A Review of Surgical Techniques for Harvesting Corneal Stem Cells From Allograft Donor Tissue

Lim L.T., Bhatt P.R., Ramaesh K.

Aim: To describe an alternative and novel technique using cyanoacrylate glue to achieve successful limbal tissue dissection, from an organ culture media stored corneoscleral button, without an artificial anterior chamber.Methods: A donor corneoscleral button (leftover from penetrating keratoplasty) was divided into two equal semicircular halves. A thick layer of tissue adhesive (N-butyl-2-cyanoacrylate) was spread on a sterile rubber block (the under surface of the donor punch). One half of the donor corneoscleral rim was placed epithelial side up on the adhesive and allowed to attach firmly to the block. This composite provided stability to the donor rim allowing lamellar dissection of the limbal tissue to be performed without damaging the limbal epithelium.Results: Regular, partial-thickness limbal tissue was obtained. There was no histological evidence of glue or cellular toxicity of the harvested limbal stem cells. This harvested tissue had been grafted successfully in patients with limbal stem cell deficiency also undergoing keratoplasty.Conclusions: Tissue adhesive can be a simple, effective and useful tool in the dissection and harvesting of corneal limbal stem cell allografts from corneoscleral buttons stored in organ culture media.

Holland E.J., Schwartz G.S.

Pfister R.R., Sommers C.I.

To determine if transplanted corneal epithelial stem cells are safely and efficiently attached to the deficient limbal niche with use of fibrin sealant. The primary outcome is measured with respect to the stability of the transplant, with secondary qualitative evaluations of inflammation, patient comfort, speed of operation, and incidence of complications.This retrospective case study examined a total of 114 corneal stem cell reconstructions performed in 95 patients from 1996 to 2004 using corneal stem cells primarily, with a minority of amnion alone, or both. Fibrin sealant was used as the only technique of stem cell adhesion for limbal reconstruction for primary or recurrent pterygia and various stem cell-deficient diseases from 2000 to 2004.The fibrin sealant group showed 1 small recurrence of pterygium but no complications. With sutures, there were 3 recurrences in the pterygia group. After completion of all surgical procedures, all patients were free of pterygia. Miscellaneous stem cell deficiencies were included to demonstrate that corneal stem cell transplants can be used in other corneal procedures in addition to pterygia.Fibrin sealant alone effectively and safely attached corneal stem cell transplants to the limbal niche. The additional qualitative observations of a reduction in operation time, postoperative pain, and inflammation augurs for more extensive use of fibrin sealants in ophthalmology.

Meisler D.M., Perez V.L., Proudfit J.

To develop a device that facilitates the procurement of corneal limbal stem cell grafts for keratolimbal allograft procedures used in the treatment of ocular surface disease associated with stem cell deficiency.Description of device design and technique for use.The device is composed of a pedestal with a convex surface mounted to a flat platform. A corneoscleral button placed endothelial side down and centrally upon the convexity is secured by suction conveyed through a hollowed core in the pedestal that connects to fenestrated openings on the convex surface. A donut-shaped stainless steel ring placed on tension by springs braces the peripheral tissue. A circular corneal incision is created of a desired thickness by a suction trephine, and a crescent blade is utilized to peripherally dissect a donut-shaped keratolimbal allograft.This device facilitated the harvesting of the keratolimbal allograft tissue from four eye bank donor practice corneoscleral buttons and was then used to successfully procure grafts from six corneoscleral buttons used in three keratolimbal allograft procedures in three patients, one each with aniridia, alkali burn, and drug-induced limbal stem cell deficiency.The described device effectively facilitates procurement of corneoscleral buttons for keratolimbal allograft procedures. It appears to offer advantages over freehanded techniques and previously described devices used for the same purpose.

Croasdale C.R., Schwartz G.S., Malling J.V., Holland E.J.

Aldave A.J., Wong I.G.

