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

Joseph S. Uzarski, Michael D. DiVito, Jason A Wertheim*, William M Miller

*Corresponding author for this work

Research output: Contribution to journalArticle

4 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)163-175
Number of pages13
JournalBiomaterials
Volume129
DOIs
StatePublished - Jun 1 2017

Fingerprint

Scaffolds
Extracellular Matrix
Assays
Cell Count
Tissue
Cells
Benchmarking
Dyes
Population
Coloring Agents
resazurin
Kidney
Experiments
Growth
resorufin

Keywords

  • AlamarBlue
  • Bioreactor
  • Decellularization
  • Extracellular matrix
  • Liver
  • Renal

ASJC Scopus subject areas

  • Bioengineering
  • Ceramics and Composites
  • Biophysics
  • Biomaterials
  • Mechanics of Materials

Cite this

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AU - Miller, William M

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AB - 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.

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