Abstract
Purpose :
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.
Methods :
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.
Results :
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.
Conclusions :
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.