the electrodes and bridges was checked byplacing the agar bridges in the same container with 1 MNaCl solution and noting the absence of a potential. The po-tentiometer accurately measured potentials of 0.125 mV.

Net Water Flux Measurements. For the measurements ofnet water flux, the chambers were evacuated of air andmaintained watertight except for open-ended calibratedcapillary tubes (Drummond Microcaps) extending horizon-tally from the center of each chamber. Water movement

1212

* Present address: Department of Medicine, Veterans Administra-tion Hospital, Palo Alto, Calif. 94304; and Stanford UniversitySchool of Medicine, Stanford, Calif. 94305. Author to whom re-quests for reprints should be sent.

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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 perpen-dicular to the filter (pore size 5 Mm) and monolayer. The cells send cytoplasmic processes into the pores of the filter basally and bear micro-villi on their apical surfaces. Bar equals 5 ,gm. X3100. Fig. 3. A junctional complex linking two cells on the filter includes an occludingjunction 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, totwo others seen here. The fracture plane includes, from the bottom of the picture, the lateral membrane (LM) of a cell at the left, the cyto-plasm (C) of a cell at the right, the ridges and grooves of the occluding junction extending across the pictures (arrows), parts of the apicalmembranes of the two cells (Lu), with microvilli broken off by the fracture, and finally the structureless medium (M). Circled arrow indi-cates shadowing direction. Bar equals 1 Am. X30,000.

was measured as the progression of the meniscus along thecapillary tubes.

Statistical Methods. The values are expressed as themean L standard error of the mean followed by the numberof observations or samples in brackets. Statistical significancewas determined by the unpaired Student's t test.

RESULTSMorphology. Confluent, polygonal MDCK cells appear as

a monolayer on a membrane filter (Fig. 1) without nuclearoverlap. Thin sections perpendicular to the culture plane(Figs. 2 and 3) confirm that the cells form a monolayer witha morphological asymmetry, the apical surface studded with

microvilli extending upward into the medium and the baso-lateral surface interdigitating with the membrane filter. In-tercellular occluding junctions are consistently present at theborder between the apical and basolateral surfaces (Fig. 3).The nuclei and cellular organelles are not asymmetricallydistributed within the cells. Freeze-fracture preparationsdemonstrate the characteristic anastomosing network ofstrands that form the occluding junctions (Fig. 4). No base-ment membrane or mesenchymal stroma is present; the mo-nolayer appears to be formed entirely of epithelial cells.

Transepithelial Electrical Potential. When the cell layeron the membrane filter was clamped between halves of aLucite Ussing chamber and bathed with identical solutions,

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20to0

0

.0

0

't 10

E= 5 -

z F

mV

nFH. . . . . . . * * * * * * * . * * * w

8 7 6 5 4 3 2 1 0 -1-2-3-4-5-6-7-8-9-10millivolts

FIG. 5. Frequency distribution of transepithelial electrical po-tential across the MDCK cell layer.

complete medium without serum or Hanks' balanced salt so-lution, spontaneous electrical potentials were recorded, asrepresented by a histogram (Fig. 5). The mean value was1.42 I 0.260 [140] mV with the apical surface negative. Themaximum potential was present initially with a gradual de-crease (over 3-10 min) of about 25% to a stable value whenthe readings were taken. The magnitude and sign of the po-tential remained identical when the agar bridges in thebathing media were exchanged. When the cell layer was re-versed in the chamber, the sign also reversed, but the poten-tial was usually lower, probably due to some damage by themanipulation. The potential dropped to zero when the celllayer and filter were intentionally punctured. Blank mem-brane filters treated in exactly the same fashion gave noelectrical potential [12]. Also, when mouse embryo fibro-blasts were cultured on a membrane filter, no electrical po-tential was detected [15].

