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.

Keywords: AlamarBlue; Bioreactor; Decellularization; Extracellular matrix; Liver; Renal.

Figure 1. Stepwise conversion of resazurin to resorufin and hydroresorufin

Resazurin (blue) diffuses into cells where it is irreversibly reduced by diaphorase enzymes to a highly fluorescent (pink) compound, resorufin. In a subsequent, reversible reaction, resorufin may be further reduced to colorless, non-fluorescent hydroresorufin [26, 27].

Figure 2. Changes in perfusate fluorescence intensity (FI) over time mediated by metabolic reduction of resazurin to resorufin within recellularized 3D kidney ECM scaffolds

Kidney ECM scaffolds (n=2 scaffolds per cell type) were repopulated with proximal RPTE cells (12.5×10⁶), distal MDCK cells (20×10⁶), or TK188 fibroblasts (10×10⁶), and cells were allowed to proliferate under perfusion culture for up to 7 days. Kidney scaffolds were perfused with working solutions containing 10% AlamarBlue or 10% stock resazurin, and samples were withdrawn periodically. A: Representative graphs show the resulting change in FI as a function of circulating time. Inset plots with dashed outlines show the same data set with an additional sample obtained at 24 hours for RPTE and MDCK-recellularized kidney ECM scaffolds. B: Representative images of RPTE-or MDCK-recellularized kidney ECM scaffolds being perfused with AlamarBlue (top row) or resazurin solutions (bottom row) are shown at the beginning (0 hours), near-maximum FI (6 hours for RPTE cells; 3 hours for MDCK cells), and end (9 hours) of each experiment.

Figure 3. Effect of incubation time and cells: volume ratio on the increase in fluorescence intensity measured during resazurin reduction

Distal MDCK cells were plated at different cell densities to produce a range of cells:volume ratios from 8 ×10³ to 10⁶ cells per mL of resazurin working solution. After allowing cells to attach for 3 hours, medium was aspirated and 200 μL resazurin working solution was incubated in each well for 1, 2, or 4 hours. A: Standard curves generated at different incubation times are shown. As expected, longer incubation periods led to an increase in FI generated by conversion of resazurin to resorufin. However, longer incubation times (i.e., 4 hours) also resulted in a loss of linearity (decrease in slope of FI vs. cells:volume ratio curve) at higher cells:volume ratios, as demonstrated by the decreased R² value associated with the linear trendline. B: FI values are plotted on a logarithmic scale as a function of incubation time for each cells:volume ratio. The rate of increase in measured FI per hour decreased between 2 and 4 hours. Data are presented as mean ± standard deviation (n=3 wells per data point).

Figure 4. Generation of standard curves by varying cell number or resazurin working volume

Proximal RPTE cells, distal MDCK cells, or TK188 fibroblasts were plated in separate 48-well plates. After allowing cells to attach to the plate for 3 hours, medium was aspirated and resazurin working solution was added to each well and incubated for 1 hour. A: Standard curves were generated by using a constant resazurin working volume (100 μL), and varying the number of cells plated in each well to produce a range of cells:volume ratios ranging from 0.05 to 1 (10⁶ cells per mL resazurin). B: Standard curves were generated by plating a constant number of cells (10⁵ cells per well), and varying the volume of resazurin working solution added to each well to produce a range of cells:volume ratios ranging from 0.1 to 1 (10⁶ cells per mL resazurin). Results are presented as mean ± standard deviation (n=3 wells per data point). Linear regression trendline equations and associated R² values are shown in each graph.

Figure 5. Comparison of cell number calculated from resazurin reduction or by direct hemocytometer counting during cell growth to and beyond confluence

Proximal RPTE cells, distal MDCK cells, or TK188 fibroblasts were plated in separate 48-well plates at a density of 5×10³ cells/cm². Three hours after plating and subsequently every day, the resazurin reduction assay was performed and cells were lifted from replicate wells for manual counting. A: Results show the average number of cells per well calculated using either a hemocytometer (n=3) or the resazurin reduction assay (n=6). Data are presented as mean ± standard deviation. The vertical arrow shows the approximate time point (day 5) at which the cells have reached confluence. Asterisks (*) indicate a significant difference in means between calculation method at each time point as determined by independent samples Student’s t-test (p<0.05). B: Representative phase contrast images at various time points show that cells continued to proliferate even after reaching confluence. By day 7, RPTE cells had begun to grow vertically atop one another, whereas MDCK and TK188 cells remained as a monolayer, despite continued proliferation. Scale bars: 100 μm.

