Banking of post-SMILE stromal lenticules for additive keratoplasty: A new challenge for eye banks?

PURPOSE: ReLEx (Refractive Lenticule Extraction) Small Incision Lenticule Extraction (SMILE), the second generation of ReLEx Femtosecond Lenticule Extraction (FLEx), is a minimally invasive, flapless procedure designed to treat refractive errors such as myopia, hyperopia, presbyopia, and astigmatism. This review aims to provide a comprehensive overview of the methods for preserving SMILE-derived lenticules and discusses their potential future applications. METHODS: A narrative literature review was conducted using PubMed, Scopus, and Web of Science databases, focusing on articles published up to January 2024 and available in English. The authors also evaluated the reference lists of the collected papers to identify any additional relevant research. RESULTS: No standardized protocols currently exist for the storage or clinical application of SMILE-derived lenticules. However, these lenticules present a promising resource for therapeutic uses, particularly in addressing the shortage of donor corneal tissues. Their potential applications include inlay and overlay additive keratoplasty, as well as other ocular surface applications. Further research is needed to establish reliable protocols for their preservation and clinical use. CONCLUSION: SMILE-derived lenticules offer significant potential as an alternative to donor corneal tissues. Standardizing their storage and application methods could enhance their use in clinical settings.

Efficacy and Safety of Preoperative Prophylaxis in Cataract Surgery with Combined Topical Antiseptics: A Microbiological Study.

INTRODUCTION: To evaluate the antimicrobial efficacy of an ophthalmic formulation containing hexamidine diisethionate (HD) 0.05%, polyhexamethylene biguanide (PHMB) 0.0001%, and edetate disodium (EDTA) 0.01% (Keratosept®, Bruschettini, Genova, Italy) on the microbial flora of a healthy ocular surface. METHODS: Patients were enrolled consecutively. Each patient applied two drops of Keratosept® in the eye scheduled for cataract surgery (study eye) three times daily in the 2 days prior to surgery and one time in the morning of surgery. The contralateral eyes were considered as control (control eye). Bilateral conjunctival swabs were collected before the first administration (T0) and the morning of surgery (T1). The swabs were processed within 3 h from sampling for the automated detection of the presence of replicating microorganisms (colony-forming units, CFU/mL) and the provision of real-time growth curves. RESULTS: Conjunctival swabs of 32 patients (n = 128) were examined. Six patients were excluded from the efficacy analysis because of microbial load < 50 CFU/mL at T0 in the study eye. No difference between study and control eyes was observed at T0 (p = 0.40). Compared with T0, 20 (76.9%) study eyes and 10 (38.5%) control eyes showed a ≥ 1 log reduction of the microbial load at T1, with a significant difference between groups (p = 0.005). Keratosept® showed good tolerability, and no adverse events or eye discomfort were recorded. CONCLUSIONS: This study showed that the low-dose combination of antiseptic agents in the Keratosept® ophthalmic solution effectively reduces the bacterial load of healthy flora on the ocular surface.

Predicting Long-Term Endothelial Cell Loss after Preloaded Descemet Membrane Endothelial Keratoplasty in Fuchs' Endothelial Corneal Dystrophy: A Mathematical Model.

(1) Background: This study offers a biexponential model to estimate corneal endothelial cell decay (ECD) following preloaded "endothelium-in" Descemet membrane endothelial keratoplasty (DMEK) in Fuchs' endothelial corneal dystrophy (FECD) patients; (2) Methods: A total of 65 eyes undergoing DMEK alone or combined with cataract surgery were evaluated. The follow-up period was divided into an early phase (first 6 months) and a late phase (up to 36 months). Endothelial cell count (ECC) and endothelial cell loss (ECL) were analyzed; (3) Results: The half time of the ECD was 3.03 months for the early phase and 131.50 months for the late phase. The predicted time-lapse interval to reach 500 cells/mm2 was 218 months (18.17 years), while the time-lapse interval to reach 250 cells/mm2 was 349 months (29.08 years). There was no statistically significant difference between the ECL in DMEK combined with cataract extraction and DMEK alone at 24 months (p ≥ 0.20). At the late phase, long-term ECL prediction revealed a lower ECC half time in patients undergoing DMEK combined with cataract surgery (98.05 months) than DMEK alone (250.32 months); (4) Conclusions: Based on the mathematical modeling, a predicted average half-life of a DMEK graft could reach 18 years in FECD. Moreover, combining cataract extraction with DMEK could result in excessive ECL in the long term.

