Corneal endothelial regeneration and tissue engineering

Human corneal endothelial cells (HCECs) have a limited proliferative capacity. Descemet stripping with automated endothelial keratoplasty (DSAEK) has become the preferred method for the treatment of corneal endothelial deficiency, but it requires a donor cornea. To overcome the shortage of donor corneas, transplantation of cultured HCEC sheets has been attempted in experimental studies. This review summarizes current knowledge about the mechanisms of corneal endothelial wound healing and about tissue engineering for the corneal endothelium. We also discuss recent work on tissue engineering for DSAEK grafts using cultured HCECs and HCEC precursor cell isolation method (the sphere-forming assay). DSAEK grafts (HCEC sheets) were constructed by seeding cultured HCECs on human amniotic membrane, thin human corneal stroma, and collagen sheets. The pump function of the HCEC sheets thus obtained was approximately 75%–95% of that for human donor corneas. HCEC sheets were transplanted onto rabbit corneas after DSAEK. While the untransplanted control group displayed severe stromal edema, the transplanted group had clear corneas throughout the observation period. The sphere-forming assay using donor human corneal endothelium or cultured HCECs can achieved mass production of human corneal endothelial precursors. These findings indicate that cultured HCECs transplanted after DSAEK can perform effective corneal dehydration in vivo and suggest the feasibility of employing the transplantation of cultured HCECs to treat endothelial dysfunction. Additionally, corneal endothelial precursors may be an effective strategy for corneal endothelial regeneration.     

Endothelial cell transplantation and growth behavior of the human corneal endothelium

Background: The human corneal endothelium has a limited proliferative capacity in vivo. Until now it has only been possible to replace damaged endothelium by transplantation of a donor cornea. After establishing methods for the isolation and in vitro cultivation of human corneal endothelial cells, transplantation of these cells my be an alternative therapeutic option. Materials and methods: In this review methods for the in vitro cultivation of human corneal endothelial cells and their transplantation on the Descemet membrane of donor corneas are described. Results: In vitro proliferation of human adult corneal endothelial cells was achieved by the development of defined cell culture conditions, including supplementation of culture medium with specified growth factors and substances. Dependent on the culture conditions, as well as independent of them, in vitro cultured endothelial cells showed phenotypic changes and different proliferative behavior. Thus, molecular biological examinations revealed a different expression pattern of growth factor receptors in fibroblast-like endothelial cells (dedifferentiated) compared to typical endothelial cells (differentiated). Moreover, the proliferative capacity of the cells differed, dependent on their corneal location. Cells isolated from the peripheral part of donor corneas have a higher proliferative capacity than cells obtained from the central part. The propagation of corneal endothelial cells in vitro offered the possibility of their transplantation on donor corneas in an in vitro model. After transplantation, these cells formed a monolayer whose morphology and cell density depended on the differentiation of the cells. DNA synthesis was predominantly detectable in cells of the corneal periphery. Conclusions: Our findings are the basis of the following hypothesis: the periphery of the cornea represents a regenerative zone of the corneal endothelium. The fact that early after transplantation corneal endothelial cells form a monolayer on the natural extracellular matrix (ECM), which shows contact inhibition, suggests that inhibitory factors are released by the Descemet membrane that influence the proliferation of the cells. Further studies on the regulation of the proliferation and differentiation of human corneal endothelial cells in vitro and after transplantation might offer the possibility to establish a selective procedure for the treatment of corneal endothelial cell loss in the near future.      

Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells

BACKGROUND: Removal of bovine serum from organ culture medium is necessary because of the variability in serum composition and the potential risk of infection. Two specific endothelial cell media (F99 and Endothelial-SFM) were compared with the routinely used medium MEM for their use in serum free cultivation of human corneal endothelial cells (HCEC) and donor corneas. METHODS: HCEC were incubated in three test media with or without increasing serum content and a growth assay was performed. Seven pairs of donor corneas were cultured in each of three media for 3 weeks, one cornea with serum supplementation and one without. Endothelial cell density was determined once each week. Trypan blue staining of the endothelium and vital staining of keratocytes was performed after 3 weeks. RESULTS: All three media promoted proliferation of cultured HCEC when supplemented with serum. Endothelial cell density of donor corneas was comparable after 3 weeks of cultivation in the different media. Only corneas cultured in medium MEM without serum exhibited a higher endothelial cell loss. Trypan blue staining of the endothelium after cultivation revealed the lowest number of damaged cells on corneas cultured in the medium Endothelial-SFM. The highest densities of keratocytes were found in corneas cultured in Endothelial-SFM and the lowest densities occurred after culture in MEM. CONCLUSION: After incubation in Endothelial-SFM even under serum free conditions corneas were found to be of higher quality with respect to endothelial cell survival, cell membrane integrity, and keratocyte density. This medium may replace MEM, which is routinely used in European eye banks but requires supplementation with serum.     

Morphological and functional analysis of immortalized human corneal endothelial cells after transplantation.

Approximately 50% of donor corneas are unsuitable for keratoplasty due to an unacceptably low endothelial cell count. One way of overcoming this problem and minimizing wastage of donor corneas may be to transplant cultured human corneal endothelial cells onto these. In this study, we examined the morphological characteristics and functional attributes of endothelial layers formed after the transplantation of immortalized cells in vitro. Cultured human corneal endothelial cells, immortalized by transfection with a plasmid encoding SV40 T-antigen, were seeded onto human corneas denuded of their own endothelium. Seven days after transplantation the newly established monolayers were examined by light, confocal and scanning electron microscopy. Endothelial pump function was gauged by monitoring changes in corneal thickness during perfusion of the endothelial face.The endothelia formed from transplanted immortalized cells had a cobblestone-like appearance, being composed of polygonal units joined by junctional complexes. The stromal hydration state of corneas bearing such endothelial layers could be controlled during perfusion. This was an active process achieved via the Na(+)/K(+)-ATPase-dependent endothelial pump, as demonstrated by inhibiting the enzyme with ouabain.Transplantation of immortalized human corneal endothelial cells onto recipient corneas led to the establishment of new monolayers which had the morphology of the native ones in organ-cultured corneas. This model provides us with a means of studying the formation and function of corneal endothelial layers in vitro.     

Transplantation of corneas reconstructed with cultured adult human corneal endothelial cells in nude rats

PURPOSE: The feasibility of corneal reconstruction with cultured adult human corneal endothelial cells (HCEC) was examined in a nude rat model. METHODS: Endothelial cells were removed from the corneas of Lewis rats using a sterile cotton swab. Cultured adult HCEC labelled with a fluorescent marker chloromethyl-benzamidodialkylcarbocyanine (CM-Dil) were seeded onto the denuded Descemet's membrane. Then the corneas were centrifuged, incubated for 2 days, and transplanted into the eyes of nude rats using the penetrating keratoplasty technique (HCEC group). Control nude received corneas denuded of endothelium and without HCEC. The operated eyes were observed for 28 days after transplantation, and then were subjected to histological and fluorescein microscopic examination. RESULTS: The mean corneal thickness was significantly smaller in the HCEC group than in the control group throughout the observation period. The corneal endothelial cell density of the grafts at 28 days postoperatively ranged from 2425 to 3250 cells mm(-2) (mean+/-sd, 2744+/-337 cells mm(-2)). Fluorescein microscopy at 28 days after surgery showed numerous DiI-labelled cells on the posterior corneal surface in the HCEC group. Frozen sections showed a monolayer of DiI-labelled cells on Descemet's membrane. CONCLUSIONS: Cultured adult HCEC function well and maintain corneal transparency for 1 month after transplantation in nude rats.     

