Comparative study of resazurin reduction and MTT assays for cytocompatibility evaluation of nanofibrous materials

Abstract

As a major approach to evaluate the cytocompatibility of materials, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay has been proven to give a false negative result for nanofibrous mats. Herein, we proposed an alternative approach, resazurin microtiter assay (REMA) to get a more reliable cytocompatibility result for electrospun nanofibers. Mouse fibroblasts (L929 cells) were used as model cells. The cell density and incubation time of REMA were optimized. A linear relationship (R² = 0.99034) between the fluorescence intensity of resorufin (cell metabolite of REMA, which is in direct proportion to cell viability) and cell density is observed when the cell density is less than 5.0 × 10⁵ cells per mL. Moreover, the fluorescence intensity of resorufin is saturated when the cell density is higher than 5.0 × 10⁵ cells per mL. On the other hand, the fluorescence intensity data fit the equation (Y = 2.72 + 6.28 × 0.26X², R² = 0.98892) well with the incubation time (up to 10 h), and in order to get the accurate cell viability, the reduction time should not exceed 6 h. In general, REMA is proven to be more convincing than MTT assay since less or no formed resorufin can be absorbed by nanofibrous mats during REMA. We anticipate that our study will provide a general insight for the use of REMA to evaluate the cytocompatibility of other nanofibrous materials.