Deep Anterior Lamellar Keratoplasty Using Dehydrated versus Standard Organ Culture-Stored Donor Corneas: Prospective Randomized Trial.

PURPOSE: To compare the outcomes of deep anterior lamellar keratoplasty (DALK) using dehydrated versus standard organ culture-stored donor corneas for eyes with keratoconus. DESIGN: Prospective, randomized, single-center trial conducted in Italy. PARTICIPANTS: Adult patients (age ≥ 18 years) with keratoconus scheduled for elective DALK. METHODS: Patients undergoing successful type 1 bubble pneumatic dissection using a standard DALK technique were randomized during surgery to receive either dehydrated (n = 30) or standard organ culture-stored (n = 30) donor corneas. MAIN OUTCOME MEASURES: The primary study outcome was best spectacle-corrected visual acuity (BSCVA) 12 months after surgery. Secondary outcomes were refractive astigmatism (RA), endothelial cell density (ECD), and complication rates. RESULTS: Postoperative BSCVA did not significantly differ between groups at both time points: mean difference at 6 months was 0.030 logarithm of the minimum angle of resolution (logMAR; 95% confidence interval [CI], -0.53 to 0.10 logMAR; P = 0.471) and at 12 months was -0.013 logMAR (95% CI, -0.10 to 0.08 logMAR; P = 0.764). No significant differences between groups were observed in terms of postoperative RA and ECD at all time points. In the first 3 days after DALK, an epithelial defect was present in 10 patients (33%) in the organ culture cornea group and in 29 patients (97%) in the dehydrated cornea group. Complete re-epithelialization was achieved by day 7 in all patients (100%) in both groups. CONCLUSIONS: The study provides evidence that the use of dehydrated corneas is noninferior to the use of standard organ culture donor corneas for DALK. Corneal tissue dehydration represents a viable solution that can allow long-term cornea preservation and avoid wastage of unused corneas. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Ocular Manifestations in Patients Affected by p63-Associated Disorders: Ectrodactyly-Ectodermal Dysplasia-Clefting (EEC) and Ankyloblepharon-Ectodermal Defects-Cleft Lip Palate (AEC) Syndromes.

BACKGROUND/AIMS: The Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) and Ankyloblepharon-ectodermal defect-cleft lip/palate (AEC) syndromes are rare autosomal dominant diseases caused by heterozygous mutations in the p63 gene. Patients are characterized by abnormalities of the skin, teeth, and hair and have limb defects, orofacial clefting and ectodermal dysplasia. In addition, they often show ocular surface alterations, leading to progressive corneal clouding and eventually blindness. Here, we present 8 cases describing patients affected by EEC (n = 6, with 5 sporadic and 1 familial cases) and AEC (n = 2, both sporadic cases) syndromes. We attempt to provide a description of the ocular disease progression over the years. METHODS: Clinical examinations and monitoring of ocular parameters for the assessment of limbal stem cell deficiency were constantly performed on patients between 2009 and 2023. Quantitative data and comparison with existing cases described in the literature are reported. RESULTS: The therapies supplied to patients were essential for the management of the symptoms, but unfortunately did not halt the progression of the pathology. CONCLUSIONS: A constant monitoring of the patients would help avoid the sudden worsening of symptoms. If the progression of the disease slows down, it would allow for the development of newer therapeutic strategies aimed at correcting the genetic defect.

Hemi-Ultrathin Descemet Stripping Automated Endothelial Keratoplasty (Hemi-UT-DSAEK) Using Pediatric Donor Corneas: A Case Series.

