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Jana D'Amato Tothova, Barbara Ferrari, Laura Giurgola, Claudio Gatto; Quantitative method for testing toxicity of ocular endo-tamponades in human retina ex-vivo model.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6613.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a quantitative method to determine the toxicity of ocular endo-tamponades using human retina ex-vivo model based on MTT viability determination according to ISO 10993-5. The study aimed at determination of retina viability time-course, sample size, positive and negative controls, and the method accuracy and repeatability.
Human eye globes were recovered and transported to the Eye Bank, Fondazione Banca degli Occhi del Veneto Onlus. The eye globe for research use, was stored in DMEM/F-12 medium at 4°C for 24, 48 and 72 hours before retina extraction. The punch with 2- and 3-mm diameters were evaluated for retina sampling. The tissue viability was determined using TOX-1 In Vitro Toxicology Assay Kit, MTT based (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 and 0.50 mg/ml were tested as positive (cytotoxic) controls. DMEM/F12 medium was used as negative control.
Retina extracted from eye globe 24 and 48 hours within donor death showed optimal viability at T0 and the mean viability increased up to 178% after 7 days of tissue culture.Incubation of the samples with 0.25 and 0.50 mg/ml SDS (cytotoxic control) induced 89 ± 4 % and 100 ± 0 % reduction in cell viability at T0, respectively. Samples incubated with DMEM/F-12 showed 2 ± 1 % viability reduction in average at T0. At least eight replicates of 3-mm diameter samples in each group were required in order to obtain the coefficient of variation ≦ 20% within each group of samples. The samples with 2-mm diameter showed significantly higher variability in viability within the group than 3-mm samples.
Quantitative method to assess toxicity of ocular endo-tamponades in vitro, based on the human retina ex-vivo model and the cell viability assay, was standardized with high sensibility a repeatability according to ISO 10993-5.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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