transplantation of tissue-cultured corneal...

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Transplantation of tissue-culturedcorneal endothelium

Marcia M. Jumblatt, David M. Maurice, and James P. McCulley

Cultured endothelial cells have been shown to regain their physiological function when re-placed in the rabbit eye. Corneas were wiped free of native endothelium and seeded withcultured cells. After an incubation period, full-thickness buttons were cut from these corneasand transplanted into recipient animals. Clear grafts were obtained only when the donor cellswere derived from cultures less than a month old. Light and scanning electron microscopyshowed the endothelial cells of these grafts to be present as a slightly irregular monolayer on theposterior surface of the cornea. In corneas made edematous by benzalkonium chloride, theclear graft remained surrounded by thick and, cloudy host tissue. In those grafts with ;lH-thymidine-labeled cells, radioactivity was limited to the host tissue.

Key words: corneal endothelium, tissue culture, corneal transplantation,endothelial physiology

I n many cases requiring keratoplasty, thecomponent of the tissue that requires re-placement is the endothelial cell layer. Therewould appear to be substantial advantages inbeing able to replace this layer alone, withendothelial cells grown in tissue culturesused as the donor material. The possibility ofgrowing pure endothelial cultures was estab-lished by Stocker et al.,' and since then, his-tochemical studies,2 as well as examination ofthe morphology2'3 with transmission electronmicroscopy, have shown the similarity of thecultured to the native cell. Furthermore, ithas been shown that cells cultured as mono-layers will re-establish their proper orienta-tion and secrete a Descemet's membrane-like material.3

It is not clear, however, whether cultured

From the Division of Ophthalmology, Stanford Univer-sity Medical Center, Stanford, Calif.

This study was supported by National Institutes ofHealth Grant EY 00431.

Submitted for publication July 5, 1978.Reprint requests: Dr. David M. Maurice, Division of

Ophthalmology, Stanford University Medical Center,Stanford, Calif. 94305.

monolayers are capable of reacquiring theirnormal physiological function, that of pump-ing fluid out of the stroma and thus keeping itthin and transparent. Unless this occurs,their use in keratoplasty would be withoutvalue. The present experiments are designedto test whether endothelial cells seeded ontothe Descemet's membrane of corneas lackingendothelium and replaced in a living rabbit'seye are capable of acquiring normal mor-phology and function. At the same time, itwas hoped to develop techniques that mightbe adapted to the clinical application of cul-tured endothelial transplantation.

These experiments were briefly reportedat the ARVO meeting in the spring of 1977.4

Materials and methods

Eyes (fresh-iced) were obtained from Pel-FreezBiologicals, Inc., Rogers, Ark., and washed in sev-eral changes of sterile saline. Fungal contamina-tion was frequently a problem. In recent experi-ments it was prevented by immersing the globesfor 1 min in 1:1000 Lugol's iodine solution beforerinsing them and dissecting the cornea. Cornealendothelial cell cultures were initiated by themethods of Perl man and Baum5 adapted from

0146-0404/78/121135+07$00.70/0 © 1978 Assoc. for Res. in Vis. and Ophthal., Inc. 1135

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1136 Jumblatt, Maurice, and McCulleyInvest. Ophthalmol. Visual Sci,

December 1978

Fig. 1. Seeding endothelial cells onto Descemet'smembrane. The conjunctiva is tied to a plastic ringso that the cornea retains its natural curvature. Aglass cylinder fits snugly over the sclera and pro-vides a watertight seal for the cell suspension.

those of Stacker et al.'; the medium employed wasEagle's Minimal Essential Medium (MEM) with10% fetal calf serum, 5% calf serum, twice-strength amino acids and vitamins, and 50 ^tg/mlgentamicin. Confluent cell layers in flasks of 75cm2 were trypsinized, and the suspended cellswere used to initiate cultures in 15 cm2 flasks.These subcultures were allowed to become conflu-ent, which occurred within 3 to 7 days, and wereused as donor material. The layers were trypsin-ized, and the suspended endothelial cells weregently centrifuged (3000 rpm; 5 min; 20° C) insterile glass centrifuge tubes. The trypsin solutionwas decanted, and the cells were suspended at afinal concentration of 2 to 5 x 105 cells/ml in thecomplete culture medium. These cells were usedto replace the native endothelium of donor corneas.