OBJECTIVE: We sought to evaluate the clinical outcomes of hemi-UT-DSAEK grafts from the pediatric donor corneas of patients affected by Fuchs Endothelial Corneal Dystrophy (FECD). METHODS: A prospective, interventional case series was conducted at the Ophthalmology Department of Venice Civil Hospital and the Veneto Eye Bank Foundation (Venice, Italy). Six eyes of six patients affected by FECD received large-diameter, semicircular hemi-UT-DSAEK grafts obtained from three pediatric donor corneas using the standard pull-through method. Endothelial cell density (ECD), central corneal thickness (CCT), best-corrected visual acuity (BCVA) and intraoperative and postoperative complications were recorded at different time intervals up to 12 months. RESULTS: The average donor age was 64.6 ± 8.6 years, and the pre-operative ECD was 3266 ± 225 cells/mm2. At 12 months postoperatively, the average ECD was 1376 ± 509 cells/mm2 with a mean decrease of 56.8 ± 19.1% from the preoperative donor count. At 12 months, four out of six eyes had significantly improved and reached a BCVA of ≥20/25 (Snellen equivalent). The mean CCT significantly decreased from 788 ± 138 µm before surgery to 576 ± 30 µm at 12 months postoperatively (p < 0.01). CONCLUSIONS: Hemi-UT-DSAEK grafts using pediatric donor corneas are surgically feasible and can provide similar clinical outcomes compared to conventional UT-DSAEK. Transplanting pediatric donor tissues with high ECD into two patients could potentially increase the donor tissue pool to treat endothelial disease.

Preoperative surgeon evaluation of corneal endothelial status: the Viability Control of Human Endothelial Cells before Keratoplasty (V-CHECK) study protocol.

INTRODUCTION: The success of keratoplasty strongly depends on the health status of the transplanted endothelial cells. Donor corneal tissues are routinely screened for endothelial damage before shipment; however, surgical teams have currently no means of assessing the overall viability of corneal endothelium immediately prior to transplantation. The aim of this study is to validate a preoperative method of evaluating the endothelial health of donor corneal tissues, to assess the proportion of tissues deemed suitable for transplantation by the surgeons and to prospectively record the clinical outcomes of a cohort of patients undergoing keratoplasty in relation to preoperatively defined endothelial viability. METHODS AND ANALYSIS: In this multicentre cohort study, consecutive patients undergoing keratoplasty (perforating keratoplasty, Descemet stripping automated endothelial keratoplasty (DSAEK), ultra-thin DSAEK (UT-DSAEK) or Descemet membrane endothelial keratoplasty) will be enrolled and followed-up for 1 year. Before transplantation, the endothelial viability of the donor corneal tissue will be evaluated preoperatively through trypan blue staining and custom image analysis to estimate the overall percentage of trypan blue-positive areas (TBPAs), a proxy of endothelial damage. Functional and structural outcomes at the end of the follow-up will be correlated with preoperatively assessed TBPA values. ETHICS AND DISSEMINATION: The protocol will be reviewed by the ethical committees of participating centres, with the sponsor centre issuing the final definitive approval. The results will be disseminated on ClinicalTrials.gov, at national and international conferences, by partner patient groups and in open access, peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT05847387.

P26-A125†Donation medicine: a change of paradigm in procuring ocular tissues for transplantation.

PURPOSE: To reflect on the implementation of the concept of 'Donation Medicine' as a substitute for 'Procurement' to describe the Foundation's activities in the procurement of ocular tissue and donor selection, considering that the prevailing connotation of procurement (the action of obtaining materials, goods and services necessary to the functioning of a productive activity) did not express satisfactorily all the social, human and medical implications of a programme aimed at promoting ocular tissue donation and recovery. Moreover, in medicine the term 'Procurement' is generally associated with the worst therapeutic outcome, which is the end of a human life. METHODS: A retrospective evaluation of the main indicators pertaining to activities in 2021 was performed, with particular regard to donor screening ,tissue recovery, and the human and professional relations with donor families and hospital staff. The results were assessed by an interdisciplinary team, composed of eye bank and healthcare personnel, regulators and experts in medical humanities. RESULTS: In 2021, in light of 2944 non-oppositions to donation (opting out system), 891 consultations of the national SIT donor registry were performed (Sistema Informativo Trapianti), with 2551 clinical charts reviewed, 4332 related phone consultations performed, and 2032 nasopharyngeal swabs for SARS-CoV-2 nucleic acid tested; as a consequence, 2213 condolence and gratitude letters were sent to donor families, of which 57%(1269) conveyed the outcome of donation, along with 115 gratitude letters sent in instances of the non-recovery. 24 families requested, and were granted, the opportunity to visit the eye bank. CONCLUSION: A consensus was reached on the evidence that the term 'Procurement' has obvious limitations in the long term nurturing and maintenance of the motivation of the eye bank and healthcare personnel. As a consequence, the concept of 'Donation Medicine' was implemented to define and develop the activities related to the promotion of donation, the recovery of ocular tissues for transplantation, and internal/external relations with healthcare personnel, thus changing the meaning of 'Procurement', from a process at the end of a life to the realization of a new pathway of care that takes into account both donor families and recipients. Donation medicine begins with the re-opening of the donor clinical chart, the interaction with donor relatives and the recovery of a precious gift for use in the restoration of sight of patients.

