Functionally polarized layers formed by epidermal cells on a permeable

transparent collagen film

KATSUTOSHI YOSHIZATO*, AKIO NISHIKAWA and TOSHIO TAIRAf

Developmental Biology Laboratory, Department of Biology, Faculty of Science, Tokyo Metropolitan University, Fukazawa 2-1-1, Setagaya-ku,Tokyo 158, Japan

• Author for correspondence•(•Present address: Koken Bioscience Institute, Nakane 2-11-21, Meguro-ku, Tokyo 152, Japan

Summary

Rat epidermal cells were cultured on a transparentcollagen film, which was permeable to low Mr

substances. Then the cells were bathed in bothmedia (apical and basal), 'which 'were separated bythe collagen membrane. The cells formed a multi-layered epidermal sheet with well-developed struc-tures of desmosomes. This sheet on a permeablesupport was found to be an effective permeabilitybarrier for glucose and amino acids. The epidermallayer showed functional polarity for the uptake andexcretion of nutrients, metabolites and newly syn-thesized proteins: glucose and amino acids weretaken up exclusively from the basal medium andlactate was secreted selectively into the same

medium, whereas ammonia was secreted into theapical medium. The apical media became moreacidic than the basal ones, presumably due to thepreferential distribution of H+ transport systems onthe apical side of the epidermal layer. The epider-mal cells that expressed functional polarities invitro as described above were able to proliferateand differentiate, and remained healthy for as longas at least 40 days even using a conventional culturemedium with foetal calf serum, but without anyspecial growth factors and feeder cells.

Key words: epidermal cells, collagen, glucose transport, H +

transport, transport polarity.

Introduction

Generally, epithelial cells are specialized to perform awide variety of vectorial functions. The apical domain ofepithelial plasma membrane is often covered with micro-villi and faces the external milieu. The basal domain isattached to basement membranes and in contact with theinternal milieu. Many studies of morphological andfunctional polarities of the plasma membrane have beenreported for some epithelial cells (Simons & Fuller,1985): adenylate cyclase (Reiketal. 1970; Schwartz et al.1974) and H-2 antigens (Parr & Kirby, 1979) are localizedin the basolateral domain, whereas leucine amino-peptidase (Desneulle, 1979; Louvard, 1980; Feraccietal. 1981; Roman & Hubbard, 1984) and S'-nucleo-tidase (George & Kenny, 1973; Colas & Maroux, 1980;Inoue et al. 1983; Meier et al. 1984) are in the apicaldomain.

To our knowledge, no study on the cell surface polarityhas been reported for epidermal cells. It is easily assumedthat many nutrients reach the epidermal layers from thebasal side, which faces the blood circulation in the dermallayers. The aim of the present study is to verify thispolarity of the epidermal cell surface.

Epidermal cells in vivo are known to adhere to

Journal of Cell Science 91, 491-499 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

basement membranes, which seem to provide a suitableenvironment for proliferation and differentiation(Vracko, 1978). We have tried to devise a new culturesystem for epidermal cells that mimics the in vivo state asdescribed above, using a permeable collagenous mem-brane as an artificial basement membrane. With thisculture method, we could demonstrate the functionalpolarity with regard to uptake and excretion of nutrients,metabolites and newly synthesized proteins through theplasma membrane. In addition to this, we found thatepidermal cells can grow and differentiate even in aconventional culture medium, when they are cultured onthe permeable collagen membrane.

