Transplantable cultivated mucosal epithelial sheet for ocular surface reconstruction

Ocular surface reconstruction by tissue engineering using somatic stem cells is a second-generation therapeutic modality. In view of future treatment of bilaterally affected, severe ocular surface disorders, two types of transplantable cultivated mucosal epithelial sheets can be used for reconstruction. One is an allogeneic corneal epithelial stem cell sheet, and the other is an autologous oral mucosal epithelial cell sheet. We first investigated the feasibility of amniotic membrane as an epithelial carrier, and found that denuded amniotic membrane was the most appropriate substrate for this purpose. Thus, cultivated corneal epithelial stem cell sheets were created by co-culturing with 3T3 fibroblast and air-lifting on amniotic membrane. These epithelial sheets demonstrated positive keratin 3 and 12 specific to in vivo corneal epithelium, light junction related proteins and proliferative activity. The transplanted allogeneic human corneal epithelial sheets existed successfully on the corneal surface, and were quite effective in achieving ocular surface stability in severe ocular surface disorders. A few cases, however, developed immunological reactions or opportunistic infections, etc. Secondly, we established transplantable autologous cultivated oral mucosal epithelial sheets in rabbits. The in vitro oral mucosal epithelial sheets showed histological characteristics similar to those of in vivo corneal epithelial sheets; for example, positive keratin 3 expression. Based on the fact that, the transplanted autologous oral mucosal epithelial sheets resembled corneal epithelium and that we achieved the recovery of corneal transparency in rabbits, we propose that cultivated oral mucosal epithelium may become the substitute for corneal epithelium in ocular surface reconstruction.     

Oberfl?chenrekonstruktion bei Limbusstammzellinsuffizienz

Various ocular surface diseases are caused by loss of corneal epithelial stem cells or dysfunction of the limbal stem cell niche. Besides conventional transplantation of autologous or allogenic limbal tissue, recent advances in tissue engineering have led to the development of new culture and expansion techniques of human limbal stem and progenitor cells (LSPC) as a new strategy to successfully treat limbal stem cell deficiency (LSCD). From a small autologous limbal biopsy with a limited amount of LSPC an epithelium ready for transplantation is achieved. Autologous grafting of cultured limbal epithelium led in most of the treated cases to a successful reconstruction of the corneal surface. Alternative methods which have recently been introduced to treat LSCD use other stem cell sources including the transplantation of oral mucosal epithelium. In this article the challenges and controversies associated with these stem cell culture techniques for ocular surface reconstruction are reviewed.     

Ocular surface reconstruction by tissue engineering

Ocular surface reconstruction by tissue engineering using somatic stem cells is a second-generation modality. In order to treat bilaterally affected, severe ocular surface disorders, we investigated the transplantation of two types of cultivated mucosal epithelia: allogenic corneal epithelial stem cells, and autologous oral mucosal epithelial cells. For this, first, we summarized the clinical results of allogenic keratoepithelioplasty and limbal transplantation. In addition, we showed that the immunological shift from Th1 to Th2 by using keyhole limpet hemocyanin was effective in suppressing the incidence of immunological rejection. Second, we investigated the transplantation of cultivated human corneal epithelial stem cells onto amniotic membrane. The cultivated sheet was created by co-culture with 3T3 fibroblasts, using the air-lift method, in cultivating the corneal epithelial stem cell on the amniotic membrane. These cultivated cells demonstrated positive keratin 3 and 12 specific to in vivo corneal epithelium, tight junction related proteins, and telomerase activity. The transplanted allogenic human corneal epithelial sheet survived on the corneal surface in all cases, and was quite effective for achieving ocular surface stability in the acute phase of Stevens-Johnson syndrome, ocular cicatricial pemphigoid, or chemical injury. However, a few cases developed immunological rejection or opportunistic infection. Third, to establish the transplantation of the autologous cultivated oral mucosal epithelial sheet, we performed animal experiments using rabbits. In vitro oral mucosal epithelial sheet showed histology similar to that of in vivo corneal epithelial sheet. It expressed positive keratin 3 as well. Since the autologous transplantation of this sheet survived on the ocular surface with the recovery of corneal transparency, a cultivated oral mucosal epithelium may become a substitute for corneal epithelium. Fourth, we created a cultivated human corneal endothelial cell sheet on amniotic membrane using a similar technique, and transplanted it to a rabbit eye as a xenograft. The transplanted corneal endothelial cell density was over 3,000 cells/mm2, and it was actively functioning even after the transplantation. Lastly, to explore cell markers for corneal epithelial stem cells, we established a technique using laser micro-capture, and introduced amplified fragment length polymorphism (AFLP), identifying several candidate molecules as stem cell markers.     