P16-A111†Factors affecting the density of corneal endothelial cell cultures obtained from donor corneas.

PURPOSE: The shortage of donor corneas represents a worldwide problem, and corneal endothelial cell (CEC) therapy might be a promising alternative approach. CEC can be implanted alone, which has shown limited efficacy, or with a scaffold that holds the cells together as a monolayer tissue, thus imitating Descemet membrane endothelial keratoplasty. We believe that endothelial cell density (ECD) >2000 cells/mm2, a cut-off value that eye banks use to provide quality tissues for transplantation to surgeons, should also be adopted as a parameter to define the quality of CECs as a new Advanced Therapy Medicinal Product for clinical applications in patients with endothelial dystrophies. METHODS: We isolated and cultured CECs from one or more corneas of elderly age donors with ECDs higher than or below 2000 cells/mm2. CEC cultures were carried out on coated plates and on hydrogels with a preformed basement membrane (from TissueGUARD, Germany). Immunofluorescence with antibodies against ZO-1 was performed to evaluate the ECDs of the CEC graft obtained. RESULTS: Our results suggest that primary cultures with ECDs>2000 cells/mm2 can be obtained on coated plated only when (1) CECs are isolated from one or more corneas of young donors; (2) CECs are isolated and pooled together from at least 2 elderly age donor corneas (if ECD>2000 cells/mm2) or 3 elderly age donor corneas (if ECD<2000 cells/mm2). Secondary cultures are all characterized by low ECDs. Hydrogels have been shown to be able to lead to increased ECDs after their release. CONCLUSION: Our protocol highlights the difficulties in obtaining cultures with ECDs>2000 cells/mm2. Despite being achievable with corneas from young donors, this becomes challenging when corneas from elderly donors are used, i.e., the overall majority of those collected by eye banks, particularly when corneas from elderly age donors with ECD<2000 cells/mm2 are considered as a source. One alternative would be to isolate CECs from more corneas, but this might raise the issue of antigenic stimulation, which could eventually lead to transplantation failure. Our strategy to overcome these challenges is the use of a preformed basement membrane as a scaffold for CECs. However, this challenging approach should be investigated more before proceeding to clinical application.

Effect of Liposomal-Lactoferrin-Based Eye Drops on the Conjunctival Microflora of Patients Undergoing Cataract Surgery.

INTRODUCTION: Postoperative endophthalmitis is typically caused by the patient's conjunctival bacterial flora. Povidone iodine solution (5%) is used perioperatively to obtain periocular and ocular antisepsis. However, an adjunctive prophylaxis procedure could further help control the conjunctival microbial load. Considering the increase in antibiotic resistance, a progressive shift toward alternative methods would be desirable. Somilux® eye drops (Alfa Intes, lactoferrin-based eye drops) are medical devices containing liposomal lactoferrin (LF). This study evaluates the effects on conjunctival microflora of LF-based eye drops used in the preoperative phase in patients scheduled for cataract surgery. METHODS: LF-based eye drops or a vehicle solution (water solution) were instilled 4 times a day starting 3 days before cataract surgery. Before the therapy (T0) and at the time of surgery (T1), a conjunctival swab was performed in both eyes and processed to detect microbial growth, microbiological isolation, and species identification. The outcome was the quantification and characterization of the local microbial flora before and after using LF-based or vehicle-based eye drops. Safety of the treatments was also evaluated. RESULTS: 88 eyes of 44 patients (mean [± SD] age 75 [± 12.6] years) were enrolled. At baseline, 54 conjunctival swabs showed only saprophytic flora, 27 showed only potential pathogenic flora, and seven showed both of them. LF-based eye drops reduced the proportion of potentially pathogenic bacteria (36% at T0 vs. 9% at T1, p = 0.008) compared with the vehicle (41% at T0 vs. 55% at T1, p = 0.302) without altering the physiological ocular microbial composition. No adverse events have been reported. CONCLUSION: Our findings provide a novel contribution to the scientific knowledge on the role of LF in the ophthalmic field, supporting the use of LF-based eye drops as a safe and selective treatment to improve the ocular surface physiological defenses and control the bacterial ocular surface contamination prior to cataract surgery.

Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture.

Recent clinical studies suggest that retinal pigment epithelial (RPE) cell replacement therapy may preserve vision in retinal degenerative diseases. Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this study was to investigate human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells survival and behaviour on a decellularized Descemet's Membrane (DM), which may be of clinical relevance in retinal transplantation. DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope, scanning electron microscopy and histology. hESC-RPE cells were seeded onto the endothelial-side surface of decellularized DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. 24 hours post-seeding, hESC-RPE cell attachment and initial proliferation rate over the denuded DM were higher than hESC-RPE cells cultured on tissue culture inserts. On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell culture showed characteristic RPE cell morphology and proper protein localization. Gene expression analysis and VEGF secretion demonstrate DM provides supportive scaffolding and inductive properties to enhance hESC-RPE cells maturation. Decellularized DM was shown to be capable of sustaining hESC-RPE cells culture, thus confirming to be potentially a suitable candidate for retinal cell therapy.

Microscopic corneal epithelial changes and clinical outcomes in simple limbal epithelial transplantation surgery after treatment with amniotic membrane eye drops (AMED): A case report.

Purpose: To describe the microscopic epithelial changes and the clinical outcomes of a patient treated with amniotic membrane eye drops (AMED) because of a persistent epithelial defect (PED) and a partial limbal stem cell deficiency (LSCD) after simple limbal epithelial transplantation (SLET) and deep anterior lamellar keratoplasty (DALK). Observations: A 72-year-old patient, who had previously undergone SLET and DALK due to a total LSCD, presented with a PED related to a partial LSCD, and was treated with AMED for one month. We evaluated the patient's visual acuity, the Oxford grading scale, the Wong-Baker Pain Rating Scale, and in vivo confocal microscopy, both at baseline and 3 months after the end of treatment. Visual acuity improved from 0.5 to 0.4 LogMAR, the Oxford grading scale changed from grade III to grade I and the Wong-Baker Pain Rating Scale from grade 4 to grade 1. The corneal surface, which initially showed conjunctival characteristics over approximately 50% of the whole area, consisted mainly (75%) of mature corneal epithelium 3 months after the end of treatment. Conclusions and importance: While improving symptoms and clinical characteristics, AMED was also able to restore the normal corneal epithelium's morphology in a case of partial LSCD after SLET and DALK.

Results of ultrathin Descemet stripping automated endothelial keratoplasty with donor corneas preserved in synthetic organ culture media.

PURPOSE: To evaluate outcomes of ultrathin Descemet stripping automated endothelial keratoplasty (UT-DSAEK) with donor corneas preserved at 31°C in Cornea Syn®, a medium formulated with recombinant human serum albumin (rHSA) to replace foetal calf serum, and deswelled-transported in the xeno-free medium Cornea Trans®. METHODS: Prospective, multicentre, open-label study. We evaluated the endothelial cell loss (ECL) as the percentage variation of the endothelial cell density (ECD, cells/mm2) between 6 and 12 months after surgery, corneal transparency and thickness at 12 months, and adverse events within 12 months. Endothelial lenticules of mean 89 µm, ECD ≥ 2300 cells/mm2, minimum signs of cell mortality or morphology alterations, were dissected by microkeratome in the eye bank, and grafted in patients ≥ 18 years without corneal neovascularisation, conjunctivalization, or blinking impairment. RESULTS: Thirty-five patients underwent UT-DSAEK, 3 showed primary failure, 1 late failure, and 2 skipped the 6-month visit. We analysed data from 29 patients, 27 with Fuchs endothelial corneal dystrophy (FECD) and 2 with pseudophakic bullous keratopathy (PBK). The median ECL between 6 and 12 months was 2.6% (p = .054, CI 0 to 12.5) and the absolute mean (SD) was 158.4 (364.1) cells/mm2. After 12 months, 96.5% of corneas were clear, with mean pachymetry of 585.9 (50.4) µm. CONCLUSIONS: The ECL rate after UT-DSAEK match overall that observed in DSAEK or UT-DSAEK models of endothelial survival and the overall safety compared that reported for similar follow-up. Corneas maintained in Cornea Syn® and Cornea Trans® did not affect the ECD and functional outcomes of UT-DSAEK up to 12 months.

