26†Development of human amniotic membrane products for regenerative medicine applications.

INTRODUCTION: Human amniotic membrane (HAM) has important biological properties that make this tissue an ideal substrate for regenerative medicine applications, including treatment of ocular diseases and wound healing. NHSBT can successfully decellularise HAM for promoting enhancement of limbal stem cell expansion in vitro more efficiently than the cellular HAM.1 In this study we present new formulations of decellularised HAM as freeze-dried powder and derived natural hydrogel. The aim was to develop a variety of GMP-compliant allografts to treat ocular diseases. MATERIALS AND METHODS: Six HAM, obtained from elective caesarian deliveries, were dissected, decontaminated and subjected to an in-house developed decellularisation protocol including a mild SDS concentration as detergent and nuclease steps. Following decellularisation, the tissue was placed in a sterile tissue culture flask and freeze dried. The freeze-dried tissue was cut into pieces of ~1g each, dipped into liquid nitrogen, then ground with a pulverisette. Ground tissue was solubilised using porcine pepsin and 0.1M HCl (stirred for 48 hours, 25oC). At the end of solubilisation, the pre-gel solution was kept on ice to adjust the pH back to 7.4. Gelation was induced when the temperature of the solution was increased to 25oC and aliquots were used for both in vitro cytotoxicity (up to 48 hours) and biocompatibility (up to 7 days) testing (MG63 and HAM cells). Cells were added into the solution before gelling and on top after gelling. RESULTS: The pre-gel solution obtained from decellularised HAM appear homogenous without undigested powder, and it was able to gel within 20 minutes at RT. Gels with a concentration of 4-8mg/mL tissue powder retained shape (including in an aqueous environment). When added on top of gels, cells were observed to attach and proliferate over time. When added into gels, the cells were observed throughout the gels and appeared to be migrating through the gel. CONCLUSION: Acellular HAM can be successfully freeze dried and converted into new formulations for topical application (powder and hydrogel). The new formulations could improve HAM delivery and provide a better scaffold for tissue regeneration. To our knowledge, this is the first time an amnion hydrogel formulation has been developed in GMP compliant setting for tissue banking purpose. Further studies will also investigate the ability of amnion hydrogel to promote stem cells differentiation into the three lineages (adipogenic, chondrogenic, osteogenic) in and/or on the gels. REFERENCES: Figueiredo GS et al. Acta Biomater 2017;61, 124-133.

29†Production of ultra-thin decellularised dermis to treat severe ocular diseases.

INTRODUCTION: The ocular surface may be damaged by several ocular conditions such as chemical trauma, infection, neoplasia or autoimmune disease causing a loss of tissue and function leading to a painful loss of vision. Tissue regeneration is needed to re-establish homeostasis of the ocular surface and to preserve vision. Present replacement strategies have limitations ranging from availability of the same type of tissue to long-term stability. NHSBT currently produces decellularised dermis (DCD) for clinical allografting; comprising a "thin" (up to 1.0 mm) and a thick (>1.2 mm) DCD, used to treat non-healing leg ulcers or in rotator cuff repair. Even the thin DCD, however, is too thick for ophthalmic purposes. The objective of this study was to develop a new ultra-thin DCD for ocular allografting. MATERIALS AND METHODS: Skin was retrieved, with consent for non-clinical use, from the back, front and back of the thighs of 3 different deceased donors, within 48 hours post-mortem. The tissue was cut into 5x5 cm squares and decellularised over 5 days as follows: decontamination with antimicrobials, de-epidermalisation (1M NaCl), hypotonic washes, detergent washes (with 0.01% SDS) and nuclease incubation. The DCD obtained was examined for integrity, handleability, residual remaining DNA and potential ultra-structural changes (by histology, DAPI and hematoxylin and eosin staining). RESULTS: We obtained an intact ultra-thin DCD using the same standard GMP protocol, regularly used to decellularise skin for clinical use. Tissue handleability was comparable to amniotic membrane, as evaluated by the ophthalmic surgeons as well as tissue bank assistants. The mean thickness of the tissue was 0.25 mm (±0.11) at the end of processing (total N=18 samples from 3 donors). Histology confirmed successful removal of epithelial cells and integrity of the extracellular matrix. CONCLUSION: We have successfully validated standard operating procedures for the production of ultra-thin DCD, in the attempt to obtain a valid alternative to amnion for the reconstruction of specific ocular regions (fornix, eye lids), where increased strength may be required. The thickness measurements at the end of processing suggest ultra-thin DCD obtained could represent a promising scaffold for regeneration of conjunctival tissue.

3†Emergency salvage of time expired clinical corneas during the COVID-19 pandemic.

