Essential design considerations for the resazurin reduction assay to noninvasively quantify cell expansion within perfused extracellular matrix scaffolds.
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.
Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
Joseph S
JS
Uzarski
Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
Michael D
MD
DiVito
Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Department of Surgery, Jesse Brown VA Medical Center, Chicago, IL 60612, USA; Simpson Querrey Institute for BioNanotechnology, Northwestern University, Chicago, IL 60611, USA; Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA. Electronic address: jason.wertheim@northwestern.edu.
Jason A
JA
Wertheim
Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, USA; Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA. Electronic address: wmmiller@northwestern.edu.
William M
WM
Miller
D000818:Animals; D019149:Bioreactors; D002452:Cell Count; D049109:Cell Proliferation / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004285:Dogs; D005109:Extracellular Matrix / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D007668:Kidney / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism*; D010477:Perfusion; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry*; D014966:Xanthenes / Q000737:chemistry / Q000378:metabolism*
D017077:Culture Media, Conditioned; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; C014180:resorufin
AlamarBlue; Bioreactor; Decellularization; Extracellular matrix; Liver; Renal