PURPOSE
Considering the growing shortage of corneal tissues for research, the present study aimed to develop and optimize a porcine cornea model with qualitative features comparable to those of human tissues.

METHODS
A new decontamination procedure of porcine eye bulbs was set up and its efficacy as well as endothelial mortality were evaluated. Human corneas unsuitable for transplant and porcine corneas were then compared after storage under hypothermic (4–8°C, Eusol-C, AL.CHI.MI.A. S.R.L) or organ-culture (31–35°C, Tissue-C, AL.CHI.MI.A. S.R.L) storage conditions for 14 days. A new method, based on the semi-automatic analysis of Trypan-blue stained endothelial areas by Fiji software, was developed to quantify the whole endothelium viability.
Corneas were assessed for central corneal thickness (CCT), corneal transparency, endothelial morphology, and endothelial cell density (ECD) at days 0, 7, and 14 of storage. Portions of lamellar tissues consisting of Descemet’s membrane and endothelial cells were prepared for histological investigations.

RESULTS
The new decontamination procedure of porcine eye bulbs resulted in 18% versus 89% (“no decontamination” control) of corneas still contaminated after 28 days of storage at 31°C. The decontamination protocol did not affect endothelium viability, as assessed by the new Fijibased method. ECD (porcine: 3156 ± 144 cells/mm2; human: 2287 ± 152 cells/mm2), CCT (porcine: 1073 ± 151 μm; human: 581 ± 39 μm), transparency (porcine: 88.6 ± 11.0 %; human: 76.3 ± 5.4 %), and morphology score (porcine: 4.0 ± 0.0; human: 3.2 ± 0.4) measured in the porcine cornea at day 0 were significantly higher than in human corneas. Nonetheless, the qualitative parameters of porcine and human corneas showed comparable trends during the storage under hypothermic (4–8°C) and organ-culture (31–35°C) conditions for 14 days.

CONCLUSIONS
The presented porcine cornea model represents a reliable and alternative model to human donor tissues for preliminary investigations and can be used for testing new media, substances, drugs, or preservation conditions and their impact on corneal tissue quality and safety. Furthermore, the quantitative method to assess whole endothelium mortality can be implemented at eye banks for the evaluation of corneas intended for transplantation.

PURPOSE
Descemet’s membrane endothelial keratoplasty (DMEK) is a frequently used treatment option for patients with corneal endothelial dysfunction.
The aim of this study was to set up a method to prepare porcine DMEK grafts and to simulate DMEK surgery in porcine eye bulbs in order to
establish an ex-vivo-model for laboratory investigations on DMEK surgery conditions.

METHODS
Ten (n=10) porcine eye bulbs from domestic pigs (Sus scrofa domestica), between 6 and 8 months old, were recovered at a local slaughterhouse, transported on ice and processed within 2 h after death. Porcine eye bulbs were decontaminated by immersion in 10 mL of 5% povidone-iodine and corneas were dissected under aseptic conditions, leaving approximately 2 mm of the scleral rim. DMEK grafts were prepared by means of mechanical stripping technique using specific surgical instruments for DMEK (Moria, France) on fresh corneas (n=2) and on corneas stored in Eusol-C (AL.CHI.MI.A. Srl, Italy) at 4°C for 7 days (n=4) and for 14 days (n=4). Endothelial cell (EC) density was compared before DMEK-preparation (specular and light microscopy on trypan blue stained tissues) and after DMEK-preparation (fluorescence microscopy on Calcein-AM stained tissues). DMEK graft injection was simulated in anterior chamber of fresh porcine eye bulbs.

RESULTS
The porcine DMEK grafts preparation resulted to be more challenging compared to human DMEK grafts. Despite similarity between human and porcine corneas, porcine Descemet membrane (DM) firmly adheres to the underlying stroma. DMEK grafts preparation was not successful at day 0; DMEK preparation was possible by mechanical stripping technique on corneas stored in Eusol-C for 7 and 14 days obtaining naturally rolled endo-out porcine DMEK grafts. An EC mortality increase up to 20% was observed on DMEK graft compared to initial whole corneal tissue. DMEK roll injection was successfully simulated in anterior chamber of the porcine eye bulb.

