a tissue culture assay of corneal epithelial wound...

A Tissue Culture Assay of CornealEpithelial Wound Closure

Marcia M. Jumblarr and Arthur H. Neufeld

Experimental assays have been developed using cultured tissue derived from rabbit corneal epitheliumto study migration of epithelial sheets during wound closure and cell-substrate adhesion. To study woundclosure, epithelial defects, 6 mm in diameter, were produced in vitro in 24 well multiplates by a localfreezing technique, and the size of the remaining defect was quantitated over time by staining. To studyadhesion, cultured cells were labeled with 3H-leucine, suspended, and added to fresh culture plates. Atvarious times, adherent cells were lysed and the radioactivity of the lysate was determined. Serumenhances the closure of experimental defects, but laminin and fibronectin have no effect. Agents whichalter mitotic rate, such as epidermal growth factor and 5-fluorouracil, do not influence the rate of woundclosure in this assay. Compounds which elevate intracellular levels of cyclic AMP inhibit wound closurebut promote cell-substrate adhesion. Thus, cultured corneal epithelial cells may be used to assay forinfluences on the migratory events governing closure of superficial epithelial wounds. Invest OphthalmolVis Sci 27:8-13, 1986

The corneal epithelium closes superficial wounds bymigration of the epithelial sheet over the denudedstroma. Mitosis ceases at the wound periphery, and acohesive layer of epithelial cells slides toward the centralwound area.l>2 During this time, cells adhere reversiblyto the corneal surface in the absence of attachmentorganelles and sustained migration is dependent uponcontinued synthesis of cell surface glycoproteins.3'4

Once coverage of the wound is complete, migrationceases, and the epithelial monolayer re-forms stablehemidesmosomal attachments to its basement mem-brane.3 Mitosis then resumes, resulting eventually ina mature stratified corneal epithelium.1

Recently, we have established and characterizedcultures of rabbit corneal epithelial cells. These cellsrequire cholera toxin for optimal growth, form partiallystratified cell layers with apical microvilli, and retaintheir ability to respond to /3-adrenergic agonists withincreased cyclic AMP synthesis.5 We have used thesecell cultures to describe aspects of corneal epithelialwound healing, to study the effects of pharmacologicalagents on wound closure and cell-substrate adhesion,and to develop an assay which may prove useful as analternative to in vivo toxicity testing.

From the Ophthalmic Pharmacology Unit Eye Research Instituteand Department of Ophthalmology, Harvard Medical School, Boston,Massachusetts.

This work was supported by National Institutes of Health grantEY02360.

Submitted for publication: November 14, 1984.Reprint requests: Dr. Arthur H. Neufeld, Eye Research Institute,

20 Staniford Street, Boston, MA 02114.

Materials and Methods

Corneal Epithelial Cultures

Rabbit corneal epithelial cell cultures were initiatedfrom Dispase II treated corneas as previously de-scribed.5 New Zealand white rabbits, used as a sourceof corneal tissue, were treated in accordance with theARVO Resolution on the Use of Animals in Research.Cultures were established in the absence of a feederlayer in a medium consisting of equal parts of Dul-beccos Modified Eagles Medium and Hams F12 sup-plemented with 5% fetal bovine serum, cholera toxin(0.1 Mg/ml), epidermal growth factor (10 ng/ml), insulin(5 n%/m\), gentamicin (5 /ig/ml) and dimethylsulfoxide(0.5% v/v). Epithelial cells derived from six corneaswere pooled and plated into the 6 wells of a 35-mmdiameter multiplate. Cultures were fed three timesweekly. After 7-10 days the confluent multilayer (cfreference 5 for morphology) were subcultured, and thecells derived from each 35-mm diameter well wereplated into a 24-well multiplate, each well of whichcontained 1 ml of the above medium, except that chol-era toxin was omitted. In some experiments, cells wereplated into 6-well multiplates in the presence or absenceof cholera toxin as indicated.

