Selective ILM Staining and Safety of Two Vital Dyes During a Human-Like Pars Plana Vitrectomy Ex Vivo in Porcine Eyes.

PURPOSE: An ideal dye for intraocular use should effectively stain the target tissue while being easy to apply and remove. Additionally, it should not have any adverse effects resulting from prolonged contact with the retinal tissue. Recently, concerns have been raised about the safety of some vital dyes during surgical procedures as they may cross the internal limiting membrane and deposit on the retina. In this study, we aimed to investigate whether commercially available vital dyes, VIEW-ILM® and TWIN® (AL.CHI.MI.A. S.r.l., Ponte San Nicolò, Padova, Italy), have the potential to cross the internal limiting membrane during vitreoretinal surgery and deposit on the retina. Furthermore, we evaluated their safety in vitro and in vivo. METHODS: A human-like pars plana vitrectomy was performed on porcine eyes ex vivo, with VIEW-ILM® or TWIN® used to stain the internal limiting membrane either with or without subsequent internal limiting membrane peeling. The two dyes were then extracted from retinal punches with or without internal limiting membrane, and quantified using high performance liquid chromatography. Safety was evaluated through in vitro cytotoxicity tests and in vivo skin sensitization and irritation tests according to ISO standards. RESULTS: High performance liquid chromatography analyses demonstrated that VIEW-ILM® and TWIN® effectively stained the internal limiting membrane without crossing the membrane. No residual dyes were found in the retinal layers after internal limiting membrane removal. Furthermore, both in vitro and in vivo safety tests confirmed the absence of cytotoxicity, skin sensitization, and irritation. CONCLUSION: The results of this study support the safety and efficacy of VIEW-ILM® and TWIN® for internal limiting membrane staining. The experimental protocol described in this study could be utilized to gain a comprehensive understanding of the characteristics of vital dyes.

Assessment of performance and safety of Corneal Chamber hypothermic storage medium and PSS-L corneal rinsing solution in human and porcine corneas.

PURPOSE: To prove the safety and performance of the hypothermic corneal storage medium "Corneal Chamber" and the rinsing solution "PSS-L" in support of the new Conformité Européenne (CE) certification process in accordance with the Medical Device Regulation. METHODS: Fifteen (n=15) human donor corneas and 11 (n=11) porcine corneas were evaluated for the following parameters: endothelial cell density (ECD) and mortality, percentage of hexagonal cells (HEX%), coefficient of cellular area variation (CV%) and corneal transparency at Day 0 and after 14±1 days of storage in Corneal Chamber medium at 2-8°C. Then, the same parameters were assessed after rinsing of corneas in PSS-L for 1 min at room temperature. Evaluation of gentamicin sulfate carryover after corneal storage and PSS-L rinsing was performed by ultra-high performance liquid chromatography analysis on human corneas homogenates. RESULTS: Human and porcine corneas stored in Corneal Chamber medium showed a good overall quality of the tissue according to the 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 to 3.1±3.3 and 7.8±3.5% in human and porcine corneas, respectively. Tissue rinsing with PSS-L did not affect the quality parameters evaluated before and gentamicin sulfate residues were absent in human corneas. CONCLUSIONS: Corneal preservation in Corneal Chamber medium at 2-8°C for 14 days and the corneal rinse with PSS-L are safe and effective procedures allowing the preservation of the corneal quality parameters as well as the complete elimination of gentamicin sulfate from the tissues before transplantation.Cite Now.

P12-A107†Porcine cornea ex vivo model as an alternative to human donor tissues for investigating new preservation conditions.

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 Fiji-based 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. CONCLUSION: 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.

P15-A112†Descemet's membrane endothelial keratoplasty (DMEK) graft preparation in porcine cornea.

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. CONCLUSION: 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.

P14-A117†Assessment of performance and safety of corneal chamber hypothermic storage and PSS-L corneal rinsing in human and porcine corneas.