INTRODUCTION: Corneas for clinical use can be stored for a maximum of 28 days in organ culture medium after death. At the beginning of the COVID-19 pandemic in 2020 it became apparent that; a rare situation was arising in that clinical operations were being cancelled and that there would be a surplus of "clinical grade" corneas. Consequently, when the corneas reached the end of the storage period, if the tissue had appropriate consent, they were transferred to the Research Tissue Bank (RTB). However, University research had also stopped due to the pandemic and there was a situation where the RTB had good quality tissue without any users. Rather than discarding the tissue, a decision was made to store the tissue for future use by cryopreservation. MATERIALS AND METHODS: An established protocol for cryopreserving heart valves was adapted. Individual corneas were placed into wax histology cassettes then inside a Hemofreeze heart valve cryopreservation bag with 100 ml cryopreservation medium (10% Dimethyl sulphoxide)). They were frozen in a controlled rate freezer (Planer, UK) to below -150oC and stored in vapour phase over liquid nitrogen (VPLN) below -190oC. To assess morphology, six corneas were cut in half, one half was processed for histology whilst the other half was cryopreserved, stored for 1 week then thawed and processed for histology. The stains used were Haematoxylin and Eosin (H&E) and Miller's with Elastic Van Gieson (EVG). RESULTS: Comparative histological examination indicated that there were no visible, major, detrimental changes in morphology in the cryopreserved group as compared to the controls. Subsequently, a further, 144 corneas were cryopreserved. Samples were assessed for handling properties by eye bank technicians and ophthalmologists. The eye bank technicians felt that the corneas may be suitable for training purposes such a DSAEK or DMEK. The ophthalmologists said that they had no preference between the fresh or cryopreserved corneas, and both would be equally suitable for training purposes. CONCLUSION: Time expired, organ-cultured corneas, can be successfully cryopreserved using an established protocol by adapting the storage container and conditions. These corneas are suitable for training purposes and may prevent discard of corneas in future.

9†Supply of non-clinical ocular tissue from a tissue and eye services research tissue bank.

INTRODUCTION: NHS Blood and Transplant Tissue and Eye Services (TES) is a human multi-tissue, tissue bank supplying tissue for transplant to surgeons throughout the UK. In addition, TES provides a service to scientists, clinicians and tissue bankers by providing a range of non-clinical tissue for research, training and education purposes. A large proportion of the non-clinical tissues supplied is ocular tissue ranging from whole eyes, to corneas, conjunctiva, lens and posterior segments remaining after the cornea is excised. The TES Research Tissue Bank (RTB) is based within the TES Tissue Bank in Speke, Liverpool and is staffed by two full-time staff. Non-clinical tissue is retrieved by Tissue and Organ Donation teams across United Kingdom. The RTB works very closely with two eye banks within TES, the David Lucas Eye Bank in Liverpool and the Filton Eye Bank in Bristol. Non-clinical ocular tissues are primarily consented by TES National Referral Centre Nurses. METHODS AND RESULTS: The RTB receives tissue via two pathways. The first pathway is tissue specifically consented and retrieved for non-clinical use and the second pathway is tissue that becomes available when tissue is found to be unsuitable for clinical use. The majority of the tissue that the RTB receives from the eye banks comes via the second pathway. In 2021, the RTB issued more than 1000 samples of non-clinical ocular tissue. The majority of the tissue, ~64% was issued for research purposes (including research into glaucoma, COVID-19, paediatrics and transplant research), ~31% was issued for clinical training purposes (DMEK and DSAEK preparation, especially after COVID-19 cessation of transplant operations, training for new eye bank staff) and ~5% was issued for in-house and validation purposes. One of the findings was that corneas are still suitable for training purposes up to 6-months after removal from the eye.In 2021, the RTB received 43 applications for ocular projects from new customers and supplied to 36 different projects, meeting 95% of all orders placed this year. DISCUSSION: The RTB works to a partial cost-recovery system and in 2021 became self-sufficient. The supply of non-clinical tissue is crucial for advancement in patient care and has contributed to several peer-reviewed publications.

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells.

PURPOSE: Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood. METHODS: Four human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs). RESULTS: scRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks. CONCLUSIONS: Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.

Co-expression of SARS-CoV-2 entry genes in the superficial adult human conjunctival, limbal and corneal epithelium suggests an additional route of entry via the ocular surface.

PURPOSE: The high infection rate of SARS-CoV-2 necessitates the need for multiple studies identifying the molecular mechanisms that facilitate the viral entry and propagation. Currently the potential extra-respiratory transmission routes of SARS-CoV-2 remain unclear. METHODS: Using single-cell RNA Seq and ATAC-Seq datasets and immunohistochemical analysis, we investigated SARS-CoV-2 tropism in the embryonic, fetal and adult human ocular surface. RESULTS: The co-expression of ACE2 receptor and entry protease TMPRSS2 was detected in the human adult conjunctival, limbal and corneal epithelium, but not in the embryonic and fetal ocular surface up to 21 post conception weeks. These expression patterns were corroborated by the single cell ATAC-Seq data, which revealed a permissive chromatin in ACE2 and TMPRSS2 loci in the adult conjunctival, limbal and corneal epithelium. Co-expression of ACE2 and TMPRSS2 was strongly detected in the superficial limbal, corneal and conjunctival epithelium, implicating these as target entry cells for SARS-CoV-2 in the ocular surface. Strikingly, we also identified the key pro-inflammatory signals TNF, NFKß and IFNG as upstream regulators of the transcriptional profile of ACE2+TMPRSS2+ cells in the superficial conjunctival epithelium, suggesting that SARS-CoV-2 may utilise inflammatory driven upregulation of ACE2 and TMPRSS2 expression to enhance infection in ocular surface. CONCLUSIONS: Together our data indicate that the human ocular surface epithelium provides an additional entry portal for SARS-CoV-2, which may exploit inflammatory driven upregulation of ACE2 and TMPRSS2 entry factors to enhance infection.