Estimating Cell Concentration in Three-Dimensional Engineered Tissues Using High Frequency Quantitative Ultrasound

Abstract

Histology and biochemical assays are standard techniques for estimating cell concentration in engineered tissues. However, these techniques are destructive and cannot be used for longitudinal monitoring of engineered tissues during fabrication processes. The goal of this study was to develop high-frequency quantitative ultrasound techniques to nondestructively estimate cell concentration in three-dimensional (3-D) engineered tissue constructs. High-frequency ultrasound backscatter measurements were obtained from cell-embedded, 3-D agarose hydrogels. Two broadband single-element transducers (center frequencies of 30 and 38 MHz) were employed over the frequency range of 13 to 47 MHz. Agarose gels with cell concentrations ranging from 1×104 to 1×106 cells mL−1 were investigated. The integrated backscatter coefficient (IBC), a quantitative ultrasound spectral parameter, was calculated and used to estimate cell concentration. Accuracy and precision of this technique were analyzed by calculating the percent error and coefficient of variation of cell concentration estimates. The IBC increased linearly with increasing cell concentration. Axial and lateral dimensions of regions of interest that resulted in errors of less than 20% were determined. Images of cell concentration estimates were employed to visualize quantitatively regional differences in cell concentrations. This ultrasound technique provides the capability to rapidly quantify cell concentration within 3-D tissue constructs noninvasively and nondestructively.

Ultrasound patterning technologies for studying vascular morphogenesis in 3D

“…Ultrasound offers unique capabilities to interact with cells and/or extracellular matrix scaffolds through localized mechanical forces (Dalecki, 2004). Furthermore, ultrasound imaging provides noninvasive, non-destructive capabilities for visualizing 3D cell-based constructs Dalecki et al, 2016;Mercado et al, 2014Mercado et al, , 2015. Investigations in this report demonstrate the versatility of ultrasound technologies for studying cells in 3D environments.…”

“…1C. Cells are the predominant scatterers within the cell-embedded collagen hydrogels (Mercado et al, 2014). Thus, in B-scan images of samples in these investigations, regions of increased brightness correspond to regions of higher cell concentration.…”

“…Ultrasound imaging provides a depth of penetration unachievable with current light-microscopy imaging techniques, thereby enabling volumetric visualization of cell patterning throughout the hydrogel volume. Immediately after USWF exposure, backscatter (B-scan) images of cell-embedded collagen gels were generated using a 38-MHz transducer (Mercado et al, 2014(Mercado et al, , 2015. A schematic of the imaging protocol is depicted in Fig.…”

“…1C). Highfrequency imaging techniques have been described previously (Mercado et al, 2014(Mercado et al, , 2015. Briefly, a pulser-receiver (5073PR, Olympus, Waltham, MA) generated a broadband pulse to excite a 38-MHz, focused PVDF transducer (PI50-2, Olympus, Waltham, MA) at a pulse repetition frequency (PRF) of 1 kHz.…”

“…Ultrasound also offers unique tools to noninvasively and non-destructively image, and quantitatively characterize 3D tissue constructs during fabrication (Dalecki et al, 2016;Mercado et al, 2014Mercado et al, , 2015. Thus, we also employed high-frequency ultrasound imaging to visualize and develop quantitative metrics in order to characterize the initial spatial organization of acoustically patterned cells throughout the depth of hydrogel constructs.…”