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. CONCLUSION: 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.

Porcine Cornea Storage Ex Vivo Model as an Alternative to Human Donor Tissues for Investigations of Endothelial Layer Preservation.

Purpose: Due to the growing shortage of human corneas for research, we developed a porcine cornea storage model with qualitative features comparable to human tissues. Methods: We established a decontamination procedure for porcine eye bulbs to ensure corneal storage at 31°C to 35°C for up to 28 days without contamination. We compared human and porcine corneas under hypothermic (2-8°C) or culture (31-35°C) conditions for central corneal thickness (CCT), corneal transparency, endothelial morphology, endothelial cell density (ECD), and a novel method to quantify whole endothelial mortality. We also examined portions of lamellar tissues consisting of Descemet's membrane and endothelial cells under the microscope after Alizarin red staining. Results: Our decontamination procedure reduced corneal contamination from 94% (control corneas without decontamination) to 18% after 28 days of storage at 31°C to 35°C. ECD, CCT, transparency, and morphology were significantly higher in porcine corneas than in human corneas at day 0. Nevertheless, the qualitative parameters of porcine and human corneas showed comparable trends under both investigated storage conditions for up to 14 days. Conclusions: The presented corneal storage model provides a reliable alternative to human tissues for preliminary corneal investigations. Translational Relevance: The porcine cornea storage model can be used to investigate the efficacy and safety of new media, substances, or storage conditions. Furthermore, the method developed to assess the percentage of endothelial mortality is tissue conservative and can be used in eye banks to monitor endothelial mortality during storage of tissues intended for transplantation.

Prospective In Vitro Comparison of Kerasave and Optisol-GS Corneal Storage Solutions.

PURPOSE: The aim of this study was to compare the performance of Kerasave and Optisol-GS for hypothermic corneal storage for 14 days. METHODS: This study was a prospective laboratory investigation. Mate corneas were recovered into Kerasave or Optisol-GS (27 pairs) and stored at 2°C to 8°C for 14 days. Corneas were evaluated by trained eye bank technicians, and study parameters were compared between the initial and final evaluations. Endothelial cell density (ECD), hexagonality (HEX), and coefficient of variation (CV) were evaluated by specular microscopy, and central corneal thickness (CCT) was examined by optical coherence tomography 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 cells/mm 2 , 57 ± 4%, and 36 ± 3%) and Optisol-GS (2623 ± 306 cells/mm 2 , 57 ± 5%, and 36 ± 4%) groups were not significantly different at day 1. There was also no difference at any other study time points (all P > 0.05). 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 µm and those stored in Optisol-GS was 580 ± 35 µm ( P < 0.01). The difference in CCT measurements was not significantly different at day 14 (Kerasave: 674 ± 46 µm vs. Optisol-GS: 647 ± 58 µm, P > 0.05). Corneal transparency was not significantly different between the 2 groups at day 1 or day 14. CONCLUSIONS: The corneal quality and clinically relevant parameters including ECD, endothelial morphometry, and corneal transparency were not different in corneas stored in Kerasave or Optisol-GS for 14 days. The initial difference in CCT between the 2 groups decreased at day 14. These results demonstrated that Kerasave corneal storage solution preserves the corneal endothelium similarly to Optisol-GS.

Comparison of Perfluorocarbon Liquids Cytotoxicity Tests: Direct Contact Versus the Test on Liquid Extracts.

