PURPOSE
The aim of the present study was to determine the killing efficacy of Kerasave (AL.CHI.MI.A Srl), a corneal cold storage medium provided with antimycotic tablet against nine contaminants associated with corneal infections.

METHODS
The killing efficacy of Kerasave was determined after 0, 3 and 14 days of incubation at 4°C in Kerasave after inoculation of the medium with 10(5)–10(6) (CFU) of Candida albicans (CA), Fusarium solani (FS), Aspergillus brasiliensis (AB), Staphylococcus aureus (SA), Enterococcus faecalis (EF), Bacillus subtilis spizizenii (BS), Pseudomonas aeruginosa (PA), Enterobacter cloacae (EC) and Klebsiella pneumoniae (KP). Log10 reductions at different time intervals were determined by the serial dilution plating technique.

RESULTS
After 3 days, Kerasave induced the highest log10 decrease in the concentrations of KP, PA, CA and EC. The 2 log10 decrease was observed for SA and EF. The lowest log10 decrease was observed in BS, AB and FS concentrations. After 14 days, the microbial count of CA, FS, SA, EF, PA and EC further decreased.

CONCLUSIONS
Corneal cold storage medium Kerasave effectively reduced the microbial concentration of almost all tested microorganisms after 3 days and represents a valuable tool to control the microbial contamination of human donor corneas intended for transplantation.

PURPOSE
To simulate pars plana vitrectomy in porcine eyes ex-vivo using intraoperative devices and to evaluate viability of retinal cells.

METHODS
25 enucleated porcine eyes were divided in following groups Group A) No surgery control: Group B) Sham surgery; Group C) Cytotoxic control; Group D) Surgery with residues; Group E) Surgery with minimal residues. The retina was extracted from each eye bulb and the cell viability was determined by MTT assay. The in vitro cytotoxicity of each used compounds was tested on ARPE-19 cells.

RESULTS
No cytotoxicity was detected in retinal samples in groups A, B and E. Samples from eye bulbs that had undergone surgery with minimal removal of residues (group D) and cytotoxic controls (group C) showed high retinal cytotoxicity. The simulation of vitrectomy indicated that the combined use of compounds does not affect retinal cells viability if all the compounds are properly removed, whereas the cytotoxicity detected in group D may suggest that the presence and accumulation of the residues of the compounds used intraoperatively could negatively impact retinal viability.

CONCLUSIONS
The present study demonstrate the crucial role of an optimal removal of the intraoperative devices used in eye surgery to ensure safety to the patient.

BACKGROUND
Globally, more than 12 million people are awaiting corneal transplantation and cornea donor reduction has been observed since the outbreak of the COVID-19 pandemic, negatively influencing the availability of human corneas for research purposes as well. Therefore, the exploitation of ex vivo animal models in this field is of great value. The present study aimed at the development of a novel experimental model of porcine cornea ex vivo and lamellar tissue preparation to investigate the effects of storage conditions on corneal preservation.

METHODS
Twelve fresh porcine eye bulbs were disinfected by immersion in 10 mL of 5% povidone-iodine under orbital mixing for 5 minutes at room temperature. The corneoscleral rims were dissected, and stored in Tissue-C (Alchimia S.r.l., n=6) at 31°C and in Eusol-C (Alchimia S.r.l., n=6) at 4°C up to 14 days. The evaluation of Endothelial Cell Density (ECD) and endothelial mortality was performed using vital dye Trypan Blue staining (TB-S, Alchimia S.r.l.). Digital 1X pictures of TB-stained corneal endothelium were acquired and percentage of stained area was quantified using FIJI ImageJ software. ECD and endothelial mortality were determined at 0, 3, 7 and 14 days. Medium turbidity detected by naked eye was considered as proof of tissue contamination. Additionally, non-vital staining of the endothelium with Alizarin Red (AR) was performed and the endothelial morphology was investigated at Day 14 in both whole corneas and dissected endothelial lamellae.

RESULTS
The contamination rate of porcine corneas corresponded to <10% and 0% in Tissue-C and Eusol-C after 14 days, respectively. Porcine corneas stored in Tissue-C and Eusol-C showed <10% and <20% mortality in Tissue-C and Eusol-C respectively at the end of storage. Preliminary ECD determination (range 3700-4100 cells/mm2) at Day 0 aligned with data present in the literature (Meltendorf et al., Graefe’s Arch Clin Exp Ophthalmol, 2007). Whole cornea and dissected lamellae stained with TB and AR showed comparable endothelial morphology after incubation in Tissue-C and Eusol-C for 14 days. The lamellar tissue allowed endothelium morphology analysis at higher magnification compared to whole cornea.