Human corneal endothelial cells: isolation, characterization and long-term cultivation

Long-term cultures of human corneal endothelial cells have been established. In culture, these cells form a dense monolayer (about 500,000 cells cm-2), similar to that found in vivo, and synthesize an extracellular matrix containing laminin, entactin, and fibronectin. Factor VIII and angiotensin-converting enzyme were not found in either the cultured or native corneal endothelium. Cells were obtained by scraping corneal buttons that had been preincubated in the culture medium supplemented with endothelial cell mitogen. The human corneal endothelium was grown under conditions virtually the same as those used for cultivation of human vascular endothelial cells, namely, on fibronectin- or gelatin-coated tissue culture plastic in Medium 199 supplemented with 20% human serum and 400 micrograms ml-1 endothelial cell growth supplement. Human corneal endothelial cells from the culture obtained can be used for transplantation onto human corneas, for studying repair of damaged corneal endothelium in situ, as well as for in vitro studies of cell growth regulation.     

Effect of donor age on morphologic variation of cultured human corneal endothelial cells

PURPOSE: To examine the effect of donor age on the morphologic variation of cultured human corneal endothelial cells (HCEC). METHODS: HCEC were obtained from the remaining corneoscleral rims of seven human corneas used for penetrating keratoplasty. The donor age ranged from 2 to 75 years. Primary cultures were established from explants of the endothelial cell layer, including the Descemet's membrane, and were propagated on culture dishes coated with bovine corneal endothelial extracellular matrix. At the fourth passage, frequency distribution of cell area in the confluent monolayer was calculated and the effect of donor age on cell area was analyzed. RESULTS: The percentage of HCEC with cell area over 2000 microm2 significantly increased with donor age (r = 0.935, p = 0.0007). CONCLUSION: Cultured HCEC established from older donor tissue display greater heterogeneity. The use of HCEC from younger donors may be preferable to maximize the benefits of HCEC transplantation.     

Cultured human corneal endothelial cell transplantation

Researchers have demonstrated the feasibility of transplanting human cultured corneal endothelial cells (HCEC) in various animal models. This review provides an overview of recent advances in our understanding of cultured corneal endothelial cell transplantation. We propose HCEC transplantation with a collagen sheet as the substitute carrier of HCEC. We also propose a novel strategy for corneal endothelial cell deficiency with the injection of adult human corneal endothelial precursors (HCEP). Using white rabbits or nude rats as keratopathy models, cultured HCEC were seeded on a collagen sheet. Descemetorhexis was performed on rabbit eyes. The HCEC collagen sheet was brought into the anterior chamber and fixed to the posterior stroma (HCEC group). Rabbit corneas with collagen sheet transplantation after descemetorhexis(collagen group) and with only descemetorhexis(no transplantation group) were the controls, respectively. As for HCEP transplantation, HCEP, isolated from rabbit corneal endothelial cells by sphere-forming assay, were injected into the anterior chamber and a face-down position was maintained for 24 hours in the rabbits (HCEP group). Pump function parameters of the HCEC sheets were 76-95% of those of human donor corneas. Mean corneal thickness in the HCEC group was significantly less than in the collagen and no transplantation groups 1, 3, 7, 14, 21, and 28 days (p< 0.05) after surgery. Cells were spread over the rear corneal surface in the HCEC group. In HE staining, marked stromal edema was present in the collagen and in the no transplantation groups, but not in the HCEC group with collagen sheets bearing monolayer cells. In the HCEP group, injected spheres were spread over the rear surface of the cornea and corneal edema was markedly suppressed. Our findings indicate that transplantation of cultured HCEC from adult human donor cornea by means of a collagen sheet can maintain the function of corneal dehydration. This suggests the feasibility of transplantation using cultured HCEC with a collagen sheet for corneal endothelial cell dysfunction. Additionally, adult precursor injection therapy can be also an effective strategy for corneal endothelial cell deficiency in place of conventional full-thickness corneal transplantation.     

