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Using MTT viability assay to test the cytotoxicity of antibiotics and steroid to cultured porcine corneal endothelial cells.

Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics

Thiazoles, Animals, Anti-Bacterial Agents, poisoning, Cell Survival, drug effects, Cells, Cultured, Coloring Agents, Endothelium, Corneal, cytology, Swine, Tetrazolium Salts

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      Abstract

      Intracameral injection (ICI) of antibiotics and steroid is an effective method to deliver drugs into eyeballs, and rapidly achieve therapeutic concentrations. The high intraocular concentrations, however, that occur by such injections can harm the corneal endothelium. The MTT assay, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], has been used to test cytotoxicity of five antibiotics (including amphotericin-B, colistin-M, sulbenicillin, amikacin, cephradine) and a steroid, betamethasone, to cultured porcine corneal endothelial cells. The third passage of porcine corneal endothelial cells were plated into 96-well microtitration plates for drugs exposure. Cytotoxicities of the six drugs in different concentrations were compared, using exposure time as an independent variable. In its original and 10-fold ICI dose, only amphotericin-B among the six tested drugs showed significant cytotoxicity; the other drugs were considered safe. In a 100-fold ICI dose, amphotericin-B, colistin-M, and sulbenicillin were all toxic. Although a cell culture system utilizing the MTT assay is an in vitro method of testing for drug toxicity to the corneal endothelium, it nevertheless offers the advantages over in vivo methods. It is rapid, convenient, and economical. Large numbers of toxic compounds can be compared simultaneously, so that relative toxicities among different drugs and concentrations can be obtained, and be a guide for determining ICI dose, or as a reference for further in vivo testing.

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