Corneal Endothelial Cell Cultures from Organotypic Preservation of Older Donor Corneas Are Suitable for Advanced Cell Therapy.

INTRODUCTION: The purpose of this work was to evaluate the in vitro growth capacity and functionality of human corneal endothelial cells (hCEC) expanded from corneas of elderly (>60 years) donors that were preserved using an organotypic culture method (>15 days, 31°C) and did not meet the clinical criteria for keratoplasty. METHODS: Cell cultures were obtained from prior descemetorhexis (≥10 mm) and a controlled incubation with collagenase type I followed by recombinant trypsin. Cells were seeded on coated plates (fibronectin-albumin-collagen I) and cultures were expanded using the dual supplemented medium approach (maintenance medium and growth medium), in the presence of a 10 µm Rho-associated protein kinase inhibitor (Y-27632). Cell passages were obtained at culture confluency (∼2 weeks). A quantitative colorimetric WST-1 cell growth assay was performed at different time points of the culture. Morphometric analysis (area assessment and circularity), immunocytochemistry (ZO-1, Na+/K+-ATPase α, Ki67), and transendothelial electrical resistance (TEER) were performed on confluent monolayers. RESULTS: There was no difference between the cell growth profiles of hCEC cultures obtained from corneas older than 60 years, whether preserved cold or cultivated organotypic corneas. Primary cultures were able to maintain a certain cell circularity index (around 0.8) and morphology (hexagonal) similar to corneal endothelial mosaic. The ZO-1 and Na+/K+-ATPase pump markers were highly positive in confluent cell monolayers at 21 days after isolation (passage 0; P0), but significantly decreased in confluent monolayers after the first passage (P1). A weak expression of Ki67 was observed in both P0 and P1 monolayers. The P0 monolayers showed a progressive increase in TEER values between days 6 and 11 and remained stable until day 18 of culture, indicating a state of controlled permeability in monolayers. The P1 monolayers also showed some functional ability but with decreased TEER values compared to monolayers at P0. CONCLUSIONS: Our results indicate that it is possible to obtain functional hCEC cultures in eye banks, using simplified and standardized protocols, from older donor corneas (>60 years of age), previously preserved under organotypic culture conditions. This tissue is more readily available in our setting, due to the profile of the donor population or due to the low endothelial count (<2,000 cells/mm2) of the donated cornea.

38†Lyophilized amniotic membrane for pterygium surgery: long-term outcomes.

PURPOSE: To investigate the tolerability, security and long-term efficacy of lyophilized amniotic membrane (LAM) as an alternative to cryopreserved amniotic membrane in pterygium surgery. MATERIAL AND METHODS: Prospective case series of patients with primary nasal pterygium who undergone pterygium surgery and LAM implant either with sutures or glue. Postoperative follow-up was until month 24. Clinical and cosmetic outcomes, quality of life (as ocular comfort), and complications were evaluated. RESULTS: LAM was stiff and easy to manipulate as well as no tearing occurred during surgery or suturing. 4 patients (3 males) had pterygium surgery and LAM implant two with sutures and the other two with glue. Ocular comfort was checked and similar among those patients with LAM glued or sutured. After 24 months, there were no issues about tolerability or adverse events. Lower cosmetic outcomes (recurrence) were stated in 3 patients. CONCLUSION: Our study showed that LAM could be an effective alternative to cryopreserved amniotic membrane for graft after pterygium excision surgery. Its main advantage, storage at room temperature, can make it of immediate availability. Further studies comparing clinical outcomes of pterygium surgery with cryopreserved amniotic membrane versus LAM would confirm the benefits of the last.

4†New strategies in the barcelona eye bank to minimize the impact of the COVID-19 pandemic.

Since the start of the pandemic, the tissue donation in Catalonia (Spain) has decreased drastically. At the beginning of the lockdown (from March to May 2020) there was a drop of around 70% in donation of corneas and of approximately 90% in donation of placentas. Despite the fast updating of standard operating procedures, we had big difficulties in different points. For instance, in the availability of the transplant coordinator for the donor detection and evaluation, in obtaining the necessary PPE (personal protective equipment), or in the resources available in the quality control laboratories for screening. This, added to the collapse that hospitals suffered due to the large number of patients hospitalized each day, made donation levels slowly rebound.In order to provide solutions to all patients, we tried to adapt quickly to these emerging changes.In the case of corneas, we found a scenario that we had never had before. Although the cornea transplant plummeted at the beginning of the confinement (decreased by 60% compared to 2019), we run out of corneas -even for emergency situations- at the end of March.This situation led us to develop a new type of therapeutic solution in our Eye Bank. The cryopreserved cornea for tectonic purposes is a tissue that is kept frozen at -196°C and can be preserved for up to 5 years. Therefore, it is a tissue that allows us to respond to possible emergencies in subsequent similar situations.Regarding amniotic membrane for ocular care indications, the strategy was completely different. For this kind of tissue, we carried out an adaptation of our processing with two different purposes. On the one hand, to make sure that we could inactivate the SARS-CoV-2 virus, if it was there. On the other hand, to increase the donation of placentas. For this, changes in the transport medium and in the antibiotic cocktail were performed. In addition, an irradiation step was added to the final product.Little by little, it seems that the donations of corneas and placentas have been recovering. However, it is necessary to think about future contingency strategies in case a stop in donation is repeated.

Eye bank and theatre factors for positive microbiological culture of corneoscleral rim and cornea storage medium in the real-world.

OBJECTIVES: To evaluate microbiological culture rate and positivity of corneoscleral rim and cornea storage media as well as possible risk factors for contamination with real-world data. METHODS: Data of consecutive cornea donors implanted in the reference centre from January 2013 to January 2018 were reviewed. Information about cornea characteristics (donor demographic data, endothelial cell density, type of cornea conservation, days of storage, and precut vs full-thickness tissue), and microbiological culture information (corneoscleral rim vs storage sample, positive result) were statistically analysed. RESULTS: During the study period, 1369 corneas (737 donors) were implanted. Cultures were performed in 76.8% (n = 1052) of them and were positive in 3.2% of cases, mainly bacteria (84.4%). Corneas preserved in hypothermia represented 61.8% of all positive microbiology results (p < 0.001). Other analysed risk factors did not reach statistically significant association with microbiological positivity. None of the 34 cases with positive microbiological cultures reported ocular infection for the recipients in at least 6 months' follow-up. CONCLUSIONS: Microbiological tests rate in real-world practice are high despite not being compulsory. Organotypic cultured corneas showed a statistically less positivity in corneoscleral and storage medium than hypothermic ones, resulting in another advantage of this kind of cornea storage. Although precut corneas are thought to present less microbiological positivity, a statistically significant association was not found in the present study.