Figure 6. Accurate estimation of cell number in kidney perfusion bioreactors depends on resazurin working volume

Kidney ECM scaffolds (n=4 scaffolds per cell type) were repopulated with proximal RPTE cells (12.5×10⁶), distal MDCK cells (20×10⁶), or TK188 fibroblasts (10×10⁶), and cells were allowed to proliferate under perfusion culture. Two days after seeding, 10 mL or 100 mL of resazurin working solution was circulated through each reservoir for 1 hour, and medium samples were withdrawn. A: The calculated number of cells varied greatly between the two volumes of resazurin circulated, with the lower volume providing a significantly lower estimate. The dashed line shows the number of cells calculated via the resazurin perfusion assay, using 50 mL of working solution, on the previous day. Data are presented as mean ± standard deviation (n=4 ECM scaffolds per data point). Asterisks (*) indicate a significant difference in means as determined by paired samples Student’s t-test (p<0.05). B: In a separate experiment, a standard curve generated by plating varying numbers of distal MDCK cells in 2D cultures (left panel) was used to derive a linear regression equation to relate FI to cells:volume ratio. One day after injecting 20 million MDCK cells into a kidney ECM, The resazurin assay was run for 1 hour each, circulating volumes of 10, 25, 50, 75, or 100 mL (middle panel), and the number of cells calculated using each volume is shown in the right panel. The 50, 75, or 100 mL volumes provided a consistent estimation at approximately 20 million cells (the number of cells injected the previous day, indicated by the dashed line), whereas the 25 mL and, especially, the 10 mL volumes provided much lower estimations of cell number.

Figure 7. Comparison of fresh vs. nutrient-depleted culture medium as a diluent for resazurin

Kidney ECM scaffolds (n=4 scaffolds per cell type) were repopulated with proximal RPTE cells (12.5×10⁶), distal MDCK cells (20×10⁶), or TK188 fibroblasts (10×10⁶), and cells were allowed to proliferate under perfusion culture. A: One day after seeding, 5 mL of resazurin stock solution was diluted in 45 mL of nutrient-depleted media obtained from the bioreactor reservoir, and the solution was recirculated through each scaffold for 1 hour. After a 10 minute wash using 20 mL of fresh medium, a new 50 mL volume of resazurin working solution prepared using fresh medium as a diluent was circulated for 1 hour. As shown, the calculated number of cells present was lower when fresh medium was used as a diluent, due to a decrease in metabolic reduction of resazurin to resorufin. Results are presented as mean ± standard deviation (n=4 scaffolds per data point). Asterisks (*) indicate a significant difference in means as determined by independent samples Student’s t-test (p<0.05). B: Four days after seeding, resazurin was diluted in nutrient-depleted medium with or without supplementation to basal concentrations of D-glucose. As shown, glucose supplementation had no inhibitory effect on resazurin reduction. Results are presented as mean ± standard deviation (n=2 scaffolds per data point). C-D: Resazurin working solutions were prepared by diluting resazurin in nutrient-depleted (C) or fresh (D) culture media, and varying numbers of plated cells were incubated with these solutions to prepare standard curves as previously described. As shown, FI generated by resazurin reduction was greater in nutrient-depleted medium (C) compared with fresh medium (D). Results are presented as mean ± standard deviation (n=3 wells per data point). Linear regression trendline equations and associated R² values are shown within each graph.

Figure 8. Proliferation trends measured within recellularized kidney scaffolds using the standardized resazurin perfusion assay

Kidney ECM scaffolds (n=2 scaffolds per cell type) were repopulated with proximal RPTE cells (12.5×10⁶), distal MDCK cells (20×10⁶), or TK188 fibroblasts (10×10⁶), and cells were allowed to proliferate for up to 7 days in perfusion bioreactors. The optimized resazurin reduction assay (100 mL of working solution created using cell-conditioned medium circulated for 1 hour) was performed in concert with a standard curve from 2D culture to quantify the number of cells present at each time point. Data are presented with normalized FI on the primary vertical axes, and the calculated number of cells on the secondary vertical axes.