Jennifer Walshe

Cultivation of corneal endothelial cells from sheep

&NA; 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. HighlightsSheep and human corneal endothelial cell cultures are highly similar.Sheep corneal endothelial cells express characteristic markers in vitro.Small, compact corneal endothelial cells form next to explants in culture.

Demonstration of P-selectin expression and potential function in human corneal epithelial cells

&NA; In response to an unexpected observation of apparent localisation by immunocytochemistry, we have investigated the potential expression and function of P‐selectin (CD62P) in human corneal epithelial cells. The SV40 immortalised cell line, HCE‐T (validated by STR profiling), along with multiple donor corneal‐limbal tissue samples, were examined for P‐selectin expression using a combination of immunocytochemistry, Western blotting, RT‐PCR and immunohistochemistry. Potential expression of the major ligand for P‐selectin (P‐selectin glycoprotein ligand‐1; PSGL‐1; CD162) was also examined by immunocytochemistry and RT‐PCR. A selective inhibitor of P‐selectin‐PSGL‐1 binding (KF38789) was subsequently tested for effects on HCE‐T cells using a cell culture gap‐closure assay. HCE‐T cells as well as primary epithelial cultures derived from donor corneal‐limbal tissue, displayed positive immunostaining for P‐selectin. Staining was particularly evident at cell‐cell boundaries and at the outer edge of expanding epithelial islands. P‐selectin expression was confirmed by Western blotting and RT‐PCR (validated by product sequencing), as well as by immunohistochemistry performed on serial sections of corneal‐limbal tissue stained for P‐selectin, keratin 3 and p63. PSGL‐1 was detected by RT‐PCR and immunocytochemistry in both corneal epithelial cells as well as human limbal fibroblasts (HLF). KF38789 (5 &mgr;M) significantly reduced closure of a 500‐&mgr;m gap between confluent sheets of HCE‐T cells over an 8‐hr period (by ˜40%, p < 0.01; paired two‐tailed T test), but had no effect on culture gap‐closure by either HLF or murine 3T3 fibroblasts. These results provide evidence of P‐selectin expression in human corneal epithelial cells and suggest a potential role for this glycoprotein in facilitating the net movement of confluent sheets of human corneal epithelial cells. HighlightsHuman corneal epithelial (HCE) cells express P‐selectin in vitro and in situ.HCE and limbal fibroblasts both appear to express the P‐selectin ligand, PSGL‐1.An inhibitor of P‐selectin binding to PSGL‐1 reduces HCE migration in vitro.These findings suggest a potential new role for P‐selectin within the cornea.

Mounting of Biomaterials for Use in Ophthalmic Cell Therapies

When used as scaffolds for cell therapies, biomaterials often present basic handling and logistical problems for scientists and surgeons alike. The quest for an appropriate mounting device for biomaterials is therefore a significant and common problem. In this review, we provide a detailed overview of the factors to consider when choosing an appropriate mounting device including those experienced during cell culture, quality assurance, and surgery. By way of example, we draw upon our combined experience in developing epithelial cell therapies for the treatment of eye diseases. We discuss commercially available options for achieving required goals and provide a detailed analysis of 4 experimental designs developed within our respective laboratories in Australia, the United Kingdom, and Belgium.

Optimization of Corneal Epithelial Progenitor Cell Growth on Bombyx mori Silk Fibroin Membranes

Scaffolds prepared from silk fibroin derived from cocoons of the domesticated silkworm moth Bombyx mori have demonstrated potential to support the attachment and growth of human limbal epithelial (HLE) cells in vitro. In this study, we attempted to further optimize protocols to promote the expansion of HLE cells on B. mori silk fibroin- (BMSF-) based scaffolds. BMSF films were initially coated with different extracellular matrix proteins and then analysed for their impact on corneal epithelial cell adhesion, cell morphology, and culture confluency. Results showed that collagen I, collagen III, and collagen IV consistently improved HCE-T cell adherence, promoted an elongated cell morphology, and increased culture confluency. By contrast, ECM coating had no significant effect on the performance of primary HLE cells cultured on BMSF films. In the second part of this study, primary HLE cells were grown on BMSF films in the presence of medium (SHEM) supplemented with keratinocyte growth factor (KGF) and the Rho kinase inhibitor, Y-27632. The results demonstrated that SHEM medium supplemented with KGF and Y-27632 dramatically increased expression of corneal differentiation markers, keratin 3 and keratin 12, whereas expression of the progenitor marker, p63, did not appear to be significantly influenced by the choice of culture medium.

Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit.

The New Zealand White rabbit has been widely used as a model of limbal stem cell deficiency (LSCD). Current techniques for experimental induction of LSCD utilize caustic chemicals, or organic solvents applied in conjunction with a surgical limbectomy. While generally successful in depleting epithelial progenitors, the depth and severity of injury is difficult to control using chemical-based methods. Moreover, the anterior chamber can be easily perforated while surgically excising the corneal limbus. In the interest of creating a safer and more defined LSCD model, we have therefore evaluated a mechanical debridement technique based upon use of the AlgerBrush II rotating burr. An initial comparison of debridement techniques was conducted in situ using 24 eyes in freshly acquired New Zealand White rabbit cadavers. Techniques for comparison (4 eyes each) included: (1) non-wounded control, (2) surgical limbectomy followed by treatment with 100% (v/v) n-heptanol to remove the corneal epithelium (1-2xa0min), (3) treatment of both limbus and cornea with n-heptanol alone, (4) treatment of both limbus and cornea with 20% (v/v) ethanol (2-3xa0min), (5) a 2.5-mm rounded burr applied to both the limbus and cornea, and (6) a 1-mm pointed burr applied to the limbus, followed by the 2.5-mm rounded burr applied to the cornea. All corneas were excised and processed for histology immediately following debridement. A panel of four assessors subsequently scored the degree of epithelial debridement within the cornea and limbus using masked slides. The 2.5-mm burr most consistently removed the corneal and limbal epithelia. Islands of limbal epithelial cells were occasionally retained following surgical limbectomy/heptanol treatment, or use of the 1-mm burr. Limbal epithelial cells were consistently retained following treatment with either ethanol or n-heptanol alone, with ethanol being the least effective treatment overall. The 2.5-mm burr method was subsequently evaluated in the right eye of 3 live rabbits by weekly clinical assessments (photography and slit lamp examination) for up to 5 weeks, followed by histological analyses (hematoxylin & eosin stain, periodic acid-Schiff stain and immunohistochemistry for keratin 3 and 13). All 3 eyes that had been completely debrided using the 2.5-mm burr displayed symptoms of ocular surface failure as defined by retention of a prominent epithelial defect (∼40% of corneal surface at 5 weeks), corneal neovascularization (2-3xa0quadrants), reduced corneal transparency and conjunctivalization of the corneal surface (demonstrated by the presence of goblet cells and/or staining for keratin 13). In conclusion, our findings indicate that the AlgerBrush II rotating burr is an effective method for the establishment of ocular surface failure in New Zealand White rabbits. In particular, we recommend use of the 2.5-mm rotating burr for improved efficiency of epithelial debridement and safety compared to surgical limbectomy.