Corneal Endothelial Transplantation

Corneal Endothelial Transplantation JAMES P. McCULLEY, MD, DAVID M. MAURICE, PhD, BARRY D. SCHWARTZ, PhD

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Abstract: Patients with visually significant corneal edema, secondary to endothelial dysfunction, before the development of scarring or vascularization, need only have the corneal endothelium replaced to restore corneal clarity. This fact, plus the lack of consistently available donor material, prompted us to evaluate tissue cultured corneal endothelium (TCCE) as a donor source. We have shown that TCCE, when transplanted, can regain normal morphology and physiologic function. To accomplish practical use of autologous stroma, a transparent gelatin membrane which can serve as substrate for endothelial growth in tissue culture, has been developed. This cellular membrane has been transplanted successfully in rabbits with good functional results. It is hoped that ultimately this technique can be developed for routine use in man. [Key words: artificial membrane, corneal edema, corneal endothelium, corneal transplant, cyanoacrylate adhesive, donor tissue, keratoplasty, tissue culture.] Ophthalmology 87:194-201, 1980

Patients with corneal edema secondary to endothelial dysfunction, prior to the development of scarring and vascularization, need only have the endothelium replaced to From the Division of Ophthalmology, Stanford University Medical School, Stanford. Presented at the Eighty-Fourth Annual Meeting of the American Academy of Ophthalmology, San Francisco, November 5-9, 1979. Supported by Grant EY 00431 from the National Institutes of Health.

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Reprint request to James P. McCulley, MD, Division of Ophthalmology, Stanford University Medical Center, Stanford, CA 94305.

have return of normal corneal clarity. This fact, plus the lack of consistently available donor material, led us to evaluate the possibility of using tissue cultured corneal endothelium as a source of donor material. There are several theoretical advantages to the use of such material. By maintaining endothelium in tissue culture, one could have consistently available donor material by combining the tissue cultured endothelium with stromal tissue from cadavers. This would allow one to use almost all acquired cadaver corneas for transplantation by replacing unhealthy or aged endothelium with cells from tissue culture. It is also probable that one would decrease the immunologic challenge to the recipient by providing an endothelial layer from multiple sources thus decreasing the challenge from any single antigen. Also, if one were able to use autologous stroma, it would be possible to transplant only the endothelial layer, further decreasing the antigenic challenge. Another advantage is that one could transplant cells which were physiologically and morphologically uniform and capable of increased mitotic activity. With the ability to maintain corneal endothelium in tissue culture established, 1 •2 one of us (DMM) suggested that endothelium from tissue culture could be used to repopulate corneas in which there was an unhealthy endothelial layer. This was proposed at a Corneal Task Force at the National Eye Institute in 1972, the proceedings of which were subsequently published. 3 This was first evaluated in rabbit eyes that had the endothelium destroyed by irrigating the anterior chamber with benzalkonium chloride. 4 After the eye recovered from the initial injury, which left an edematous cornea, a suspension of 5 x 10 5 cells was

0161-6420/80/0300/0194/$00.90 ©American Academy of Ophthalmology

Fig 1. Injection of 5 x 10 5 cells into the anterior chamber of an eye which has had the corneal endothelium chemically destroyed with benzalkonium chloride. The cornea is held dependent for four hours, however, cells tended to attach as random clumps, not only to cornea but to iris and lens.

injected into the anterior chamber. The corneas were then held dependent for approximately four hours in the hopes that the endothelial cells would attach to the denuded Descemet's membrane (Fig 1). Unfortunately only scattered clumps of endothelial cells attached not only to cornea, but to iris and lens. 5 This approach was therefore abandoned. The next approach was to seed rabbit corneas, after mechanically removing the native endothelium, with homologous endothelial cells from tissue culture 6 •7 (Fig 2). When these corneas were used as donor material in a standard penetrating keratoplasty in rabbits, it was possible to demonstrate the recovery of normal endothelial morphology and physiologic function. This has since been confirmed by others using heterologous cells. 8 There was an 80% success rate when the endothelial cells were maintained in tissue culture for one month or less; however, when cells were maintained in tissue culture without ·growth factors for longer than one month, the success rate was poor. This reflected either a lack of a required factor or nutrient in the growth medium or an inherent limited mitotic capability of the rabbit corneal endothelium. It was felt that a system such as this, using homologous stroma and tissue

