Cultivation Of Corneal Endothelial Cells Research Articles

Optimization of polycaprolactone - based nanofiber matrices for the cultivation of corneal endothelial cells

Posterior lamellar transplantation of the eye’ s cornea (DSAEK, DMEK) currently is the gold standard for treating patients with corneal endothelial cell and back surface pathologies resulting in functional impairment. An artificial biomimetic graft carrying human corneal endothelium could minimize the dependency on human donor corneas giving access to this vision-restoring surgery to large numbers of patients, thus reducing current long waiting lists. In this study, four groups of electrospun nanofibrous scaffolds were compared: polycaprolactone (PCL), PCL/collagen, PCL/gelatin and PCL/chitosan. Each of the scaffolds were tissue-engineered with human corneal endothelial cells (HCEC-B4G12) and analyzed with regard to their potential application as artificial posterior lamellar grafts. Staining with ZO-1 and Na+/K+-ATPase antibodies revealed intact cell functionalities. It could be shown, that blending leads to decreasing contact angle, whereby a heterogeneous blend morphology could be revealed. Scaffold cytocompatibility could be confirmed for all groups via live/dead staining, whereby a significant higher cell viability could be observed for the collagen and gelatine blended matrices with 97 ± 3% and 98 ± 2% living cells respectively. TEM images show the superficial anchoring of the HCECs onto the scaffolds. This work emphasizes the benefit of blended PCL nanofibrous scaffolds for corneal endothelial keratoplasty.

Cultivation of corneal endothelial cells from sheep

Research is currently under way to produce tissue engineered corneal endothelium transplants for therapeutic use in humans. This work requires the use of model animals, both for the supply of corneal endothelial cells (CECs) for experimentation, and to serve as recipients for test transplants. A variety of species can be used, however, a number of important advantages can be gained by using sheep as transplant recipients. The purpose of the present study was therefore to develop a method for culturing sheep CECs that would be suitable for the eventual construction of corneal endothelium grafts destined for sheep subjects. A method was established for culturing sheep CECs and these were compared to cultured human CECs. Results showed that cultured sheep and human CECs had similar growth characteristics when expanded from corneal endothelium explants on gelatin-coated plates, and achieved similar cell densities after several weeks. Furthermore, the markers zonula occludens-1, N-cadherin and sodium potassium ATPase could be immunodetected in similar staining patterns at cell boundaries of cultured CECs from both species. This work represents the first detailed study of sheep CEC cultures, and is the first demonstration of their similarities to human CEC cultures. Our results indicate that sheep CECs would be an appropriate substitute for human CECs when developing methods to produce tissue engineered corneal endothelium transplants.

Cell-based approach for treatment of corneal endothelial dysfunction.

Decompensation of the corneal endothelium causes severe visual impairments that lead to blindness. Although corneal transplantation is a well-known effective therapy for corneal endothelial dysfunction, many patients are not afforded that therapeutic opportunity owing to the worldwide shortage of donor corneas. Thus, a tissue engineering-based therapy for treating corneal endothelial dysfunction is highly anticipated. Obstacles associated with the development of tissue engineering therapy include in vitro culture of corneal endothelial cells (CECs) and the techniques used to transplant those cells. Limited proliferation ability, cellular senescence, and fibroblastic transformation during culture are all problems associated with the cultivation of CECs. In addition, transplantation of cultured CECs is technically difficult because the corneal endothelium is composed of a fragile monolayer sheet of cells located at the posterior cornea. In this review article, we present our recent findings using a novel cell culture protocol and show that modulation of CEC adhesion properties through a Rho-kinase inhibitor enables transplantation of CECs in the form of a cell suspension without the use of a carrier. Finally, we provide an update on the clinical application status of a cell-based therapy for treating corneal endothelial dysfunction.

Cultivation of Corneal Endothelial Cells on a Pericellular Matrix Prepared from Human Decidua-Derived Mesenchymal Cells

The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs) is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM) as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs) via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na+/K+-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction.

Die Transplantation des kornealen Endothels – Möglichkeiten und Grenzen

Endothelial keratoplasty is a promising surgical procedure which may replace penetrating keratoplasty in cases of endothelial cell diseases of the cornea. This method may thereby help to prevent postoperative astigmatism and transplant rejection. A survey of publications reporting about results after endothelial keratoplasty shows that the main problem of this transplantation technique is a postoperative endothelial cell loss which is comparable to or even higher than that observed in penetrating keratoplasty. Improving surgical techniques led to a reduction of the endothelial cell loss, however, cell-based strategies to prevent postoperative cell loss or to enhance the cell densities of donor corneas or endothelial lamellae are rare. This review presents an overview of clinical results after endothelial keratoplasty. Current strategies in the field of cell biology and tissue cultivation of corneal endothelial cells, genetic manipulation of the corneal endothelium and tissue engineering strategies aiming at the production of transplantable endothelial cell sheets are described. The limited availability of donor corneas makes it mandatory to develop methods in the field of tissue engineering in order to improve corneal endothelial cell survival or to increase corneal endothelial cell density, using interdisciplinary approaches.