Materials and methods

Chemicals and culture materialsMedium 199 (M199) and Dulbecco's modified Eagle's medium(DMEM) were purchased from GIBCO (Grand Island, NY);foetal calf serum (FCS) from Nihon Biotest Laboratory Inc.(Tokyo); EDTA (ethylenediamine tetraacetic acid) and Hepes(iV-2-hydroxyethylpiperazine-A''-2-ethanesulphonic acid) fromDojin Chemical Institute (Kumamoto); penicillin and strepto-mycin from Meiji Seika Kaisha (Tokyo); dispase from GodoShushei Co., Ltd (Tokyo); Percoll from Pharmacia Fine

491

Chemicals (Uppsala); Falcon tissue culture dishes from Bec-ton-Dickinson Labware (Oxnard, CA); trypsin from SigmaChemical Co. (St Louis, MO); crude bacterial collagenase fromWako Pure Chemical Industries (Tokyo); cycloheximide fromNakarai Chemicals (Kyoto); [3H]proline (sp. act., 272Cimmol"1), [HC]proline (sp. act., 273Cimmor ' ) , [14C]glycine(sp. act. 113Cimmol~'), and a scintillation cocktail Atomlightfrom New England Nuclear (Boston, MA); omeprazol fromYoshitomi Pharmaceutical Inc. (Osaka); amiloride from MerckSharp Dohme Res. Lab. (Rahway, NJ); glucose C-test kitsfrom Wako Chemical Industries, Ltd (Osaka); and F-kits ofurea/NH4 and of lactate from Boehringer-Manheim-Yamanou-chi (Tokyo).

— 232K

— 140K

— 67K

Preparation of collagen film and its assembly into a cellculture vesselCollagen was extracted from calf skin and purified to homo-geneity as described (Nishikawa et al. 1987). This preparationcontained type I and type III collagens at a ratio of 49: 1. A 1 %solution of collagen in HC1 (pH3) was poured onto hydro-phobic plastic at 0-2 ml per cm2 of the surface, air-dried, andirradiated with a 15 W ultraviolet (u.v.) lamp (maximumabsorbance at 254 nm; the distance between collagen and thelamp was 10 cm) for 20min in order to introduce cross-linksamong the collagen fibrils (Yoshizato et al. 1988). Collagen waspeeled from the plastic with forceps as a thin (20 ̂ m) trans-parent membrane (collagen film). This type of collagen waspermeable to low Mr but not to high Mr substances. Thepermeability was tested by inserting the membrane into adialysis cell as described below. For the preparation of filmspermeable to high MT substances, air-dried films were soaked inHanks' solution at 37CC for 30min before u.v. irradiation inorder to polymerize collagen. This type of collagen film wastranslucent. Collagen films were cut in a disc form of 44 mmdiameter and sandwiched between circular pieces of plastic.The final diameter of collagen films assembled in this equip-ment was 34 mm. The equipment was placed in a plastic culturedish of 50 mm diameter. A suspension of epidermal cells(1-5 ml) was inoculated into the upper side of a collagen film.Culture media (5-5 ml) were placed also in the basal compart-ment of the equipment. Cells on both types of film could beobserved with a phase-contrast microscope.

Collagen films were assembled into a cylindrical dialysis cellmade of transparent acrylic resin, which had two compart-ments, A and B. Each compartment had a volume of 3-2ml.Phosphate-buffered saline (PBS) containing substances to bechecked for permeability was placed into A and PBS alone intoB. Dialysis of the substance through a collagen film wasperformed for 3 days at room temperature. The formation of aconcentration gradient in compartment B was inhibited bygently stirring a bar magnetically. After dialysis, solutions fromcompartments A and B were subjected to polyacrylamide gel(7-5%) electrophoresis in the presence of sodium dodecylsulphate (SDS) according to Laemmli (1970) for proteinsamples, or were analysed spectrophotometrically for concen-trations of non-protein samples, glucose and protamine.