组织工程角膜上皮移植重建眼表面的临床研究

目的 观察组织工程角膜上皮移植重建眼表面治疗完全性角膜上皮干细胞缺乏的短期临床效果.方法 系列病例研究.6例(6眼)单眼全角膜缘干细胞缺乏患者,包括碱烧伤3例、爆炸伤2例、热烧伤1例.采用去上皮羊膜组织作为载体,体外扩增患者自体健眼角膜上皮干细胞,构建组织工程角膜上皮.然后进行组织工程角膜上皮移植重建眼表面.术前及术后检查指标包括裸眼视力、裂隙灯显微镜、超声生物显微镜、泪液分泌试验.结果 患者自体来源的角膜上皮干细胞在去上皮羊膜上培养3周后均可形成直径15 mm的复层上皮片.组织工程角膜上皮均成功移植于所有受体眼表面.移植术后3个月,所有患者角膜上皮完整光滑,角膜瘢痕及纤维血管翳明显减轻,6例患者视力均有不同程度的提高.结论 患者自体来源的角膜上皮干细胞构建的组织工程角膜上皮移植可以成功重建全角膜缘干细胞缺乏患者的眼表面,为这类患者的复明带来希望.     

The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane

PURPOSE: To determine the feasibility of using human amniotic membrane (AM) as a substrate for culturing oral epithelial cells and to investigate the possibility of using autologous cultivated oral epithelial cells in ocular surface reconstruction. METHODS: An ocular surface injury was created in one eye of each of eight adult albino rabbits by a lamellar keratectomy, and a conjunctival excision was performed, including and extending 5 mm outside the limbus. Oral mucosal biopsy specimens were obtained from these eight adult albino rabbits and cultivated for 3 weeks on a denuded AM carrier. The cultivated epithelium was examined by electron microscopy (EM) and immunohistochemically labeled for several keratins. At 3 to 4 weeks after the ocular surface injury, the conjunctivalized corneal surfaces of the eight rabbits were surgically reconstructed by transplanting the autologous cultivated oral epithelial cells on the AM carrier. RESULTS: The cultivated oral epithelial sheet had four to five layers of stratified, well-differentiated cells. EM revealed that the epithelial cells were very similar in appearance to those of normal corneal epithelium, had numerous desmosomal junctions, and were attached to a basement membrane with hemidesmosomes. Immunohistochemistry confirmed the presence of the keratin pair 4 and 13 and keratin-3 in the cultivated oral epithelial cells. Corneas that were grafted with the cultivated oral epithelial cells on an AM carrier were clear and were all epithelialized 10 days after surgery. CONCLUSIONS: Cultures of oral epithelial cells can be generated to confluence on AM expanded ex vivo from biopsy-derived oral mucosal tissue. Autologous transplantation was performed with these cultivated oral epithelial cells onto the ocular surfaces of keratectomized rabbit eyes. Autologous transplantation of cultivated oral epithelium is a feasible method for ocular surface reconstruction. The long-term outcome of such transplantation is not yet clear, and its feasibility in clinical use should be evaluated further.     

Progress in the development of tissue engineering of the cornea in Japan

Recently, regenerative medicine focusing on tissue-engineering techniques has been developed and established as a new clinical field. In Japan, two types of cultivated mucosal epithelial transplantation have been clinically applied to ocular surface reconstruction of severe stem cell deficiencies; one is allogeneic/autologous cultivated corneal epithelial transplantation and the other is autologous cultivated oral mucosal epithelial transplantation. These new techniques enabled us to treat severe stem cell deficiencies by covering the whole damaged ocular surface with cultivated mucosal epithelial sheets. In the field of corneal endothelial research, reconstruction of damaged corneal endothelium utilizing cultivated human corneal endothelial cells or their precursors has been developed and its usefulness confirmed in animal models. This review will provide an overview of recent results, as well as an insight into the future of research on corneal regenerative medicine in Japan.     