The purpose of this study is to compare the in vitro cytotoxicity tests according to the ISO 10993-5 (2009) standards using direct contact and the test on liquid extracts of compounds previously identified as possible toxic impurities in perfluorocarbon liquids (PFCLs) for use in vitreoretinal surgery. Compounds including perfluorooctanoic acid (PFOA), 1H-perfluorooctane (1H-PFO), 2H-tridecafluoro-2-methylpentane, 1H,2H-octafluorocyclopentane, and 2H,3H-decafluoropentane were analyzed by 19F NMR before and after extraction using an aqueous solution and tested by both the direct contact and liquid extract tests in L929, BALB 3T3, and ARPE-12 cells. The concentration that reduced in vitro cell viability by 30%, the cytotoxicity concentration threshold (CC30), was determined for each compound. 19F NMR spectroscopy confirmed the immiscibility of perfluoro-n-octane (PFO) and 1H-PFO and the solubility of PFOA with the extraction vehicle. The other samples reacted with the extraction vehicle, releasing fluoride ions. Using the direct contact test, the CC30 of PFOA, 1H-PFO, 2H-tridecafluoro-2-methylpentane, 1H,2H-octafluorocyclopentane, and 2H,3H-decafluoropentane corresponded to 48 124, 50, 14, 8035, and 46 ppm, respectively. The method on liquid extracts did not detect cytotoxicity in three out of five tested compounds, and CC30 could not be determined. In conclusion, the in vitro cytotoxicity test by direct contact revealed a positive correlation between cell toxicity and the concentration of the tested substance. Conversely, the test on liquid extracts hardly detected the cytotoxicity of toxic impurities in PFCLs. Thus, only the cytotoxicity test by direct contact, according to ISO 10993-5 (2009), is a sensible and reliable method to detect possible cytotoxic impurities in PFCLs to guarantee patient safety.

A new storage medium containing amphotericin B versus Optisol-GS for preservation of human donor corneas.

BACKGROUND/AIM: We compared the quality of human donor corneas stored in a cold storage medium containing 2.5 µg/ml of amphotericin B (Kerasave, AL.CHI.MI.A. S.R.L., Ponte San Nicolò, Italy) and Optisol-GS (Bausch & Lomb Inc., Bridgewater, NJ, USA) for 14 days. METHODS: Sixteen pairs of human donor corneas were collected in Eusol-C (AL.CHI.MI.A. S.R.L., Ponte San Nicolò, Italy). Next, all tissues underwent the first evaluation that included the assessments of central corneal thickness (CCT), endothelial cell density (ECD) measured using both trypan blue staining and specular microscopy, endothelial cell (EC) mortality and morphology, and corneal transparency within 24 hours from recovery (Day 1). Afterwards, one cornea of each pair was transferred into Kerasave or Optisol-GS. ECD and CCT were also assessed at Day 7, and all the metrics were evaluated again at the end of the storage period (Day 14). RESULTS: At all tested time points, no differences were found in the qualitative (corneal transparency, EC morphology) and quantitative metrics (ECD, CCT, EC mortality) between the Kerasave and the Optisol-GS storage groups. At Day 14, the corneas stored in Kerasave and Optisol-GS showed ECD of 2312±98 and 2335±128 cells/mm2 (p=0.886), CCT of 717±17 and 697±19 µm (p=0.454) and central EC mortality of 0.54%±0.40% and 0.14%±0.14% (p=0.719), respectively. CONCLUSIONS: The new amphotericin B-containing medium Kerasave was comparable to Optisol-GS in terms of preservation of corneal characteristics at 2-8°C for 14 days.

18†A porcine cornea and lamellar tissue model to investigate effects of storage conditions on corneal preservation.

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.

25†Simulation of eye surgery in porcine eye globes and evaluation of retinal cytotoxicity.

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.

Ex vivo evaluation of retinal cytotoxicity after the use of multiple medical devices in pars plana vitrectomy in porcine eyes.

