Design considerations to ensure accuracy when using the resazurin reduction assay to noninvasively quantify cell expansion within perfused extracellular matrix scaffolds

Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Precise measurement of cell numbers within bioartificial tissues and extracellular matrix scaffolds is necessary to provide measurement assurance and rigorous characterization of cell behavior within three-dimensional (3D) scaffolds. Accurate benchmarking of tissue function and biosynthetic activity to cell number facilitates comparison of data across experiments and between laboratories to increase rigor and reproducibility in tissue engineering and biofabrication. Soluble, fluorescent indicators of metabolic activity are valuable, noninvasive tools for estimating viable cell number. We investigated experimental conditions in which resazurin is a reliable indicator of cell content within 3D extracellular matrix kidney and liver scaffolds, and we present recommendations on experimental methodology for its optimal use. Resazurin is reduced to resorufin in proportion to metabolic activity of viable cells. Using three renal cell lines and one hepatic cell line, we show that correlation of viable cell number with the rate of resorufin generation may deviate from linearity at higher cell density, low resazurin working volumes, and/or longer incubation times – all of which contribute to depleting the working pool of resazurin. Importantly, we also show that the resazurin reduction rate in cell-conditioned medium is about double that in fresh culture medium. This finding has the potential to increase assay sensitivity, while saving expensive media. In conclusion, while the resazurin reduction assay provides a powerful, noninvasive readout for cell growth within extracellular matrix scaffolds, assay conditions may strongly influence its applicability for accurate quantification of cell number. The approach and recommendations developed in this study to maintain the pool of reducible resazurin may be used as a guide for application-specific optimization of the resazurin reduction assay to obtain accurate measurements of cell content in bioengineered tissues

Adaptation of Endothelial Cells to Physiologically-Modeled, Variable Shear Stress

<div><p>Endothelial cell (EC) function is mediated by variable hemodynamic shear stress patterns at the vascular wall, where complex shear stress profiles directly correlate with blood flow conditions that vary temporally based on metabolic demand. The interactions of these more complex and variable shear fields with EC have not been represented in hemodynamic flow models. We hypothesized that EC exposed to pulsatile shear stress that changes in magnitude and duration, modeled directly from real-time physiological variations in heart rate, would elicit phenotypic changes as relevant to their critical roles in thrombosis, hemostasis, and inflammation. Here we designed a physiological flow (PF) model based on short-term temporal changes in blood flow observed <em>in vivo</em> and compared it to static culture and steady flow (SF) at a fixed pulse frequency of 1.3 Hz. Results show significant changes in gene regulation as a function of temporally variable flow, indicating a reduced wound phenotype more representative of quiescence. EC cultured under PF exhibited significantly higher endothelial nitric oxide synthase (eNOS) activity (PF: 176.0±11.9 nmol/10⁵ EC; SF: 115.0±12.5 nmol/10⁵ EC, p = 0.002) and lower TNF-a-induced HL-60 leukocyte adhesion (PF: 37±6 HL-60 cells/mm²; SF: 111±18 HL-60/mm², p = 0.003) than cells cultured under SF which is consistent with a more quiescent anti-inflammatory and anti-thrombotic phenotype. <em>In vitro</em> models have become increasingly adept at mimicking natural physiology and in doing so have clarified the importance of both chemical and physical cues that drive cell function. These data illustrate that the variability in metabolic demand and subsequent changes in perfusion resulting in constantly variable shear stress plays a key role in EC function that has not previously been described.</p> </div

HL-60 cell adhesion to flow-conditioned endothelial cells.

<p>Endothelial cell monolayers were grown to confluence and cultured under steady flow (A,C,E,G) or physiological flow (B,D,F,H) for 24 hours. During the last four hours, endothelial cells were activated with 1 U TNF-a to stimulate adhesion molecule expression. At hour 24, monolayers were removed from flow chambers and incubated for 10 minutes with a bolus of GFP+ HL-60 cells (1000 cells/mm²) and stained as described (A,B: DAPI; C,D: F-actin; E,F: GFP+ HL-60 cells; G,H: overlay). Shown are representative images (40x) from each condition. Scale bar: 20 microns. (I): HL-60 cell adhesion in 15 predetermined locations per monolayer was quantified. Results are displayed as mean±SEM (n = 5–6). Asterisk denotes significant difference in means between groups as determined by Student’s t-test.</p

eNOS function in flow-conditioned endothelial cells.

<p>(A): eNOS mRNA expression (presented as mean±S.E.M.) was upregulated under either perfusion condition, but no statistical difference between flow groups was observed. (B): After 0, 12, or 24 hours of conditioning, media was collected and samples analyzed using a fluorometric assay. Total NO byproduct accumulation was normalized by the mean cell count at the end of each period. Results are displayed as mean±SEM (n = 4–6). Asterisks denote significant differences in individual means between groups at each time point. Dagger (†) denotes a significant difference in mean with respect to t = 0 hours. Double dagger (‡) denotes significance with respect to t = 0 and t = 12 hours.</p

Inflammation-associated gene expression in flow-conditioned endothelial cells.

<p>Shown is fold mRNA expression (with respect to static-cultured endothelial cells) of cell adhesion molecules (A) and genes with roles in recruitment of inflammatory cells (B). Results are presented as mean±SEM. An embedded asterisk indicates a significant difference with respect to static controls; an asterisk over a bracket indicates a significant difference between flow groups. Abbreviations: <i>PSGL-1</i>: P-selectin glycoprotein ligand-1; <i>ICAM-1</i>: intercellular adhesion molecule-1; <i>VCAM-1</i>: vascular cell adhesion molecule-1; <i>PECAM-1</i>: platelet-endothelial cell adhesion molecule-1; <i>ALOX5</i>: arachidonate 5-lipoxygenase; <i>PLA2G4C</i>: cytosolic phospholipase A2 gamma; <i>ADAM17</i>: ADAM metallopeptidase domain 17; <i>MCP-1</i>: monocyte chemoattractant protein-1; <i>CX3CL1</i>: fractalkine.</p

Parallel plate culture system.

<p>Endothelial cell monolayers were grown to confluence on glass coverslips, then affixed to parallel plate flow chambers (A) using a vacuum pump. Peristaltic pumps (B) are used to impose pulsatile flow of culture media (C) over the endothelial monolayers. The rotational speed of the pumps is controlled by an external computer (D) via RS-232 linkage.</p

Cytoskeletal morphology of flow-conditioned endothelial cells.

<p>Endothelial cell monolayers were grown to confluence and cultured under static conditions (A), steady flow (B), or physiological flow (C) for 24 hours. Monolayers were subsequently fixed and co-stained with rhodamine phalloidin (middle row) and DAPI (top row) in order to visualize f-actin and cell nuclei, respectively. Shown are representative images (40x) from each condition. Applying shear stress resulted in cytoskeletal alignment of endothelial cells in the flow direction (horizontally left to right). Scale bar: 20 microns.</p

Cardio-protective gene expression in flow-conditioned endothelial cells.

<p>Shown is fold mRNA expression (with respect to static-cultured EC) of genes that promote or inhibit cardiovascular disease progression. Results are presented as mean±SEM. An embedded asterisk indicates a significant difference with respect to static controls; an asterisk over a bracket indicates a significant difference between flow groups. Abbreviations: <i>EDN1</i>: endothelin-1; <i>PTGIS</i>: prostacyclin synthase; <i>SOD1</i>: superoxide dismutase-1.</p