Concept and clinical application of cultivated epithelial transplantation for ocular surface disorders

Corneal epithelial replacement using a tissue engineering technique holds much promise for ocular surface reconstruction in cases of corneal epithelial stem cell deficiency. However, even though an autologous cultivated corneal epithelial stem cell sheet is the safest and most reliable form of sheet, bilaterally affected ocular surface disorders cannot be treated by this method. To treat bilateral cases, we must choose either an allogeneic cultivated corneal epithelial sheet or an autologous cultivated oral mucosal epithelial sheet. In the case of the former, immunological reaction is a threat. Thus, understanding of the immunological background of ocular surface reconstruction using allogeneic tissues is essential. In the case of the latter, the transplanted sheet is not exactly the same as corneal epithelium, and understanding ocular surface epithelial biology is important. In this review, we summarize and explain the concept and clinical application of cultivated mucosal epithelial transplantation for ocular surface disorders.     

Towards a defined,serum‐ and feeder‐free culture of stratified human oral mucosal epithelium for ocular surface reconstruction

Purpose: Ocular surface reconstruction with cultivated oral mucosal epithelial transplantation technique is a viable treatment option for severe ocular surface injuries and diseases with limbal stem cell deficiency. Currently, this technique is based on utilization of xenogenic, allogenic or undefined components such as murine 3T3 feeders, serum and amniotic membrane. In this study, we aimed to find a more defined culture method to generate stratified human oral mucosal epithelium. Methods: In this study, we have examined the formation of stratified cell sheets from human oral mucosal epithelial cells under serum‐free culture environment both in the absence and presence of fibroblast‐conditioned culture medium and elevated epidermal growth factor (EGF) concentration. Results: In all examined culture conditions, the cultivated oral epithelial cells formed a stratified tissue, which was positive for keratins K3/12, K4 and K13. The tissue‐engineered oral epithelia also expressed proliferation and progenitor markers Ki67 and p63 in the basal layer of the cell sheets, suggesting that the epithelia still had regenerative capacity. The cultures presented expression of tight junction proteins ZO‐1 and occludin and high transepithelial electrical resistance values. Conclusion: In this culture method, we have been able to produce stratified cell sheets successfully without serum, conditioning of the medium or increased EGF concentration. We provide a novel protocol to produce tight multi‐layered epithelium with proliferative potential, which can be easily adapted for cultivated oral mucosal epithelial transplantation.     

Midterm results on ocular surface reconstruction using cultivated autologous oral mucosal epithelial transplantation

PURPOSE: To perform a midterm assessment of the integrity and reproducibility of cultivated autologous oral mucosal epithelial sheets, and to evaluate the clinical efficacy of their transplantation in ocular surface. DESIGN: Observational case series. METHODS: Cultivated autologous oral mucosal epithelial sheets were created using amniotic membrane and buccal mucosal epithelium from 12 patients with Stevens-Johnson syndrome, chemical and thermal injury, pseudo-ocular cicatricial pemphigoid, and idiopathic ocular surface disorder. They were transplanted onto 15 eyes from these patients who were then followed up for a mean of 20 months; with the longest follow-up being 34 months. We assessed their clinical outcomes with special reference to neovascularization. RESULTS: Cultivated autologous oral mucosal epithelial sheets could be generated from all patients. On the second postoperative day, 14 of 15 sheets transplanted demonstrated total re-epithelialization on the cornea. During the follow-up, the ocular surface was stable and transparent without any major complications in 10 of 15 eyes (67%), and the transplanted epithelium survived for at least 34 months. There were five eyes (33%) with small but long-standing epithelial defects, three of these healed spontaneously, and two (13%) required reoperation. In 10 eyes, postoperative visual acuity was improved by more than 2 lines. All eyes manifested some peripheral corneal vascularization. CONCLUSIONS: We established a successful tissue-engineering technique to generate cultivated autologous oral mucosal epithelial sheets and succeeded in reconstructing the ocular surface. We suggest that this surgical modality may be both safe and useful, especially in younger patients with the most severe ocular surface disorders.     