Donor corneas were obtained from young NewZealand White (NZW) rabbits killed by barbitu-rate overdose. The orbit was exenterated, the con-junctival sac flooded with 1 ml of Neosporin, andsterile technique used thereafter. The cornea wasisolated by the method of Dikstein and Maurice6

and was placed endothelial side up attached to itsmounting ring in a plastic dish. The native en-dothelium was thoroughly wiped from Descemet's

membrane by means of a cotton swab. A glass cyl-inder of 13.5 mm inside diameter and 20 mm highwas fitted snugly over the scleral "gasket." Thechamber thus formed was filled with 1 ml of cellsuspension and an additional 1 ml of medium(Fig. 1). The cylinder was covered with the top ofthe plastic dish, and the preparation was incu-bated from 1 to 3 days at 35° C in a humidifiedincubator with an atmosphere of 95% air, 5% CO2.

During incubation, corneas became too edema-tous for keratoplasty, and therefore they were os-motically thinned by exposure to dextran-contain-ing medium. Twenty-four hours before surgery,0.5 ml of incubation medium was removed andreplaced with complete medium containing 5%dextran 40 (Sigma Chemical Co., St. Louis, Mo.).Three hours later, 1.0 ml of medium was removedand replaced with 5% dextran medium. After an-other 3 hr, all the medium was removed andreplaced with dextran medium. Incubation wascontinued as previously described. Immediatelybefore surgery, the incubation medium was de-canted, and the glass cylinder was removed fromthe mounted cornea. The cornea, surrounded byits scleral rim, was cutfiee from the mounting ringand washed gently in two changes of dextranmedium in order to remove unattached cells.

Control corneas of two types were prepared andincubated as described above. In one, the nativecell layer was left intact, and in the other, the cellswere wiped off and Descemet's membrane leftbare.

Recipient rabbits (5 to 6 kg NZW) were tran-quilized with 50 ing of thorazine and received1000 U of intravenous heparin 1 hr before surgery.The iris was dilated with a 0.15 ml of subconjunc-tival injection of a combination of 1% procaine,0.5% phenylephrine, and 0.4% homatropine hy-drobromide.7 General anesthesia was inducedwith sodium pentobarbital (100 to 150 ing, intra-venous), and the cornea was anesthetized withproparacaine HCl. Fixation sutures of 4-0 silkwere placed beneath two of the extraocularmuscles.

A 7.0 mm trephine was used to cut the hostcornea, and the section was completed with scis-sors. The open anterior chamber was irrigatedwith 100 U/ml heparin (purified; Sigma) in bal-anced salt solution. An identical button was cutfrom the donor tissue and positioned in the recip-ient eye. Four cardinal sutures of 8-0 silk held thebutton in place. A continuous 10-0 nylon monofil-ament suture was used to join host and graft tis-sues. The cardinal sutures were removed, as were


Volume 17Number 12 Tissue-cultured corneal endothelium transplant 1137

Fig. 2, Micrographs of endothelium. The cornea was stained with Weigert's hematoxylin, andthe layer was then isolated on Descemet's membrane. (X500.) A, Native endothelium stored 3days in tissue culture medium. The cells are intact, have distinct borders, and retain someregularity. The nuclei stain in this case. B, Endothelium of a cornea seeded with cultured cellsand incubated for 3 days. The cells lack clear boundaries and are nonuniformly stained andvery irregularly distributed. The nuclei are unstained.

the fixation sutures. The eye was injected subcon-junctivally with a further 0.15 ml of mydriatic so-lution, 6.6 ing of triamcinolone diacetate, and 0.15ml of gentamicin (40 ing/ml) and then taped shutto prevent evaporation until the animal recoveredfrom the anesthesia. The operated eye receiveddaily applications of antibiotic-hydrocortisone andatropine ointments and twice weekly injections of6.6 mg of triamcinolone diacetate.

Keratoplasty was performed on both eyes withnormal corneas and those where the endotheliumhad been totally destroyed by previous treatmentwith benzalkonium chloride (BAK).8 In the latter,there is no possibility of host cells re populating thegraft.