Apoptosis in the endothelium of human corneas for transplantation

PURPOSE: To determine whether endothelial cell loss of human corneas stored in organ culture before transplantation is due to apoptosis. METHODS: The corneal endothelium of human corneas, stored in organ culture at 34 degrees C for varying periods of time, were analyzed for the presence of apoptotic cells using the TdT-mediated dUTP nick-end labeling (TUNEL) technique. Corneal endothelial cell apoptosis was confirmed by Hoechst staining and immunolabeling with anti-caspase 3 active antibody. RESULTS: Apoptotic cells were identified in the corneal endothelium of human organ cultured corneas: their number and distribution demonstrated a close correlation with corneal folding and overall quality of the corneal endothelium. TUNEL-positive labeling of cells was confirmed as apoptotic by the presence of morphologic nuclear alterations identified by Hoechst staining and the presence of immunostaining for caspase-3 activity. Corneal endothelial cell apoptosis was independent of cause of donor death, death to enucleation time, and death to culture times. CONCLUSIONS: Corneal endothelial cell apoptosis appears to determine the suitability of a cornea for transplantation.     

Properties of Corneas Reconstructed with Cultured Human Corneal Endothelial Cells and Human Corneal Stroma

Purpose

To examine the properties of corneas tissue-engineered with cultured human corneal endothelial cells (HCEC) and human corneal stroma.

Methods

Primary HCEC cultures were established from endothelial cell layer explants and propagated on culture dishes coated with bovine corneal endothelial extracellular matrix. A cell suspension of HCEC at the fifth passage was transferred onto human corneal stroma deprived of endothelial cells, and the corneas were gently centrifuged to enhance cell attachment. The cell density of the tissue-engineered corneas was examined after staining with alizarin red and trypan blue. The tissue-engineered corneas were histologically examined by light and electron microscopy. The pump function of the tissue-engineered corneas was measured using an Ussing chamber.

Results

The mean endothelial cell density of four tissue-engineered corneas was 2380 ± 264 cells/mm² (mean ± SD). HCEC on the tissue-engineered corneas had a morphology similar to HCEC in vivo. The pump function parameters of the tissue-engineered corneas were 55%–75% of those of normal corneas.

Conclusions

HCEC on the tissue-engineered corneas have morphology and cellular density similar to HCEC in vivo, whereas the pump function of the tissue-engineered corneas was lower than in normal corneas. Jpn J Ophthalmol 2005;49:448–452 © Japanese Ophthalmological Society 2005     

Proliferative capacity of corneal endothelial cells

The corneal endothelial monolayer helps maintain corneal transparency through its barrier and ionic "pump" functions. This transparency function can become compromised, resulting in a critical loss in endothelial cell density (ECD), corneal edema, bullous keratopathy, and loss of visual acuity. Although penetrating keratoplasty and various forms of endothelial keratoplasty are capable of restoring corneal clarity, they can also have complications requiring re-grafting or other treatments. With the increasing worldwide shortage of donor corneas to be used for keratoplasty, there is a greater need to find new therapies to restore corneal clarity that is lost due to endothelial dysfunction. As a result, researchers have been exploring alternative approaches that could result in the in?vivo induction of transient corneal endothelial cell division or the in?vitro expansion of healthy endothelial cells for corneal bioengineering as treatments to increase ECD and restore visual acuity. This review presents current information regarding the ability of human corneal endothelial cells (HCEC) to divide as a basis for the development of new therapies. Information will be presented on the positive and negative regulation of the cell cycle as background for the studies to be discussed. Results of studies exploring the proliferative capacity of HCEC will be presented and specific conditions that affect the ability of HCEC to divide will be discussed. Methods that have been tested to induce transient proliferation of HCEC will also be presented. This review will discuss the effect of donor age and endothelial topography on relative proliferative capacity of HCEC, as well as explore the role of nuclear oxidative DNA damage in decreasing the relative proliferative capacity of HCEC. Finally, potential new research directions will be discussed that could take advantage of and/or improve the proliferative capacity of these physiologically important cells in order to develop new treatments to restore corneal clarity.     