Recent advances in cultivated epithelial transplantation

Currently, cultivated epithelial transplantation usually uses ex vivo-expanded epithelial cells, with or without biological carriers. Our group has carried out approximately 100 such procedures and has conducted research in this area. The results of a retrospective study indicate that cultivated limbal epithelial transplantation with amniotic membrane (AM) and airlifting may be beneficial at avoiding sight-threatening complications in patients with severe chronic cicatricial keratoconjunctivitis. Our experience in cultivated oral mucosal epithelial transplantation indicates that this technique is useful in achieving a stable ocular surface. However, the treatment for severe ocular surface diseases such as Stevens-Johnson syndrome remains unsatisfactory. In addition to using epithelial sheets with AM, we have developed a technique for generating carrier-free sheets using fibrin sealants. These sheets seem to contain more differentiated epithelium than those obtained with AM while retaining similar levels of colony-forming progenitor cells. A clinical trial of this technique is currently underway. We have also generated epithelial sheets using as biological carrier silk fibroin film, which offers a more transparent medium than conventional sheets. In terms of isolation and cultivation of corneal epithelial stem/progenitor cells, we found that single murine limbal cells exhibited clonal growth and generated stratified epithelial sheets. We also reported that hypoxic culture (2%) enhanced proliferation in human limbal epithelial cells while inhibiting differentiation. This technique may help maintain progenitor cells during ex vivo expansion of epithelial cells. Although these advances are expected to improve clinical outcomes in patients with ocular surface disorders, further improvements, such as the development of cultivation methods that do not require 3T3 feeder cells or real-time assessment of cultivated sheets, are required in the near future.     

利用组织工程方法治疗角膜缘干细胞缺失的相关研究进展

角膜缘干细胞在角膜上皮更新和创伤愈合中起重要作用,角膜缘干细胞缺失可导致各种严重眼表面疾患。因为治疗上存在自体移植的健眼损伤问题,同种异体移植的排斥反应以及供体不足等问题,近年来随着对干细胞研究的深入以及组织工程学的发展,有利于克服和解决上述问题,为角膜缘干细胞缺乏的治疗带来了曙光。本文通过近年来组织工程治疗角膜缘干细胞缺失的相关研究的回顾,归纳了种子细胞和支架材料选择两方面的研究进展,以探索该领域存在的问题和未来的研究方向。（国际眼科纵览, 2017,  41:   346-351）     

Transplantation of cultivated autologous oral mucosal epithelial cells in patients with severe ocular surface disorders

BACKGROUND/AIMS: To determine outcomes of transplants of cultivated autologous oral epithelial cells in patients with severe ocular surface disorders. METHODS: The eyes (n = 6) of four patients with Stevens-Johnson syndrome (three eyes) or chemical burns (three eyes) were studied. Autologous oral epithelial cells, grown for 2-3 weeks on a denuded amniotic membrane carrier in the presence of 3T3 fibroblasts, were air lifted. The resultant sheet was transplanted onto the damaged eye, and acceptance of the sheet by the corneal surface was confirmed 48 hours after surgery. The success of ocular surface reconstruction, graft survival, changes in visual acuity, and postoperative complications were assessed and the quality of the cultivated oral epithelial sheet was evaluated histologically. RESULTS: At 48 hours after transplant, the entire corneal surface of all six eyes was free of epithelial defects indicating complete survival of the transplanted oral epithelium. Visual acuity was improved in all eyes. During follow up (mean 13.8 (SD 2.9) months), the corneal surface remained stable, although all eyes manifested mild peripheral neovascularisation. CONCLUSIONS: Autologous oral epithelial cells grown on denuded amniotic membrane can be transplanted to treat severe ocular surface disorders.     

UV absorption in human oral mucosal epithelial sheets for ocular surface reconstruction

BACKGROUND: Ocular surface reconstruction with autologous oral mucosal epithelium has attracted attention as a novel treatment strategy that avoids allograft rejection. OBJECTIVES: To evaluate the absorption of ultraviolet (UV) A or B irradiation by human oral mucosal epithelium cultured on human amniotic membrane. METHODS: Human oral mucosal and limbal epithelial cells were co-cultured on amniotic membrane with inactivated 3T3 fibroblasts. The cell sheets were also subjected to UV-A (365 nm) or UV-B (302 nm) irradiation at energy levels ranging from 50 to 800 microW/cm2, and the UV absorption rate was measured with a UV irradiation meter. RESULTS: Cultured oral mucosal epithelium had a structure with 3-5 layers of cells, consistent with the histological features of cultured corneal limbal epithelium after 4 weeks. The decrease in UV-A absorption of cultivated oral mucosal epithelium ranged from 25 to 36% of that for cultured corneal epithelium. The increase in UV-B absorption by cultured oral mucosal epithelium between 200 and 800 microW/cm2 was approximately 145% of that for cultured corneal limbal epithelium. CONCLUSION: Our data demonstrated that cultured oral mucosal epithelium has low UV-A and high UV-B absorption capacity as compared with those of cultured corneal epithelium, suggesting that oral mucosal epithelium can compensate for UV absorption of corneal epithelium.     