In other cases endothelial cells were labeledwith :iH-thymidine before seeding onto donorcorneas by adding it at 2.5 ju,Ci/ml to the mediumin the final culture flask. These corneas were thentransplanted into eyes with normal host en-dothelium. When these animals were killed, thecornea was divided into host and graft parts. Thetissue was dissolved in NCS (Amersham/SearleCorp., Arlington Heights, 111.), and radioactivitywas determined in a scintillation counter. In four

eyes autoradiography of flat preparations of en-dothelium was carried out. Sections were fixed inCarnoys fluid, air-dried, dipped in Kodak NTBemulsion diluted 1:1 with distilled water, and de-veloped after 1 month.

The grafted corneas were examined daily byslit-lamp microscopy, and the thickness of thegraft was measured with a pachometer. All hostanimals were killed within 1 month of surgery.The morphology of endothelial cells from bothdonor tissues and grafted eyes was examined in flatpreparations. Excised corneas were stained bydropping freshly prepared Weigert's hematoxylininto the corneal cup. After 3 to 5 min the stain wasremoved, and excess stain was rinsed off with 70%ethanol. Stained corneas were placed in dishescontaining 70% ethanol, and the Descemet'smembrane with attached cellular layer was dis-sected free from both donor and host areas of thecornea. These fragments were dehydrated in al-cohol and toluene, mounted with Permount, andexamined by light microscopy. Some graftedcorneas were fixed in 4% glutaraldehyde in phos-phate buffer and prepared for scanning electronmicroscopy (SEM).


1138 Jumblatt, Maurice, and McCalley Invest. Ophthalmol. Visual Sri.December 1978

0.6 h

DAYS AFTER SURGERY

Fig. 3. Variation in the thickness of a normal control (•) and an experimental (o) corneal graftvs. postoperative time. A tarsorrhaphy was performed on each animal (arrows) and released24 hr later. The peak in the cun'e was a measurement made immediately after the cutting ofthe lid suture when the effect of preventing evaporation was at its greatest.

Results

Morphology of donor tissue. After incuba-tion with culture medium for 3 days, intactcorneas showed a continuous but rather ir-regular endothelium (Fig. 2, A). Controlcorneas whose endothelia had been removedby wiping showed only the bare surface ofDescemet's membrane and a few cellularremnants; no viable endothelial cells werepresent. Corneas seeded with cultured en-dothelium showed a complete monolayer ofcells, but their arrangement was irregular,and they stained more vividly than intact na-tive cells (Fig. 2, B).

Graft evaluation. Keratoplasty was per-formed into normal eyes and into those withchemically destroyed endothelia. In bothcases, in addition to the experimental opera-tions using cultured endothelial cells, con-trols were carried out in which intact corneasor those without an endothelial layer wereused for donor material after being incubatedand thinned as previously described.

Caution must be exercised in evaluatingthe results of keratoplasty in rabbits, sinceevaporation can thin a graft in which the en-dothelium is lacking and make it appear suc-cessful by casual slit-lamp examination.9 Pre-

venting evaporation in these cases leads to amarked swelling. In the present series, twoconditions had to be met for the graft to beconsidered successful. First, the presence ofa continuous endothelial cell layer had to beconfirmed by biomicroscopy or histology,and second, the corneal thickness had to re-main below 0.5 mm during the final 10 to 30days postoperatively and not rise above 0.65mm after evaporation had been prevented for24 hr by tarsorrhaphy. The criteria of successand failure were clear-cut in the controlseries, which will be described first.

Several grafts failed because of postopera-tive accidents or infection, and these will notbe discussed further.

Intact controls. All grafts with native en-dothelium were successful whether the hostshad normal corneas (two cases) or BAK-treated corneas (three cases). These grafts re-covered a mean thickness of 0.35 mm a fewdays after surgery, and this rose by 0.07 mm(0.04 to 0.13) after 24 hr tarsorrhaphy(Fig. 3). The endothelial reflex was normal inthe slit lamp, and the cell mosaic was regularwhen viewed under the microscope.

Cell-free controls. Every graft made from acornea in which the endothelium had been


Volume 17N limber 12 Tissue-cultured corneal endothelium transplant 1139

rubbed away became edematous and cloudyafter a few days and ultimately swelled to athickness of 1 mm or more even in the ab-sence of tarsorrhaphy. Fibrous tissue wasfound to be covering the posterior surface ofthese graft buttons, the fibroblast-like cellsapparently arising from the wound margin,where a ring of fibroblastic scar tissue waspresent.