Wound Closure Assay

Discs, 6 mm in diameter, were cut from MilliporeHA filters, rinsed in 6 changes of distilled water, boiledin distilled water, and dried in a laminar flow hoodprior to use. A disc was placed on the surface of each

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No. 1 CORNEAL EPITHELIAL WOUND CLOSURE / Jumblorr and Neufeld

Fig. 1. Experimental woundclosure assay: Corneal epithelialcells were subcultured into 24-well multiplates and grown toconfluency. Wounds were pro-duced in quadruplicate by freez-ing at the indicated times beforefixing and staining. Control(CON) were not wounded. Darkareas are remaining cellularareas; light areas are remainingcell-free wound areas.

Con

culture of a 24-well multiplate, gently tapped down,and a stainless steel probe (6 mm in diameter) wascooled in liquid nitrogen and placed against the plasticsurface opposite the disc for 5 sec. The probe was thenremoved, medium consisting of Dulbeccos ModifiedMinimal Essential Medium and Hams F12 with theaddition of gentamycin and dimethylsulfoxide and withor without other components as indicated was added,and the disc carefully lifted out leaving a discrete cir-cular defect in the ceil layer.

To determine the time course of wound closure,wounding was done in replicate fashion at staggeredintervals, usually 0, 6, 24, 32 and 48 hr before the fix-ation step. At the time the earliest set of replicate con-trol wounds would be almost closed, all cultures weredrained of medium, fixed with neutral buffered for-malin, and stained in situ with full strength Giemsa(see Fig. 1 for an example). The size of the remainingdefect at the staggered time intervals, as revealed bythe unstained cell-free area, was determined by pro-jecting the plates onto a screen with an overhead pro-jecter at a fixed distance, tracing the unstained areaonto paper, and cutting out and weighing the remainingwound area. The weight was normalized, as a per-centage, to that of the initial wound size or convertedto area in mm2 for comparison of wound sizes. Todetermine the effects of various agents on wound clo-sure, all 24 wounds were made at 0 hr and fixed andstained at 42 hr. Relative wound size was determinedas above.

24 32 48

hrsMitotic Rate

Cells derived from primary culture were plated in a24-well multiplate at 1 X 104 cells/well. One ml ofculture medium containing 5-fluorouracil (5-FU) and/or epidermal growth factor (EGF) at the indicated con-centration was added to each well. At the indicatedtimes, cell number was determined by hemacytometercount.

Cyclic AMP Synthesis

Synthesis of cyclic AMP by cultured corneal epithe-lial cells was measured in cells subcultured in both thepresence and absence of stimulators of adenylate cy-clase. Medium was removed from the 6- or 24-wellculture dishes and replaced with physiological buffercontaining 0.5 mM isobutylmethylxanthine, a phos-phodiesterase inhibitor.5 After 20 min, the buffer wasremoved and the adherent cell layer was lysed in 0.1M KOH at 100°C. This alkaline extract was neutralizedwith an equal volume of 0.1 N HC1, and the resultingprotein precipitate was removed by centrifugation(2000 X G, 15 min, 4°C), solubilized with 1 N NaOHand quantitated by the method of Lowry et al.6 CyclicAMP concentration in the supernatant was determinedby radioimmunoassay using commercially availablereagents. Rates of cyclic AMP synthesis are expressedas the mean ± SEM (number of observations) of pmolescyclic AMP/mg protein/20 min.


10 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1986 Vol. 27

T3

O

18 24 30 36 42Hours

Fig. 2. Time course of wound closure in vitro: Wounded cultureswere incubated in the presence (•) or absence (O) of 5% fetal bovineserum and the size of the wounds determined and plotted as a functionof time after healing. Mean ± SEM (n = 4).

Cell-Substrate Adhesion Assay

Corneal epithelial cells were grown to confluencyand then labeled with 1 ^Ci/ml 3H-leucine (300 mCi/mmol) for 18 hr. Cells were removed from the culture

10--CM

ECOCD

C3O 5--

0 0.1 1

% FCS

Fig. 3. Effect of fetal calf serum (FCS) on wound closure. Cultureswith experimental wounds were incubated in the presence of theindicated serum concentrations and at 42 hr were fixed and stained.Mean ± SEM (n = 6).

