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 language | English (US) |
|---|---|
| Pages (from-to) | 163-175 |
| Number of pages | 13 |
| Journal | Biomaterials |
| Volume | 129 |
| DOIs | |
| State | Published - Jun 1 2017 |
Funding
Research reported in this publication was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (under award numbers 1F32DK103499 to J.S.U. and K08DK101757 to J.A.W.), the U.S. Department of Veterans Affairs (Merit Review Award I01BX002660, Biomedical Laboratory Research and Development Service), a Postdoctoral Research Grant (PDR-070 to J.S.U.) from the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust, the Human Organ Project/AST TIRN Research Grant, the Zell Family Foundation, the Robert R. McCormick Foundation and the American Society of Transplant Surgeon's Faculty Development Grant. J.A.W. is a member of the NIDDK ReBuilding a Kidney Consortium (U01DK107350) and we gratefully acknowledge input from the RBK consortium. M.D.D. was supported in part by the National Institutes of Health's National Center for Advancing Translational Sciences, Grant Number TL1TR001423. Funding sources played no role in study design or analysis. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the U.S. Department of Veterans Affairs, or the United States Government. This research was supported by the Analytical BioNanoTechnology Core Facility of the Simpson Querrey Institute at Northwestern University. Imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. TK188 fibroblasts [35] were kindly provided by Dr. Michael Zeisberg and Dr. Gerhard Muller (Göttingen University Medical Center). The authors would like to acknowledge the Northwestern University Microsurgery Core for rodent organ procurements.
Keywords
- AlamarBlue
- Bioreactor
- Decellularization
- Extracellular matrix
- Liver
- Renal
ASJC Scopus subject areas
- Mechanics of Materials
- Ceramics and Composites
- Bioengineering
- Biophysics
- Biomaterials