Experimental graftsSuccessful grafts. Nineteen grafts into

normal eyes were made with donor tissuecovered with cultured endothelium. Four ofthese were clear and thin 1 week after sur-gery and remained so through the 1-monthobservation period. In those grafts labeledwith 3H-thymidine, radioactivity could befound only in the graft area by scintillationcounting. Autoradiography showed labelednuclei only in the endothelial cell layer of thegraft.

To provide further evidence that thesource of functional graft endothelium is thedonor cells and is not due to migration ormitosis of host cells, we performed a series ofgrafts into animals whose endothelium hadbeen previously destroyed by irrigation with0.05% BAK.8 Two of eight grafts were clearby 1 week after surgery and remained clearand thin until the animal was killed. The hostcorneas remained edematous throughout theobservation period (Fig. 4, A).

In these successful grafts, the endothelialcell layer was visible with the slit lamp, andspecular microscopy in vivo indicated cells ofslightly irregular boundaries with a slightlygray rather than a normal yellow color. Astained preparation of graft endotheliumshowed compact cells with well-defined cellborders (Fig. 5, A) and appeared similar to apreparation of the host endothelium {Fig. 5,B). SEM revealed the endothelial cells of thegraft to be of somewhat irregular geometrybut to exhibit normal surface morphology andjunctional interdigitation (Fig. 4, B). Epithe-lial defects healed within a week, and thislayer remained intact in all experimentalanimals as shown by fluorescein staining.

The thickness of the graft tended to show aday-by-day variation, especially in the two

Fig. 4. A, Graft into an edematous cornea. Thehost endothelium was destroyed by irrigation withBAK before keratoplasty. One month after surgerythe graft with cultured endothelium was clear andcompact. The host tissue was edematous and vas-cularized. B, Scanning election micrograph ofgraft endothelium. A continuous inonolayer ofcells is present. The cells are irregular in shapebut show surface microvilli and junctional in-terdigitations characteristic of endothelial cells(X4000.)

BAK-treated eyes (Fig. 3), but from medianvalues, the average of the six eyes was 0.44mm. The graft stroma came from randomlyavailable animals, so that no value can be at-tached to its original thickness; however, inthe normal control grafts the average thick-ness was measured at 0.35 mm. After 24 hrtarsorrhaphy, the thickness of the experi-mental grafts rose by an average of 0.11 mm(range: 0.07 to 0.16) compared to the value of0.07 mm found for intact controls.

Failed grafts. The greater number of graftswith replaced endothelial cells cleared ini-


1140 Jumblatt, Maurice, and McCulleyInvest, Ophtlutlmo!. Visual Sci.

December 1978

Fig. 5. Micrographs of endothelium stained and treated as in Fig. 2. (x500.) A, Graft en-dothelium from cultured graft into normal eye 1 month after operation. The cells are denselypacked in a monolayer but are slightly irregular in outline. The small stained particles are redblood cells. B, Host endothelium from the same eye appears normal.

tially after surgery but became edematouswithin 7 to 10 days and remained so through-out the observation period, like control graftslacking endothelium. No inflammatory signswere seen in the eyes, nor was an endothelialrejection line noted.10 The epithelium re-mained intact in these eyes.

Stained preparations of such grafts showedthe posterior surface to be covered withfibroblast-like cells, apparently originatingfrom the wound margin, where a dense ringof fibrous tissue was often present. Auto-radiography of failed 3H-thymidine-labeledgrafts showed occasional dark nuclei in theregion of the fibrous tissue. It is not clearwhether these nuclei belong to endothelialcells that have transformed to fibroblast-likecells11 or have remained unchanged. In eyespreviously treated with BAK, fibroblast-likecells often covered the posterior surface ofthe host cornea as well.

Examination of the data reveals that onlycells which have been in culture longer than

1 month yielded consistently failing grafts.Clear and compact grafts were obtained onlywhen cells in culture for less than 1 monthwere used as donor material.

Discussion

These experiments show that endothelialcells seeded onto the bare Descemet's mem-brane of corneas which are used for kerato-plasty are capable of maintaining the graft ina thin and transparent state. These trans-planted cells acquire a morphology similar tothat of native cells and can regain somemeasure of physiological function. Even inclear grafts, the stroma remained somewhatedematous, indicating that the endothelialpump had not acquired its full activity. How-ever, for visual function the graft probablywould have been adequate. This degree ofsuccess encourages a continuation of researchwith the technique.