Transplantation of adult human or porcine corneal endothelial cells onto human recipients in vitro. Part I: Cell culturing and transplantation procedure

PURPOSE: To develop a method for grafting endothelial cells isolated from organ-cultured adult human corneas onto the denuded Descemet's membrane of human recipients. METHODS: Adult human or porcine corneal endothelial cells were isolated and maintained in monolayer cultures before seeding. Recipient corneas were stripped of their own endothelium by one of three different methods (mechanical, chemical, or physical) and the completeness of removal assessed after vital staining. The utility of each method was evaluated by monitoring the quality of attachment of the seeded-cell population. The seeding density of transplanted cells required for optimal results also was determined and the final numeric cell density achieved on recipient corneas after culturing for 7-20 days ascertained. The influence of incubating source cells with fibroblast growth factor (FGF), both on this latter parameter and on cell morphology, also was evaluated. The functional integrity of regrafted endothelium was assessed in 24-h perfusion experiments. RESULTS: The seeding of between 150,000 and 700,000 cells onto recipient corneas, followed by gentle centrifugation to improve attachment, yielded maximal final numeric cell densities of 3,450/mm2 and 1,850/mm2 in porcine and human lines, respectively. Recipient corneas were most effectively denuded of their own endothelium by freezing-and-thawing. The newly established endothelial monolayer remained stable for up to 20 days in organ culture (longest period monitored). FGF treatment did not enhance the final numeric density of cells attained on recipient corneas, but it did have a beneficial effect on their morphology. Only those recipient corneas that exhibited a well-differentiated monolayer of seeded endothelial cells underwent stromal deswelling near to physiologic levels. CONCLUSION: A practical working model has been developed, whereby recipient corneas stripped of their own endothelium can be furnished with a "new," near-normal endothelium by appropriate manipulations of the seeded-cell population. This now paves the way for a realistic tackling of the problem of endothelial cell paucity in donor corneas destined for transplantation.     

角膜内皮细胞治疗研究进展

人角膜内皮细胞(HCECs)是一种有丝分裂后的单层内皮细胞,因此,其在体内和体外的增殖能力十分有限。HCECs在严重受损的情况下会发生内皮失代偿,极易引起失明。目前,唯一有效的治疗方法是使用含健康角膜内皮的供体植片进行角膜移植。因此,世界范围内供体材料的严重短缺推动了对角膜内皮替代来源的研究。随着HCECs的细胞培养研究的不断开展,细胞治疗为角膜内皮失代偿提供了希望。本文对角膜内皮细胞治疗方面的最新研究进展进行综述。     

Heterogeneous induction of major histocompatibility complex class II antigens on corneal endothelium by interferon-gamma

The expression and distribution of major histocompatibility complex (MHC) class II gene products, HLA-DR, HLA-DQ, and the HLA-DR invariant chain, were studied on flat mounts of human corneal endothelial cells (HCEC) after in vitro incubation of donor corneas with interferon-gamma (IFN-gamma), interleukin-1 (IL-1), and IL-6, using a sensitive immunoperoxidase technique with monoclonal antibodies. Control HCEC and endothelium treated with IL-1 or IL-6 completely lacked MHC class II antigens. After treatment with 50 U/ml, 100 U/ml, 500 U/ml, and 5000 U/ml of human IFN-gamma, a mosaic-like, patchy staining for all MHC class II products was observed: part of the HCEC showed membranous and/or cytoplasmic positivity; other endothelial cells were negative. In addition, a dose-dependent response to IFN-gamma was observed: the proportion of cells expressing class II products rose with increasing doses of IFN-gamma. The induction of MHC class II antigen expression on HCEC by IFN-gamma was completely inhibited by the addition of a neutralizing antibody directed to IFN-gamma but not by IL-1 beta. The significance of these findings with respect to corneal transplantation immunology is discussed.     