Transepithelial Electrical Resistance. The transepithel-ial electrical resistance, as calculated by Ohm's Law fromthe linear relation of external current passed through the po-tential drop and recorded across the cell layer, was 83.7 I1.2 [175] ohm-cm2. The blank membrane filter or mem-brane filter with a fibroblast cell layer registered a resistancemuch less, 6.25 i 0.70 [22] ohm-cm2, which is the same asthe resistance of the fluid-filled chamber, bridges, and elec-trodes when no filter was present. The transepithelial resis-tance measurements were taken when the electrical poten-tial was stable. The electrophysiological measurements weremade at room temperature, 22-25°; however, when the Uss-ing chamber and bathing solutions were maintained at 370,the transepithelial potential was 1.57 I 0.82 [8] mV and theresistance 61 + 10 [14] ohm-cm2.

Transepithelial Net Water Flux. Water in the absence ofan osmotic gradient flowed in an apical-to-basolateral direc-tion and was measured at a rate of 7.2 k 3.5 [15] ,Al cm-2hr-'. There was no net flux of water across the blank filteror fibroblast-covered filter or in the absence of any separa-tion of the chambers. Water flux measurements were madeat 370.

Metabolic Inhibitors. MDCK cells exposed to 1 mM io-doacetate became refractile and separated from adjacentcells within 90 min; after 4 hr the cells were floating in themedium. The transepithelial potential and net water trans-port dropped to zero within 20 min [18] and the resistance tozero by 45 min [11]. In contrast, the addition of potassiumcyanide, 1 mM final concentration, had no effect on the ap-pearance, transepithelial potential, or resistance after 2 hr ofexposure [10].

V - RT (TNa -TCI)F

In [NaCI] a[NaCI] bl

y -b + mx

'mmRT (T TFT Na-TCI)1 - TNa + TCIb - 0.83m - 15.46r = .999

TNa -.63, TCI -.37

TNa/TCI = PNa/pC, - 1.7

10

log [NaCI]a[NaCI ] bi

30

FIG. 6. Graphic representation of the magnitude of transepi-thelial electrical potential change as a function of the sodium chlo-ride concentration gradient. The slope represents the differencebetween TNa and Tci. Direction of gradient is from. apical to baso-lateral side of cell layer.

Dilution Potentials. The electrical potential arising froma transepithelial concentration gradient indicates that thereis a permeability difference between sodium and chloride.The transference number, the relative amount of currentcarried by each ion, provides a quantitative estimate of per-meability; determinations were made considering the mem-brane separating the different concentrations as a liquidjunction. Sodium chloride solutions of 150, 75, 15, and 5 mMwere prepared by dilution of a 150 mM NaCl solution with a300 mM sucrose solution so that no osmotic gradient waspresent. The electrical potential plotted against the loga-rithm of the concentration gradient, a graphic representa-tion of the Nernst equation, allows determination of TNa andToi from the slope of the relationship. Observations were re-corded on ten different filter preparations of cell layermembranes. The mean potentials at each concentration gra-dient were plotted against the gradient, and the slope wasdetermined by a least squares regression analysis. Thegraphic representation, correlation coefficient, and calcula-tions are summarized on Fig. 6, for an apical-to-basolateralgradient.

Osmotically Induced Streaming Potentials. Bulk flow

-2

mV

-1

+100 +50

1[14] +1i [13]

i [17]

j[17]

-50 -100

+2L

[mOsm]a-[m sm]bl

FIG. 7. Graphic representation of the magnitude of transepi-thelial "streaming potentials" resulting from bulk water flow in-duced by an osmotic gradient. Plotted are the mean values; theSEM is indicated by the vertical line and the number of observa-tions in brackets. Each value differs from the other P < 0.05.There is increased resistance when the bulk flow is from basolater-al (bl) to apical (a) as discussed in the text.

