Quantitative Reduction of Methyl Tetrazolium by Fresh Vein Homograft Biopsies in vitro Is an Index of Viability

Abstract

Objective: The color density of the methyl tetrazolium (MTT) test is proportional to mitochondrial enzyme activity thus reflecting cellular viability. The aim was to evaluate the MTT test as a viability assay for vein homograft studies. Materials and Methods: Fresh intact vein samples were harvested during multi-organ procurement. The reliability of the MTT assay was tested by a fluorescent dye combination (1 µg/ml propidium iodide PI and 4 µM/ml SYTO-16 stains). The enzyme kinetics of the reaction was also investigated. The optimal reagent concentration, biopsy size and incubation period were established. Results: There was a linear relationship between the vein homograft’s weight and the pigment production activity. A nonspecific reaction (8.6%) was observed in negative controls. The MTT cleavage up to 0.1% (w/v) follows the Michaelis kinetics. The Michaelis constant (2,805 ± 130 µM), the maximal velocity (196 ± 2.2 × 10^–5µM s^–1) and the velocity constant (6.98 ± 0.2 × 10^–7 s^–1) was calculated. The viability assessed by fluorescent dyes simultaneously visualized the live/dead cell ratio, which can be calculated by image analysis software. Conclusion: The use of MTT in colorimetric assays offers high sensitivity. The assay is simple, inexpensive, and reproducible in vein homograft studies.