dish with Dispase, centrifuged, resuspended in HEPESbuffered Dulbeccos Modified Eagles Medium, tritu-rated with a flame polished pipet, and passed through2 layers of 120 u mesh nylon cloth. The resulting singlecell suspension was adjusted to 1-5 X 104 cells/ml inthe same medium at 4°C. Aliquots containing1-5 X 103 cells were placed in the 24 wells of a mul-tiplate, each well containing 1 ml of medium. Attach-ment proceeded for various times up to 30 min at 22°Cin room atmosphere. At the end of the attachmentperiod, medium and unattached cells were removedand the adherent cells were rinsed twice with 1 ml me-dium. Adherent cells were lysed with 1 ml 0.1 N NaOHcontaining 1% Triton X-100 and samples of the lysate,as well as aliquots of the original cell suspension, wereplaced in scintillation fluid to determine radioactivity.Radioactivity in the adherent cells was expressed as apercentage of that present in the initial cell aliquot.

Materials, Drugs, and Supplies

Tissue culture multiplates were obtained from Fal-con (Oxnard, CA); medium and serum from Micro-biological Associates (Walkersville, MD), Dispase IIfrom Boehringer-Mannheim (Indianapolis, IN); insulinand epidermal growth factor from Collaborative Re-search (Waltham, MA); 5-fluorouracil, cholera toxinand forskolin from Calbiochem (San Diego, CA); 3H-leucine and components of cyclic AMP radioimmu-noassay from New England Nuclear (Boston, MA);type HA filters from Millipore (Bedford, MA); lamininand fibronectin from Bethesda Research Labs (Gaith-ersburg, MD) and isoproterenol, isobutylmethylxan-thine, Giemsa stock solution, and Triton X-100 fromSigma (St. Louis, MO).

Results

Cyclic AMP Levels in Cultured Cells

Primary corneal epithelial cultures synthesize cyclicAMP at the rate of 22 ± 3 (6) pmol cyclic AMP/mgprot/20 min. The continued presence of cholera toxinin the primary tissue culture medium greatly enhancesthe rate of epithelial cyclic AMP synthesis to 386 ± 47(6) pmol cyclic AMP/mg prot/20 min. However, cellsoriginally grown in cholera toxin-containing mediumand subcultured into cholera toxin-free medium forone wk have essentially basal rates of cyclic AMP syn-thesis 46 ± 8 (6) pmol cyclic AMP/mg prot/20 min.We have, therefore, used cholera toxin-free subcultures,derived from cholera toxin-containing primary cul-tures, to assay the effects of drugs on wound closureand cell-substrate adhesion.


No. 1 CORNEAL EPITHELIAL WOUND CLOSURE / Jumblorr and Neufeld 11

Wound Closure Model

The wounds obtained by local freezing are discreteand of uniform size with an area of approximately 28± 0.2 mm2 (mean ± SEM) for 24 wounds. The epi-thelial cells in the wound that are damaged adhere tothe filter disc as it is lifted away, leaving a clearly de-marcated cell-free wound area (Fig. 1). The remainingepithelial cells, moving as a cohesive sheet, graduallycover the easily visualized and quantitated centralwound area. When wounds are made in replicate fash-ion and sequentially over time, a time curve can beconstructed by relating the remaining wound area tothe original wound area and the rate of closure deter-mined (Fig. 2). When wounded cultures are incubatedat 37°C in the presence of medium containing 5% fetalbovine serum, the central area is re-covered at a rateof 0.8 mm2/hr over the first 30 hr, and closure is gen-erally complete before 48 hr (Fig. 2).

Because a discrete and measurable wound remainsafter 42 hr of closure, the size of the wound at thatinterval was used as a measure to define the basal con-ditions for wound closure. In serum-free medium, ep-ithelial wounds closed slowly; over 30% of the originalwound remained at 42 hr (Fig. 2) and continued in-cubation for up to 72 hr did not lead to further closure.The effect of fetal bovine serum is dose dependent (Fig.3) and cannot be mimicked by 5 or 20 mg of the serumattachment factor fibronectin or by 10 mg laminin(data not shown). Thus, serum is necessary for com-plete wound closure in this assay and is included at aconcentration of 5% (v/v) in the medium in experi-ments designed to determine the effect of drugs onwound closure.