Serum proteins were placed in compartment A of thecylindrical dialysis cell and dialysed through a collagen filmagainst PBS from compartment B. When transparent collagenfilms were used, no proteins passed through into compartmentB, indicating that this film is permeable only to low Mr

substances. Glucose (Mr 180) passed the film and reachedequilibrium after 8h. Protamine (average jWr6000) reachedequilibrium after 7 days, indicating that the upper limit of MT

for membrane permeability is around 6000. The electrophoreticpatterns of serum proteins after dialysis through translucent

A B

Fig. 1. A permeability test for translucent collagen films.PBS containing 2-5 % FCS was injected into compartment Aand PBS into compartment B. FCS was dialysed for 3 days atroom temperature with constant and gentle stirring incompartments B. Samples of the solutions from bothcompartments were subjected to SDS (1 %)-polyacrylamidegel (75%) electrophoresis. Lane A, compartment A. LaneB, compartment B. Molecular weights determined by markerproteins (thyroglobulin, ferritin, catalase, lactatedehydrogenase and serum albumin) are shown on the right.

collagen films are presented in Fig. 1. The results show that thisfilm passes high MT substances with values up to about 200-300(X103).

CellsEpidermal cells were dissociated from newborn rat skin usingdispase and trypsin as described (Yoshizato et al. 1986; Nishi-kawa et al. 1987). They were cultured in M199 or DMEMcontaining 10% foetal calf serum (FCS), 10mM-NaHCO3,20mM-Hepes, lOOi.u.mP1 penicillin, and 100/.igml"' strepto-mycin. The cells were maintained at 37°C in a humidifiedatmosphere of 5 % CO2 and 95 % air. Usually 2x 106 cells wereinoculated in 9 cm2 of growth area. The number of cells wascounted in a chamber with Neubauer rulings. Viable cells weredetermined by the Trypan Blue dye-exclusion method. Cellswere cultured on collagen-coated plastic dishes according toYoshizato et al. (1985). Human dermal fibroblasts were ob-tained and cultured as described (Yoshizato et al. 1980). Theexperiments shown in Figs 2—10 were done using transparentcollagen films, with the exception of Fig. 7, in which trans-lucent films were used.

Assays for metabolitesGlucose was determined in culture media with a glucose C-testkit. Free NH4"1" and lactate were determined with an F-kit forurea/NH4+ and an F-kit for lactate, respectively.

Radioisotope experimentsThe incorporation of amino acids into epidermal cell layers wasdetermined as follows. Epidermal cells (4xl0 6 to 8X106 in1-5 ml of DMEM with 10% FCS) were inoculated into theupper side of a transparent collagen film, which had beenassembled in the equipment, and 5 5 ml of DMEM-10% FCSwas added in the basal compartment. After confirming micro-scopically that the cells had become confluent (usually 3-4 daysafter the inoculation), the media were replaced with fresh mediacontaining radioactive proline. [3H]proline was added to the

492 K. Yoshizato et al.

apical compartment at a final concentration of 1 [iC\ ml and[ C]proline in the basal compartment at 01/iCiml"1 . Cellswere labelled for 12 h and the labelling was terminated byadding 1000 times excess amounts of unlabelled proline. Thecollagen films with cell layers were washed three times withHanks' solution, transferred into vials with 5 ml of Atomlightand assayed for radioactivity.

The secretion of proteins newly synthesized by epidermalcells was analysed as follows. Epidermal cells were cultured oncollagen films that were permeable to high M, substances(translucent membranes) and were allowed to reach a conflu-ence as described above. [14C]glycine was added to serum-freemedia of both apical and basal compartments at a concentrationof 1 /*Ciml~' and the cells were cultured for an additional 24 h.The labelling was terminated by adding 1000 times excessamounts of non-labelled glycine. The media from the apical andbasal compartments were collected separately and dialysedexhaustively against distilled water. Samples of the retentateswere used to determine the radioactivity.

Transmission electron-microscopic (TEM) observationsEpidermal cells cultured for 3 days on collagen films were fixedin 2-5% glutaraldehyde in a 0-1 M-phosphate buffer (pH7-4)containing 1 % tannic acid for 2h at 4°C. The specimens werepost-fixed with 1 % OsO4 in a 0-1 M-phosphate buffer (pH7-4)for 2 h at 4°C, dehydrated in a graded series of ethanol andembedded in Epoxy resin. Ultrathin sections were double-

stained with uranyl acetate-lead acetate and examined in aHitachi HU-12A electron microscope.

