Controlling the Spatial Organization of Cells and Extracellular Matrix Proteins in Engineered Tissues Using Ultrasound Standing Wave Fields

Abstract

Tissue engineering holds great potential for saving the lives of thousands of organ transplant patients who die each year while waiting for donor organs. However, to successfully fabricate tissues and organs in vitro, methodologies that recreate appropriate extracellular microenvironments to promote tissue regeneration are needed. In this study, we have developed an application of ultrasound standing wave field (USWF) technology to the field of tissue engineering. Acoustic radiation forces associated with USWF were used to non-invasively control the spatial distribution of mammalian cells and cell-bound extracellular matrix proteins within three-dimensional collagen-based engineered tissues. Cells were suspended in unpolymerized collagen solutions and were exposed to a continuous wave USWF, generated using a 1 MHz source, for 15 min at room temperature. Collagen polymerization occurred during USWF exposure resulting in the formation of three-dimensional collagen gels with distinct bands of aggregated cells. The density of cell bands was dependent on both the initial cell concentration and the pressure amplitude of the USWF. Importantly, USWF exposure did not decrease cell viability, but rather enhanced cell function. Alignment of cells into loosely clustered, planar cell bands significantly increased levels of cell-mediated collagen gel contraction and collagen fiber reorganization as compared to sham-exposed samples with a homogeneous cell distribution. Additionally, the extracellular matrix protein, fibronectin, was localized to cell banded areas by binding the protein to the cell surface prior to USWF exposure. By controlling cell and extracellular organization, this application of USWF technology is a promising approach for engineering tissues in vitro.

Ultrasound assisted particle and cell manipulation on-chip

“…Further, for certain cell types there is evidence that the microenvironment provided by ultrasonic excitation can modify and enhance the production of the cells' extra cellular matrix [85,86]. A detailed study of the response of chondrocytes to culture within polymer scaffolds under ultrasonic excitation [87] observed positive changes in viability, proliferation, and gene expression, and modified expression of a large number of proteins, in comparison with control samples in the absence of ultrasonic excitation.…”

“…Garvin et al have used ultrasonic fields to pattern cells within collagen hydrogels [86] and suggest that the ultrasound-induced cell patterning itself has a significant influence on the structure of the extracellular matrix. They later used this technique to organise endothelial cells so as to form a vascular-like bed within the matrix [93].…”

Spatial patterning of endothelial cells and vascular network formation using ultrasound standing wave fields

“…The experimental setup used for ultrasound standing wave field exposures has been described previously (Garvin et al, 2010;Garvin et al, 2011). A plastic exposure tank was filled with degassed, deionized water.…”

“…A waveform generator (Hewlett Packard, Model 33120A, Palo Alto, CA), an attenuator (Kay Elemetrics, model 837, Lincoln Park, NJ), and a radio-frequency power amplifier (ENI, Model 2100L, Rochester, NY) were used to generate the ultrasound signal driving the transducer. Samples were contained within acoustically transparent, silicone elastomer-bottomed cell culture plates (FlexCell International Corporation, BioFlex V R plates, Hillsborough, NC) that had been modified to reduce the well diameter to 1 cm using elastomer molds (Sylgard V R 184 silicone elastomer; Dow Corning Corporation, Midland, MI) (Garvin et al, 2010). Previous work (Garvin et al, 2010) characterized the attenuation of the sample holder and measured the standing wave fields generated within the sample holder volume.…”

“…The diameter of endothelial tubes was shown to increase linearly with increasing microchannel diameter (Raghavan et al, 2010), suggesting that spatial cues can also be utilized to shape vessel morphology. In previous studies, we demonstrated that acoustic radiation forces associated with ultrasound standing wave fields can rapidly and non-invasively organize cells spatially into distinct multicellular planar bands within three-dimensional (3D) collagen gels (Garvin et al, 2010). Ultrasound standing wave field-induced alignment of endothelial cells led to the formation of lumen containing, branching vessel networks throughout the complete volume of the collagen construct (Garvin et al, 2011).…”