Essential design considerations for the resazurin reduction assay to noninvasively quantify cell expansion within perfused extracellular matrix scaffolds

Abstract

Precise measurement of cellularity within bioartificial tissues and extracellular matrix (ECM) scaffolds is necessary to augment rigorous characterization of cellular behavior, as accurate benchmarking of tissue function to cell number allows for comparison of data across experiments and between laboratories. Resazurin, a soluble dye that is reduced to highly fluorescent resorufin in proportion to the metabolic activity of a cell population, is a valuable, noninvasive tool to measure cell number. We investigated experimental conditions in which resazurin reduction is a reliable indicator of cellularity within three-dimensional (3D) ECM scaffolds. Using three renal cell populations, we demonstrate that correlation of viable cell numbers with the rate of resorufin generation may deviate from linearity at higher cell densities, lower resazurin working volumes, or longer incubation times that all contribute to depleting the pool of resazurin. In conclusion, while the resazurin reduction assay provides a powerful, noninvasive readout of metrics enumerating cellularity and growth within ECM scaffolds, assay conditions may strongly influence its applicability for accurate quantification of cell number. The approach and methodological recommendations presented herein may be used as a guide for application-specific optimization of this assay to obtain rigorous and accurate measurement of cellular content in bioengineered tissues.

Comparative In Vitro Toxicology of Novel Cytoprotective Short-Chain Naphthoquinones

“…Based on the induction of γ-H 2 AX, indicative of DNA damage by some test compounds, their potential to transform substrate-dependent growth of HepG2 cells into substrate-independent cell growth by DNA mutations was assessed. To quantify transformation potential of our test compounds, a high throughput variant of the traditional semi-solid agar invasion assay was employed [ 33 ] using resorufin fluorescence as the indicator of cell growth [ 34 ]. The mutagenic compound 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was used as a positive control [ 35 ].…”

“…The plates were left to solidify for 1 h at room temperature and incubated overnight at 37°C before cells were treated for 21 days with reference and test compounds (10–40 μM in 15 µL DMEM/well). Subsequently, the cells were stained with resazurin for 4 h (440 µM in 7 µL PBS/well) [ 34 ] before fluorescence (Ex/Em 545/600 nm) was quantified using a plate reader (Fluoroskan Ascent, Thermo Fisher Scientific, Scoresby, VIC, Australia). RFUs were standardized on the non-treated control and expressed as fold-induction.…”

Does the Mass Balance of the Reactive Tracers Resazurin and Resorufin Close at the Microbial Scale?

“…matches findings by O'Brien et al (2000) and Chen et al (2018), who demonstrated that a secondary reaction from Rru to hydroresorufin is favored after all Raz has been reduced (reducing total detectable Raz + Rru) and that this reaction can happen both inside and outside cells. Another study using recellularized kidney scaffolds observed a fluorescence peak that gradually decreased throughout the assay, decaying to zero by 24 hr, demonstrating that cells were capable of secondary reduction of Rru to hydroresorufin (Uzarski et al, 2017). Moreover, Vehniäinen et al (2012) demonstrated that in the liver S9 fraction of some fish species, Rru was enzymatically reduced to a less fluorescent form.…”

Residual sodium dodecyl sulfate in decellularized muscle matrices leads to fibroblast activation in vitro and foreign body response in vivo

“…Cell viability was measured using a resazurin assay based on our previous protocol with some modifications (Uzarski, DiVito, Wertheim, & Miller, 2017). A 440 μM stock solution of resazurin sodium salt (Sigma Aldrich, St. Louis, MO) was prepared in PBS and then diluted 1 to 10 in fresh cell culture media for each cell type to create a working solution.…”