Results

Electivn-microscopic observations

Epidermal cells on transparent collagen films were exam-ined electron-microscopically (Fig. 2). The cells prolifer-ated and formed well-organized sheets of colonies six toseven layers thick. The uppermost layer was keratinizedand anucleate, representing a terminal differentiated state(a stratum corneum). Cells were connected by well-developed desmosomes. However, hemidesmoses werenot formed between the lowermost cells and the collagenfilms. Instead, these cells protruded microvilli-like struc-tures towards the collagen films. Keratohyalin granuleswere not observed in the cells and tight junctions werenot noticed between the cells, although an extensivesearch for these structures was not made in the presentstudy.

Polaritv of the membrane transport system in epidennalcells

In the following experiments transparent collagen filmsthat are permeable to substances with Mt values less than

V .. \

Fig. 2. TEM observations of epidermal cellscultured on collagen films. A total of 8X 106

epidermal cells were inoculated on transparentcollagen films (9 cm2 in growth area) andcultured in M199-10% FCS for 3 days. Thearrowheads show the structures of desmosomesand arrows the cytoplasmic protrusions seen inthe bottommost layers of cells, cf, collagen film.X5800.

Epidennal cell polarity 493

1-0

0-8

"Si

0-6

5 0-2

10

r+1 rtl

A l-5mlB5-5ml

I

B l-5mlA 5-5 ml

II

Fig. 3. Polarity in glucose uptake. A total of 8xlO6

epidermal cells were inoculated on transparent collagen films(9 cm2) and cultured in DMEM-10% FCS for appropriatetimes (usually 3-4 days) to reach a confluence. Media werereplaced with fresh media and glucose uptake was measured.The cells were allowed to utilize glucose for 3 h and amountsof glucose in the media were determined. The volume ofmedia was 1*5 ml in the apical compartment and 5'5 ml in thebasal compartment. Experiment I was performed in thenormal position and experiment II in the up-side-downposition to experiment I. Each value represents the average oftwo determinations and the bars give their range. A. Apicalcompartment; B, basal compartment.

6000 were used if not specified otherwise. First, weconfirmed that collagen films are not permeable even toglucose when the film is fully covered with epidermalcells, indicating that a sheet of epidermal cells can be aneffective permeability barrier to diffusion of solutes. Incontrast to epidermal cells, fibroblasts grown to conflu-ence did not act as a barrier to glucose permeability.

The uptake of glucose by epidermal cells is shown inFig. 3 (exp. I). Epidermal cells were inoculated, allowedto grow for 3-4 days to reach confluence and exposed tofresh media. The concentrations of glucose were deter-mined for media in the apical and basal compartmentsafter an additional 3 h. The cells took up glucose from thebasal medium, but not from the apical side, indicatingthat there are functional differences between the apicaland basal sides of epidermal sheets. However, there is apossibility that gravity somehow exerts effects on trans-port through the membrane. To exclude this possibility,the cells were cultured up-side-down (with the apicalcompartment down and the basal one up) as shown inFig. 3 (exp. II). The same preferential uptake of glucosefrom the side attached to collagen was observed also inthis culture.

The same polarity was observed for the excretion oflactate (Fig. 4). Lactate was produced from the basal sideof the epidermal sheet at a five times higher rate thanfrom the apical side. In contrast to the transport of

0-5

•o•cO'5.3

rh

BA 1-5 mlB 5-5 ml

I

B A

B

AB 1-5 mlA 5-5 ml

II

Fig. 4. Polarity in lactate release. This experiment and theexpression of the results were done in an identical manner tothe experiment of Fig. 3, except that amounts of lactate weredetermined instead of glucose.

40-i

20-

Apical Basal

Fig. 5. Polarity in NH 4+ release. The experimental

conditions and the way of expressing the results are the sameas for Fig. 3 except that the concentrations of ammonia weredetermined instead of glucose. Release of NH 4

+ into thebasal compartment was not detected. Each value is theaverage of two measurements and bars show their range.

glucose and lactate, ammonia was excreted only from theapical sides of epidermal sheets (Fig. 5).