Effect of Mitosis on Wound Closure

Corneal epithelial mitotic rates were determined incultures grown in the presence of an epithelial growthpromoter, EGF, and an antimitotic agent, 5-FU. EGFat 10 /ig/ml enhanced the cellular mitotic rate; whereas,5-FU at 10 Mg/ml totally suppressed EGF stimulatedmitosis. Nevertheless, when added to cultures at thetime of wounding, these agents had no effect on woundclosure (Table 1).

Effect of Cyclic AMP Synthesis on Wound Closure

Corneal epithelial cultures were wounded and ex-posed to test agents for 42 hr, at which time cyclicAMP synthesis and wound closure were measured.Cholera toxin (0.1 Mg/ml) and forskolin (10~5 M) in-creased cyclic AMP synthesis and slowed wound clo-sure as shown in Figure 4. Neither of these agents,however, completely prevented migration, and sub-stantial closure occurred in all cases. Cultures treated

Table 1. Effects of growth modulators on celldensity and wound closure

ModulatorCell Number*

(X104)Wound Area\

(mm2)

ControlEGFEGF + 5FU

1.8 ±0.28.9 ± 1.11.4 ±0.2

4.3 ± 0.54.2 ± 0.44.2 ± 0.7

* Determined in triplicate 7 days after innoculation of 1 X I04 cells.\ Determined in quadruplicate 42 hr after wounding.

with the /3-subunit of cholera toxin (data not shown)or isoproterenol (10~5 M) had unchanged rates of cyclicAMP synthesis and wound closure.

Cell Adhesion

After 18 hr in medium containing 3H-leucine, con-fluent cultures of epithelial cells had incorporated labelat a rate that produced 1-3 counts/min/cell, providinga simple method for subsequent estimation of thenumber of adherent cells. Thirty minutes after additionof suspended cells to HEPES buffered medium at am-

5--

220

I 2000 180

^ 160

1 140°> 120

100

80a

<o 60-

| 40Q 20-

CON ISOP CTX FS

CON ISOP CTX FS

Fig. 4. Effect of cyclic AMP synthesis on wound closure. Cultureswere wounded and exposed for 42 hr to cholera toxin (CTX), isobutyl-methylxanthine (IBMX), forskolin (FS), or isoproterenol (ISOP)added to medium containing 5% FCS. At this time, remaining woundarea and cyclic AMP synthesis were determined. Mean ± SEM(n = 4).


12 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1986 Vol. 27

7 0 T

MinutesFig. 5. Effect of cholera toxin on cell substrate adhesion. Epithelial

cell cultures grown in cholera toxin free medium were exposed tocholera toxin (1 Mg/ml) and labelled with 3H-leucine for 18 hr. Cellswere then suspended and added to replicate tissue culture wells. Atthe indicated times, medium and unattached cells were removed andthe adherent cells lysed. Radioactivity of the lysate was determinedas a percentage of the total radioactivity of the cell suspension. Choleratoxin treated (•), control (O). Mean ± SEM (n = 6).

bient temperature, 30-50% of cells derived from chol-era toxin supplemented cultures had adhered to theplastic substrate; whereas, during the same time only10-15% of the cells derived from cholera toxin-freecultures adhered (Fig. 5). When collagen coated wellswere used as a substrate, 30% of cells grown in theabsence of cholera toxin adhered in the 30 min-assayperiod. When cells grown in the presence of choleratoxin were exposed to the collagen substrate, there wasno further increase in adhesion of these cells as com-pared to cells exposed to the plastic substrate.

Discussion

Superficial epithelial defects close by epiboly, mi-gration of the epithelial cell sheet over the basal lamina.The corneal epithelium, which consists of stratifiedsquamous cells and rests on an avascular matrix, is aparticularly simple system in which to study epithelialmigration. Corneal epithelial wound closure has beenstudied both in vivo and in organ culture.''2A7>8 In thepresent study, we have demonstrated that cultured cellsof the corneal epithelium in culture retain the abilityto re-cover a model defect. The rate of wound closurein vitro in the presence of serum, as determined bylinear regression analysis of the rate curve in Figure 2,is 0.8 mm2/hr and is similar to that observed in ex-perimental wounds in vivo.7-9

For wound closure to occur in vitro, serum is a nec-essary component of the medium. Recently, a serum

protein, epibolin, was isolated which, when added toepidermal organ cultures, promotes migration of theepithelial sheet.10 Whole serum, added to corneal organcultures, promotes migration of the epithelium, andthe attachment factor fibronectin, a serum component,has been identified in the basement membrane zoneof healing corneas."12 However, our results demon-strate that the permissive factor for migration in serumis unlikely to be fibronectin or laminin.

