Articles. Transplantation of tissue-cultured corneal endothelium

Transcription

1 Articles Transplantation of tissue-cultured corneal endothelium Marcia M. Jumblatt, David M. Maurice, and James P. McCulley Cultured endothelial cells have been shown to regain their physiological function when replaced in the rabbit eye. Corneas were wiped free of native endothelium and seeded with cultured cells. After an incubation period, full-thickness buttons were cut from these corneas and transplanted into recipient animals. Clear grafts were obtained only when the donor cells were derived from cultures less than a month old. Light and scanning electron microscopy showed the endothelial cells of these grafts to be present as a slightly irregular monolayer on the posterior surface of the cornea. In corneas made edematous by benzalkonium chloride, the clear graft remained surrounded by thick and, cloudy host tissue. In those grafts with ;l H- 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, the component of the tissue that requires replacement is the endothelial cell layer. There would appear to be substantial advantages in being able to replace this layer alone, with endothelial cells grown in tissue cultures used as the donor material. The possibility of growing pure endothelial cultures was established by Stocker et al.,' and since then, histochemical studies, 2 as well as examination of the morphology 2 ' 3 with transmission electron microscopy, have shown the similarity of the cultured to the native cell. Furthermore, it has been shown that cells cultured as monolayers will re-establish their proper orientation and secrete a Descemet's membranelike material. 3 It is not clear, however, whether cultured From the Division of Ophthalmology, Stanford University Medical Center, Stanford, Calif. This study was supported by National Institutes of Health Grant EY Submitted for publication July 5, Reprint requests: Dr. David M. Maurice, Division of Ophthalmology, Stanford University Medical Center, Stanford, Calif monolayers are capable of reacquiring their normal physiological function, that of pumping fluid out of the stroma and thus keeping it thin and transparent. Unless this occurs, their use in keratoplasty would be without value. The present experiments are designed to test whether endothelial cells seeded onto the Descemet's membrane of corneas lacking endothelium and replaced in a living rabbit's eye are capable of acquiring normal morphology and function. At the same time, it was hoped to develop techniques that might be adapted to the clinical application of cultured endothelial transplantation. These experiments were briefly reported at the ARVO meeting in the spring of Materials and methods Eyes (fresh-iced) were obtained from Pel-Freez Biologicals, Inc., Rogers, Ark., and washed in several changes of sterile saline. Fungal contamination was frequently a problem. In recent experiments it was prevented by immersing the globes for 1 min in 1:1000 Lugol's iodine solution before rinsing them and dissecting the cornea. Corneal endothelial cell cultures were initiated by the methods of Perl man and Baum 5 adapted from /78/ $00.70/ Assoc. for Res. in Vis. and Ophthal., Inc. 1135

2 1136 Jumblatt, Maurice, and McCulley Invest. Ophthalmol. Visual Sci, December 1978 Fig. 1. Seeding endothelial cells onto Descemet's membrane. The conjunctiva is tied to a plastic ring so that the cornea retains its natural curvature. A glass cylinder fits snugly over the sclera and provides a watertight seal for the cell suspension. those of Stacker et al.'; the medium employed was Eagle's Minimal Essential Medium (MEM) with 10% fetal calf serum, 5% calf serum, twicestrength amino acids and vitamins, and 50 ^tg/ml gentamicin. Confluent cell layers in flasks of 75 cm 2 were trypsinized, and the suspended cells were used to initiate cultures in 15 cm 2 flasks. These subcultures were allowed to become confluent, which occurred within 3 to 7 days, and were used as donor material. The layers were trypsinized, and the suspended endothelial cells were gently centrifuged (3000 rpm; 5 min; 20 C) in sterile glass centrifuge tubes. The trypsin solution was decanted, and the cells were suspended at a final concentration of 2 to 5 x 10 5 cells/ml in the complete culture medium. These cells were used to replace the native endothelium of donor corneas. Donor corneas were obtained from young New Zealand White (NZW) rabbits killed by barbiturate overdose. The orbit was exenterated, the conjunctival sac flooded with 1 ml of Neosporin, and sterile technique used thereafter. The cornea was isolated by the method of Dikstein and Maurice 6 and was placed endothelial side up attached to its mounting ring in a plastic dish. The native endothelium was thoroughly wiped from Descemet's membrane by means of a cotton swab. A glass cylinder of 13.5 mm inside diameter and 20 mm high was fitted snugly over the scleral "gasket." The chamber thus formed was filled with 1 ml of cell suspension and an additional 1 ml of medium (Fig. 1). The cylinder was covered with the top of the plastic dish, and the preparation was incubated from 1 to 3 days at 35 C in a humidified incubator with an atmosphere of 95% air, 5% CO 2. During incubation, corneas became too edematous for keratoplasty, and therefore they were osmotically thinned by exposure to dextran-containing medium. Twenty-four hours before surgery, 0.5 ml of incubation medium was removed and replaced with complete medium containing 5% dextran 40 (Sigma Chemical Co., St. Louis, Mo.). Three hours later, 1.0 ml of medium was removed and replaced with 5% dextran medium. After another 3 hr, all the medium was removed and replaced with dextran medium. Incubation was continued as previously described. Immediately before surgery, the incubation medium was decanted, and the glass cylinder was removed from the mounted cornea. The cornea, surrounded by its scleral rim, was cutfiee from the mounting ring and washed gently in two changes of dextran medium in order to remove unattached cells. Control corneas of two types were prepared and incubated as described above. In one, the native cell layer was left intact, and in the other, the cells were wiped off and Descemet's membrane left bare. Recipient rabbits (5 to 6 kg NZW) were tranquilized with 50 ing of thorazine and received 1000 U of intravenous heparin 1 hr before surgery. The iris was dilated with a 0.15 ml of subconjunctival injection of a combination of 1% procaine, 0.5% phenylephrine, and 0.4% homatropine hydrobromide. 7 General anesthesia was induced with sodium pentobarbital (100 to 150 ing, intravenous), and the cornea was anesthetized with proparacaine HCl. Fixation sutures of 4-0 silk were placed beneath two of the extraocular muscles. A 7.0 mm trephine was used to cut the host cornea, and the section was completed with scissors. The open anterior chamber was irrigated with 100 U/ml heparin (purified; Sigma) in balanced salt solution. An identical button was cut from the donor tissue and positioned in the recipient eye. Four cardinal sutures of 8-0 silk held the button in place. A continuous 10-0 nylon monofilament suture was used to join host and graft tissues. The cardinal sutures were removed, as were