CONCLUSIONS
Naturally rolled DMEK endo-out grafts were successfully prepared by mechanical stripping technique on porcine corneal tissues stored in Eusol-C at 4°C (up to 14 days). DMEK Surgery including the tissue injection in anterior chamber could be simulated. Further studies will be performed to improve ex-vivo-porcine DMEK surgery model.

PURPOSE
To prove the safety and performance of the hypothermic corneal storage medium Corneal Chamber, containing Eusol-C solution (AL.CHI.MI.A. S.r.l.) and of the rinsing solution PSS-L (AL.CHI.MI.A. S.r.l.) in support to the new CE certification process in accordance to the EU 2017/745 Medical Device Regulation.

METHODS
Fifteen (n=15) human donor corneas unsuitable for transplantation and n=11 porcine corneas were evaluated for the following quality parameters: ECD, HEX%, CV%, endothelial morphology, endothelial mortality and transparency at day 0 and after 14±1 days (day 14) of storage in Corneal Chamber at 2-8°C. Then, corneas were rinsed in PSS-L for 1’ at room temperature (RT) and the same parameters were assessed Post Rinsing (Day 14PR). In order to evaluate the antimicrobial carryover after the corneal storage in Corneal Chamber(14 days at 4°C), gentamicin sulphate was quantified in human and porcine corneas homogenates by UHPLC.

RESULTS
Human and porcine corneas stored in Corneal Chamber at 2-8°C for 14 days showed a good overall quality of the tissue according to quality parameters evaluated. In particular, mean ECD, HEX% and CV% did not show statistically significant changes at the end of storage and endothelial mortality increased of 3.1±3.3 % in human corneas and 7.8±3.5 % in porcine corneas. Slight variations in endothelial morphology score and corneal transparency were observed. Rinsing with PSS-L did not negatively affect the quality parameters evaluated before and after rinsing and gentamicin sulfate residues were completely removed.

CONCLUSIONS
The storage of corneal tissues in Corneal Chamber at 2-8°C for 14 days and the corneal rinse with 30 ml of PSS-L at RT for 1 min are safe and
effective procedures allowing the preservation of the corneal quality parameters including ECD, endothelial mortality, endothelial morphology,
HEX%, CV%, and corneal transparency and the elimination of gentamicin sulfate from the tissues before transplantation.

PURPOSE
To compare endothelial cell density and morphometry, thickness, and transparency of corneas stored in Kerasave vs. Optisol-GS for 14 days.

METHODS
Mate corneas were recovered into Kerasave or Optisol-GS (27 pairs) and stored at 2-8°C for 14 days.
Endothelial cell density (ECD), hexagonality (HEX), and coefficient of variation (CV) were evaluated by specular microscopy, and central corneal thickness (CCT) was examined by OCT after 1, 3, 7, and 14 days of storage. Corneal transparency was scored using slit-lamp examination at days 1 and 14.

RESULTS
Average ECD, HEX, and CV for the Kerasave (2653±303, 57±4, 36±3) and Optisol-GS (2623±306, 57±5, 36±4) groups were not significantly different at day 1. There was also no difference at any other study timepoints (P>0.05 for all). ECD did not significantly change from day 1 to day 14 in either group (P>0.05), but a statistically significant change in HEX and CV was observed between day 1 and day 14 in both groups (P<0.01). Average CCT measured at day 1 for corneas stored in Kerasave was 622±49 and those stored in Optisol-GS was 580±35 (P<0.01). The difference in CCT measurements was not significant different at day 14 (674±46 v. 647±58, P>0.05). Corneal transparency was not significantly different between the two groups at day 1 or day 14.

CONCLUSION
Endothelial cell density, endothelial morphometry, and corneal transparency were not different in corneas stored in Kerasave or Optisol-GS for 14 days.
Optisol-GS was initially better at thinning the cornea than Kerasave. These results suggest that Kerasave is a viable alternative to Optisol-GS for corneal storage.

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.