The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowman’s layer, stroma, Descemet’s membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified expression of CAV1, HOMER3 and CPVL in the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, CXCL14 was expressed exclusively in the suprabasal/superficial limbus, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.

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Purpose: This study compares the effect of the transport of conventionally prestripped Descemet membrane endothelial keratoplasty (DMEK) tissue with the DMEK revolutionary advanced Preloadable Injection Device (RAPID) preloaded transport system from Geuder AG (Heidelberg, Germany). Endothelial cell loss, tissue integrity, endothelial cell phenotype, and viability were assessed and compared. Methods: Twelve DMEK grafts were prestripped by the cornea bank and transported using the following 2 conditions: conventional flask (n = 6) or a preloaded transport cartridge (DMEK RAPID, n = 6). After transport, tissues were analyzed for cell density; denuded areas; immunolocalization of corneal endothelial markers, such as ZO-1, CD166, and Na+/K+ ATPase; histology analysis; and cell viability staining with Hoechst, calcein AM, and ethidium homodimer. Results: Endothelial cell loss (10.35% vs. 9.15%) did not differ between transport conditions. Histological analysis confirmed the integrity of the Descemet membrane and endothelial cell layer with both transport conditions. Similarly, the corneal endothelial cell mosaic was conserved in both conditions. The ZO-1 tight junctions confirmed the integrity of the confluent corneal endothelial cell monolayer. CD166 and Na+/K+ ATPase detection with immunofluorescence was also comparable. A similar percentage of dead cells was reported in both conditions (18.1% vs. 16.73%). Moreover, the surface covered with calcein-positive cells (59.02% vs. 61.95%) did not differ between transport conditions. Conclusions: Our results suggest that DMEK grafts can be prestripped or preloaded into a novel transport cartridge and shipped to the clinic with comparable endothelial cell loss, phenotypical marker expression, and viability to the conventional prestripped donor tissue.

The corneal endothelium is the innermost layer of the cornea that selectively pumps ions and metabolites and regulates the hydration level of the cornea, ensuring its transparency. Trauma or disease affecting human corneal endothelial cells (hCECs) can result in major imbalances of such transport activity with consequent deterioration or loss of vision. Since tissue transplantation from deceased donors is only available to a fraction of patients worldwide, alternative solutions are urgently needed. Cell therapy approaches, in particular by attempting to expand primary culture of hCECs in vitro, aim to tackle this issue. However, existing cell culture protocols result in limited expansion of this cell type. Recent studies in this field have shown that topographical features with specific dimensions and shapes could improve the efficacy of hCEC expansion. Therefore, potential solutions to overcome the limitation of the conventional culture of hCECs may include recreating nanometer scale topographies (nanotopographies) that mimic essential biophysical cues present in their native environment. In this review, we summarize the current knowledge and understanding of the effect of substrate topographies on the response of hCECs. Moreover, we also review the latest developments for the nanofabrication of such bio-instructive cell substrates.