3 Volume 17 Number 12 Tissue-cultured corneal endothelium transplant 1137 Fig. 2, Micrographs of endothelium. The cornea was stained with Weigert's hematoxylin, and the layer was then isolated on Descemet's membrane. (X500.) A, Native endothelium stored 3 days in tissue culture medium. The cells are intact, have distinct borders, and retain some regularity. The nuclei stain in this case. B, Endothelium of a cornea seeded with cultured cells and incubated for 3 days. The cells lack clear boundaries and are nonuniformly stained and very irregularly distributed. The nuclei are unstained. the fixation sutures. The eye was injected subconjunctivally with a further 0.15 ml of mydriatic solution, 6.6 ing of triamcinolone diacetate, and 0.15 ml of gentamicin (40 ing/ml) and then taped shut to prevent evaporation until the animal recovered from the anesthesia. The operated eye received daily applications of antibiotic-hydrocortisone and atropine ointments and twice weekly injections of 6.6 mg of triamcinolone diacetate. Keratoplasty was performed on both eyes with normal corneas and those where the endothelium had been totally destroyed by previous treatment with benzalkonium chloride (BAK). 8 In the latter, there is no possibility of host cells re populating the graft. In other cases endothelial cells were labeled with :i H-thymidine before seeding onto donor corneas by adding it at 2.5 ju,ci/ml to the medium in the final culture flask. These corneas were then transplanted into eyes with normal host endothelium. When these animals were killed, the cornea was divided into host and graft parts. The tissue was dissolved in NCS (Amersham/Searle Corp., Arlington Heights, 111.), and radioactivity was determined in a scintillation counter. In four eyes autoradiography of flat preparations of endothelium was carried out. Sections were fixed in Carnoys fluid, air-dried, dipped in Kodak NTB emulsion diluted 1:1 with distilled water, and developed after 1 month. The grafted corneas were examined daily by slit-lamp microscopy, and the thickness of the graft was measured with a pachometer. All host animals were killed within 1 month of surgery. The morphology of endothelial cells from both donor tissues and grafted eyes was examined in flat preparations. Excised corneas were stained by dropping freshly prepared Weigert's hematoxylin into the corneal cup. After 3 to 5 min the stain was removed, and excess stain was rinsed off with 70% ethanol. Stained corneas were placed in dishes containing 70% ethanol, and the Descemet's membrane with attached cellular layer was dissected free from both donor and host areas of the cornea. These fragments were dehydrated in alcohol and toluene, mounted with Permount, and examined by light microscopy. Some grafted corneas were fixed in 4% glutaraldehyde in phosphate buffer and prepared for scanning electron microscopy (SEM).

