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Globally there is a shortage of available donor corneas with only 1 cornea available for every 70 needed. A large limitation to corneal transplant surgery is access to quality donor tissue due to inadequate eye donation services and infrastructure in many countries, compounded by the fact that there are few available long-term storage solutions for effectively preserving spare donor corneas collected in countries with a surplus. In this study, we describe a novel technology termed low-temperature vacuum evaporation (LTVE) that can effectively dry-preserve surplus donor corneal tissue, allowing it to be stored for approximately 5 years, shipped at room temperature, and stored on hospital shelves before rehydration prior to ophthalmic surgery. The dry-preserved corneas demonstrate equivalent biological characteristics to non-dried donor tissue, with the exception that epithelial and endothelial cells are removed and keratocytes are rendered non-viable and encapsulated within the preserved extracellular matrix. Structure and composition of the dried and rehydrated corneas remained identical to that of non-dried control corneas. Matrix-bound cytokines and growth factors were not affected by the drying and rehydration of the corneas. The ability to preserve human donor corneas using LTVE will have considerable impact on global corneal supply; utilisation of preserved corneas in lamellar keratoplasties, corneal perforations, ulcers, and tectonic support, will allow non-preserved donor tissue to be reserved for where it is truly required., Competing Interests: Declarations. Conflict of interests: OM, LB and LS declare they have no relevant financial or non-financial interests to disclose. AH declares a relationship with NuVision Biotherapies Ltd, Nottingham that includes employment and equity or stocks. EB declares a relationship with NuVision Biotherapies Ltd, Nottingham that includes employment. Ethical approval and consent to participate: Anonymised human corneas surplus to transplant requirement were obtained from SightLife (now CorneaGen, Seattle, WA, USA) under a materials transfer agreement. All work was performed in a laboratory under a research license from the UK Human Tissue Authority, UK. Informed consent was obtained from donors/relative prior to collection. Institutional ethical approval was not required as samples arrived anonymised, and consent was held at SightLife., (© 2025. The Author(s).)

Amniotic membrane (AM) is used to treat a range of ophthalmic indications but must be presented in a non-contaminated state. AM from elective caesarean sections contains natural microbial contamination, requiring removal during processing protocols. The aim of this study was to assess the ability of antibiotic decontamination of AM, during processing by innovative low-temperature vacuum-drying. Bioburden of caesarean section AM was assessed, and found to be present in low levels. Subsequently, the process for producing vacuum-dried AM (VDAM) was assessed for decontamination ability, by artificially loading with Staphylococcus epidermidis at different stages of processing. The protocol was highly efficient at removing bioburden introduced at any stage of processing, with antibiotic treatment and drying the most efficacious steps. The antibacterial activity of non-antibiotic treated AM compared to VDAM was evaluated using minimum inhibitory/biocidal concentrations (MIC/MBC), and disc diffusion assays against Meticillin-resistant Staphylococcus aureus, Meticillin-resistant S. epidermidis, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis. Antibacterial activity without antibiotic was low, confirmed by high MIC/MBC, and a no inhibition on agar lawns. However, VDAM with antibiotic demonstrated effective antibacterial capacity against all bacteria. Therefore, antibiotic decontamination is a reliable method for sterilisation of AM and the resultant antibiotic reservoir is effective against gram-positive and -negative bacteria.

Background: An in vitro injury model mimicking a corneal surface injury was optimised using human corneal epithelial cells (hCEC)., Aim: To investigate whether corneal-stroma derived stem cells (CSSC) seeded on an amniotic membrane (AM) construct manifests an anti-inflammatory, healing response., Methods: Treatment of hCEC with ethanol and pro-inflammatory cytokines were compared in terms of viability loss, cytotoxicity, and pro-inflammatory cytokine release, in order to generate the in vitro injury. This resulted in an optimal injury of 20% (v/v) ethanol for 30 s with 1 ng/mL interleukin-1 (IL-1) beta. Co-culture experiments were performed with CSSC alone and with CSSC-AM constructs. The effect of injury and co-culture on viability, cytotoxicity, IL-6 and IL-8 production, and IL1B , TNF , IL6 , and CXCL8 mRNA expression were assessed., Results: Co-culture with CSSC inhibited loss of hCEC viability caused by injury. Enzyme linked immunosorbent assay and polymerase chain reaction showed a significant reduction in the production of IL-6 and IL-8 pro-inflammatory cytokines, and reduction in pro-inflammatory cytokine mRNA expression during co-culture with CSSC alone and with the AM construct. These results confirmed the therapeutic potential of the CSSC and the possible use of AM as a cell carrier for application to the ocular surface., Conclusion: CSSC were shown to have a potentially therapeutic anti-inflammatory effect when treating injured hCEC, demonstrating an important role in corneal regeneration and wound healing, leading to an improved knowledge of their potential use for research and therapeutic purposes., Competing Interests: Conflict-of-interest statement: There are no potential conflicts of interest relevant to this study reported by the authors.