This study aimed to evaluate viability of retinal cells after the use of multiple intraoperative devices, namely a vitreal dye (triamcinolone acetonide,TA), a ERM/ILM dye (solution of trypan blue 0.15% and brilliant blue 0.025%), and two intraocular tamponades, namely perfluoro-n-octane, (PFO) and silicone oil (SO 1000 cSt), with minimal and maximal removal of their residues, during a simulated pars plana vitrectomy (PPV) in porcine eyes ex-vivo. The in vitro cytotoxicity of each of these compounds was verified on ARPE-19 cells by direct tests according to the ISO 10993-5 (2009). Pars plana vitrectomy was performed on 25 enucleated porcine eyes divided in five groups according to the following conditions: Group A) No surgery control: eye bulbs were kept at room temperature for 40 min; Group B) Sham surgery: PPV with the sole use of BSS for 40 min; Group C) Cytotoxic control: PPV with BSS infusion (20 min) followed by intravitreal injection of 1H-PFO (contact time: 20 min); Group D) Surgery with residues: PPV with BSS infusion and sequential intravitreal injection of TA, ERM/ILM dye, PFO and SO, with minimal removal of each compound after a specified contact-time (overall duration: 40 min); Group E) Surgery with minimal residues: PPV performed as in group D, but with maximal removal of each compound (overall duration: 40 min). All the experimental procedures were performed at room temperature. Immediately after surgery, the retina was extracted from each eye bulb and samples of 3-mm diameter were prepared. Retinal viability was determined for each sample by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay. A cell viability <70% was considered the cytotoxicity threshold. Kruskal-Wallis test was used to evaluate the differences in retinal viability between groups. 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 tested conditions indicated that the combined use of TA, ERM/ILM dye, PFO and SO during PPV does not affect retinal cells viability if all the devices 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 due to a cumulative and/or synergistic cytotoxic effect between them, supporting the crucial role of an optimal removal of the intraoperative medical devices to ensure a safe vitrectomy to the patient.

Toxicity Threshold of Perfluorocarbon Liquids for Intraocular Use: Dose-Response Assessment of In Vitro Cytotoxicity of Possible Contaminants.

Purpose: This study assessed the cytotoxicity of the impurities detected in the perfluorooctane (PFO) batches for vitreoretinal surgery that were associated with serious adverse incidents of ocular toxicity, namely, the perfluorooctanoic acid (PFOA), 1H,1H,7H-dodecafluoro-1-heptanol (DFH), 1H-perfluorooctane (1H-PFO), ethylbenzene, anhydrous p-xylene, and perfluoro-2-butyltetrahydrofurane, and two additional substances 1H,1H,1H,2H,2H-perfluorooctane (5H-PFO) and hexafluoro-1,2,3,4-tetrachlorobutane. Methods: Serial dilutions were tested by in vitro direct contact cytotoxicity test, validated in accordance with the ISO 10993-5:2009 standard using BALB3T3 and ARPE-19 cell lines, after sample application for 24 hours. Results: Six of the eight tested substances were cytotoxic according to the above-mentioned ISO standard. Anhydrous p-xylene, ethylbenzene, and PFOA were the most cytotoxic impurities as traces 1.55 ppm, 1.06 ppm, and 28.4 ppm reached the cytotoxicity limit, respectively. Hexafluoro-1,2,3,4-tetrachlorobutane, DFH, and 1H-PFO were cytotoxic at 980, 22,500, and 123,000 ppm, respectively. Both 5H-PFO and perfluoro-2-butyltetrahydrofuran were non-cytotoxic at the highest available concentrations (≥970,000 ppm). The dose-response curves allowed to calculate the cytotoxic concentration (CC30) for each tested substance that would reduce 30% of cell viability and corresponding to the cytotoxicity threshold according to ISO 10993-5. Conclusions: Our study determined the in vitro cytotoxicity of several impurities in PFO associated with serious adverse incidents in retinal surgery patients. Translational Relevance: Severe cytotoxicity of some impurities previously found in toxic perfluorocarbon liquids was confirmed. The cytotoxicity test validated according to the ISO 10993-5:2009 standard is a sensible and fast method for reliable detection of the cytotoxicity in perfluorocarbon liquids to guarantee maximal safety for the patients.

Killing efficacy of a new hypothermic corneal storage medium against the micro-organisms frequently found in human donor cornea intended for transplantation.