4 1138 Jumblatt, Maurice, and McCalley Invest. Ophthalmol. Visual Sri. December h DAYS AFTER SURGERY Fig. 3. Variation in the thickness of a normal control ( ) and an experimental (o) corneal graft vs. postoperative time. A tarsorrhaphy was performed on each animal (arrows) and released 24 hr later. The peak in the cun'e was a measurement made immediately after the cutting of the lid suture when the effect of preventing evaporation was at its greatest. Results Morphology of donor tissue. After incubation with culture medium for 3 days, intact corneas showed a continuous but rather irregular endothelium (Fig. 2, A). Control corneas whose endothelia had been removed by wiping showed only the bare surface of Descemet's membrane and a few cellular remnants; no viable endothelial cells were present. Corneas seeded with cultured endothelium showed a complete monolayer of cells, but their arrangement was irregular, and they stained more vividly than intact native cells (Fig. 2, B). Graft evaluation. Keratoplasty was performed into normal eyes and into those with chemically destroyed endothelia. In both cases, in addition to the experimental operations using cultured endothelial cells, controls were carried out in which intact corneas or those without an endothelial layer were used for donor material after being incubated and thinned as previously described. Caution must be exercised in evaluating the results of keratoplasty in rabbits, since evaporation can thin a graft in which the endothelium is lacking and make it appear successful by casual slit-lamp examination. 9 Preventing evaporation in these cases leads to a marked swelling. In the present series, two conditions had to be met for the graft to be considered successful. First, the presence of a continuous endothelial cell layer had to be confirmed by biomicroscopy or histology, and second, the corneal thickness had to remain below 0.5 mm during the final 10 to 30 days postoperatively and not rise above 0.65 mm after evaporation had been prevented for 24 hr by tarsorrhaphy. The criteria of success and failure were clear-cut in the control series, which will be described first. Several grafts failed because of postoperative accidents or infection, and these will not be discussed further. Intact controls. All grafts with native endothelium were successful whether the hosts had normal corneas (two cases) or BAKtreated corneas (three cases). These grafts recovered a mean thickness of 0.35 mm a few days 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 in the slit lamp, and the cell mosaic was regular when viewed under the microscope. Cell-free controls. Every graft made from a cornea in which the endothelium had been

5 Volume 17 N limber 12 Tissue-cultured corneal endothelium transplant 1139 rubbed away became edematous and cloudy after a few days and ultimately swelled to a thickness of 1 mm or more even in the absence of tarsorrhaphy. Fibrous tissue was found to be covering the posterior surface of these graft buttons, the fibroblast-like cells apparently arising from the wound margin, where a ring of fibroblastic scar tissue was present. Experimental grafts Successful grafts. Nineteen grafts into normal eyes were made with donor tissue covered with cultured endothelium. Four of these were clear and thin 1 week after surgery and remained so through the 1-month observation period. In those grafts labeled with 3 H-thymidine, radioactivity could be found only in the graft area by scintillation counting. Autoradiography showed labeled nuclei only in the endothelial cell layer of the graft. To provide further evidence that the source of functional graft endothelium is the donor cells and is not due to migration or mitosis of host cells, we performed a series of grafts into animals whose endothelium had been previously destroyed by irrigation with 0.05% BAK. 8 Two of eight grafts were clear by 1 week after surgery and remained clear and thin until the animal was killed. The host corneas remained edematous throughout the observation period (Fig. 4, A). In these successful grafts, the endothelial cell layer was visible with the slit lamp, and specular microscopy in vivo indicated cells of slightly irregular boundaries with a slightly gray rather than a normal yellow color. A stained preparation of graft endothelium showed compact cells with well-defined cell borders (Fig. 5, A) and appeared similar to a preparation of the host endothelium {Fig. 5, B). SEM revealed the endothelial cells of the graft to be of somewhat irregular geometry but to exhibit normal surface morphology and junctional interdigitation (Fig. 4, B). Epithelial defects healed within a week, and this layer remained intact in all experimental animals as shown by fluorescein staining. The thickness of the graft tended to show a day-by-day variation, especially in the two Fig. 4. A, Graft into an edematous cornea. The host endothelium was destroyed by irrigation with BAK before keratoplasty. One month after surgery the graft with cultured endothelium was clear and compact. The host tissue was edematous and vascularized. B, Scanning election micrograph of graft endothelium. A continuous inonolayer of cells is present. The cells are irregular in shape but show surface microvilli and junctional interdigitations characteristic of endothelial cells (X4000.) BAK-treated eyes (Fig. 3), but from median values, the average of the six eyes was 0.44 mm. The graft stroma came from randomly available animals, so that no value can be attached to its original thickness; however, in the normal control grafts the average thickness was measured at 0.35 mm. After 24 hr tarsorrhaphy, the thickness of the experimental grafts rose by an average of 0.11 mm (range: 0.07 to 0.16) compared to the value of 0.07 mm found for intact controls. Failed grafts. The greater number of grafts with replaced endothelial cells cleared ini-