PurposeFibrin glue has been used successfully in numerous ophthalmic surgical procedures. Recently, fibrin glue has been used in limbal stem cell transplantation to reduce both operative time and to negate the need for sutures. The aim of this study was to determine the effects of fibrin glue on epithelial cell migration in vitro.MethodsCorneoscleral rims were split to retain the epithelial layer, Bowman's layer, and anterior stroma. Rims were cut into eight equal-sized pieces and were placed directly on culture plates or affixed with fibrin glue. Rims were maintained in culture for 25 days and epithelial cell growth was monitored. Cells were photographed to measure area or growth and immunofluorescence staining of explants for fibrin was performed.ResultsExplants that were glued demonstrated significantly delayed epithelial cell growth and migration as compared with explants without glue. By day 16, all fibrin glue had dissolved and coincided with onset of cell growth from glued explants. Cell growth commenced between days 3 and 4 for control explants without glue and around days 14-16 for explants with fibrin glue.ConclusionsFibrin glue delays epithelial cell migration by acting as a physical barrier and can potentially interfere with explant-derived limbal epithelial cell migration on to the corneal surface. We propose that glue should be used to attach the conjunctival frill of the limbal explant but care should be taken to ensure that the glue does not wrap around the explant if used to secure the explant as well. Strategic use of glue, to attach the recessed conjunctiva, can be advantageous in delaying conjunctival cell migration and reducing the need for sequential sector conjunctival epitheliectomy.

Purpose: Cells of the corneal epithelium and stroma can be distinguished in vivo by different intermediate filaments, cytokeratins for corneal epithelial cells (CEC) and vimentin for keratocytes. Isolated and cultured keratocytes change phenotype, losing expression of keratocyte markers and gaining markers associated with mesenchymal stromal cells (MSC). This study investigates this change in phenotype in relation to intermediate filament expression in cultured corneal stromal cells (CSC) compared to CEC., Methods: Expression of epithelial markers (CK3, CK12, CK19, pan cytokeratin, E-cadherin), keratocyte markers (CD34, vimentin), and MSC markers (CD73, CD90, and CD105) were compared in CEC and CSC by immunocytochemistry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Expression was evaluated at different stages of CSC culture and compared to another stromal cell type, extracted from Wharton's jelly (WJ-MSC)., Results: In vivo keratocytes did not express cytokeratins. However, cultured CSC expressed epithelial-associated CK3, CK12, and CK19, but not other cytokeratins. Expression of cytokeratins increased as CSC were passaged and decreased as CSC were induced to become quiescent. Comparatively, WJ-MSC expressed lower levels of CK3, CK12, and CK19, but also stained for pan cytokeratin and expressed KRT5., Conclusions: Cultured CSC undergo phenotypic change during culture, expressing specific cytokeratin filaments normally associated with CEC. Cytokeratin expression begins as cells are cultured on plastic and increases with passage. This discovery may influence the way in which differences are discerned between cultured CEC and CSC. Investigators need to be aware that the expression of cytokeratins does not necessarily represent epithelial contamination, and that CEC and CSC may be more related than previously recognized.

Purpose: Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material., Methods: AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays., Results: Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM., Conclusions: Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors.

Introduction: The corneal stroma is being increasingly recognized as a repository for stem cells. Like the limbal and endothelial niches, stromal stem cells often reside in the peripheral cornea and limbus. These peripheral and limbal corneal stromal cells (PLCSCs) are known to produce mesenchymal stem cells in vitro. Recently, a common corneal stromal and epithelial progenitor was hinted at. This study aims to examine the stem cell potential of corneal stromal cells and to investigate their epithelial transdifferentiation ability., Methods: PLCSCs were grown in traditional Dulbecco modified Eagle medium (DMEM)-based keratocyte culture medium and an M199-based medium and analyzed for a profile of cell-surface markers by using flow cytometry and differentiated into mesenchymal phenotypes analyzed with quantitative polymerase chain reaction (qPCR) and histologic staining. PLCSCs in M199 were subsequently divided into subpopulations based on CD34 and CD105 expression by using fluorescence- activated cell sorting (FACS). Subpopulations were characterized by marker profile and mesenchymal differentiation ability. Both whole PLCSCs and subpopulations were also cultured for epithelial transdifferentiation., Results: Cells cultured in M199 demonstrated a more stem-like cell-surface marker profile, and the keratocyte marker CD34 was retained for several passages but absent in cells cultured in DMEM. Cells cultured in M199 also exhibited a greater mesenchymal differentiation potential, compared with DMEM. PLCSCs could be divided into CD34(+)CD105(+), CD34-CD105(+), and CD34-CD105- subpopulations, of which CD34(+)CD105(+) cells were the most stemlike with regard to marker expression and mesenchymal differentiation potential. Subpopulations of PLCSCs exhibited differing abilities to transdifferentiate into epithelial phenotypes. Cells that were initially CD34(+)CD105(+) showed the greatest differentiation potential, producing CK3(+) and CK19(+) cells, and expressed a range of both epithelial progenitor (HES1, FRZB1, DCT, SOD2, ABCG2, CDH1, KRT19) and terminally differentiated (DSG3, KRT3, KRT12, KRT24) genes., Conclusions: Culture medium has a significant effect on the phenotype and differentiation capacity of PLCSCs. The stroma contains a heterogeneous cell population in which we have identified CD34(+) cells as a stem cell population with a capacity for mesenchymal and epithelial differentiation.