cultured endothelium, would allow one to have a modular system. The availability of donor material could be more predictable. However, the overall efficiency and ability to use autologous stroma would be limited by the seeding time of the endothelial cells t() stroma. If the seeding time of cells to stroma exceeds 10 to 15 minutes, the potential for complications in the form of infection, hemorrhage, choroidal detachment, hypotony, and cataracts would far outweigh the potential benefits. In order to use autologous stroma and have the eye open for a minimal period of time, a thin membrane was developed on which corneal endothelial cells could be grown 5 •9 (Fig 3). The principal problems with an approach such as this included wrinkling of the membrane, attachment of the membrane to autologous stroma, and fibroblastic overgrowth either between the membrane and stroma or on the posterior surface of the membrane. Initially the membrane was formed within a platinum loop and cells were grown on its surface. The membrane was then attached to autologous stroma using liquid gelatin. 5 The initial success rate was poor' however the technique has now evolved such that there is a high success rate, the preliminary results of which have been reported. 10 The

Fig 2. Homologous stroma seeded with a suspension of homologous corneal endothelial cells from tissue culture. Cells settle by gravity onto the stromal surface and attach. Seeding time ranged from 12 hours to four days. The corneal stroma seeded with endothelial cells can then be used as the donor source for standard penetrating keratoplasty.

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Fig 3. A thin gelatin membrane cross-linked with gluteraldehyde and detoxified, serves as substrate for growth of tissue cultured corneal endothelial cells. The membrane with cells attached is then transplanted into an eye using autologous stroma.

current approach and results obtained with it are reported.

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Rabbit corneal endothelial cells were obtained andmaintained by methods previously described. 7 •10 The cells were labeled with tritiated thymidine. Each membrane was inoculated with 400 cells/mm 2 which results, after two to three doublings, in a final cell density of approximately 2500 cells/mm. 2

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MEMBRANE FORMATION

Initially thick gelatin membranes, three to five microns thick, crosslinked with gluteraldehyde and detoxified, were used; however, we currently are using membranes that are one micron thick. This results in a strong, transparent, permeableto-water flow and solute flux, wrinkle-free

membrane which supports the growth of tissue cultured corneal endothelial cells. 9 These membranes are formed within a 13-mm cut-out in millipore filter (Fig 4) which is fashioned so that it can be mounted in a modified Sykes-Moore chamber (Fig 5). ATTACHMENT OF MEMBRANE TO AUTOLOGOUS STROMA

The surgical procedure described below provides a 6.5-mm corneal button from which the endothelium is mechanically removed. Various substances including gelatin, platelets-fibrinogen-thrombin, polysaccharides, amino acids, fibronectin, and cyanoacrylates have been used in attempts to attach the membrane to autologous stroma. Various dilutions of and methods of applying cyanoacrylate have been utilized including undiluted and diluted methyl-,

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Fig 4. A 1-~m thick gluteraldehyde cross-linked gelatin membrane is formed within a 13 mm cut-out in millipore filter. The millipore filter is of appropriate outside diameter so that it can be mounted in a modified Sykes-Moore tissue culture chamber.

Fig 5. A modified Sykes-Moore chamber with mounted membrane is shown. The inside surface of the membrane has been seeded with corneal endothelial cells and maintained in tissue culture until a cellular density of 2500 cells/mm 2 is attained prior to mounting in the chamber. The chamber is filled with tissue culture medium identical to that in which the cells were grown. The chamber with mounted membrane is shown with a corneal button placed on its center.