OBJECTIVE: To study the in vitro killing efficacy of Kerasave (AL.CHI.MI.A Srl), a medium provided with amphotericin B tablet for hypothermic storage of human donor corneas, against relevant contaminants associated with postkeratoplasty infections. METHODS AND ANALYSIS: The antimicrobial activity of Kerasave was determined after 0, 3 and 14 days of incubation at 2°C-8°C, inoculating Kerasave and the control medium with 105-106 colony forming units (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 assessing the number of viable CFU using the serial dilution plating technique. RESULTS: After 3 days, Kerasave induced the highest log10 decrease in the concentrations of KP, PA, CA and EC (5.37, 4.15, 2.97 and 2.67, respectively; all p<0.001). The log10 decreases of SA and EF were 2.27 and 2.11, respectively (all p<0.001). The lowest log10 decrease was observed in BS, AB and FS concentrations (0.25, 0.30 and 0.67, respectively; p<0.001 for BS and AB and p=0.004 for FS). After 14 days, the microbial count of CA, FS, SA, EF, PA and EC further decreased (p=0.006 for FS; p<0.001 for the others). CONCLUSION: Kerasave effectively reduced or kept unchanged the microbial concentration of almost all tested strains after 3 days. Thus, this novel medium represents a valuable tool to control the microbial contamination of human donor corneas during hypothermic storage for up to 14 days before transplantation.

Safety of silicone oils as intraocular medical device: An in vitro cytotoxicity study.

This study aimed to assess the cytotoxic effect of low molecular weight components (LMWC) and conventional silicone oils (SOs) 1000 cSt with different degree of purification (raw, intermediate, and purified) using in vitro cytotoxicity tests. Direct contact cytotoxicity tests were performed in BALB 3T3 and human retinal pigment epithelial cells (ARPE-19) using quantitative and qualitative evaluation according to the ISO 10993-5 (2009) standards. Conventional SOs 1000 cSt in form of raw, intermediate (intermediate product obtained during distillation process), and purified SO (final product after distillation) and a concentrate of LMWC (including siloxane chains with molecular weight up to 1557 g/mol) were directly applied to 100% of cell layer area for 24 h. Cell viability was quantified using 3-(4,5-dimethylthiazole-2-yl)-2,5-28 diphenyltetrazolium bromide (MTT) and neutral red uptake assays in ARPE-19 and BALB3T3, respectively. All tested samples, including the concentrate of LMWC, resulted to be not cytotoxic according to ISO 10993-5 in both qualitative and quantitative evaluations. However, the cellular viability was significantly higher in the intermediate and purified SO compared with the raw SO in ARPE-19 cells. No reduction in cell viability was detected by LMWC. The absence of cytotoxicity was observed for all tested samples in both BALB3T3 and ARPE-19 after 24 h of application. A direct cytotoxic effect is not likely to be involved in the potential complications related to SO and LMWC. Long-term potential adverse effects of SO could be related to the raw material and to different concentrations of LMWC.

Application of Circular Dichroism and Fluorescence Spectroscopies To Assess Photostability of Water-Soluble Porcine Lens Proteins.

The eye lens is mainly composed of the highly ordered water-soluble (WS) proteins named crystallins. The aggregation and insolubilization of these proteins lead to progressive lens opacification until cataract onset. Although this is a well-known disease, the mechanism of eye lens protein aggregation is not well understood; however, one of the recognized causes of proteins modification is related to the exposure to UV light. For this reason, the spectroscopic properties of WS lens proteins and their stability to UV irradiation have been evaluated by different biophysical methods including synchrotron radiation circular dichroism, fluorescence, and circular dichroism spectroscopies. Moreover, dynamic light scattering, gel electrophoresis, transmission electron microscopy, and protein digestion followed by tandem LC-MS/MS analysis were used to study the morphological and structural changes in protein aggregates induced by exposure to UV light. Our results clearly indicated that the exposure to UV radiation modified the protein conformation, inducing a loss of ordered structure and aggregation. Furthermore, we confirmed that these changes were attributable to the generation of reactive oxygen species due to the irradiation of the protein sample. This approach, involving the photodenaturation of proteins, provides a benchmark in high-throughput screening of small molecules suitable to prevent protein denaturation and aggregation.