The relatively large proportion of failuresin the cultured grafts requires some com-ment. The endothelial cells appeared to havebeen replaced by fibroblasts in these eyes.


Volume 17Number 12 Tissue-cidtured corneal endothelium transplant 1141

The behavior of the eye does not suggest thatan immune reaction was the original cause ofcell loss. Possibly the cells were not suf-ficiently well attached to Descemet's mem-brane or each other in the first place to resistfibroblast invasions. The fibroblast-like cellson the posterior surface of failed grafts ap-peared to originate from the wound margin,although we have not ruled out the possibil-ity that they arose from the transformation ofendothelial cells.

Cells in culture for longer than 1 monthinvariably failed to provide functional en-dothelial layers. This fact may reflect thelimited capacity of the rabbit endothelial cellto divide, or it may reflect some metabolicneed not provided in our tissue culture sys-tem. It has recently been demonstrated thatsome growth factors promote endothelialmitosis and differentiation in tissue cul-ture. 12' 13 These factors may prolong the use-ful life of donor cultures and permit moreuniformly successful surgery.

The relative degree of success in the re-covery of physiological function in trans-planted cells encourages further researchinto their clinical use as monolayer trans-plants. In those cases where endothelial in-volvement in corneal edema is the primaryindication for transplant surgery, monolayersof cells could be introduced either by seedingin the host button or by implanting a perme-able membrane and an attached endothelialcell layer. We are currently investigating thislatter possibility.

Scanning electron micrographs were prepared by Mr.Neal Burstein.

REFERENCES1. Stocker, F. W., Eiring, A., Georgiade, R., and

Georgiade, N.: A tissue culture technique for grow-ing corneal epithelial, stromal and endothelial tis-sues separately, Am. J. Ophthalmol. 46:294, 1958.

2. Lowry, G. M.: Comeal endothelium in vitro: char-acterization by ultrastnicture and histochemistry,INVEST. OPHTHALMOL. 5:355, 1966.

3. Perlman, M., Bauni, J., and Kaye, G.: Fine struc-ture and collagen synthetic activity of monolayercultures of rabbit corneal endothelium, J. Cell Biol.63:306, 1974.

4. Maurice, D. M., McCulley, J. P., and Perlman,M. M.: Donor endothelium from tissue culture,INVEST. OPHTHALMOL. VISUAL SCI. 16(ARVO

Suppl.):103, 1977 (abst).5. Perlman, M., and Baum, J.: The mass culture of

rabbit comeal endothelial cells, Arch. Ophthalmol.92:235, 1974.

6. Dikstein, S., and Maurice, D.: The metabolic basisto the fluid pump in the cornea, J. Physiol. 221:29,1972.

7. Brockhurst, R., Schepens, C. L., Okamura, I. D.,Regan, C. D. J., and McMeel, J. W.: Scleral buck-ling procedures. VIII. Preoperative complications,Arch. Ophthalmol. 74:792, 1965.

8. Maurice, D., and Perlman, M.: Permanent de-struction of the corneal endothelium in rabbits, IN-VEST. OPHTHALMOL. VISUAL SCI. 16:647, 1977.

9. Sherrard, E. S.: Full thickness keratoplasty in therabbit: an unsatisfactory index of donor integrity,Exp. Eye Res. 18:135, 1974.

10. Khodadoust, A. A., and Silverstein, A. M.: Trans-plantation and rejection of individual cells of thecornea, INVEST. OPHTHALMOL. 8:180, 1969.

11. Silbert, J. E., and Baum, J. L.: Origin of the retro-corneal membrane, INVEST. OPHTHALMOL. VISUAL

SCI. 17(ARVO Suppl.):253, 1978 (abst.).12. Perlman, M. M.: Growth stimulation of corneal en-

dothelial cells in vitro, INVEST. OPHTHALMOL. VI-SUAL SCI. 17(ARVO Suppl.):253, 1978 (abst).

13. Gospodarowicz, D., Mescher, A. L., and Birdwell,C. R.: Stimulation of corneal endothelial cell prolif-eration in vitro by fibroblast and epidermal growthfactors, Exp. Eye Res. 25:75, 1977.


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