Epidermal cells incorporated amino acids into cellularproteins preferentially from the basal side of the epider-mal sheet (Fig. 6). The apical side of the epidermal sheetwas exposed to medium containing [3H]proline and thebasal side containing [l4C]proline. The basal side incor-porated the amino acid at a more than eight times higherrate than the apical side. The incorporation of [14C]- and[3H]proline was sensitive to cycloheximide.

Release of newly synthesized proteins from epidermalcells was determined (Fig. 7). In this experiment epider-mal cells were cultured on translucent film. The basalsurface of the epidermal sheet was exposed to [14C]gly-cine for 24 h and the media were collected from both

494 K. Yoshizato et al.

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nem

br

8

rolin

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corp

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B14C

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3H

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Fig. 6. Polarity in proline uptake. The methods of culture ofepidermal cells were the same as those described for in Fig. 3unless otherwise specified below. [3H]proline was added inthe apical compartment (2 ml) at l^Ciml"1 and [14C]prolinein the basal compartment (6 ml) at 01/ iCiml"1 . Theconcentration of proline (radioactive and non-radioactive) inboth compartments was made 0-366mmolml" by addingnon-radioactive proline. The cells were labelled forl2h in thepresence and absence of 3^gml~' cycloheximide and thelabelling was terminated by adding 1000 times excessamounts of non-radioactive proline. The radioactivitiesincorporated into the cell layers were determined. Values arethe average of two determinations and bars show their range.Open columns are without cycloheximide, and hatched oneswith the drug.

compartments. Radioactivities that were not dialysablewere determined for both media. 14C-labelled proteinswere secreted twofold more to the apical side than to thebasal one, indicating the presence of polarity for thesecretion of proteins in the epidermal sheet. The incor-poration of the amino acid and the excretion of proteinswere temperature-sensitive.

200

100

U

Fig. 7. Polarity in secretion of newly synthesized proteins.Epidermal cells were cultured as for Fig. 3, except that thecollagen films were translucent. The cells were bathed with[14C]glycine in the basal compartment at 1 /iCiml"1

(8-8nmolmr ' ) for 24h at 37°C or 4°C. The labelling wasterminated by adding 1000 times excess amounts of non-radioactive glycine. Media from both compartments (A andB) were collected and dialysed exhaustively against distilledwater. Radioactivities in the retentates were determined.Values were the average of the two determinations and barsshow their range. A. Apical compartment. B. Basalcompartment.

7-2-

7 1 -

7-0

6-9

basal

iiapical

-

0-3

3ex

£i

None Omeprazol Amiloride

t—tt-

0-2

0 1

0 4 40 400Concentration of omeprazol

Fig. 8. Difference in pH produced in the apical and basalcompartments during culture. Rat epidermal cells werecultured as for Fig. 3. On day 4, media were replaced withfresh ones and culture was continued for an additional 8 h inthe presence and absence of inhibitors. The value of pH wasdetermined for both media. A. Open triangles show the pHof basal compartments and closed triangles show the pH ofapical compartments: omeprazol, 0 4 mM; amiloride, 2mM.B. The same experiment was performed with differentconcentrations of omeprazol. Values are the pH differencebetween the apical and basal compartment and are theaverage of duplicate determinations. Bars show their range.

20

~ 15

'•6

V 10ef 53

05 9

Days in culture16

Fig. 9. Growth curves of epidermal cells. Rat epidermal cells(2xl06) were inoculated on transparent collagen films(growth area 9 cm2) assembled in the culture vessel or onplastic (the same growth area). The cells were cultured inM199 with 10% FCS. Media were changed every second dayduring culture. Cells were dissociated at days indicated andviable ones counted. Cells on plastic were harvested with0-25 % trypsin-1 mM-EDTA in PBS for 15 min at 37°C. Cellson collagen films were dissociated by the following threesuccessive treatments: 1000 units ml~' of dispase inM199-10% FCS for 6h at 37°C, 0-3 % collagenase in Hanks'solution for 2-4 h at 37°C and 0-25 % trypsin-1 mM-EDTAfor 15 min at 37 °C. Each point represents the average of twodeterminations and bars show their range. (O O) Cells oncollagen films; ( • • ) cells on plastic.

