Laboratory of Advanced Microfluidic Systems  
Elastomeric Microposts integrated into Microfluidics for Endothelial Mechanotransduction Analysis  

Mechanotransduction is known to represent the collective mechanisms converting mechanical factors (e.g. material rigidity and force) into intracellular signals to determine genetic and phenotypic cell behaviours. While microfluidic technology supports precise and independent control of the soluble microenvironment (e.g. nutrients, reagents and dissolved gases), the regulation of insoluble mechanical factors, especially the substrate rigidity, is yet to be achieved.

In this research we successfully integrated elastomeric micropost arrays, which acted as both rigidity regulators and sub-cellular force sensors, into the conventional soft lithography-based microfluidic devices with the arbitrarily definable cell attachment capability. Manufacturing procedures compatible with the soft lithography-based fabrication were developed to enable the micro-contact printing of patterned proteins and subsequent cell attachment in microchannels. We further validated the protein patterning by seeding and culturing cells in the devices. To demonstrate the general applicability of our methodology, the microdevices have been applied to study the flow-mediated endothelial mechanotransduction process and, specifically, the involvement of subcellular contractile force dynamics correlating to the cellular morphological realignment. We have discovered the active cellular modulation of cell morphology and intracellular traction force mediated by shear both as directional and transient mechanotransductive response (Fig. 1). Overall, this research provides a microfluidic platform with both defined soluble and insoluble microenvironment to analyze the endothelial mechanoresponse, and its applications can be extended to quantitatively investigate the mechanotransduction of other cell types in general.


Figure 1. Scanning electron microscopic image of a fixed vascular endothelial cell attaching on an micropost array. The cell realigned both its morphology and contractility to the shear direction mediated by constant liquid flow.


  Stretchable Microengineered Substrates for Characterization of Sub-Cellular Mechanical Properties  

Cell stiffness together with other cell functional responses are recognized for their sensitiveness to external stimuli including biochemical and mechanical signals. It has been shown that the stiffness over the cell body is heterogeneous, and the subcellular stiffness may correlate with other intracellular activities such as the cytoskeleton reorganization and the contractility dynamics.

We report a novel subcellular stiffness measurement strategy implemented by a cell stretching platform we recently developed. Our strategy involved a microfabricated array of the micropost structure integrated onto a stretchable silicone-based elastomeric membrane in order to apply global equibiaxial cell stretch. The simultaneous subcellular cell deformations under a stretching force profile reflected the mechanical properties of the adherent cells. Furthermore, we devised a computational scheme based on the finite element theory and the optimization techniques to convert the measurement results to the corresponding spatial stiffness of live-cells (Fig. 2). We quantified the correlations between cell area, contractile force and the cell stiffness, and we examined the significance of intracellular components such as actin filaments and myosin molecules for the whole-cell stiffness. Collectively, this work has provided a novel measurement strategy for the subcellular live-cell stiffness profile that will help elucidate the intracellular mechanical responses and the related pathogenesis and developmental processes.

Figure 2. Measurement of subcellular cell stiffness of human cells adhering on a stretcheable micropost array. Changes in the local cell body areas and traction forces (left) can be converted to cell stifness profiles with subcellular resolutions (right).
  Stretchable Microengineered Substrates for Cell Mechanotransduction Study  

External forces are increasingly recognized as major regulators of cell structure and function, yet the underlying mechanism by which cells sense force and transduce it into intracellular biochemical signals and behavioral responses (‘mechanotransduction’) is largely undetermined. We have developed a membrane stretching device that incorporates a biocompatible elastomeric micropost array to obtain real-time observation of single live cell behaviors in response to both changes in matrix rigidity and local stretching forces. Additionally, this platform can be used to investigate the dynamic responses of focal adhesions and cytoskeleton of single live cells, and to help extend the current understanding of the mechanisms driving force transmission between cell and extracellular matrix.

Using this device, we studied the subcellular dynamic responses of contractile force and adhesion remodeling of vascular smooth muscle cells to stretch (Fig. 3a and 3b). We have observed that the cells could acutely enhance their contraction to resist rapid cell deformation, but they could also allow slow adaptive inelastic cytoskeletal reorganization in response to sustained cell stretch (Fig. 3c). Our study may help elucidate the mechanotransduction system in smooth muscle cells, and thus contribute to our understanding of pressure-induced vascular disease processes. Continued study will further reveal the adaptation mechanism of cells under stress and its role in hypertension-related disease.


Figure 3. Vascular smooth cell seeded on elastomeric micropost array (a) before and (b) after equibiaxial stretch. (c) The cell contractility dynamics was reflected by the total micropost bending of the substrate at different time points after stretch.



Laboratory of Advanced Microfluidic Systems | Department of Mechanical and Biomedical Engineering | City University of Hong Kong
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