Experimental gene transfer to the corneal endothelium

Transfer of cDNA to corneal endothelial cells has been demonstrated in cell monolayers in?vitro, in endothelium of whole thickness corneas ex?vivo and following intracameral injection. Studies examining the feasibility and optimal methods for gene transfer to the cornea have used viral and non-viral vectors, initially histochemical or fluorescent marker genes, and in endothelium of numerous species ranging from mouse to man. As the feasibility of genetic modification of corneal endothelial cells has been successfully demonstrated in a number of cell culture and animal models, there is significant potential for gene transfer in the treatment of human corneal endothelial disease. The two most widely studied applications of gene transfer to endothelium are (i) transduction of immunomodulatory genes to donor corneal endothelium prior to transplantation as a strategy to delay allogeneic rejection and (ii) modulation of apoptosis or induction of replication in human corneal endothelial cells to increase cell density. Although continued improvements in vectors for gene transfer will improve the efficacy and safety of gene therapy, more detailed understanding of the altered biology of endothelium in disease will be necessary to allow selection of appropriate targets for a gene-based treatment approach.     

Repopulation of denuded murine Descemet’s membrane with life-extended murine corneal endothelial cells as a model for corneal cell transplantation

· Background: Corneal endothelial cell transplantation has been an intriguing concept as an alternative to full-thickness penetrating keratoplasty. Using a murine corneal transplantation model, we sought to establish the optimal conditions to repopulate, ex vivo, denuded murine Descemet’s membrane with life-extended cell cultures of murine corneal endothelial cells. These ex vivo repopulated corneas were used as donor corneas in a murine orthotopic corneal transplantation model to assess, in vivo, the function of the transplanted, life- extended murine corneal endothelial cells (MCEC). · Methods: Mouse corneas were surgically trephined and the native corneal endothelium was removed mechanically using a sterile cotton swab. Cultured murine corneal endothelial cells (life extended by expression of the SV40 large T antigen) were added onto the denuded Descemet’s membrane and allowed to attach in culture at 37°C. Evidence of corneal cell attachment to Descemet’s membrane was determined between 1 and 8 h by scanning and transmission electron microscopy. Donor life-extended corneal endothelial cells were labeled with a fluorescent dye to allow tracking of the donor cells following seeding onto denuded Descemet’s membrane. In four independent experiments, the Descemet’s repopulated corneas were placed into syngeneic mice and evaluated for corneal clarity after 6 weeks. · Results: We could detect attachment of the life-extended murine CEC by scanning and transmission electron microscopy to denuded Descemet’s membrane. The optimal time for adherence was 2 h and these repopulated corneas were used as donors in a murine model of penetrating keratoplasty. Of 20 mice evaluated after 6 weeks, 4 displayed corneal clarity, and fluorescent evaluation demonstrated that only the donor corneal endothelial cells were present. · Conclusions: This experimental protocol establishes that ”life- extended” MCEC can bind to Descemet’s membrane ex vivo and form a distinct monolayer. The repopulated Descemet’s membrane allowed us to directly test the hypothesis that cultured life-extended corneal endothelial cells are functional when reintroduced into an in vivo milieu and provides evidence that specific corneal endothelial cell transplantation may be a viable alternative to pentrating keratoplasty. Received: 21 July 1999 Accepted: 22 September 1999     

Isolation and characterization of a mouse monoclonal antibody against human corneal endothelial cells.

