I tested my drug, by MTT assay, on different cell lines (HeLa, Caco-2, HepG2, WI38, J774, Raw264.7) and I obtained different results in terms of cytotoxicity on these cell lines.
Can someone kindly explain the possible reasons for this difference in cytotoxicity on these cell lines?
Thanks in advance.
Hello Abdullah Abdullah
MTT reduction can be significantly affected by several factors which include metabolic and energy perturbations, changes in the activity of oxidoreductases, endo-/exocytosis and intracellular trafficking. Over or underestimation of cell viability by the MTT assay may be due to adaptive metabolic and mitochondrial reprogramming of cells subjected to drug treatment-mediated stress and inhibitor off-target effects.
So, it is recommended that tetrazolium salt-based assays should be supplemented with other non-metabolic assays in order to avoid result misinterpretation.
Best.
Toxicity depends on both mechanism of drug and cell type . Each type of cell has its special gene expression level according to the requirements and its specialisation that determines its function within the body. This effects cellular metabolism and biochemical actiities.Drug metabolism of a cell is also varies according to cell types due to different metabolism and gene expression.
This may be the reason of different toxicity values in different cells.
Results of MTT are dependent on many factors as mentioned by other above.
In simple words, MTT works on how energy active the cells are i.e., number of mitochondria present in the cells. More mitochondria mean more conversion of the dye (3,4,5-dimethyl.......tetrazolium bromide to formazan), results in higher color and hence different reading. It is known that metabolically active cells like heart cells have higher mitochondrial content as compared to HEK or 293 cells. Further in your list, you have mouse macrophage line (Raw264) that would be different from other human cell lines. Macrophages are relatively more active then say HepG2 cells. So the reason of your variability with the same drug is highly dependent on the mitochondria content in these cell lines.
Secondly, cell densities would also affect MTT results. You may have seeded for instance, 10K cells for each cell line but each cell line has different growth kinetics, so one cell line would be at 11K whereas another at 15K at the time of the assay, this would also alter the readouts. This is one of the reasons now days MTT needs to be supported by other non-metabolic cytotoxicity evaluation methods.
Best of luck!
MTT assay is not really a cytotoxicity assay but rather a test of cell respiration and mitochondrial activity which may differ drom one cell type to another according to the organ and the species from which the cell is derived . Even the same line may respond differently to a given drug within different hands. Cell lines, even initially originating from a given cancerous cell, may progressively diverge after numerous cell passages and under different culture conditions (serum, growth factor, stirring, nature of the support, freezing and decongelation....) in different labs and become in fact truely different lines which respond differently to drugs.
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