Fig 6. After removal of endothelium from the cornea, it is placed posterior surface down on a membrane mounted in a Sykes-Moore chamber. The cornea is then glued to the membrane by sequentially lifting small segments of the cornea and applying cy a noacrylate adhesive between membrane and cornea. The cyanoacrylate is delivered with a specially designed "glue gun".

ethyl-, and butylcyanoacrylate. The diluent used to date, is methylene chloride. The cyanoacrylate has been applied using a specially devised glue gun which allows the application of minute quantities of the diluted or undiluted cyanoacrylate . The glue is applied by sequentially lifting small segments of the cornea off the mounted membrane and applying a small amount of cyanoacrylate to the membrane surface (Fig 6). The cornea is then replaced before the glue has an opportunity to polymerize, thus creating an almost complete peripheral adhesion between membrane and cornea (Fig 7).

SURGICAL PROCEDURE

Ten- to twelve-pound New Zealand albino rabbits are placed under general anesthesia

after dilation of the pupil and systemic heparinization as previously described. 7 Tubing is placed in the anterior chamber so that there is a continuous flow of tissue culture medium, with heparin added, throughout the entire surgical procedure. A 6.5-mm trephine is used to obtain an autologous keratoplasty button. The endothelium or endothelium and Descemet's membrane is then mechanically removed. The cornea is then placed, posterior surface down, on a membrane with endothelial cells attached which has been mounted in a modified Sykes-Moore chamber. Cornea and membrane are then glued together as described (Figs 5, 6). The chamber is then partially dismantled and a 7-mm trephine is used to free the cornea with attached membrane from the remainder of the membrane. The button thus obtained, with attached membrane and cells, is then replaced in the

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Fig 7. Schematic representation of corneal button glued to underlying membrane by an almost continuous peripheral ring of cyanoacrylate adhesive .

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rabbit eye and sutured in place using a continuous 10-0 nylon suture. Postoperatively, mydriatic cycloplegics, topical antibiotics, and subconjunctival steroids are given for a two- to three-week period. Postoperatively, the animals are examined two to three times per week at the slit-lamp biomicroscope at which time corneal thickness is determined on the transplanted as well as the contralateral normal cornea by pachometry. To rule out thinning from evaporation as a major corneal deturgescing factor, lids have been closed mechanically by tarsorrhaphy or head wraps and the thickness remeasured after several hours. SCINTILLATION COUNTS

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The portion of the membrane covered with labeled cells which is not transplanted is counted in a scintillation counter at the time of the initial surgical procedure. At variable time intervals, animals which have been transplanted are killed, corneal scleral rims removed, membrane dissected from the cornea, and counts obtained for comparison to the initial cell counts present on the membrane at the time of surgery. CONTROLS

Two different types of controls have been performed. A complete autograph has been done omitting only the removal of endothelium and placement of a membrane with tissue cultured endothelium. A second control has been done utilizing a membrane free of endothelium.

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ATTACHMENT OF MEMBRANE TO AUTOLOGOUS STROMA

The use of gelatin, platelets-fibrinogenthrombin, polysaccharides, amino acids, or fibronectin resulted in a very tenuous attachment of membrane to stroma. Therefore, during the course of the surgical procedure, the membrane tended to slip either partially or totally off the cornea. A peripheral, almost continuous rim, of butylcyanoacrylate diluted with two parts methylene chloride, resulted in the most successful attachment of membrane to cornea (Fig 7). When the cyanoacrylate was diluted with methylene chloride as indicated above, it was possible to demonstrate fewer toxic effects from the cyanoacrylate. When endothelium alone was removed and the membrane glued (as described above), the membrane would remain detached from the cornea centrally for 10 to 12 days before finally attaching to the

overlying stroma. Attempts to close this space earlier by applying gelatin or a central spot of cyanoacrylate resulted in either no difference in the closure of the space or tearing of the membrane respectively. It was found that by removing not only endothelium, but Descemet's, the membrane would attach at the time of glue application and remain attached without the formation of a fluid space. The total time lapse from removal of autologous keratoplasty button; with subsequent removal of endothelium or endothelium and Descemet's, attachment of membrane with tissue cultured cells to autologous stroma, dismantling of chamber and trephination, providing an autologous button with attached membrane and tissue cultured endothelial cells; to replacement of this button in the eye, averaged ten minutes with a range of eight to twelve minutes. RESULTS OF SURGERY