Epidermal cell polarity 495

Fig. 10. Epidermal cells on collagen films. Rat epidermal cells were inoculated at 2-5 XlO5 cells cm 2 and were cultured inM199-10% FCS for 24h (A-C) and 10 days (D-F). A,D. On transparent collagen films; B,E, on plastics; C,F, on plasticscoated with 2mgcm~ of collagen. Bar, 200 fim.

During culture on transparent film, the pH in theapical medium became more acidic than that in the basalmedium. The pH difference after 4 days in culture wasaround 0-2 (Fig. 8A). This fact was rather surprising,because epidermal cells produce more lactate in the basalmedium and more ammonia in the apical medium (Figs 4and 5, respectively). If the pH in the medium isdetermined by the quantity of lactate and ammonia, theapical medium should have a more alkaline pH. Toovercome this discrepancy, we postulated a localizeddistribution of H + transport systems in the plasmamembrane. To verify this possibility, we utilized inhibi-tors for H+-involved transport systems. Omeprazol (aspecific inhibitor of H+/K+-ATPase) and amiloride (aninhibitor of N a + / H + antiporter) were effective in abo-lishing the pH difference between the two compartmentsdescribed above (Fig. 8A). The effect of omeprazol wasdose-dependent with a critical concentration between4/iM and 40 fXM (Fig. 8B). The inhibitor experimentssupport the idea that the K + / H + and N a + / H + transportsystems are localized on the apical side of epidermalcells.

Gmwth of epidermal cells on collagen filmsRat epidermal cells were cultured on the upper sides oftransparent collagen films and their growth was deter-mined (Fig. 9). Cells on plastic culture dishes were notable to grow in the conditions adopted in the presentstudy and were completely detached from the disheswithin 9 days. On the other hand, cells on collagen filmsgrew steadily up to 16 days. Microscopical changes inepidermal cells are compared among three differentcultures at days 5 and 10 in Fig. 10. Typical epidermalcolonies with cobblestone-like patterns were well sus-tained and all the surface of the growth area was coveredwith epidermal cells in the collagen-film culture, but notin cultures on plastic or collagen-coated surfaces.

In our experience, epidermal cells can survive in ahealthy state for at least 40 days on the collagen films.This improved growth and survival of epidermal cellscannot be ascribed to collagen itself, because no improve-ment was observed when the cells were cultured oncollagen-coated plastic (Fig. 10E). Therefore, it wassuggested that culture on collagen films, where thesupply of nutrients and essential factors from medium to

496 K. Yoshizato et al.

cells was possible to both the apical and basal sides ofepidermal sheets, has a beneficial effect on the physiologyof epidermal cells.

Discussion

Little is known about the functional polarity of epidermalcells both in vivo and in vitro. The present study clearlydemonstrates that epidermal cells on a permeable col-lagen film can easily express cell surface polarity ofnutrient uptake and metabolite excretion as they mightdo /;/ vivo. They take up glucose and amino acids andexcrete lactate exclusively from the basal side of the cellsheet and produce H + and ammonia preferentially fromthe apical side.

Epidermal layers formed on permeable collagen mem-branes can be a permeability barrier, as demonstrated inthe present study on the permeability to glucose and toamino acids (Fig. 6). This fact suggests that epidermalcells in the layers contact neighbouring cells, formingtight connections between them, which presumably aretight junctions and desmosomes. We demonstrated thepresence of desmosome structures electron-microscopi-cally. We have not made an extensive search for tightjunctions. Kitajima et al. (1983) reported the presence oftight junctions in primary cultures of human epidermalcells. Polarized monolayers formed by MDCK cells on apermeable support are also known to be an effectivepermeability barrier with the morphological and func-tional properties of transporting epithelia (Misfeldt et al.1976).