CONCLUSION
The presented ex vivo porcine model allows evaluation of the performance and safety of storage conditions. Future perspectives of this method will be the extension of the porcine corneas storage up to 28 days.

Purpose
To extract crystalline proteins from porcine eye lenses and to evaluate their relation with oxidative stress.

Methods
10 lenses were extracted from porcine eyes. The lenses were washed with phosphate-buffered saline (PBS) and homogenized in extraction buffer (20 mM Tris HCl, 5 mM EDTA, 2.6 ml/g of lenses) using Polytron homogenizer. The water insoluble residues were eliminated from the homogenate by subsequent centrifugations at 12000 rpm for 10 minutes. Crystalline proteins concentration of the extract was assessed with Bradford assay using Bradford reagent (Sigma Aldrich) and HPLC using Jupiter 5 μm C18 300 Å column.
The reactive oxygen species (ROS) probe Dihydrorhodamine-123 (DHR123, Sigma Aldrich) was added to the lens homogenate solution containing 3 mg/ml of crystalline proteins and irradiated by UV light (254 nm) for 0, 5, 10, 15 minutes. Irradiated samples were analyzed both by HPLC for protein characterization
and fluorimetry recording emission fluorescence spectra from 510 to 700 nm, using a Perkin Elmer luminescence spectrophotometer with excitation at 505 nm to determine ROS production.

Results
HPLC analysis showed the presence of a specific peak pattern of crystalline proteins in the porcine eye lens extract corresponding to 21% of α, 66% of β, and 13% of γ crystalline proteins. The concentration of each crystalline protein decreased after 15 minutes of UV irradiation. The emission fluorescence spectra showed a peak at 527 nm corresponding to the presence of Rhodamine 123, as a result of the oxidation of DHR123 probe induced by the presence of ROS.

Conclusions
α, β and γ crystalline proteins were extracted from porcine eye lens and quantified. UV irradiation of crystalline proteins solution induced the protein degradation that could be related to ROS production.
Additional studies are necessary to evaluate the oxidative stress mechanism that induce crystalline proteins degradation.

Purpose
To develop a quantitative method to assess the toxicity of intraocular endotamponades using human retina ex-vivo model. The study aimed at determination of retina viability, positive and negative controls, method accuracy and repeatability.

Methods
Human donor eye globes for research use were transported to FBOV and stored in DMEM/F-12 medium at 4°C for a maximum of 48 hours. 2-mm and 3-mm retina samples were evaluated for viability using TOX-1 In Vitro Toxicology Assay Kit (Sigma-Aldrich, Italy) immediately (T0) and 24 h, 48 h, 72 h and 7 days after retina extraction. Sodium Dodecyl Sulfate (SDS) concentrations of 0.25 mg/ml, 0.50 mg/ml and 1 mg/ml were tested as positive (cytotoxic) controls. DMEM/F-12 medium was used as a negative control. The toxicity of perfluorooctane samples (PFO) and the same PFO containing toxic residues (1H-PFO and PFOA, positive controls) was assessed in a comparison between donor retina ex vivo model and cytotoxicity test in vitro model using ARPE-19 cell line.

Results
Retina extracted within 24 and 48 hours post mortem showed optimal viability at T0. Incubation of the samples with 0.25, 0.50 mg/ml and 1 mg/ml SDS (cytotoxic control) induced 75.3 ± 4 %, 98.6 ± 2 % and 98.5 ± 2 % mortality at T0, respectively. In average 53 ± 2 samples with 3-mm diameter were prepared from one donor retina. 3-mm sample size was suitable to allow good method repeatability. Both donor retina ex vivo and cell line models showed comparable cytotoxic results; not cytotoxic samples resulted in similar % of cell viability corresponding to 92 ± 3 % and 97 ± 1 % for donor retina ex vivo and cell line models respectively. Cytotoxic samples induced higher mortality in donor retina ex vivo model compared to the cytotoxicity test in vitro in cell line model.

Conclusion
A quantitative method to assess the toxicity of intraocular liquid devices in human retina ex-vivo model was standardized with high sensibility and repeatability.