A control autologous penetrating keratoplasty using anterior chamber irrigation with removal of the corneal button for a 10-minute period, during which time it was placed in tissue culture medium, then replaced and sutured in place, resulted in the rapid thinning of the button to normal (Fig 8). When a membrane which was not seeded with cells was transplanted, either with or without Descemet' s being removed in addition to the endothelium; the cornea rapidly thickened and remained so throughout the entire period of observation (Fig 8). In eyes in which the endothelium alone was removed, prior to the transplantation of a membrane seeded with tissue cultured cells, the membrane was noted to be attached in the periphery; however, a fluid space would persist centrally between the membrane and Descemet' s for approximately 10 to 12 days, after which time it would disappear. After the space disappeared, the cornea would rapidly thin to normal and remain so for the entire period of observation which ranged from one to seven months (Fig 8). In eyes where not only endothelium, but Descemet's was removed prior to attachment of membrane with cells, no fluid space developed between membrane and stroma; however, these corneas did not reach normal thickness any more rapidly than those which did not have Descemet's removed (Fig 8). Ten of thirteen attempts have been successful using the technique of removing endothelium mechanically with or without the additional removal of Descemet's membrane, followed by attachment of membrane to autologous stroma using a peripheral rim of diluted butylcyanoacry-

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Fig 8. Graphic representation of corneal thickness of penetrating keratoplasty button relative to corneal thickness of contralateral normal cornea. ( - - - - - - - - ) autograft without removal of native endothelium or transplantation of gelatin membrane (G.M.) or tissue cultured corneal endothelial cells (T.C.C.E.C.). ( · - - - - k - - - - ) control stromal autograft after removal of native endothelium and application of G.M. not seeded with T.C.C.E.C. ( - - - - I R I - - - ) control stromal autograft after removal of native endothelium and Descemet's membrane and application of G.M. not seeded with T.C.C.E.C. (-----•-----) stromal autograft after removal of native en) stromal autograft after removal of dothlium and application of G.M. seeded with T.C.C.E.C. ( native endothelium and Descemet's and application ofG.M. seeded with T.C.C.E.C. ('f) indicates time of apposition of central membrane to overlying stroma.

late. Transplants have been judged successful when the corneal thickness has returned to normal and remained so after occlusion to rule out evaporation as the principal deturgescing force. Transplants appear clear even when there is moderate stromal edema in the early postoperative period. Normal thickness has been achieved in those which were judged successful. The eyes have been followed for

up to seven months without evidence of rejection or delayed failure being observed. The animals have been killed at variable periods of time in order to obtain the cellular membrane so that the origin of the cells on the posterior surface of the membrane could be established. At the time of sacrifice, histology has confirmed the presence of normal appearing corneal endothelium. The origin of the cells has been

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confirmed as being from tissue culture using the comparative tritiated thymidine counts. Forty-eight to 78% of the original count has been retained on the membrane in eyes which have returned to normal thickness. There have been equally good results with both the thicker membrane (3- 5 microns thick), and the more recently developed thin membrane ( 1 micron thick). There have also been equally good results with or without the additional removal of Descemet' s at the time of removal of native corneal endothelium. There is theoretical advantage to removal of Descemet' s along with endothelium since the membrane attaches immediately to the stroma and remains attached. Therefore, the possibility of mechanical damage to the endothelial layer is decreased. (Y)

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It has been, shown in the past by us 5 - 7 • 10 and otherss· 12 that tissue cultured endothelium can be seeded onto homologous stroma and transplanted successfully with the return of normal morphology and physiological function. Such a technique could provide a ready source of donor material using homologous stroma, stored over long periods of time, which could be seeded with endothelial cells on demand. This approach would solve some of the problems with donor availability; however, it would not approach the ideal of using autologous stroma. To apply this technique in patients with endothelial dysfunction and resultant corneal edema prior to scarring, it would be necessary to decrease the seeding time for these cells onto the stromal surface to an acceptable period. It would seem unlikely at the present time that the seeding time can be decreased to less than 30 minutes which, theoretically, would lead to an increase in complications as outlined above. There would also be an associated uncertainty as to the attachment of the cells to the autologous stroma. These problems may be overcome in the future, if the seeding time can be shortened to 10-15 minutes. The ideal of transplanting the endothelial layer alone to autologous stroma is approached in the procedure outlined above. The total time lapse from removal of cornea to replacement after the attachment of a membrane lined with tissue cultured endothelial cells, averages 10 minutes. The success rate is approaching 80% and should improve with further refinement in surgical techniq~e. On first evaluation, the use of cyanoacrylate would not appear to be ideal,

