An untargeted metabolomics approach to study changes of the medium during human cornea culture.

INTRODUCTION: Two main approaches (organ culture and hypothermia) for the preservation and storage of human donor corneas are globally adopted for corneal preservation before the transplant. Hypothermia is a hypothermic storage which slows down cellular metabolism while organ culture, a corneal culture performed at 28-37 °C, maintains an active corneal metabolism. Researchers, till now, have just studied the impact of organ culture on human cornea after manipulating and disrupting tissues. OBJECTIVES: The aim of the current work was to optimize an analytical procedure which can be useful for discovering biomarkers capable of predicting tissue health status. For the first time, this research proposed a preliminary metabolomics study on medium for organ culture without manipulating and disrupting the valuable human tissues which could be still used for transplantation. METHODS: In particular, the present research proposed a method for investigating changes in the medium, over a storage period of 20 days, in presence and absence of a human donor cornea. An untargeted metabolomics approach using UHPLC-QTOF was developed to deeply investigate the differences on metabolites and metabolic pathways and the influence of the presence of the cornea inside the medium. RESULTS: Differences in the expression of some compounds emerged from this preliminary metabolomics approach, in particular in medium maintained for 10 and 20 days in presence but also in the absence of cornea. A total of 173 metabolites have been annotated and 36 pathways were enriched by pathway analysis. CONCLUSION: The results revealed a valuable untargeted metabolomics approach which can be applied in organ culture metabolomics.

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.

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.

Antioxidant Nutraceutical Strategies in the Prevention of Oxidative Stress Related Eye Diseases.

This review aims to discuss the delicate balance between the physiological production of reactive oxygen species and the role of antioxidant nutraceutical molecules in managing radicals in the complex anatomical structure of the eye. Many molecules and enzymes with reducing and antioxidant potential are present in different parts of the eye. Some of these, such as glutathione, N-acetylcysteine, α-lipoic acid, coenzyme Q10, and enzymatic antioxidants, are endogenously produced by the body. Others, such as plant-derived polyphenols and carotenoids, vitamins B2, C, and E, zinc and selenium, and omega-3 polyunsaturated fatty acids, must be obtained through the diet and are considered essential nutrients. When the equilibrium between the production of reactive oxygen species and their scavenging is disrupted, radical generation overwhelms the endogenous antioxidant arsenal, leading to oxidative stress-related eye disorders and aging. Therefore, the roles of antioxidants contained in dietary supplements in preventing oxidative stress-based ocular dysfunctions are also discussed. However, the results of studies investigating the efficacy of antioxidant supplementation have been mixed or inconclusive, indicating a need for future research to highlight the potential of antioxidant molecules and to develop new preventive nutritional strategies.

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.

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.

Biocompatibility of intraocular liquid tamponade agents: an update.

Intraocular liquids tamponade agents, such as perfluorocarbon liquids (PFCLs), semifluorinated alkanes (SFAs), silicone oils (SOs) and heavy silicone oils (HSOs), are a crucial intraoperative and/or postoperative tool in vitreoretinal surgery, in particular for the management of complex vitreoretinal diseases. However, their use is not without complications, which are potentially severe. Consequently, a growing interest has been devoted to the biocompatibility of these compounds and the adequacy of current regulations that should guarantee their safety. Obviously, an updated knowledge on research findings and potential risks associated to the use of intraocular liquid compounds is essential, not only for vitreoretinal surgeons, but also for any ophthalmologist involved in the management of patients receiving intraocular liquid tamponades. In light of this, the review provides a comprehensive characterisation of intraocular liquid tamponades, in terms of physical and chemical properties, current clinical use and possible complications. Moreover, this review focuses on the safety profile of these compounds, summarising the existing regulation and the available evidence on their biocompatibility.

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.

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.

A new approach to extend the storage of donor corneas after 28 days of corneal culture in an Italian eye bank.

