Measurement of cell forces using a microfabricated polymer cantilever sensor

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Abstract

A polymer MEMS sensor was developed for measuring the mechanical forces generated by single adherent cells. Mechanical forces are known to play a role in cell regulation, and measuring these forces is an important step in understanding cellular mechanotransduction. The sensor consisted of four polystyrene microcantilever beams with cell adhesion pads at the end of each beam. Finite element analysis was used to guide the design of a compound cantilever to allow measurement of forces in any direction in the plane of the sensor. The device was used to measure the forces generated by WS1 human skin fibroblasts under a microscope. Single cells were placed on the sensor using a custom micromanipulator. Forces were calculated by optically measuring the deflection of each probe during cell attachment and spreading. Measurements were performed on normal WS1 fibroblast cells and those treated with cytochalasin D to disrupt the actin cytoskeleton. Cytochalasin D treated cells showed a significant decrease in force, with time information about the rate of force change obtained from the sensor. This device can be used to evaluate the mechanical response of cells to a variety of chemical, mechanical, and other environmental stimuli.

Introduction

When adherent cells are attached to the extracellular matrix (in vivo) or a tissue culture substrate (in vitro), stresses are generated within the cytoskeleton. Molecular linkages between the cytoskeleton and the surface transmit these forces to the extracellular space [1], [2]. Previous research has shown that the forces are critical in regulating a host of cellular processes [3], [4], [5]. Cytoskeletal components (primarily the actin network in mesenchymal cells) are responsible for regulating cellular shape and spreading. Integrin-rich focal adhesions provide a molecular linkage between the intracellular and extracellular spaces that is imperative for establishing bi-directional signal transduction pathways [4], [6]. Adhesion forces at the cell membrane–substrate interface are also responsible for remodeling the extracellular matrix. Transient changes in the adhesion forces and the intracellular stress field are the primary mechanism of cellular motility [7], [8], [9]. In addition to normal cell function, abnormalities in the cytoskeleton have been implicated in a wide range of pathologies [10] and in would healing [11], [12]. Several classes of chemicals, including the cytochalasins, have been shown to disrupt actin network formation as well as the functions of other cytoskeletal components [13], [14], [15], [16]. A better understanding of the cytoskeletal mechanics, with respect to both regulation and disruption, could lead to improved diagnostic and therapeutic approaches to a number of diseases.

Some of the seminal work studying forces at the cell-surface interface was done by Harris et al. [17], who grew cells on cross-linked silicone rubber sheets and observed the wrinkling of the polymer film. The major limitation of this method was the difficulty in quantifying the forces due to an inability to directly calculate a force vector from the wrinkle geometry. Since this initial work, improved methods have been developed that incorporate fluorescent microbeads into polyacrylamide gels [18]. The displacement of the beads and the stiffness of the gel were used to calculate forces exerted by the cell. This method is commonly referred to as traction force microscopy [19]. While calculation of the forces is still not straightforward, this method does allow quantification of spatially resolved forces applied to the substrate.

Some interesting work has been done looking at the effects of substrate mechanical properties on cell behavior. Pelham and Wang [20] used polyacrylamide gels with a range of stiffness to show that the formation of focal adhesions and motility of epithelial (normal rat kidney) and fibroblast (3T3) cells is dependent on the stiffness of the substrate. Lo et al. [21] took this work a step further and showed that 3T3 cells preferentially migrate from the softer regions to the stiffer regions of the substrate. These experiments demonstrate several important issues. First, the cells sense the mechanical properties of their surroundings and respond accordingly. Second, this work demonstrates a link between substrate properties and cytoskeletal mechanics given that the forces exerted on the soft materials were less than those on the stiffer material.

Several BioMEMS devices, both silicon and polymer, have been developed for measuring cell mechanics. Galbraith and Sheetz developed a silicon-based cantilever device for measuring fibroblast cell forces [22]. Using this device, they were able to measure traction forces exerted by fibroblasts at various locations on the cell (i.e. lead edge, nucleus, and trailing edge) as they moved over the cantilever to determine the spatial distribution of cell forces. They were also able to measure the time-varying force. One disadvantage of this device was that deflection of the beam could only be measured in the direction orthogonal to the cantilever direction. The force had to be calculated by making the assumption that the measured force was oriented in the direction of the long axis of the cell (i.e. the direction of movement). More recently, microfabricated polymer structures have been developed for measuring cell forces. Tan et al. [23] developed an array of PDMS micropillars that act as vertical cantilevers. They were able to measure the time dependence of the force, and by micropatterning adhesion molecules on the surface of the pillars, they showed that the force per pillar is proportional to the amount of cell spreading.

Here we describe a polystyrene cantilever sensor for measuring forces generated by fibroblast cells. A cantilever array sensor was developed using finite element analysis to guide the design of a compound cantilever beam capable of measuring forces in two dimensions. The device was fabricated from polystyrene and treated with oxygen plasma to provide a culture surface identical to cell culture plates, that is well-studied and does not require coating with cell adhesion proteins. Time-dependent and spatially resolved forces were measured with normal WS1 fibroblasts. Cells were treated with cytochalasin D and demonstrated that the sensor could detect changes in cell force generation based on disruption of cytoskeletal actin.

Section snippets

Design and finite element simulation

The force sensor consists of four compound cantilever beams that converge in the center of the device as shown in Fig. 1. Each cantilever has a pad at the end to allow cell attachment and spreading. A stationary adhesion pad at the center of the device provides a location for initial cell attachment and a reference point for image analysis. The shaded black areas in Fig. 1(a) indicate regions that are fixed to the substrate, and gray areas of the figure are suspended above the substrate. The

Results and discussion

A map of the cantilever deflection for forces of 50, 100, and 200 nN is shown in Fig. 2(a). From the plots, it can be seen that a force in a given direction, along the 0° axis for example, does not lead to a deflection exactly in that specific direction. However, the difference between the applied force and resultant deflection is minor (∼3°) and a relatively constant offset, and the monotonic relationship between them allows for easy back-calculation of the applied force from the measured

Conclusion

A polystyrene cantilever sensor was developed for measuring forces generated by single fibroblasts. Finite element analysis aided in the iterative design of the compound cantilever beam and was used to simulate the behavior of the final beam design. WS1 skin fibroblasts were used as a model cell and forces generated by these cells were measured during cell attachment and spreading. The device was able to detect changes in force generation caused by disruption of the cytoskeleton using the actin

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