Comparison of human amniotic membrane decellularisation approaches for hESC-derived RPE cells culture.

OBJECTIVE: Recent clinical studies have shown that the transplantation of functional retinal pigment epithelium (RPE) cells can prevent the onset of RPE degeneration in age-related macular degeneration. This study aimed to investigate the potential of human amniotic membrane (hAM) as a viable scaffold for the growth and proliferation of pluripotent-derived RPE cells. METHODS AND ANALYSIS: Three enzymatic hAM de-epithelialisation methods (thermolysin, trypsin-EDTA and dispase II) were assessed by histological analysis and optical coherence tomography (OCT). We generated RPE cells from a human embryonic stem cell (hESC) line subjected to spontaneous differentiation in feeder-free conditions. The hESC-derived RPE cells were seeded over denuded hAM at a density of 2.0×105 cells/cm2 and maintained in culture for up to 4 weeks. Immnofluorescence was carried out to evaluate the development of a confluent monolayer of RPE cells on the top of the hAM. Conditioned medium was collected to measure pigment epithelium-derived factor (PEDF) concentration by ELISA. RESULTS: Laminin α5 and collagen IV staining confirmed the efficiency of the de-epithelialisation process. In particular, thermolysin showed good retention of tissue integrity on OCT images and greater preservation of the hAM basement membrane. The hESC-derived RPE cells formed patches of pigmented cells interspersed along the denuded hAM, but failed to form a regular sheet of RPE cells. These cells expressed typical RPE markers, such as PMEL17 and RPE65, but they secreted low levels of PEDF. CONCLUSION: The biological variability of the hAM could influence the adhesion and the expansion of hESC-derived RPE cells. Further studies are required to verify whether a non-confluent monolayer might represent a limit to transplantation.

Analysis and pharmacological modulation of senescence in human epithelial stem cells.

Human epithelial stem cells (ESCs) are characterized by long-term regenerative properties, much dependent on the tissue of origin and varying during their lifespan. We analysed such variables in cultures of ESCs isolated from the skin, conjunctiva, limbus and oral mucosa of healthy donors and patients affected by ectrodactyly-ectodermal dysplasia-clefting syndrome, a rare genetic disorder caused by mutations in the p63 gene. We cultured cells until exhaustion in the presence or in the absence of DAPT (γ-secretase inhibitor; N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine T-butyl ester). All cells were able to differentiate in vitro but exhibited variable self-renewal potential. In particular, cells carrying p63 mutations stopped prematurely, compared with controls. Importantly, administration of DAPT significantly extended the replicative properties of all stem cells under examination. RNA sequencing analysis revealed that distinct sets of genes were up- or down-regulated during their lifetime, thus allowing to identify druggable gene networks and off-the-shelf compounds potentially dealing with epithelial stem cell senescence. These data will expand our knowledge on the genetic bases of senescence and potentially pave the way to the pharmacological modulation of ageing in epithelial stem cells.

Autologous simple conjunctival epithelial transplantation for primary pterygium.

PURPOSE: To evaluate the feasibility of a new method of conjunctival transplantation to achieve recovery of the normal conjunctival epithelium over the bare sclera after pterygium excision and prevent its recurrence. METHODS: After excision of the primary pterygium, we performed simple conjunctival epithelial transplantation (SCET) in which we glued an amniotic membrane patch pre-loaded with tiny autologous conjunctival tissue fragments over the scleral defect. Slit-lamp evaluation was performed at 2 and 7-10 days, and then at 1, 3, 6, and 12 months after surgery, together with confocal microscopy at 3, 6, and 12 months. RESULTS: Surgical excision and SCET for nasal primary pterygium were performed in 6 eyes (6 patients). No graft detachment occurred. An inflammatory granuloma was excised without sequelae in one patient 2 months after surgery. No signs of recurrence or sight-threatening complications were recorded at 12 months, and in vivo confocal microscopy showed progressive expansion of the conjunctival cell population and formation of a clear corneal-conjunctival transition. CONCLUSIONS: SCET takes advantage of the ability of the amniotic membrane and conjunctival cells to renew. Outcomes after SCET are comparable to conventional conjunctival flap surgery and can be achieved in less surgical time and with less donor tissue to be removed.

