A Device to Study the Effects of Stretch Gradients on Cell Behavior

Abstract

Mechanical forces are key regulators of cell function with varying loads capable of modulating behaviors such as alignment, migration, phenotype modulation, and others. Historically, cell-stretching experiments have employed mechanically simple environments (e.g., uniform uniaxial or equibiaxial stretches). However, stretch distributions in vivo can be highly non-uniform, particularly in cases of disease or subsequent to interventional treatments. Herein, we present a cell-stretching device capable of subjecting cells to controllable gradients in biaxial stretch via radial deformation of circular elastomeric membranes. By including either a defect or a rigid fixation at the center of the membrane, various gradients are generated. Capabilities of the device were quantified by tracking marked positions of the membrane while applying various loads, and experimental feasibility was assessed by conducting preliminary experiments with 3T3 fibroblasts and 10T1/2 cells subjected to 24 h of cyclic stretch. Quantitative real-time PCR was used to measure changes in mRNA expression of a profile of genes representing the major smooth muscle phenotypes. Genes associated with the contractile state were both upregulated (e.g., calponin) and downregulated (e.g., α-2-actin), and genes associated with the synthetic state were likewise both upregulated (e.g., SKI-like oncogene) and downregulated (e.g., collagen III). In addition, cells aligned with an orientation perpendicular to the maximal stretch direction. We have developed an in vitro cell culture device that can produce non-uniform stretch environments similar to in vivo mechanics. Cells stretched with this device showed alignment and altered mRNA expression indicative of phenotype modulation. Understanding these processes as they relate to in vivo pathologies could enable a more accurately targeted treatment to heal or inhibit disease, either through implantable device design or pharmaceutical approaches.

Device-Based In Vitro Techniques for Mechanical Stimulation of Vascular Cells: A Review

“…To produce equiaxial strains, a circular clamped membrane is stretched by use of an indenter [90,93,114,[116][117][118] or vacuum [91,119]. Systems may be modified to provide additional device features, such as live-cell imaging, protein analysis, and higher throughput [98,[114][115][116][117]119]. Also of note are commercially available systems that use a vacuum to stretch the membrane (Flexcell International, Hillsborough, NC), which can be used for equiaxial strain studies [120][121][122].…”

“…Recently, Lau et al implemented a system using the Flexcell equiaxial device that was computer-controlled to generate arbitrary stretch waveforms based on physiologically relevant data [123]. As with uniaxial systems, equiaxial systems can be modified to intentionally produce a less homogeneous strain, if desired [91,117]. For example, Richardson et al developed an equiaxial stretching method in which a circular defect or fixation was introduced into the center of the membrane as a means of establishing strain gradients to facilitate study of heterogeneous stretch, which more closely replicates in vivo conditions than a homogeneous strain field [117].…”

“…A variety of techniques (Northern blot, immunofluorescence assays, etc.) have been used to investigate biochemical responses, such as the expression of important signaling proteins [35,91,117,130]. Magnetic cytometry has also been employed to determine cell rheological properties [97].…”

Altered phenotypic gene expression of 10T1/2 mesenchymal cells in nonuniformly stretched PEGDA hydrogels

“…The cell-stretching device employed here is based on circular membrane deformation analysis and has been previously described in detail (11,38). Briefly, a computer-controlled stepper motor drives a rack-and-pinion mechanism that radially deforms circular constructs by vertically displacing the clamped outer circumference past a stationary circular platen (Teflon).…”

“…To investigate SMC behavior in nonuniform stretch environments representative of in vivo conditions, we have previously designed an experimental stretching device capable of subjecting cells in two (2D)-and three (3D)-dimensional cultures to gradients in biaxial stretch (38). A proper investigation of SMC phenotype change in response to mechanical stimuli should come as close as possible to the fully 3D in vivo case.…”