Proc. Nat. Acad. Sci. USA 73 (1976)

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Proc. Nat. Acad. Sci. USA 73 (1976) 1215

FIG. 8. Graphic representation of typical change in electricalpotential with the continuous passage of a measured current. Be-tween measurements the current was increased and the potentialallowed to stabilize over 10-15 sec. The line through zero providesa reference for the constant linear relation of potential to currentwhen water flow in an apical-to-basolateral direction was induced.In contrast, water flow in a basolateral-to-apical direction pro-

duces an increasing transepithelial resistance and a nonlinear rela-tion with current over 800,MA.

across the MDCK epithelial layer was created by the addi-tion of sucrose to the complete medium without serum tocreate gradients of 50 and 100 mOsm. The reflection coeffi-cient for sucrose was assumed to be 1.0 for this cell layer. Os-motic gradients produced streaming potentials of -0.0027mV/mOsm with a 6% average decrease in resistance whenflow was from apical to basolateral and +0.0012 mV/mOsmwith a 45% average increase in resistance when the flow was

from basolateral to apical. The potential present with no os-

motic gradient was subtracted and the absolute change rep-

resented graphically in Fig. 7.By electroosmosis, current passed across the cell layer in-

duced water flow toward the surface which became nega-

tively charged. As continuous current was increased in a

stepwise fashion, the basolateral surface negative, transepi-thelial potential increased linearly. In contrast, currentpassed in the opposite direction, apical surface negative andwater flow in the basolateral-to-apical direction, was associ-ated with a nonlinear increase in potential when currentdensities exceeded 800 AA, about 250,MA cm-2 (Fig. 8).

DISCUSSIONWhen the epithelial cell layer is bathed on both surfaces byidentical solutions, the presence of an electrical potential in-dicates that the cell layer can effect a separation of opposite-ly charged ions. The magnitude of the potential is a functionnot only of the rate of ion transport, but also of the relativepermeability to the oppositely charged ions (17). In general,the rate of ion transport in "leaky" low-potential and "tight"high-potential epithelia is not markedly different (18). Con-sidering the high resistance of cellular membranes and therelatively low resistance, 84 ohmcm2, of the MDCK epithe-lial sheet, the paracellular shunt may attenuate the transepi-thelial potential many-fold (19). Rather than the magnitude,it is the existence of an electrical potential and its sign whichare significant, although they do not indicate the mecha-nism, rate, direction, or ions transported. Net water flux byvertebrate epithelia is presumptive evidence of net solutetransport. The positive charge on the basolateral side towardwhich water was transported suggests that net sodium trans-

port may be responsible for water transport. However, theMDCK cell layer is cation selective and bulk flow of waterin an apical-to-basolateral direction, whatever the mecha-nism, will generate a streaming potential positive on the ba-solateral side of the cell layer. It is not possible to state towhat extent active solute transport is responsible for thetransepithelial potential.

Both the electrical potential and net water flux fell to zeroin the presence of 1 mM iodoacetate, an inhibitor of glycol-ysis (20); however, potassium cyanide (1 mM), an inhibitorof ATP generation by mitochondrial electron transport inaerobic metabolism (21), had no effect on the electrical po-tential. The MDCK cells in culture are in this respect verysimilar to the anaerobically adapted fresh water turtle,which is highly resistant to cyanide (22) and can maintainsodium transport across the urinary bladder during anaero-biosis and cyanide exposure (23). Indeed, a linear relationwas noted between lactate formation and net aerobic sodiumtransport (24). The anaerobic nature of the metabolism ofcells in culture is documented (25-27), and the resistance ofMDCK to cyanide exposure would suggest that this cell line,long adapted for growth in culture, is also highly anaerobic.

Resistance to the flow of ions is a measure of passive per-meability and a sensitive measure of the integrity of the epi-thelial cell layer. The resistance across the MDCK cell sheetwas 84 ohm-cm2, which places it among the "leaky" epithel-ia (18). -For other mammalian renal epithelium, resistancevalues of 4-6 ohm'cm2 in the proximal tubule (28, 29),350-600 ohm-cm2 in the distal tubule (28, 30), and 1200ohm-cm2 in the collecting duct (31, 32) have been obtained.