Use of autologous cultured limbal and conjunctival epithelium in a patient with severe bilateral ocular surface disease induced by acid injury: a case report of unique application

PURPOSE: Reconstruction of the ocular surface in a case of severe bilateral partial limbal stem cell deficiency (LSCD) with extensive symblephara using autologous cultured conjunctival and limbal epithelium. CASE REPORT: A 31-year-old woman presented with severe bilateral ocular surface disease with partial limbal stem cell deficiency, symblephara, lid and facial scarring, with a vision of 20/400 and counting fingers at 1 m in both eyes. Limbal and conjunctival tissue was harvested from the healthy-appearing left eye and used to generate two sheets of composite epithelium consisting of central limbal and peripheral conjunctival cells. The limbal tissues were explanted in the central region while the conjunctival tissues were explanted on the periphery of the deepithelialized human amniotic membrane (HAM) and nurtured using human corneal epithelial cell medium. After successful generation of a monolayer from both tissues had been confirmed, the composite of cultivated limbal and conjunctival epithelium with HAM was transplanted in each eye after excision of fibrous tissue and release of symblephara. One year postoperatively, the patient had a best spectacle-corrected visual acuity of 20/40 in the right eye (preoperative acuity 20/400) and counting fingers at 1 m in the left eye (same as preoperative) with a stable ocular surface. CONCLUSIONS: Autologous cultured epithelial transplantation is as an excellent option in selected patients with bilateral partial LSCD with small area(s) of healthy limbus in either eye and avoids the attendant risk of rejection and cost and potential toxicity of immunosuppression in allogeneic tissue transplantation. This case also highlights the feasibility of generating a composite culture of limbal and conjunctival epithelium using a single amniotic membrane.     

The use of autologous serum in the development of corneal and oral epithelial equivalents in patients with Stevens-Johnson syndrome

PURPOSE: To evaluate the use of autologous serum (AS) from patients with severe ocular surface disease (OSD) in the development of transplantable corneal and oral epithelial tissue equivalents and to compare it with the use of conventional culture methods by using fetal bovine serum (FBS). METHODS: AS was obtained from patients with severe OSD secondary to Stevens-Johnson syndrome. Corneal and oral epithelial cells were cultivated in medium supplemented with either AS or FBS. Corneal and oral epithelial equivalents were constructed on denuded amniotic membranes. The bromodeoxyuridine (BrdU) ELISA cell proliferation assay and colony-forming efficiency (CFE) of cells cultivated in AS- or FBS-supplemented media were compared. The morphologic characteristics and the basement membrane assembly of cultivated epithelial equivalents were analyzed by light and electron microscopy, as well as by immunohistochemistry. RESULTS: BrdU proliferation assay and CFE analysis showed that human corneal and oral epithelial cells cultivated in AS-supplemented media had comparable proliferative capacities compared with FBS-supplemented media. The corneal and oral epithelial equivalents cultivated in AS- and FBS-supplemented media were morphologically similar and demonstrated the normal expression of tissue-specific keratins and basement membrane assembly. The presence of a well-formed stratified epithelium, a basement membrane, and hemidesmosomal attachments was confirmed by electron microscopy. CONCLUSIONS: AS-supplemented cultures were effective in supporting the proliferation of human corneal and oral epithelial cells, as well as the development of transplantable epithelial equivalents. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.     

Ex vivo expansion of corneal limbal epithelial/stem cells for corneal surface reconstruction