Impact of the COVID-19 Pandemic on Corneal Transplantation: A Report From the Italian Association of Eye Banks.

Purpose: To analyze the impact of COVID-19 on Italian corneal transplantation from March-2020 to February 2021 compared to the same timeframe of the 2 previous years, in order to identify potential consequences of a global pandemic on corneal procurement and transplantation services during this time. Methods: This national, multicentric, retrospective cohort study evaluated data collected from 12 (100%) Italian eye banks from March 2020 to February 2021 (Group A). The number of tissues collected, distributed and discarded were compared with the same time-frame of the 2 previous years: 2019 and 2018 (group B and C, respectively). The different type of transplants performed were reported. Data were analyzed using a non-parametric Friedman test. Results: Corneal procurement and the percentage of distributed tissues reduced in 2020 by more than 30 and 15%, respectively, compared to the 2 previous years. During the pandemic corneal transplant surgery showed only a modest drop: the number of the penetrating keratoplasties (PKs) and the anterior lamellar keratoplasties (ALKs) decreased by about 30 and 20% in comparison with groups B and C, respectively; between the Endothelial Keratoplasties (EKs), the Descemet membrane endothelial keratoplasty (DMEK) increased slightly from March 2020 to February 2021. Conclusions: Italy was one of the first countries most affected by the outbreak of COVID-19, and the Italian government adopted severe measures to limit viral transmission. The pandemic generated several implications in corneal transplant activity during the first lockdown. Then an efficacious reaction with constant, vigorous work led to a resumption of transplant surgery to a near-normal standard. The increase of EKs, despite the pandemic, is a sign that the advance in corneal transplantation has gone ahead and it continues to evolve.

21†Denuded descemet's membrane as potential tool to support human embryonic stem cell derived retinal pigment epithelial cells culture.

INTRODUCTION: Recent clinical studies suggest that RPE-cell replacement therapy may preserve vision and restore retinal structure in retinal degenerative diseases. New developments enabled the differentiation of RPE cells from pluripotent stem cells. Scaffold-based methods are being tested in ongoing clinical trials for delivering these cells to the back of the eye. Borrowed materials from donor tissues can be used as cell supports in subretinal transplantation. These biological matrices resemble the extracellular matrix microenvironment of the native tissue. The Descemet's membrane (DM) is an example of high collagen-rich basement membrane (BM). The potential of this tissue in retinal repair remains to be uncovered. AIMS: To investigate human embryonic stem cell-retinal pigment epithelium (hESC-RPE) cells survival and behaviour on a decellularized DM, which may be of clinical relevance in retinal transplantation. MATERIALS: DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope and histology. hESC-RPE cells were seeded onto the endothelial-side surface of acellular DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene, protein expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. RESULTS: Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. hESC-RPE cell attachment 6 days post-seeding and proliferation rates over the acellular DM were similar to hESC-RPE cells cultured on tissue culture inserts.On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell graft showed the characteristic RPE morphology. The expression of typical RPE genes, proper protein localization and key growth factor secretion further confirmed the correct RPE phenotype. The viability of the cells was maintained for up to 4 weeks in culture. CONCLUSION: Acellular DM was shown to be capable of sustaining hESC-RPE cells growth, thus confirming to be potentially a valid alternative to the Bruch's membrane.Further in vivo studies will need to verify if this product can represent a feasible tool to deliver RPE cells in the back of the eye.Our study highlights the possibility of recycling unsuitable corneal tissues, which would otherwise be discarded by the eye banks for clinical application.

23†Cryopreservation of human amniotic membrane (HAM) for ocular surface reconstruction: a comparison beetween protocols.

