Background The risk of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission through corneal graft is an ongoing debate and leads to strict restrictions in corneas procurement, leading to a major decrease in eye banking activity. The aims of this study are to specifically assess the capacity of human cornea to be infected by SARS-CoV-2 and promote its replication ex vivo, and to evaluate the real-life risk of corneal contamination by detecting SARS-CoV-2 RNA in corneas retrieved in donors diagnosed with Coronavirus Disease 2019 (COVID-19) and nonaffected donors. Methods and findings To assess the capacity of human cornea to be infected by SARS-CoV-2, the expression pattern of SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE-2) and activators TMPRSS2 and Cathepsins B and L in ocular surface tissues from nonaffected donors was explored by immunohistochemistry (n = 10 corneas, 78 ± 11 years, 40% female) and qPCR (n = 5 corneas, 80 ± 12 years, 40% female). Additionally, 5 freshly excised corneas (80 ± 12 years, 40% female) were infected ex vivo with highly concentrated SARS-CoV-2 solution (106 median tissue culture infectious dose (TCID50)/mL). Viral RNA was extracted from tissues and culture media and quantified by reverse transcription quantitative PCR (RT-qPCR) (viral RNA copies) 30 minutes (H0) and 24 hours (H24) after infection. To assess the risk of corneal contamination by SARS-CoV-2, viral RNA was tested by RT-qPCR (Ct value) in both corneas and organ culture media from 14 donors diagnosed with COVID-19 (74 ± 10 years, 29% female) and 26 healthy donors (79 ± 13 years, 57% female), and in organ culture media only from 133 consecutive nonaffected donors from 2 eye banks (73 ± 13 years, 29% female). The expression of receptor and activators was variable among samples at both protein and mRNA level. Based on immunohistochemistry findings, ACE-2 was localized mainly in the most superficial epithelial cells of peripheral cornea, limbus, and conjunctiva, whereas TMPRSS2 was mostly expressed in all layers of bulbar conjunctiva. A significant increase in total and positive strands of IP4 RNA sequence (RdRp viral gene) was observed from 30 minutes to 24 hours postinfection in central cornea (1.1 × 108 [95% CI: 6.4 × 107 to 2.4 × 108] to 3.0 × 109 [1.4 × 109 to 5.3 × 109], p = 0.0039 and 2.2 × 107 [1.4 × 107 to 3.6 × 107] to 5.1 × 107 [2.9 × 107 to 7.5 × 107], p = 0.0117, respectively) and in corneoscleral rim (4.5 × 109 [2.7 × 109 to 9.6 × 109] to 3.9 × 1010 [2.6 × 1010 to 4.4 × 1010], p = 0.0039 and 3.1 × 108 [1.2 × 108 to 5.3 × 108] to 7.8 × 108 [3.9 × 108 to 9.9 × 108], p = 0.0391, respectively). Viral RNA copies in ex vivo corneas were highly variable from one donor to another. Finally, viral RNA was detected in 3 out of 28 corneas (11%) from donors diagnosed with COVID-19. All samples from the 159 nonaffected donors were negative for SARS-CoV-2 RNA. The main limitation of this study relates to the limited sample size, due to limited access to donors diagnosed with COVID-19 and concomitant decrease in the procurement corneas from nonaffected donors. Conclusions In this study, we observed the expression of SARS-CoV-2 receptors and activators at the human ocular surface and a variable increase in viral RNA copies 24 hours after experimental infection of freshly excised human corneas. We also found viral RNA only in a very limited percentage of donors with positive nasopharyngeal PCR. The low rate of positivity in donors diagnosed with COVID-19 calls into question the utility of donor selection algorithms. Trial registration Agence de la Biomédecine, PFS-20-011 https://www.agence-biomedecine.fr/.

Fresh corneal donation is essential for basic and preclinical research, but more unknown to public and the medical teams than donation for transplantation: it may raise concerns. We prospectively compared the acceptance rates and the characteristics of targeted corneal donation for research versus donation for transplantation during one year. The Agence de la Biomé decine authorized us to procure fresh corneas targeted for research, only from the donors with medical contraindications for transplantation, in order not to increase grafts shortage. Three nurses from the hospital coordination team of Saint-Etienne University Hospital, obtained consent for research and transplantation in parallel, screening all intra-hospital deaths cases, following standard protocol to check no refusal from families, despite the French opt-out system. They contacted 127 families for research and 244 for transplantation, in 71% of cases by telephone. Consent was obtained in 62% of cases for research and 54% for transplantation (P = 0.135). The main contraindication for transplantation was the cognitive disorders (66%) followed by the blood cancers (8%). This new specific activity, providing new source of fresh corneas for research immediately usable without any eyebank storage steps, didn't reduce the number of corneas procured for transplantation versus previous years (P = 0.998). Donors in the research group were 10 years older (P<0.001) without difference regarding endothelial cell quality (P = 0.071), allowing maximal clinical relevance for protocols using these fresh human scientific corneas provided by targeted donation.

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Purpose The organ culture (OC) is a passive corneal storage, gold standard in Europe for decades. The bioreactor (BR) is a sterile transparent device that allows active storage: transcorneal pressure gradient and fluid renewal. Aim: To compare quality of human corneas stored for 1 month in our BR versus (vs) OC. Methods Randomized controled preclinical study, authorized by the French Biomedicine Agency and an IRB, including only fresh scientific paired human corneas >2000cells/mm2 and <10% difference between both corneas at day (D)2. Immediately at retrieval, one cornea was randomized and stored in OC and the other in BR, both with the same commercial medium (CorneaMax). At D26 only OC corneas were transferred to a deswelling medium (CorneaJet). We calculated that 49 pairs were needed to show a difference of 500 viable cells/mm2 between groups. Assessments were performed at D2, 26, 28. Main criterion: viable ECD (vECD) determined by triple staining Hoechst/Ethidium/Calcein at D28, blind of storage method. Secondary criteria: average transparency (T), central corneal thickness (CCT) and microbiology. Results We retrieved 60 pairs, included 52 pairs at D2, analyzed 50 pairs (1 contamination per group). At D2 ECD in BR was 2607±340 vs 2576±357cells/mm2 in OC (p=0.243). At D26 ECD was >2000cells/mm2 for 92% of corneas in BR vs 58% in OC (p<0.001). At D28 vECD was higher in BR 2188±379 vs 1687±389cells/mm2 in OC, with 76% of corneas >2000 viable cells/mm2 in BR vs 22% in OC (p<0.001); T didn't differ and CCT was higher in BR with 684±52 vs 608±53μm in OC (p<0.001). Conclusions Restoring IOP and renewing medium significantly improves endothelial cell survival while avoiding deleterious deswelling necessary in OC: after 1 month‐storage, the BR provides much more corneas suitable for transplantation and higher quality grafts that could survive longer in recipients.