6 1140 Jumblatt, Maurice, and McCulley Invest, Ophtlutlmo!. Visual Sci. December 1978 Fig. 5. Micrographs of endothelium stained and treated as in Fig. 2. (x500.) A, Graft endothelium from cultured graft into normal eye 1 month after operation. The cells are densely packed in a monolayer but are slightly irregular in outline. The small stained particles are red blood cells. B, Host endothelium from the same eye appears normal. tially after surgery but became edematous within 7 to 10 days and remained so throughout the observation period, like control grafts lacking endothelium. No inflammatory signs were seen in the eyes, nor was an endothelial rejection line noted. 10 The epithelium remained intact in these eyes. Stained preparations of such grafts showed the posterior surface to be covered with fibroblast-like cells, apparently originating from the wound margin, where a dense ring of fibrous tissue was often present. Autoradiography of failed 3 H-thymidine-labeled grafts showed occasional dark nuclei in the region of the fibrous tissue. It is not clear whether these nuclei belong to endothelial cells that have transformed to fibroblast-like cells 11 or have remained unchanged. In eyes previously treated with BAK, fibroblast-like cells often covered the posterior surface of the host cornea as well. Examination of the data reveals that only cells which have been in culture longer than 1 month yielded consistently failing grafts. Clear and compact grafts were obtained only when cells in culture for less than 1 month were used as donor material. Discussion These experiments show that endothelial cells seeded onto the bare Descemet's membrane of corneas which are used for keratoplasty are capable of maintaining the graft in a thin and transparent state. These transplanted cells acquire a morphology similar to that of native cells and can regain some measure of physiological function. Even in clear grafts, the stroma remained somewhat edematous, indicating that the endothelial pump had not acquired its full activity. However, for visual function the graft probably would have been adequate. This degree of success encourages a continuation of research with the technique. The relatively large proportion of failures in the cultured grafts requires some comment. The endothelial cells appeared to have been replaced by fibroblasts in these eyes.

7 Volume 17 Number 12 Tissue-cidtured corneal endothelium transplant 1141 The behavior of the eye does not suggest that an immune reaction was the original cause of cell loss. Possibly the cells were not sufficiently well attached to Descemet's membrane or each other in thefirstplace to resist fibroblast invasions. Thefibroblast-likecells on the posterior surface of failed grafts appeared to originate from the wound margin, although we have not ruled out the possibility that they arose from the transformation of endothelial cells. Cells in culture for longer than 1 month invariably failed to provide functional endothelial layers. This fact may reflect the limited capacity of the rabbit endothelial cell to divide, or it may reflect some metabolic need not provided in our tissue culture system. It has recently been demonstrated that some growth factors promote endothelial mitosis and differentiation in tissue culture. 12 ' 13 These factors may prolong the useful life of donor cultures and permit more uniformly successful surgery. The relative degree of success in the recovery of physiological function in transplanted cells encourages further research into their clinical use as monolayer transplants. In those cases where endothelial involvement in corneal edema is the primary indication for transplant surgery, monolayers of cells could be introduced either by seeding in the host button or by implanting a permeable membrane and an attached endothelial cell layer. We are currently investigating this latter possibility. Scanning electron micrographs were prepared by Mr. Neal Burstein. REFERENCES 1. Stocker, F. W., Eiring, A., Georgiade, R., and Georgiade, N.: A tissue culture technique for growing corneal epithelial, stromal and endothelial tissues separately, Am. J. Ophthalmol. 46:294, Lowry, G. M.: Comeal endothelium in vitro: characterization by ultrastnicture and histochemistry, INVEST. OPHTHALMOL. 5:355, Perlman, M., Bauni, J., and Kaye, G.: Fine structure and collagen synthetic activity of monolayer cultures of rabbit corneal endothelium, J. Cell Biol. 63:306, 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, Dikstein, S., and Maurice, D.: The metabolic basis to the fluid pump in the cornea, J. Physiol. 221:29, Brockhurst, R., Schepens, C. L., Okamura, I. D., Regan, C. D. J., and McMeel, J. W.: Scleral buckling procedures. VIII. Preoperative complications, Arch. Ophthalmol. 74:792, Maurice, D., and Perlman, M.: Permanent destruction of the corneal endothelium in rabbits, IN- VEST. OPHTHALMOL. VISUAL SCI. 16:647, Sherrard, E. S.: Full thickness keratoplasty in the rabbit: an unsatisfactory index of donor integrity, Exp. Eye Res. 18:135, Khodadoust, A. A., and Silverstein, A. M.: Transplantation and rejection of individual cells of the cornea, INVEST. OPHTHALMOL. 8:180, Silbert, J. E., and Baum, J. L.: Origin of the retrocorneal membrane, INVEST. OPHTHALMOL. VISUAL SCI. 17(ARVO Suppl.):253, 1978 (abst.). 12. Perlman, M. M.: Growth stimulation of corneal endothelial 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 proliferation in vitro by fibroblast and epidermal growth factors, Exp. Eye Res. 25:75, 1977.