Electrical potential arising from a NaCl concentrationgradient across an MDCK epithelial layer indicates the rela-tive membrane permeability to sodium and chloride. Themembrane side of lower chemical potential became electri-cally positive, indicating that sodium was permeating morereadily than chloride. Permeability of MDCK cell layers toNaCl, as for some mammalian proximal tubules, is sodium-selective (28, 29) despite the greater free-solution mobility ofchloride (33). The permeability ratio PNa/PCG of 1.7 in thebasolateral-to-apical gradient is comparable to 1.38 in thedog proximal tubules (28) and 1.58 in the rat proximal tu-bule (34).Water flow, whether caused by hydrostatic or osmotic

pressure, may create an electrical potential by the establish-ment of a diffusion potential secondary to solute concentra-tion changes in unstirred layers which generate boundarydiffusion potentials. Water flow tends to concentrate solutesin the unstirred boundary layer on the membrane side fromwhich the water is flowing and to dilute the solutes in theunstirred layer on the opposite side (35). A diffusion poten-tial results from the differences in the transference numbersof the ions as they permeate the membrane; Barry and Hopehave termed this the "transport number effect" (36, 27). Forthe MDCK epithelial membrane, as with a variety of otherepithelia (38-41), the side of the membrane bathed by thehyperosmolar solution became positive, indicating a cationselective route of water permeation.

Osmotically induced bulk water flow in an apical-to-baso-lateral direction increased transepithelial resistance by 45%,whereas in the opposite direction the resistance change wasbut 6%. Similar qualitative changes in resistance and stream-ing potential are reported in dog proximal tubules (28), ratproximal tubules (42), and rabbit gall bladder (43).

Current that renders the apical surface negative across acation-selective epithelium will result in a solute depletion at

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1216 Cell Biology: Misfeldt et al.

the basolateral surface and a basolateral-to-apical osmoticwater flow. With increased current, osmotic flow increasesand an increase in transepithelial electrical resistance results(Fig. 8); current-induced water flow apical-to-basolateralhas no effect on resistance as with the osmotically inducedwater flow. Smulders et al. observed similar current-inducedresistance changes in rabbit gall bladder (43).The study of epithelial transport in cell culture allows the

exploitation of several technical advantages. The epithelialcell layer in culture is the least complex epithelium possible,a single cell thick without stromal elements or basementmembrane. Culturing the epithelial cell layer on a strong yetfreely permeable membrane substratum allows easy han-dling and adaptability to the standard techniques of epithe-hal transport study. The relative durability of cells in culturein comparison to other preparation of mammalian epithelialtissues in vitro permits experiments of longer duration. Thedisruption of tissue architecture to isolate cells for culture al-lows the formation of a sheet of transporting epitheliumfrom epithelial tissues that do not exist in situ as convenientsheets, tubes, or sacks. Potentially the most significant tech-nical advantage of studying epithelial transport in cell cul-ture is the use of homogeneous cell populations by enrich-ment of primary isolated cells prior to culture or by cloningcell lines.

These experiments indicate that epithelial cells dissociatedand placed in cell culture reform two-sided asymmetricalsheets with distinct apical and basolateral surfaces. A contin-uous sheet is formed by circumferential intercellular occlud-ing junctions with cation-selective permeability characteris-tics and an electrical resistance of 84 ohm cm2. In the ab-

sence of a chemical gradient, the cell layer produces an elec-trical potential of 1.4 mV with the apical surface negativeand vectorially transports water 7.3 gl hr-1 cm2 apical-to-basolateral; both activities are eliminated by iodoacetate, a

metabolic inhibitor.

We gratefully acknowledge our indebtedness to Dr. DanielFriend, University of California, San Francisco, for use of freeze-fracture equipment, and to John Underhill for technical assistance.The work was supported by U.S. Public Health Service Grants CA-05388 and by Research Fellowship CA-00639 to D.S.M.

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