PURPOSE: The management of severe ocular surface disease due to limbal stem cell deficiency has changed dramatically. The concept of limbal stem cells, as the source of corneal epithelium revolutionised the therapeutic approach of ocular surface reconstruction. Deficiency of limbal stem cells results in blinding ocular surface diseases. Grafting viable limbal tissue, from either fellow healthy eye or a donor eye, with the resident stem cell population may replenish limbal stem cells and can restore the corneal surface to normality. Transplanting the limbal tissue can be achieved through a variety of procedures that include cadaveric keratolimbal allograft (KLAL), live or living related conjunctival limbal allograft (Ir-CLAL) and limbal autograft. Advances in tissue engineering techniques have offered a viable alternative to overcome the limitation of limbal tissue available for transplantation. Epithelial stem cells harvested from a small limbal biopsy can be expanded in vitro on a suitable carrier and then transplanted to the diseased cornea to successfully restore the corneal surface. This article is a chronological review of the important steps that brought ex vivo expanded stem cell transplantation in ocular, particularly corneal surface reconstruction. METHODS: The MEDLINE data base was searched for the years 1966-2002, using key words cornea, cell culture, ex-vivo expansion, limbus, stem cell, ocular surface and transplantation. Several articles that were not found by MEDLINE search were taken from references from other articles. Inclusion or exclusion of article was based on the relevance to the subject. CONCLUSIONS: Corneal epithelial reconstruction with ex vivo expanded limbal cells is a potential tool in ocular surface reconstruction, although the technique is currently investigational. Strategies to achieve conjunctival epithelial restoration and tear film replenishment will allow ophthalmic surgeons to truly reconstruct the ocular surface.     

Cultivated limbal and oral mucosal epithelial transplantation

Stem cells located at the limbus are the ultimate source for regeneration of the corneal epithelium in normal and traumatized states. When limbal stem cells are dysfunctional or deficient, limbal stem cell deficiency (LSCD) develops. Its surgical management depends on laterality and severity of corneal-limbal involvement. Conventional methods of stem cell transplantation are conjunctival-limbal autograft (CLAU), conjunctival-limbal allograft (CLAL), and kerato-limbal allograft (KLAL) surgeries. Cultivated limbal epithelial transplantation (CLET) and cultivated oral mucosal epithelial transplantation (COMET) on a carrier such as amniotic membrane are current surgical alternatives. These new surgical procedures are effective in stabilizing the ocular surface. The theoretical advantage of ex-vivo expansions over conventional methods is that only a small limbal or mucosal biopsy is needed, thus minimizing the risk to the donor eye; there is also a lower risk of rejection. They can be used in cases with unilateral or bilateral total stem cell deficiency. In the unilateral cases, the source for CLET is a healthy fellow eye and in bilateral cases the source can be living-related or cadaveric eyes. The oral explants do not have limbal stem cells, but they seem to be a source of limbal stem cell equivalents that are able to generate cornea-like epithelium under the proper culture conditions. The main advantage of COMET is that patients with bilateral LSCD can be treated with grafts derived from their own autologous oral mucosal cells. The long-term outcomes of COMET have to be elucidated.     

Oral epithelial cells transplanted on to corneal surface tend to adapt to the ocular phenotype

To understand the response of oral epithelial cells, transplanted on corneal surface to the ocular cues in vivo. The corneal button obtained after penetrating keratoplasty (PK) of an eye of a patient with total limbal stem cell deficiency (LSCD), previously treated with cultured oral mucosal epithelial transplantation (COMET) was examined by immunohistochemistry for the expression of keratins, p63, p75, PAX6, Ki-67, CD31, and CD34. COMET followed by optical-PK has improved visual acuity to 20/40 and rendered a stable ocular surface. The excised corneal tissue showed the presence of stratified epithelium with vasculatures. The epithelial cells of the corneal button expressed K3, K19, Ki-67, p63, p75 and the cornea-specific PAX6 and K12. This study confirms that the oral cells, transplanted to corneal surface, survive and stably reconstruct the ocular surface. They maintain their stemness at the ectopic site and acquire some of the corneal epithelial-like characters.     

Successful primary culture and autologous transplantation of corneal limbal epithelial cells from minimal biopsy for unilateral severe ocular surface disease

BACKGROUND: Patients with severe unilateral ocular surface disease require reconstruction of the damaged ocular surface. We succeeded in culturing primary corneal limbal epithelial cells taken from minimal biopsy and, once grown, transplanting them on denuded amniotic membrane (AM). METHODS: Autologous corneal limbal epithelial cells from a 3 mm(2) biopsy of the uninjured eye were grown for 3 weeks on a denuded AM carrier. The resultant sheet was then transplanted onto the unilateral severely chemically injured eye. RESULTS: Minimal biopsy showed the autologous cultivated corneal epithelial cells to have 4-5 layers of sufficient stratification and to be well differentiated. At 19 months post-transplantation, the ocular surface epithelium was stable and there were no epithelial defects. CONCLUSION: We document that it is possible to produce sufficiently stratified, well differentiated, autologous cultivated corneal limbal epithelium on AM from a minimal biopsy of the donor eye and to transplant it onto the injured eye.