In vivo, wounds close initially by migration of epi-thelial cells from a peripheral site rather than prolif-eration within the wound site. The addition of EGF,which is mitogenic for the cultured epithelial cells, tothe experimental medium has a negligible effect onclosure. Conversely, the antimitotic agent, 5-FU, addedin a concentration sufficient to suppress EGF-inducedmitosis, has no effect on wound closure. We thereforeconclude that migration, and not mitosis, is the dom-inant process by which wound closure occurs in thisin vitro model.

To cover superficial defects, migratory cells mustcontinuously make and break cell-substrate adhesions.The "stickiness" of cells keeps them adherent to theirsubstrate and prevents inadvertent damage to the heal-ing tissue by mechanical trauma, such as the movementof the eyelid over the surface of the cornea duringblinking. Only after migration ceases do epithelial cellsre-form stable hemidesmosomal attachments to theirbasement membrane.3

Cell substrate adhesion during migration is probablydependent upon glycoproteins on the cell surface. Inthe cornea, glycoproteins are normally synthesized bymigrating cells, and tunicamycin prevents both gly-coprotein synthesis and epithelial migration.413 Thepresent study shows that cholera toxin stimulated cyclicAMP synthesis increases the ability of cultured cornealepithelial cells to adhere to both plastic and collagenoussubstrates. Recent evidence demonstrates that dermalepithelial cells cultured in the presence of cholera toxinproduce a specific adhesion protein, and that stimulatedcyclic AMP synthesis increases the rate of secretion ofepithelial adhesion glycoproteins.1415 Synthesis of theglycoprotein fibronectin by full thickness corneas isenhanced by cyclic AMP.16 Thus, cholera toxin andforskolin, via cyclic AMP synthesis, may increase thesecretion of adhesive cell glycoproteins, thereby in-creasing cell substrate adhesion. This may act to slowthe rate of migration in culture as cyclic AMP, eitheradded exogenously or synthesized endogenously, slowsmigration of several cell types.17 The lack of effect ofisoproterenol is probably due to the inability of thisdrug to sustain elevated cyclic AMP levels.

The paradoxical effect of cholera toxin to stimulatewound closure in vivo may therefore be consistent with


No. 1 CORNEAL EPITHELIAL WOUND CLOSURE / Jumblorr ond Neufeld 13

its inhibitory effect in the culture model. Originally,we observed that pretreatment in vivo with topicalcholera toxin produced an increased rate of closure ofsuperficial corneal defects; whereas, we now report that,in vitro, cholera toxin slows closure.56 We also observedearlier that cholera toxin slows wound closure in a fullthickness corneal organ culture model in which theepithelium migrates over native substrate to close asuperficial defect.8 Thus the differences observed in vivoand in vitro are unlikely to reflect the sensitivity ofepithelial cells to differing substrates. If cyclic AMPpromotes adhesiveness of migrating cells in vivo, as itdoes in vitro, this mechanism will protect respondingcells from mechanical disruption due to blinking andtherefore appear to promote wound closure. In vitro,this mechanism will slow but not prevent migration.An alternative explanation for cholera toxin stimula-tion of wound closure in vivo may include enhancedepithelial proliferation. Cholera toxin does potentiatethe mitogenic effects of insulin and EGF.5 Choleratoxin may potentiate the influences of other cell typesin vivo or mediators that are absent in epithelial tissueculture. Our data does not permit us to distinguishbetween these hypothetical mechanisms.

