A Novel Cell Force Sensor for Quantification of Traction during Cell Spreading and Contact Guidance

Abstract

In this work, we present a ridged, microfabricated, force sensor that can be used to investigate mechanical interactions between cells exhibiting contact guidance and the underlying cell culture substrate, and a proof-of-function evaluation of the force sensor performance. The substrates contain arrays of vertical pillars between solid ridges that were microfabricated in silicon wafers using photolithography and deep reactive ion etching. The spring constant of the pillars was measured by atomic force microscopy. For time-lapse experiments, cells were seeded on the pillared substrates and cultured in an on-stage incubator on a microscope equipped with reflected differential interference contrast optics. Endothelial cells (ECs) and fibroblasts were observed during attachment, spreading, and migration. Custom image analysis software was developed to resolve cell borders, cell alignment to the pillars and migration, displacements of individual pillars, and to quantify cell traction forces. Contact guidance classification was based on cell alignment and movement angles with respect to microfabricated ridges, as well as cell elongation. In initial investigations made with the ridged cell force sensor, we have observed contact guidance in ECs but not in fibroblast cells. A difference in maximal amplitude of mechanical forces was observed between a contact-guided and non-contact-guided, but mobile, EC. However, further experiments are required to determine the statistical significance of this observation. By chance, we observed another feature of cell behavior, namely a reversion of cell force direction. The direction of forces measured under rounded fibroblast cells changed from outwards during early cell attachment to inwards during further observation of the spreading phase. The range of forces measured under fibroblasts (up to 138 nN) was greater than that measured in EC (up to 57 nN), showing that the rigid silicon sensor is capable of resolving a large range of forces, and hence detection of differences in traction forces between cell types. These observations indicate proof-of-function of the ridged cell force sensor to induce contact guidance, and that the pillared cell force sensor constructed in rigid silicon has the necessary sensitivity to detect differences in traction force vectors between different cell phenotypes and morphologies.

Engineered ridge and micropillar array detectors to quantify the directional migration of fibroblasts

“…Such comparisons are essential to compute tractions for pillars that undergo large deformations due to cell adhesions and have different aspect ratios which do not permit the use of standard bending models, with linear elastic assumptions, for non-slender beams. Contrary to earlier reports, 12 we demonstrate that topographical cues on the engineered mPAD substrates inuence broblasts morphologies and result in persistent cellular migrations in the direction of the ridges. Cell morphologies have oriented features similar to the anisotropic in vivo cellular milieu without constraining effects produced within channels.…”

“…33,34 Engineered substrate topography is a useful method to create desired cellular adhesions to substrates. 2,12,25 Reports on single-cell migration conne cell movements using either 2D microuidic channels or chemo attractants that are created using patterning of differential areas through protein modulators and inhibitors. 11,35,36 Few studies have however quantied the role of substrate anisotropies in cell migration which are essential in several physiological processes.…”

Focal adhesion kinase as a regulator of cell tension in the progression of cancer

“…However, FAK knockdowns in fibroblasts, epithelial, or endothelial cells, and fibroblasts or neurons derived from mice with a conditional knockout of FAK, exhibit an elongated or spindle-shaped morphology consistent with the inability of the cells to contract [65][66][67]62]. These new methods for inhibiting FAK expression along with recently-developed small molecules that inhibit FAK kinase activity [68,69], combined with methods to monitor the ability of the cells to contract [70,71], will shed new light on the role of FAK in regulating cell contraction and adhesion turnover.…”

Modeling Morphogenesisin silicoandin vitro: Towards Quantitative, Predictive, Cell-based Modeling

“…Although pillared substrates may yield more detailed measurements of cell mechanics, their major disadvantage is the fact that mammalian cells can respond to substrate structure and will thus affect cell behavior [37]. However, good use for pillared substrates can be found in characterizing mechanical cell-substrate interaction, including contact guidance [54].…”