A novel method for assessing adherent single-cell stiffness in tension: design and testing of a substrate-based live cell functional imaging device

Abstract

Various micro-devices have been used to assess single cell mechanical properties. Here, we designed and implemented a novel, mechanically actuated, two dimensional cell culture system that enables a measure of cell stiffness based on quantitative functional imaging of cellsubstrate interaction. Based on parametric finite element design analysis, we fabricated a soft (5 kPa) polydimethylsiloxane (PDMS) cell substrate coated with collagen-I and fluorescent micro-beads, thus providing a favorable terrain for cell adhesion and for substrate deformation quantification, respectively. We employed a real-time tracking system that analyzes high magnification images of living cells under stretch, and compensates for gross substrate motions by dynamic adjustment of the microscope stage. Digital image correlation (DIC) was used to quantify substrate deformation beneath and surrounding the cell, leading to an estimate of cell stiffness based upon the ability of the cell to resist the applied substrate deformation. Sensitivity of the system was tested using chemical treatments to both "soften" and "stiffen" the cell cytoskeleton with either 0.5 μg/ml Cytochalasin-D or 3% Glutaraldehyde, respectively. Results indicate that untreated osteosarcoma cells (SAOS-2) exhibit a 1.5±0.7% difference in strain from an applied target substrate strain of 8%. Compared to untreated cells, those treated with Cyochalasin-D passively followed the substrate (0.5±0.5%, p<0.001), whereas Glutaraldehyde enhanced cellular stiffness and the ability to resist the substrate deformation (2.9 ± 1.6%, p < 0.001). Nanoindentation testing showed differences in cell stiffness based on culture treatment, consistent with DIC findings. Our results indicate that mechanics and image analysis approaches do hold promise as a method to quantitatively assess tensile cell constitutive properties.

Development of Polydimethylsiloxane Substrates with Tunable Elastic Modulus to Study Cell Mechanobiology in Muscle and Nerve

“…S1). Summarizing the results from ten different studies shows that the standard Sylgard 184 formulation with 1∶10 curing agent to base ratio has reported elastic moduli ranging from 1 to 2.5 MPa [32], [56], [57], [60]–[62], [66], [70], [71], though most are similar to our value of E ∼1.7 MPa. Reducing the curing agent ratio produces varying results that makes it difficult to choose the optimum formulation to achieve a PDMS with a specific elastic modulus.…”

“…Reducing the curing agent ratio produces varying results that makes it difficult to choose the optimum formulation to achieve a PDMS with a specific elastic modulus. For example, the ratio of 1∶50 has reported elastic moduli of 8, 12, 30 and 48 kPa, a 600% difference between the lowest and highest values [32], [57], [61], [62]. Similarly, to tune the elastic modulus to ∼10 kPa reveals conflicting reports on whether 1∶50, 1∶55 or 1∶67 is the appropriate curing agent to base ratio [32], [57], [58], [62].…”

Tendon injury and repair – A perspective on the basic mechanisms of tendon disease and future clinical therapy

“…5) [108][109][110]. A cell can rapidly respond to tension and shear by adjusting its physical coupling to its local matrix [111,112], or by remodeling its cytoskeleton [113]. Such adjustments affect not only the loading of mechanosensory elements of the cell, but also affect sensory proteins within the cell membrane and nucleus that are mechanically coupled [114].…”

“…shear stresses from fluid flow and fascicle sliding, tensile stresses from direct elongation of collagen structures and hydrostatic stresses from the volumetric changes with external loading [116,138,139]. These mechanical stresses are responsible for the activation of candidate ''vectors" by which tendon cells can potentially ''transduce" mechanical forces within the tissue to regulate cell signaling and behavior: 1) stretch activated ion channels (SACs) as mechanosensitive ion channels, 2) focal adhesion-mediated mechanical signal transduction, 3) the primary cilium and 4) nuclear deformations [109][110][111][112][113]116]. Fig.…”

Non-invasive single-cell biomechanical analysis using live-imaging datasets

“…Biomechanical measurements using whole-cell deformation approaches, such as micropipette aspiration (Key and Robinson, 2013), optical tweezers (Ekpenyong et al, 2012), substrate stretching (Bartalena et al, 2011) or the microplate stretcher (Hoffman and Crocker, 2009), yield global easy-tointerpret single-cell measurements of biomechanical stiffness; however, such methods require detaching otherwise adherent cells or placing cells on a less biologically relevant substrate. Furthermore, techniques such as atomic force microscopy (AFM) (Haase and Pelling, 2015) and magnetic twisting cytometry (Massiera et al, 2007) utilize nano-sized physical probes to acquire biomechanical readings at subcellular regions of interest, acquiring information of superior spatial resolution; at this scale, however, intracellular non-uniformity causes considerable variability in measured biomechanical responses, exacerbating the already present heterogeneity in many cell populations.…”