Hi! I want to perform the MTT assay to assess the cytotoxicity of my hydrogels. I have read literature. It is reported that either use the extract of the hydrogels by dipping them into the DMEM media for 24 h and then replace the media of cells with the extract media. The other is by mixing hydrogels with the cells in media. I am concerned how can I remove media from the wells containing hydrogels. Cells have already made the monolayer after 24 h and before adding the MTT reagent I need to remove the media. But the hydrogels are also in wells. How to remove the hydrogels?
Hello Noor Ul Ain
Instead of mixing hydrogels with the cells in the media, your problem may be solved by making use of a semi-direct contact assay with trans well inserts as an alternative approach. In a semi-direct contact assay, the cells are cultured in situ with the hydrogel sample placed inside trans well inserts above the cell monolayer. Leachable substances from the hydrogel permeate through the porous membrane of trans well inserts to the reach the cell monolayer underneath. After the specified time point (for instance, 24h, 48h, and 72h) of incubation, the metabolic activity of the cells may be determined by MTT assay.
This semi-direct contact assay can serve as a reliable alternative to the method that you have used which is in addition to the conventional extract assay in evaluating the potential cytotoxicity of hydrogels.
Best.
Malcolm Nobre thank you so much for your response. But wouldn't this semi-direct contact similar to the extract. I performed one assay directly adding the MTT reagent to the well containing cells+hydrogel and in one well I removed the hydrogel and then added the MTT reagent but due to removal of hydrogel some of the cells also scraped alongwith it.
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