Purpose: This study aimed at validating the method for sterility testing of the corneal culture medium, TISSUE-C (AL.CHI.MI.A. S.r.l.), and the transport/deswelling medium, CARRY-C (AL.CHI.MI.A. S.r.l.), according to the method suitability test, as defined by the European Pharmacopoeia (EP), using RESEP (AL.CHI.MI.A. S.r.l), which is a new medical device for removal of antimicrobial agents, and HB&L (Alifax) automated culture system.

Materials and Methods: The six EP reference strains were inoculated in TISSUE-C and CARRY-C. Half of the samples were treated with RESEP (RESEP+ group) prior to the sterility testing, whereas the remaining samples were untreated (RESEP− group). Growth controls were obtained by direct inoculation of the microorganisms in the culture broths. Microbial growth was read by HB&L automated light scattering culture system within 48 h.

Results: The use of RESEP allowed detection of microbial growth in 100% of the tested samples, with a mean time to detection (TTD) comparable with that of the growth control group. Significantly lower sensitivity (38.83% ± 20.03% for both media, p < 0.05) and TTD variability, depending on the tested microorganism, were observed in the RESEP− group. The method specificity was 100% for both groups.

Conclusion: The use of RESEP increased the sensitivity of the sterility testing method to 100% and, for the first time, allowed validation of the method for sterility testing of corneal storage media according to the EP method suitability test. This further increases the safety of the corneas intended for transplantation.

Purpose: The aim of this study was to validate the method for sterility testing of corneal storage media Tissue-C and Carry-C according to the “Method suitability test” (EP) using BACTEC (Becton Dickinson) automated system in a multicentric study.

Material & Methods: The validation study was performed at the Eye Bank of Rome and Eye Bank of Monza, Italy. Samples of organ culture medium (Tissue-C, AL.CHI.MI.A. S.r.l.), deswelling/transport medium (Carry-C, AL.CHI.MI.A. S.r.l.), and optimal growth media (growth control) were inoculated with 6 EP reference strains to obtain final microbial concentration of 10 cfu/ml, and tested at least in triplicate with BACTEC automatized system.
Method sensitivity, specificity and robustness were determined for each medium, with and without antibiotic removal from samples with RESEP (AL.CHI.MI.A. S.r.l.).

Results: Both eye banks obtained the same method sensitivity and specificity results. The method for sterility testing of Tissue-C and Carry-C samples after RESEP-treatment using BACTEC system showed 100% sensitivity and specificity. Samples treated with RESEP showed similar times to detection as compared to growth controls.

Conclusions: BACTEC system can be considered validated with 100% sensitivity and specificity, and robustness for samples of corneal storage media contaminated with 1-10 cfu/ml, and treated with RESEP.

Purpose: This study aimed at assessing the antimycotic activity of the new cold storage medium, Kerasave, and at evaluating the quality of donor corneas preserved in the medium at 4°C for 14 days in comparison with Optisol GS.

Material and Methods: Kerasave antimycotic activity was determined by in vitro time-kill studies using sterile porcine corneal tissues, contaminated with 10⁴ cfu/ml of C. Albicans (ATCC10231 and clinical isolate). The killing rate of the microorganisms was monitored at 4°C after 5 and 10 days of incubation in Kerasave.

Kerasave performance was assessed on 16 pairs of human corneas not suitable for transplantation, procured and evaluated according to standard procedures of Monza Eye Bank, Italy. One cornea was transferred in Kerasave and the contralateral in Optisol GS. Endothelial cell density (ECD), measured by specular microscopy (Keratoanalyzer, Konan), was evaluated pre-processing, and after 7 and 14 days of storage at 4°C. Endothelial cell morphology and mortality were determined according to Stocker method, and epithelial integrity, and corneal transparency were evaluated using a Slit lamp.

Results: In vitro time-kill studies showed a 3 to 4 log₁₀ reduction for both Candida strains within 10 days of incubation at 4°C.
Kerasave- and Optisol-GS-treated tissues showed similar ECD, mortality and endothelial morphology after 7 and 14 days of cold storage. Slit lamp analysis showed comparable corneal transparency and epithelial integrity in both groups.

Conclusions: The new cold storage medium with antimycotic tablet, Kerasave, exhibited an excellent antimycotic activity and biocompatibility with donor corneas after corneal storage at 4°C for up to 14 days.