however in practice it has proved to be safe. Ideally, one would like to have a completely non-toxic adhesive to attach the membrane to autologous stroma. It would appear from previous experience, that the adhesive used will have to be effective for an extended period of time to allow the edges of the membrane to be incorporated in scar. If this is not attained, the membrane begins to slip or detach. Fibroblast also tend to grow between membrane and stroma if there is not a tight attachment between the two. 5 Work still continues in search of an ideal adhesive. Another area of potential development is that of the use of autologous vascular endothelium as a source for an endothelial covering of the cornea. 12 It will require years of observation however to establish the safety and efficacy of this tissue. Another major area which requires work is that of the actual maintenance and growth of corneal endothelial cells in tissue culture. There are considerable species differences. In our laboratory we have found that beef is easier to grow and stimulate to divide than is rabbit corneal endothelium, both of which are considerably easier to grow and maintain than human corneal endothelium (unpublished work). Much work therefore, needs to be done to determine the best growth factors and the methods by which these should be incorporated into tissue culture. Our plans for the near future include the growth of human corneal endothelium in tissue culture which we hope to initially transplant into rabbits. Once this has been accomplished, it is our plan to transplant human corneal endothelium from tissue culture into monkeys prior to considering the transplantation of human corneal endothelium into humans.

ACKNOWLEDGMENT Technical assistance was provided by Frances A. Johnson.

REFERENCES 1. Stocker FW, Eiring A, Georgiade R, Georgiade M. A tissue culture technique for growing corneal epithelial, stromal, and endothelial tissue sepa· rately. Am J Ophthalmol 1958; 46:294-98. 2. Lowry GM. Corneal endothelium in vitro: characterization by ultrastructure and histochemistry. Invest Ophthalmol 1966; 5:355-66. 3. National Eye Institute: Summary report on the cornea task force. Invest Ophthalmol 1973; 12:391-97. 4. Maurice 0, Perlman M. Permanent destruction of the corneal endothelium in rabbits. Invest Ophthalmol Vis Sci 1977; 16:646-49.

5. Maurice D, McCulley J, Perlman M. Development in use of cultured endothelium in corneal transplantation. Doc Ophthalmol Proc Ser 1 979; 20:151-53. 6. Maurice D, McCulley J, Perlman M. Donor endothelium from tissue culture. Invest Ophthalmol Vis Sci 1977 (abstract); 16 (ARVO suppl) 103. 7. Jumblatt M, Maurice M, McCulley J. Transplantation of tissue-cultured corneal endothelium. Invest Ophthalmol Vis Sci 1978; 17:1135-41. 8. Gospodarowicz D, Greenburg G, Alvarado J. Transplantation of cultured bovine corneal endothelial cells to rabbit cornea: clinical implications for human studies. Proc Nat I Acad Sci 1979; 76:464-68.

9. Jumblatt M, Maurice D, Schwartz B. A gelatin membrane substrate for the transplantation of tissue cultured cells. Transplantation (in press). 10. Maurice D, McCulley J, Schwartz B. Keratoplasty with cultured endothelium on thin membranes. Invest Ophthalmol Vis Sci 1979 (abstract); 18(suppl):1 0. 11. Perlman M, Baum J. The mass culture of rabbit corneal endothelial cells. Arch Ophthalmol 1974; 92:235-37. 12. Gospodarowicz D, Greenburg G, Alvarado J. The transplantation in vivo of cultured bovine corneal and vascular endothelial cells in rabbit and cat corneas. Invest Ophthalmol 1979 (abstract); 18(suppl) 9.

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