Efficacy and Safety of Various Amphotericin B Concentrations on Candida albicans in Cold Storage Conditions.

PURPOSE: To determine the concentration of amphotericin B that would be both effective against Candida albicans contamination and safe for corneal endothelial cells (CECs) in cold storage conditions. METHODS: Triplicate media cultures were inoculated with 10 colony-forming units (CFUs)/mL of C. albicans (American Type Culture Collection 10231), supplemented with amphotericin B (0-20 µg/mL), stored in cold conditions (2°C-8°C) for 72 hours, and analyzed quantitatively for CFUs. C. albicans concentration in each sample was determined initially and after 6, 24, 48, and 72 hours of storage. CEC mitochondrial function (oxygen consumption rate), apoptosis, and necrosis were examined in donor corneas after 7 days of amphotericin B exposure and compared with untreated controls. CEC viability was also examined by calcein-AM staining and Fiji segmentation after 72 hours or 2 weeks of amphotericin B exposure to mimic potential eye bank practices. RESULTS: Amphotericin B concentrations of 1.25, 2.5, and 5.0 µg/mL resulted in 0.47, 1.11, and 1.21 log10 CFU reduction after only 6 hours of cold storage and continued to decrease to 3.50, 3.86, and 4.49 log10 reductions after 72 hours, respectively. By contrast, amphotericin B 0.255 µg/mL showed only 1.01 log10 CFU reduction after 72 hours of incubation. CEC mitochondrial function and viability did not differ in donor corneas exposed to amphotericin B ≤2.59 µg/mL compared with the controls. CONCLUSIONS: Optimal efficacy of amphotericin B against C. albicans is achieved in cold storage conditions at concentrations ≥1.25 µg/mL, and 2.5 µg/mL reduces Candida contamination by >90% after 6 hours of cold storage without sacrificing CEC health.

Evaluation of Cytotoxicity of Perfluorocarbons for Intraocular Use by Cytotoxicity Test In Vitro in Cell Lines and Human Donor Retina Ex Vivo.

PURPOSE: To validate the cytotoxicity test of perfluorocarbon liquids (PFCLs) for intraocular use according to the ISO 10993-5 standard. METHODS: BALB/3T3, ARPE-19 cell lines, and 3-mm human retina ex vivo samples were cultured in 96-well plates. Contact areas of 22%, 59%, and 83% and 2.5-, 12-, and 24-hour contact times were tested in cell lines. Cell viability was quantified by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in ARPE-19 and neutral red uptake (NRU) viability assay for BALB/3T3. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ARPE-19 cells. 1-H perfluorooctane (1H PFO) and purified perfluorooctane (PFO) were used as cytotoxic and not cytotoxic controls, respectively. Cell viability was assessed by MTT assay in retina ex vivo samples. RESULTS: Qualitative evaluation showed that cytotoxic control induced apoptosis, severe reactivity zones, and cytotoxicity according to ISO 10993-5 in all tested conditions. Quantitative evaluation of 1H PFO showed no cytotoxicity according to ISO 10993-5 on 22% areas, whereas cytotoxicity was detected on 59%, and 83% contact areas. The PFO was confirmed not to be cytotoxic in all tested conditions. Quantitative evaluation in retina ex vivo samples confirmed no cytotoxicity with PFO and cytotoxicity with 1H PFO. CONCLUSIONS: The direct contact cytotoxicity test according to ISO 10993-5 is a suitable method to detect the cytotoxicity of PFCLs and was validated using quantitative and qualitative approaches in ARPE-19 and BALB/3T3 cells covering 59% of the cell surface areas for 24 hours. TRANSLATIONAL RELEVANCE: Direct contact cytotoxicity test using specific conditions was validated, whereas different test conditions could not be validated.

