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

Abstract

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.

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

“…The ISO 10993-5 reference cell line, murine fibroblast cells BALB3T3 clone A31 (ATCC CCL163), and the human retinal pigment epithelial cell line ARPE-19 (ATCC CRL-2302) were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA, USA) and used as previously described. 15 , 18 In particular, after thawing, BALB3T3 and ARPE-19 cells were grown as a monolayer in Dulbecco's modified Eagle medium (DMEM) with high glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate (Sigma-Aldrich Corp) and Dulbecco's modified Eagle medium/Nutrient Mixture F-12 medium with L-glutamine without 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Gibco, Waltham, MA, USA), respectively, each supplemented with 10% of newborn calf serum (Sigma-Aldrich Corp) and 1% penicillin–streptomycin (Sigma-Aldrich Corp), in 75 cm 2 culture flasks in an incubator (Memmert, Schwabach, Germany) at 37°C ± 1°C, 90% ± 10% humidity, 5.0% ± 1.0% CO 2 /air. The cultures were used after two passages.…”

“…As described by Romano et al, 15 in vitro direct contact cytotoxicity test, validated in accordance with the ISO 10993-5 standards, 12 was performed. Briefly, BALB3T3 and ARPE-19 cell suspension containing 2.0 to 3.0 × 10 5 cells/mL were seeded into 96-well microtiter plates and incubated at 37°C ± 1°C, 90% ± 10% humidity, and 5.0% ± 1.0% CO 2 /air for 24 hours to reach 70% to 80% confluence.…”

“…In the absence of ISO 10993-5 12 indications for ARPE-19 cells, the MTT cytotoxicity test in these cells was performed using the TOX-1 In Vitro Toxicology Assay Kit, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-phenyltetrazolium bromide) (Sigma-Aldrich Corp) as previously described by Romano et al 15 Briefly, the viable cells are metabolically reduced to a blue-violet insoluble formazan by the yellow water-soluble MTT; therefore the extent of cell viability correlates with the intensity of the color that is determined by photometric measurement after dissolving the formazan in alcohol.…”

“… 8 , 11 The test failed to detect the cytotoxicity of the medical device, probably because of the incorrect selection of the method that does not consider the hydrophobic and volatile characteristics of the PFCLs 11 , 14 and lack of evaluation of its critical steps. 15 …”

Chemical Transformation of Astaxanthin from Haematococcus pluvialis Improves Its Antioxidative and Anti-inflammatory Activities

“… 48 , 49 If viability was reduced to less than 70% of the blank, the compound was considered to have cytotoxic potential ( Figure S4 ). 50 The statistical differences were calculated by one-way ANOVA followed by the Dunnett’s test (Minitab, version 18). A two-sided p -value less than 0.05 was considered as statistically significant.…”