To describe a method for obtaining partial-thickness keratolimbal allografts from corneoscleral buttons to be used in corneal limbal stem cell transplantation.Description of device design and technique for use.Cyanoacrylate tissue adhesive, placed on the posterior side of a trephinated corneoscleral rim, is used to secure the allograft to a disposable acrylic sphere that is attached to a cylindrical base.After fixation of the corneoscleral rim to the acrylic sphere, keratolimbal allograft harvesting is performed as a continuous strip dissection with a 65 Beaver blade. This technique minimizes trauma to the epithelial stem cells, provides excellent stability of the corneoscleral rim during harvesting, and preserves the central corneal button.This method for obtaining keratolimbal allografts effectively provides tissue stabilization during harvesting, minimizing epithelial stem cell trauma and eliminating the need for whole donor globes.

Chuck R.S., Behrens A., McDonnell P.J.

To develop a mechanical device to perform limbal transplantation. This procedure is a valuable surgical technique for management of limbal stem cell deficiency. However, the freehand dissection to obtain donor tissue is laborious and time-consuming.A manual microkeratome (LSK One; Moria/Microtech, Doylestown, Pennsylvania) using a redesigned head (200 microm thickness, 16-mm blade) was used to create partial thickness corneoscleral caps from human donor globes.Corneoscleral caps obtained from human donor globes included a ring of approximately 1 mm of perilimbal sclera in contiguity to the cornea. Scanning electron microscopy showed a smooth cut surface with some chatter at the margins.This instrument ensures an effective and straightforward method to dissect the area where stem cells have been indirectly localized.

Mannis M.J., McCarthy M., Izquierdo L.

To report a method for obtaining thin keratolimbal lenticules from cadaveric corneoscleral buttons for corneal epithelial stem cell allografts.A standard silicone orbital sizing sphere and three 25-gauge needles were employed to fix the corneoscleral button to facilitate excision of keratolimbal lenticules.Limbal stem cells in the form of keratolimbal lenticules were obtained without difficulty.With this technique, thin keratolimbal lenticules of good quality can be obtained efficiently, inexpensively, and without specialized instruments.

Dua H.S.

BACKGROUND/AIMS During the healing of corneal epithelial wounds with limbal involvement, conjunctival epithelium often migrates across the denuded limbus to cover the corneal surface. It is believed that, over a period of time, conjunctival epithelium covering the cornea assumes characteristics of corneal epithelium by a process referred to as conjunctival transdifferentiation. The purpose of this study was to examine, clinically, the fate of conjunctival epithelial cells covering the cornea and to assess the healing of corneal epithelial wounds when the conjunctival epithelium was removed or actively prevented from crossing the limbus and extending onto the cornea.METHODS 10 patients with conjunctivalisation of the cornea were followed for an average of 7.5 months. Five patients in this group had their conjunctival epithelium removed from the corneal surface and allowed to heal from the remaining intact corneal epithelium. In another four patients with corneal epithelial defects, the conjunctival epithelium was actively prevented from crossing the limbus by mechanically scraping it off.RESULTS The area of cornea covered by conjunctival epithelium appeared thin, irregular, attracted new vessels and was prone to recurrent erosions. Conjunctivalisation of the visual axis affected vision. Removal of conjunctival epithelium from the cornea allowed cells of corneal epithelial phenotype to cover the denuded area with alleviation of symptoms and improvement of vision. It was also established that migration of conjunctival epithelium onto corneal surface could be anticipated by close monitoring of the healing of corneal epithelial wounds, and prevented by scraping off conjunctival epithelium before it reached the limbus.CONCLUSION This study shows that there is little clinical evidence to support the concept that conjunctival transdifferentiation per se, occurs in humans. “Replacement” of conjunctival epithelium by corneal epithelial cells may be an important mechanism by which conjunctival “transdifferentiation” may occur. In patients with partial stem cell deficiency this approach can be a useful and effective alternative to partial limbal transplantation, as is currently practised.

Tsubota K., Toda I., Saito H., Shinozaki N., Shimazaki J.

Although penetrating keratoplasty is an established surgical procedure, it often is ineffective for severe ocular surface diseases such as alkali burns or limbal deficiency. The authors have performed limbal allograft transplantation for the reconstruction of the corneal epithelium.A total of nine patients (3 with chemical injury, 3 with limbal deficiency with unknown etiology, 2 with moderate ocular pemphigoid [OCP], and 1 with traumatic limbal deficiency) were treated by limbal allograft transplantation. Penetrating keratoplasties were performed in all patients with the exception of one with OCP. Patients received cyclosporine both systemically (10 mg/kg) and topically (0.05%) as well as high-dose intravenous dexamethasone (8 mg).The corneal epithelium was reconstructed in all patients, although two showed partial increased fluorescein permeability and two others required a second surgery. The other five epithelia remained clear at mean follow-up of 12.3 months, with two episodes of graft rejection which were controlled successfully by medication.Limbal allograft transplantation with intensive immunosuppression by cyclosporine and high-dose steroids appears to be a promising surgical intervention for the reconstruction of corneas affected by severe ocular surface disease.

Keivyon K.R., Tseng S.C.

Limbal autograft transplantation is presented in 26 consecutive cases comprising both acute and chronic chemical injury (20 cases), thermal burns (2 cases), contact lens-induced keratopathy (3 cases), and ocular surface failure after multiple surgical procedures (1 case), with follow-up ranging from 2 to 45 months (mean, 18 months). The operative technique usually involved transfer of two free grafts of limbal tissue from the uninjured or less injured donor eye to the severely injured recipient eye, the latter having been prepared by limited conjunctival research and superficial dissection of fibrovascular pannus without keratectomy. Clinical results in 21 patients with follow-up of 6 months or more have consistently shown improved visual acuity (17 cases), rapid surface healing (19 cases), stable epithelial adhesion without recurrent erosion or persistent epithelial defect (20 cases), arrest or regression of corneal neovascularization (15 cases), and probable increased success for lamellar or penetrating keratoplasty (8 cases). No intraoperative complications were encountered, and no adverse reactions developed in donor eyes. Impression cytology in selected cases showed restoration of the corneal epithelial phenotype and regression of goblet cells from the recipient cornea. Therefore, limbal autograft transplantation is recommended for treatment of widespread ocular surface damage with loss of limbal epithelial stem cells and, specifically, for chemical or thermal burns, contact lens-induced keratopathy, and selected persistent corneal epithelial defects.

Thoft R.A.

Keratoepithelioplasty is a new surgical procedure for the treatment of persistent epithelial defects in patients without healthy donor tissue in their fellow eyes. After a total superficial keratectomy, lenticules of donor cornea covered by epithelium are placed at the corneoscleral limbus. The epithelium spreads from the lenticules and covers the center of the cornea. In three of four patients with persistent defects of long duration, healing was successful and vision improved. The three successful procedures were in patients who had had chemical injuries. They retained useful visual function without recurrence of epithelial healing problems. In the fourth case, it was impossible to determine whether the procedure was useful because the patient, who had atopic keratoconjunctivitis, required a penetrating keratoplasty two months after the keratoepithelioplasty.

DAVANGER M., EVENSEN A.

THE human cornea is covered by a five-layered epithelium. Cells are continually shed from its surface and replaced by division of the basal cells, which has a mean generation time estimated to be about 4 days1. Because of the papillae in the skin, the relation between the area of the basal cell layer and the surface is about 20 : 1. Because it must be refractive, there can be no papillae on the cornea, and the relation between the basal cell layer and the surface is accordingly 1 : 1. This should correspond to higher demands on the generative capacity of the corneal basal cells compared with skin. The epidermal basal cells are in close contact with a well developed capillary network. There are no vessels in the cornea, and so it can be assumed that the supply of its epithelium is poorer. Corneal epithelium, nonetheless, has considerable healing capacity, which is achieved primarily by migration of epithelial cells.