"in the toxicity evaluation of nanofibrous scaffold via MTT test, scaffolds with thick fibers showed maximum amount of cell infiltration and least amount of cell viability on the opposite side finer nanofibers showed least amount of cell infiltration to depth of scaffold and highest MTT value" based on this, selection of microfibrous or nanofibrous scaffold as better one to use in tissue engineering seem to be difficult!!
Dear Mojtaba,
I think MTT assay is a proper test to evaluate such variables. However, since rapidly replicating cells display high rate MTT reduction, one should be ascertain if cells in these different experimental settings display similar replication rate. Can you evaluate it? Direct reading of your experimental data might help me in my judgment. Please let me know if somebody maintains an opinion different from mine.
I agree with Piero. MTT assays are based on the conversion of MTT to its metabolite. It is dependent not only on cell viability but also on cell population. Cells may proliferate faster on the finer nanofibers. If you are truly looking to assess viability it will need to be normalised against cell numbers. One method is to perform a DNA extraction (following the MTT assay) and use quantitative PCR. This will give you an idea of how may cells are present in the assay. I am sure there are other assays possible.
It depends on what type of toxicity you trying to measure. MTT is NOT a viability assay, rather a mitochondrial enzymatic assay. Typically if you are trying to show viability you will need to use MTT in combination with other assays, such as crystal violet, trypan blue, cellular/nuclear morphology or other indirect measures such as SRB.
Yeah.. I agree to the previous answers. Cytocompatibility is a broader term. MTT assay is sufficient when you're actually testing for the cytotoxicity of the scaffold material. Depending upon my experience, MTT is just an indirect measure of viable cell count. Any material that just supports cell viability (perhaps proliferation), but if negatively affects the differentiation of the cell, then such material cannot be a good scaffolding material. I suggest you to carry out other experiments that will identify/estimate the differentiation and function (eg. For bone cells-Alizarin res S staining will give details of calcium deposition, which is the prime function of a healthy bone cell). Likewise, choose tests depending on your type of application (scaffold for what organ?? skin, bone, cartilage??) I guess those results will definitely fetch you reliable results! Good Luck for ur work!
Hi
Dear Mojtaba
Due to the strong dye sorption capability of porous scaffolding materials, it has been proposed that the cell viability determined from the MTT assay in nanofibrous scaffolds is likely to give a false negative result. I have attached a paper which strongly support this. There are other cytotoxicity assays like LDH test that could be used instead.
Hope this help.
Bear in mind that MTT assay is a metabolic assay and so can show high activity when cells are under stress. I edit a journal and get many papers incorrectly citing MTT assay as a cell number or proliferation assay which it isn't. There is a CCK kit which is better for cell number.
Thank u Dr Jonathan.. Its a new information to me. But all the papers I referred from high impact factor journals all start off the MTT assay stating that it is an assay to test cytotoxicity. It is an indirect measure of cell proliferation!!!! Thank u anyway ;)
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