Cultured corneal epithelial cells provide a usefulmodel system for investigating the events governingclosure of superficial epithelial defects and for assayingthe actions of exogenous agents on these events. Tissueculture offers several advantages over in vivo and organculture models of wound closure: (1) The extracellularmilieu including nutrients, growth factors and substrateis easily manipulated; (2) Closure of defects occurs inthe absence of other cell types, such as nerves, mes-enchymal fibroblasts, or inflammatory cells; (3) A largenumber of cultures, in this case 24, can be derivedfrom a single cornea; and (4) Cells can be easily labelledand used for cell-substrate adhesion determinations.Multiplate cultures of corneal epithelium should proveuseful for investigating the influence of a variety ofmodulators, drugs and chemicals on wound closureunder defined conditions. Establishment and routineassay of corneal epithelial wound closure in multiwellplates may also prove useful as an in vitro, toxicologicaltest and an alternative to animal testing.

Key words: cornea, epithelium, adenosine cyclic monophos-phate, wound closure, migration, tissue culture

Acknowledgments

We thank Gail Raymond and Nancy McLaughlin for theirexcellent technical assistance.

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corneal wound repair in the rabbit and the rat. Am J Ophthalmol61:55, 1966.

2. Kuwabara T, Perkins DG, and Cogan DG: Sliding of the epi-thelium in experimental corneal wounds. Invest Ophthalmol 15:4, 1976.

3. Buck RC: Hemidesmosomes of normal and regenerating mousecorneal epithelium. Virchows Arch (Cell Pathol) 41:1, 1982.

4. Gipson IK, Kiorpes TC, and Brennan SJ: Epithelial sheet move-ment: effects of tunicamycin on migration and glycoprotein syn-thesis. Dev Biol 101:212, 1984.

5. Jumblatt MM and Neufeld AH: /3-Adrenergic and Serotonergicresponsiveness of rabbit corneal epithelial cells in culture. InvestOphthalmol Vis Sci 24:1139, 1983.

6. Lowry OH, Rosenbrough NJ, Farr AL, and Randall RJ: Proteinmeasurement with the folin phenol reagent. J Biol Chem 193:265, 1951.

7. Jumblatt MM, Fogle JA, and Neufeld AH: Cholera toxin stim-ulates adenosine 3',5'-monophosphate synthesis and epithelialwound closure in the rabbit cornea. Invest Ophthalmol Vis Sci19:1321, 1980.

8. Jumblatt MM and Neufeld AH: Effects of cyclic AMP and cal-cium on corneal epithelial wound closure in vitro. ARVO Ab-stracts. Invest Ophthalmol Vis Sci 22(Suppl):25, 1982.

9. Jumblatt MM and Neufeld AH: Characterization of cyclic AMP-mediated wound closure of the rabbit corneal epithelium. CurrEye Res 1:189, 1981.

10. Stenn KS: Epibolin: a protein of human plasma that supportsepithelial cell movement. Proc Natl Acad Sci (USA) 78:6907,1981.

11. Nishida T, Nakagawa S, Ohasi Y, Awata T, and Manabe R:Fibronectin in corneal wound healing: appearance in culturedrabbit cornea. Jpn J Ophthalmol 26:410, 1982.

12. Fujikawa LS, Foster CS, Harris TJ, Lanigan JM, and Colvin RB:Fibronectin in healing rabbit corneal wounds. Lab Invest 45:120, 1981.

13. Gipson IK and Kiorpes TC: Epithelial sheet movement: proteinand glycoprotein synthesis. Dev Biol 92:259, 1982.

14. Okamoto Y, Sakai H, Sato J, and Akamatsu N: Effects of di-butyryl cyclic AMP on the synthesis of dolichol-linked saccharidesand glycoproteins in cultured hepatoma cells. Biochem J 212:859, 1983.

15. Roberts GP and Jenner L: Glycoproteins and glycosaminoglycanssynthesized by human keratinocytes. Biochem J 212:355, 1983.

16. Nishida T, Tanaka H, Nakagawa S, Sasahe T, Awata T, andManabe R: Fibronectin synthesis by the rabbit cornea: effects ofmouse epidermal growth factor and cyclic AMP analogs. Jpn JOphthalmol 28:196, 1984.

17. Dunlap MK: Cyclic AMP levels in migrating and nonmigratingnewt epidermal cells. J Cell Physiol 104:367, 1980.


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