The term 'polarity of the cell surface' should be usedwith caution in the case of epidermal cells, with which thepresent study has been performed. The experiments thatdemonstrated the in vitro expression of cell surfacepolarity have been developed using other epithelial cells,like MDCK cells or A6 cells (Simons & Fuller, 1985).These cells form monolayered epithelia on a permeablesupport and constitute a permeability barrier. For cells ina monolayer the term cell surface polarity is withoutambiguity. The upper half of the surface, which is notattached to a substratum, is the apical domain, and theother half, attached to the substratum, is the basaldomain. However, in the present study, the polarity hasbeen measured with multilayered not with monolayeredepithelia. Polarity in this case means the polarity of thecell layer as a whole, not of the cell surface: for example,glucose is taken up by epidermal cells exclusively fromthe 'basal side' of the cell layer. There is no doubt that thebasal surface of cells in the lowermost layer, i.e. the cellsattached to the collagen membrane, plays a crucial role inthis selective uptake of glucose. However, it is not clear atpresent whether the basal surface of cells in the otherupper layers is involved in this selective uptake or not.

The physiological pH values for skin in vivo are knownto be pretty acidic, for example between 4-2 and 6-2 forhuman skin (Draize, 1942). The present study suggests amechanism for this acidity. The behaviour of the meta-bolic end-products, lactate and ammonia, may not causethe acidity in vivo. If this is the case, the pH in skin

should be alkaline: lactate is excreted from the lowermostlayer of the epidermis (Fig. 4) to the dermis and thenremoved from the skin via the blood circulation. Incontrast to this, ammonia is released into the upper layersof the epidermis (Fig. 5) and remains there because thereis no circulation in the epidermis, resulting in an alkalinepH in skin. Therefore, H + transport systems should bethe cause of the acidity. In fact, the results shown inFig. 8 clearly demonstrate that H + is produced from theupper layers of the epidermis, probably due to thelocalization of the transport system. This system issensitive to omeprazol and amiloride. Several reportshave been published that show the localized distributionof amiloride-sensitive Na+/H+-antiporter in the apicaldomain of epithelia of: colon (Stoner, 1979; Turnheim etal. 1978), frog skin (Cuthbert & Schum, 1974), renaltubule (O'Neil & Boulpaep, 1979), salivary gland(Schneyer, 1970), and urinary bladder (Lewis et al.1976). Cultures of epidermal cells on a transparent andpermeable collagen membrane are a reliable experimentalmodel of epidermis and will be useful for investigations ofthe physiology and morphology of the skin.

Generally, growth in culture of normal mammalianepithelial cells has not been easy compared with mes-enchymal cells like fibroblasts, which can proliferateeasily and can be cultivated serially in a commerciallyavailable basal medium with FCS. Epithelial cells requireseveral factors or specific substances for successful pri-mary culture, in which they can grow and express normaldifferentiated functions (Tsao et al. 1982). Severalspecific methods have been reported for primary epider-mal cultures. Eisingeref al. (1979) recommended the useof an acidic culture medium for growth of cultured cells.Rheinwald & Green (1975) introduced feeder cells forprimary and passaged cultures of human epidermal cells.With this elegant technique, epidermal cells are capableof making clonal growth and forming colonies withmorphological and functional differentiation. Boyce &Ham (1983) demonstrated that normal epidermal cellscan grow in vitro even in the absence of FCS if adequategrowth factors are included in the medium.

The present study clearly demonstrates the importanceof cell surface polarity in keeping epidermal cells in ahealthy state in culture. Epidermal cells were cultured onpermeable collagen membranes and bathed in media onboth surfaces (basal and apical). In this culture, the cellsproliferated, made multi-layered epidermal sheets andsurvived for more than a month in a commerciallyavailable medium to which FCS was added, but nospecial hormones or growth factors were included.

