PurposeTo validate the cytotoxicity test of perfluorocarbon liquids (PFCLs) for intraocular use according to the ISO 10993-5 standard.MethodsBALB/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.ResultsQualitative 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.ConclusionsThe 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 RelevanceDirect contact cytotoxicity test using specific conditions was validated, whereas different test conditions could not be validated.

No abstract

In recent years, cases of retinal toxicity occurred in some European, Middle Eastern, and South American countries following the use of perfluorocarbon liquids (PFCLs) on vitreoretinal surgeries owing to impurities in the product. Moreover, Spanish ophthalmologists reported several toxic cases on the use of perfluoro- n -octane Ala Octa (Alamedics, Dornstadt, Germany), raising the necessity of reviewing the current validated methods used for assessing the safety of PFCLs. We proved that in samples of PFCLs contaminated on purpose with impurities previously detected in Ala Octa devices, the determination of the so-called H-content using a 1 H NMR quantitative assay implemented with the electronic reference to access in vivo concentrations 2 technology failed to demonstrate a correlation between the H-content and in vitro cytotoxicity test in ARPE-19 and BALB 3T3 cell lines. Therefore, direct information on the safety of PFCLs was provided only by the cytotoxicity test in vitro validated according to ISO 10993-5, and the H-content was not predictive of perfluorocarbon ocular endotamponade cytotoxicity in vitro.

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 105 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.

We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives.