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.

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.

Abstract: Elimination of Me2SO from cryopreserved allografts is one of the crucial steps in tissue processing phases in order to guarantee the tissue safety and quality. The aim of the study was to monitor the Me2SO removal from cryopreserved pig skin and heart valves and validate a rinsing procedure with BASE to eliminate Me2SO.