This study aimed to evaluate the possibility to extend the storage of unused organ-cultured donor corneas. After 28 days of corneal culture in TISSUE-C (AL.CHI.MI.A. S.R.L., Italy) and 5-day storage in transport/deswelling medium CARRY-C (AL.CHI.MI.A. S.R.L., Italy), 25 corneas that were deemed suitable for transplantation were transferred in fresh TISSUE-C at 31 °C for additional 7 days and then in fresh CARRY-C at room temperature for 24 h. Tissues were assessed for endothelial cell density (ECD), endothelial mortality and morphology after the standard and the extended corneal storage. In addition, the effect of donor age < 85 years and ≥ 85 years on corneal characteristics was assessed. After the extended storage, 6 out of 25 tested corneas (24%) showed ECD values below the acceptance limit (< 2000 cells/mm2). 19 corneas (76%) were still suitable for transplantation and showed a 5.9% loss in ECD, which was not statistically significant (P = 0.0949) compared to standard storage period. The two donor age groups did not show statistically significant differences in any tested parameter, although a trend for lower ECD and higher mortality in Descemet's folds after standard storage was observed in the ≥ 85 age donor group. Thus, the attempt of the current study to provide new sight-restorative options for unused tissues and increasing the availability of corneas in case of shortage gave encouraging results. Although a higher vulnerability of corneas from very old donors could not be statistically demonstrated in the present study, higher sample size could be required for prolonging the shelf life of these tissues.

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.

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.

Increased sensitivity of microbiological testing of cornea organ culture medium by additional resin treatment.

OBJECTIVE: This validation study investigates the treatment of cornea organ culture medium (Modified Eagle Medium, Biochrom GmbH, Berlin, Germany) with RESEP, a new medical device for antibiotics removal, before microbiological testing with BACTEC TM blood culture bottles. METHODS AND ANALYSIS: 10-100 colony forming units of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtillis, Aspergillus brasiliensis, Clostridium sporogenes, Enterobacter cloacae and Staphylococcus epidermidis were inoculated in 9mL of cornea organ culture medium. In group A, the medium was withdrawn with RESEP and treated for 20 min at room temperature, and then inoculated in BACTEC Plus Aerobic/F/Anaerobic/F blood culture bottles. In group B, the medium, spiked by the inoculation of microorganism, was injected directly. For each strain, a growth control was performed, by direct inoculation of the microorganisms in BACTECTMvials (positive control). All samples were incubated in the automated BACTECTMblood culture system at 36°C ±1°C for maximum of 14 days or until a positive reading. The elimination of antibiotics from the medium by RESEP was determined by high-performance liqiud chromatography. RESULTS: After 20 min of RESEP treatment, 100% (n=9) of streptomycin, 100% (n=9) of amphotericin B and 99.7% (n=9) of penicillin G were eliminated. In group A, all microorganisms were detected within 3 days of incubation with a sensitivity of 100% (n=99) and showed no significant delay compared with the positive controls. In group B, the overall sensitivity was 67.9% (n=96) with a significant delay until detection of microbial growth for all tested microorganisms except for A. brasiliensis. CONLCLUSION: The use of RESEP to eliminate the antibiotics from cornea organ culture medium increases the sensitivity of the microbiological testing with BACTECTMPlus blood culture bottles significantly and fulfils the requirements of the European Pharmacopoeia method suitability test.

Method for sterility testing of corneal storage and transport media after removal of interfering antimicrobials: prospective validation study in compliance with the European Pharmacopoeia.

OBJECTIVE: This study aimed at validating the method for sterility testing of the corneal culture medium, TISSUE-C, and the transport/deswelling medium, CARRY-C, according to the method suitability test, as defined by the European Pharmacopoeia (EP), using RESEP, which is a new medical device for removal of antimicrobial agents and an automated culture system. METHODS AND ANALYSIS: 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 micro-organisms in the culture broths. Microbial growth was read by an automated light scattering culture system within 48 hours. 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 micro-organism, 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.