In vitro cultivation of human corneal endothelial cells (HCEC) is associated with loss of typical cobblestone-like appearance during successive passages. Thus far morphology was the sole criterion for the cell's endothelial nature. Mouse monoclonal antibodies (mabs) to human corneal endothelial cells were raised using standard immunization and hybridoma isolation procedures. The specificity of mabs for human corneal endothelial cells was tested in comparison to other endothelial cell types, to fibroblasts, corneal keratocytes and to human retinal pigmented epithelial cells. In addition immunofluorescence or immunoperoxidase staining was performed with frozen tissue sections of human corneas and with various other human tissues. The mab 9.3.E reacts with cultured human corneal endothelial cells, but not with cultured human fibroblasts and human keratocytes. In frozen sections selective positivity of corneal endothelium in contrast to negativity of the other corneal cell types was confirmed. In investigated extraocular tissues positivity was observed in smooth muscle cells including related cells (i.e. Ito and mesangial cells) and in Schwann's cells and adipocytes, but apparently not in vascular endothelial cells. The mab is human-specific and binds to a protein with a molecular weight of 130 kDa mainly accumulating along cell membranes. A mouse monoclonal antibody against human corneal endothelial cells was established in vitro and was shown to be capable of differentiating corneal endothelial cells from other corneal cell types, especially from corneal keratocytes. It is, however, not cornea-specific, but also reacts with certain extraocular cell types.     

Regeneration with proliferation of the endothelium of cultured human donor corneas with extended postmortem time

PURPOSE: To examine the endothelium of donor corneas with extended postmortem time for survival and reparative mechanisms in an eye bank organ culture storage system. METHODS: We obtained 14 pairs of donor corneas with a postmortem time ranging from 29 to 163 hours. One cornea of a pair was immediately fixed for the study of structural changes postmortem and to serve as a control. The second was stored in organ culture for 3 days and thereafter fixed to be studied for reparative processes. Examination was done with light microscopy and scanning electron microscopy. Immunohistochemical staining with antibodies against proliferating cell nuclear antigen, Ki-67, and n-cadherin was performed to examine for cell proliferation and to characterize the cells. RESULTS: The control corneas showed increasing endothelial cell damage with increasing postmortem time. After 5-7 days postmortem, most cells were structurally damaged. After 3 days in organ culture, all corneas acquired an endothelial covering of the posterior surface, with cells, suggesting proliferation in both scanning preparations and in cross-sections. Positive endothelial cell staining with proliferating cell nuclear antigen was found in all cultured corneas. Ki-67 staining of the endothelium was found in 9 of the cultured corneas. CONCLUSIONS: The study showed survival of the corneal endothelium up to 7 days postmortem, and accordingly, the potential clinical use of donor corneas with extended postmortem time. Our results furthermore suggest that repair of the endothelium in donor corneas during organ culture storage occurs also by proliferation and not only by migration and enlargement of existing cells. If we uncover the mechanisms regulating cell proliferation in corneal endothelium, it should be possible to develop better storage methods of corneal transplants to improve quality and supply.     

Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model

PURPOSE: To evaluate the function of cultured human corneal endothelial cells (HCECs) in vivo and the feasibility of HCEC transplantation with a collagen sheet as the substitute carrier of HCECs. METHODS: Adult human donor cornea derived from cultured HCECs was labeled with the fluorescent tracker DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and seeded on a collagen sheet. The pump function of the HCEC sheet was evaluated by measurement of the potential difference and short-circuit current. A 6-mm sclerocorneal incision and Descemetorhexis were performed on rabbit eyes. The HCECs on a collagen sheet was brought into the anterior chamber and fixed to the posterior stroma (HCEC group). Rabbit corneas with collagen sheet transplantation after Descemetorhexis (collagen group) and with only Descemetorhexis (no-transplantation group) were the control. Each group, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations. RESULTS: Pump function parameters of the HCEC sheets were 76% to 95% of those of human donor corneas. Mean corneal thickness in the HCEC group was significantly less than in the collagen and no-transplantation groups 1, 3, 7, 14, 21, and 28 days (P < 0.05) after surgery. DiI-labeled cells were spread over the rear corneal surface in the HCEC group. Marked stromal edema was present in the collagen and no-transplantation groups with hematoxylin-eosin staining, but none in the HCEC group with collagen sheets bearing monolayer cells. CONCLUSIONS: The findings indicate that cultured HCECs transplanted from adult human donor cornea by means of a collagen sheet can retain their function of corneal dehydration in a rabbit model and suggest the feasibility of transplantation for CEC dysfunction using cultured HCECs with a collagen sheet.