Antimycotic Efficacy and Safety of a New Cold Corneal Storage Medium by Time-Kill and Toxicity Studies.

PURPOSE: To evaluate a new corneal cold storage medium including an antimycotic tablet (Kerasave, AL.CHI.MI.A. S.r.l.). METHODS: Kerasave and tryptone soy broth (control) were inoculated with 10 and 10 colony-forming units (CFU)/mL of 6 Candida isolates (Candida albicans [n = 4], Candida tropicalis [n = 1], and Candida glabrata [n = 1]). Minimum inhibitory concentrations (MICs) were determined using amphotericin B Etest strips. Sterile porcine corneas contaminated with 10 CFU/mL of each isolate were incubated in Kerasave and control at 4°C. Growth rate and Log10 reduction at 4°C at different time intervals were determined for liquid samples and tissue homogenates. Kerasave biocompatibility was assessed according to ISO 10993-5 and ISO 10993-10. RESULTS: No C. albicans or C. tropicalis colonies were recovered from Kerasave inoculated with 10 CFU/mL after incubation for 3 days at 4°C. C. glabrata was inhibited but not killed after 3 days at 4°C. Four of the 6 strains contaminated with 10 CFU/mL demonstrated a significant ≥ 3 Log10 reduction in media and tissue homogenates within 5 days as compared to controls (p < 0.01). Amphotericin B MICs ranged from 0.19 to 0.38 µg/mL for C. albicans (n = 3) and C. tropicalis (n = 1). C. glabrata showed reduced susceptibility (0.5 µg/mL) and 1 C. albicans was resistant to amphotericin B (≥ 1 µg/mL). Kerasave was not cytotoxic, irritating, or sensitizing according to the ISO standards. CONCLUSIONS: Kerasave showed high antifungal efficacy against susceptible fungal strains at 4°C in the presence and absence of corneal tissue. Resistant strains to amphotericin B were not eliminated by Kerasave. Kerasave is not cytotoxic, irritating, or sensitizing.

Monitoring the microbial contamination of donor cornea during all preservation phases: A prospective study in the Eye Bank of Rome.

BACKGROUND: In most European eye banks, human donor corneas are microbiologically tested after storage in organ culture conditions, and the tissues that are free of contamination are distributed for transplantation. In this prospective study, 100 donor corneas were tested for microbial contamination after cold storage, corneal culture and corneal deswelling at the Eye Bank of Rome. METHODS: Samples of cold storage medium (EUSOL-C), corneal culture medium (TISSUE-C) and deswelling medium (CARRY-C) were tested after three, seven and one days of corneal storage, respectively. The CARRY-C medium, used to transport the cornea to the operation theatre, was retested 1 day after transplantation. The TISSUE-C and CARRY-C media were also tested after removing antimicrobial and antifungal agents using a dedicated device. RESULTS: We found 67% of the EUSOL-C samples were contaminated mainly by Staphylococcus spp, 14% of TISSUE-C media were contaminated by bacteria and fungi and 3% of CARRY-C media by Staphylococcus spp The analysis performed after removing the antimicrobial and antifungal agents showed growth in three additional TISSUE-C samples (S viridans, S haemolyticus and E faecalis) and one CARRY-C (S cerevisiae and P acnes). CONCLUSION: Tissue contamination was unexpectedly high on arrival to the eye bank, indicating the need to review and update decontamination procedures during tissue recovery, and renew training for the recovery teams. Storing donor corneas in organ culture conditions significantly reduced the microorganism burden. Using devices to remove antimicrobial and antifungal agents from samples before testing can increase the sensitivity of the standard microbiological method, and thus help further reduce the risk of microbial transmission.