There should be several reasons why epidermal cellscan be maintained in good condition when they arecultured on collagen films. At present, one of the crucialreasons seems to be that epidermal cells can easily expresscell surface polarity of nutrient uptake and metaboliteexcretion as they might do in vivo. In vivo, manynutrients reach the epithelial sheet from the basolateralside, which faces the blood circulation, as described bySimons & Fuller (1985). In the present study, we haveverified that glucose is taken up exclusively from the basalsides of epidermal sheets. As pointed out by the above-

Epidemial cell polarity 497

mentioned authors, when epithelial cells are cultured in aconventional manner, e.g. on glass or plastic, the cells areforced to feed from the apical surface, which faces theculture medium; this could be a reason why epidermalcells cannot grow and survive when they are cultured inthe conventional manner (Fig. 9). Hull et al. (1983)observed improvements in culture morphology and ex-pression of terminal differentiation markers when ker-atinocytes were grown on collagen gels and fed frombelow.

Epidermal cells require several factors from serum fortheir growth: e.g. insulin, epidermal growth factor andtransferrin (Boyce & Ham, 1983). It is reasonable toconsider the possibility that the receptors for serumfactors mentioned above are also localized in the basalsurface of the cell layer. The location of EGF receptors inthe epidermis has been studied by Nanney et al. (1984)and Green & Couchman (1985), who demonstrated thatEGF receptors are present on epidermal basal cells. Theobservations by Handler et al. (1984) are noteworthy inthis regard. They cultured an epithelial cell line desig-nated A6, which was derived from the kidney olXenopuslaevis, on a permeable collagen membrane. The cellsformed an epithelium and showed a transport response tovasopressin only when access of hormone-containingmedium to the collagen membrane was not impeded.

In the present study, permeable collagen membraneswere used for successful primary culture of epidermalcells, where the cells are allowed to express the functionalpolarities of their plasma membranes. There have beenseveral studies that demonstrated the importance ofpermeable supports for other epithelial cells. Misfeldt etal. (1976) cultured Madin-Darby canine kidney(MDCK) cells on a freely permeable nitrocellulose filterand found that the cells re-form two-sided asymmetricalsheets with distinct apical and basolateral surfaces. Thesame epithelial cell line was cultured on collagen-coatednylon cloth disks by Cereijido et al. (1978), where thecells formed a monolayer sheet and were bathed fromboth apical and basolateral cell surfaces. MDCK mono-layers cultured in this way remained functional forseveral weeks.

The authors are grateful to Dr Noboru Yamamoto for takingTEM photographs of cultured epidermal cells. A.N. was therecipient of a postdoctoral fellowship from the Japan Society forthe Promotion of Science.

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CEREUIDO, M., ROBBINS, E. S., DOLAN, W. J., ROTUNNO, C. A. &SABATINI, D. D. (1978). Polarized monolayers formed by epithelialcells on a permeable and translucent support. J. Cell Biol. 77,853-880.

COLAS, B. & MAROUX, S. (1980). Simultaneous isolation of brushborder and basolateral membrane from rabbit enterocytes:Presence of brush border hydrolases in the basolateral membraneof rabbit enterocytes. Biochim. biophys. Acta 600, 406-420.

CUTHBERT, A. W. & SCHUM, W. K. (1974). Amiloride and the

sodium channel. Naunvn-Schmiedesbergs Arch. exp. Path.Phannak. 281, 261-269.

DESNEULLE, P. (1979). Intestinal and renal aminopeptidaae: A modelof a tran8membrane protein. Eur.J. Biochem. 101, 1 — 11.

DRAIZE, J. H. (1942). The determination of the pH of the skin ofman and common laboratory animals. J. invest. Derm. 5, 77-85.

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(Received 10 June 1988 - Accepted 1 August 19S8)

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