PURPOSE: In the past decades, the human amniotic membrane has been largely applied for several surgical and non-surgical procedures. It has been farther demonstrated that both hAM and cornea share similar patterns of expression of structural components of the basement membrane (like laminin 5 and collagen IV) making hAM an useful tissue for ocular surface reconstruction. Since 1996 in fact, amniotic membrane transplantation has been applied to a large number of ocular surface diseases including Stevens-Johnson syndrome, pterygium, corneal ulceration, ocular surface reconstruction after chemical/thermal burns and in the reconstruction after excision of ocular surface neoplasia. During the previous decades, hAM has achieved a pivotal role in regenerative medicine too.The possibility to preserve human amniotic membrane, without affecting membrane's features, has become pivotal, allowing virological and microbiological analyses to be carried out before grafting. The purpose of the present study is to investigate an easier and cheaper protocol for human amniotic membrane preservation without affecting its properties and structure, ensuring the safety profile of the tissue. We compared the effects on adhesive and structural properties of newer preservation conditions to those obtained with an established, standardized protocol (dimethyl sulfoxide at -160°C). In attempt to simplify and enhance the safety of the procedure, we tested dextran-based freezing medium and a dry condition (no medium) at temperatures of -80°C. METHODS: Five patches of human amniotic membrane were obtained from three different donors. For each donor, five preservation condition were tested: dimethyl sulfoxide at -160°C, dimethyl sulfoxide at -80°C, dextran-based medium at -160°C, dextran-based medium at -80°C and dry freezing at -80°C (no medium). At the end of four months storage period, adhesive properties and structure were analyzed. RESULTS: None of the newer preservation protocols showed differences in adhesive and structural properties of the tissues. The stromal layer always kept its adhesiveness, while both structure and basement membrane were not altered by any the preservation protocol. CONCLUSIONS: Switching from liquid nitrogen cryopreservation to -80°C would reduce manipulation, simplify the procedure, making it also cheaper. The use of dextran-based freezing medium or no medium at all (dry condition) would avoid the potential toxicity of the dimethyl sulfoxide-based freezing media.

22†Renewal of conjunctival epithelium over amniotic membrane to perform autologous simple conjunctival epithelial transplantation (SCET): in vitro validation and results of clinical application for primary pterygium.

BACKGROUND: Transplantation of ex vivo cultured conjunctival cell layers, generated on amniotic membrane or other scaffolds, provides a viable option in treating heterogeneous ocular surface conditions. By comparison, cell therapy is costly, labour-intensive and subject to good manufacturing practice requirements and regulatory approval; no conjunctival cell-based therapy is currently available. Several techniques are available after primary pterygium excision to recover the ocular surface anatomy by restoring healthy conjunctival epithelium and preventing recurrence and complications. However, application of conjunctival free autograft or transpositional flap to cover the bared scleral area is limited when the conjunctiva are to be spared for future glaucoma filtering surgery, in patients with large or double-headed pterygia, in recurrent pterygia, or when the harvesting of donor conjunctival is precluded by scarring. AIM: To develop a simple technique to obtain expansion of the conjunctival epithelium when applied in vivo in diseased eyes. METHODS: We evaluated in vitro the best way of gluing conjunctival fragments over the AM, the efficiency of the fragments to generate conjunctival cell outgrowths, the molecular marker expression, and the feasibility of shipping preloaded AM.We performed simple conjunctival epithelial transplantation (SCET) in which we glued an amniotic membrane patch pre-loaded with autologous conjunctival tissue fragments over the scleral defect after pterygium excision and evaluated the recovery of the normal conjunctival epithelium and the disease recurrence up to 12 months after surgery. RESULTS: 65-80% of fragments generated outgrowth 48-72h after gluing, without differences between type of AM preparation and fragment size. Within 6-13 days, a full epithelium covered the surface of the amniotic membrane. Specific marker expression (Muc1, K19, K13, p63, ZO-1) was detected. The shipping test showed after 24h the 31% of the fragments glued over the AM epithelial side, compared to more than 90% of fragments stayed attached in the remaining conditions (stromal side, stromal without spongy layer, epithelial side without epithelium).Surgical excision and SCET for nasal primary pterygium were performed in 6 eyes/patients. No graft detachment and recurrence occurred within 12 months. In vivo confocal microscopy showed progressive expansion of the conjunctival cell population and formation of a clear cornea-conjunctiva transition. CONCLUSIONS: We established the most suitable conditions for a novel strategy based on in vivo expansion of conjunctival cells from conjunctival fragments glued over the AM. The application of SCET seems to be effective and replicable for the renewal of conjunctiva in patients requiring ocular surface reconstruction.