In Vitro Profiling of the Extracellular Matrix and Integrins Expressed by Human Corneal Endothelial Cells Cultured on Silk Fibroin-Based Matrices.

Developing a scaffold for culturing human corneal endothelial (HCE) cells is crucial as an alternative cell therapeutic approach to bridge the growing gap between the demand and availability of healthy donor corneas for transplantation. Silk films are promising substrates for the culture of these cells; however, their tensile strength is several-fold greater than the native basement membrane which can possibly influence the dynamics of cell-matrix interaction and the extracellular matrix (ECM) secreted by the cells in long-term culture. In our current study, we assessed the secretion of ECM and the expression of integrins by the HCE cells on Philosamia ricini (PR) and Antheraea assamensis (AA) silk films and fibronectin-collagen (FNC)-coated plastic dishes to understand the cell-ECM interaction in long-term culture. The expression of ECM proteins (collagens 1, 4, 8, and 12, laminin, and fibronectin) on silk was comparable to that on the native tissue. The thicknesses of collagen 8 and laminin at 30 days on both PR (4.78 ± 0.55 and 5.53 ± 0.51 µm, respectively) and AA (4.66 ± 0.72 and 5.71 ± 0.61 µm, respectively) were comparable with those of the native tissue (4.4 ± 0.63 and 5.28 ± 0.72 µm, respectively). The integrin expression by the cells on the silk films was also comparable to that on the native tissue, except for α3 whose fluorescence intensity was significantly higher on PR (p ≤ 0.01) and AA (p ≤ 0.001), compared to that on the native tissue. This study shows that the higher tensile strength of the silk films does not alter the ECM secretion or cell phenotype in long-term culture, confirming the suitability of using this material for engineering the HCE cells for transplantation.

Use of Decellularized SMILE (Small-Incision Lenticule Extraction) Lenticules for Engineering the Corneal Endothelial Layer: A Proof-of-Concept.

PURPOSE: To demonstrate the suitability of using decellularized SMILE (Small-incision Lenticule Extraction) lenticules for culturing and transplanting the corneal endothelium (CE). METHODS: The SMILE lenticules, obtained during refractive surgery, were decellularized by incubating in CE culture medium and fetal bovine serum. Decellularization was confirmed by hematoxylin and eosin staining, DAPI staining, and gel electrophoresis. The amount of DNA per milligram of dry tissue weight was calculated to quantify the residual nuclear content. The transparency of the decellularized lenticules was determined by calculating the modulation transfer function. Immunostaining for stromal collagens and glycosaminoglycan was performed using specific antibodies. Engineered tissue was constructed by culturing the CE cells on lenticules and staining for ZO-1, Na/K ATPase, and N-cadherin. The functionality of the engineered tissues was assessed by transplanting them onto edematous human donor corneas and perfusing for 10 days ex-vivo. RESULTS: The residual DNA per milligram of dry tissue weight was found to be significantly reduced (p < 0.0001) in serum (0.255 µg/mg) and Opti-MEM (0.140 µg/mg) when compared to fresh lenticules (3.9 µg/mg). Decellularization did not alter the arrangement of the collagen fibers or the transparency of the lenticules. CE cells attached and matured to express ZO-1, Na/K ATPase, and N-cadherin at two weeks after seeding. The engineered tissue upon transplantation significantly reduced the corneal edema (p < 0.05) and the transplanted cells remained intact on the SMILE lenticule post-transplantation. CONCLUSION: This study demonstrates the suitability of using SMILE lenticules decellularized using a simple, chemical-free method for engineering the corneal endothelium for transplantation.

In Vitro Expansion of Corneal Endothelial Cells for Clinical Application: Current Update.

ABSTRACT: Endothelial dysfunction is one of the leading causes of corneal blindness and one of the common indications for keratoplasty. At present, the standard of treatment involves the replacement of the dysfunctional endothelium with healthy tissue taken from a donor. Because there is a paucity of healthy donor tissues, research on the corneal endothelium has focused primarily on expanding these cells in the laboratory for transplantation in an attempt to reduce the gap between the demand and supply of donor tissues for transplantation. To expand these cells, which are nonmitotic in vivo, various mitogens, substrates, culture systems, and alternate strategies have been tested with varying success. The biggest challenge has been the limited proliferative capacity of these cells compounded with endothelial to mesenchymal transition that alters the functioning of these cells and renders them unsuitable for human transplantation. This review aims to give a comprehensive overview of the most common and successful techniques used in the culture of the cells, the current available evidence in support of epithelial to mesenchymal transition (EMT), alternate sources for deriving the corneal endothelial cells, and advances made in transplantation of these cells.

In Vitro Culture of Human Corneal Endothelium on Non-Mulberry Silk Fibroin Films for Tissue Regeneration.

Purpose: The purpose of this study was to determine if non-mulberry varieties of silk are suitable for the culture of corneal endothelium (CE). Methods: Aqueous silk fibroin derived from Philosamia ricini (PR), Antheraea assamensis (AA), and Bombyx mori (BM) were cast as approximately 15 µm films with and without pores on which human CE cells were cultured. Tensile strength, elasticity, transmittance in visible range, and degradation properties of the films were characterised. Adhesion of CE to the silk films was quantified using MTT assay in addition to quantifying the number and area of focal adhesions using paxillin. Expression of CE markers was determined at the gene and protein levels using PCR and immunostaining, respectively. Barrier integrity of the cultured cells was measured as permeability to FITC dextran (10 kDa) in the presence or absence of thrombin. Results: The films exhibited robust tensile strength, >95% transmittance and a refractive index comparable to the native cornea. BM degraded significantly faster when compared to PR and AA. A comparison between the three varieties of silk showed that significantly more cells were adhered to PR and AA than to BM. This was also reflected in the expression of stable focal adhesions on PR and AA, thus enabling the formation of intact monolayers of cells on these varieties unlike on BM. Treatment with thrombin significantly increased cellular permeability to dextran. Conclusions: Our data shows that PR and AA varieties sufficiently support the growth and function of CE cells. This could be attributed to the presence of natural cell binding motifs (RGD) in these varieties. Translational Relevance: Development of a suitable carrier for engineering the CE to address a major clinical requirement of healthy donor tissues for transplantation.