Micromachined PDMS elastic post arrays for studying vascular smooth muscle cells

This thesis describes the design, modeling, fabrication and characterization of a micromachined array of high density 3-dimensional microposts (100×100) made of flexible material (silicone elastomers) for use to measure quantitatively the cellular traction force generated by vascular smooth muscle cell (VSMC) with high sensitivity and accuracy. The performance of the microposts with base and without base has been carefully analyzed by finite element analysis (FEA) software Coventorware™. The micropost arrays were then fabricated with diameters ranged from 3 to 10 µm, with edge to edge spacing of 5 and 7 µm, and with a height to diameter aspect radio up to 13 using microfabrication techniques and replica molding. The mechanical properties of the Polydimethylsiloxane (PDMS) microposts with various geometries used in the cell culture experiment were determined experimentally including detailed measurements of Young's modulus (E) and the corresponding spring constant. We have found that microposts with different sizes and geometries have different Young's modulus and spring constant values, ranged between 0.534-1.38 MPa, and 0.44 pN/nm-11.72 nN/nm, respectively.;Vascular smooth muscle cells isolated from male Sprague-Dawley rats (250-350 g) were cultured on top of the micropost arrays and incubated for 2 days before an image acquisition experiment. The micropost array was then scanned by phase contrast microscope from top to bottom. The 'top' and 'bottom' images were obtained by focusing on either top or bottom of the micropost. The outlines of the micropost covered by VSMC were successfully recorded as optical images and extracted using image processing in Matlab. The direction and the amplitude of microposts deflections were determined by comparing center location of micropost in 'top' and 'bottom' images. Hence, the corresponding force generated by cell can be calculated from deviation of the central axis of the micropost (i.e. the top vs. bottom). The force distribution map of a single VSMC was generated using the PDMS micropost array. A minimum displacement of 200 nm can be detected from optical images captured by confocal microscope. For example, micropost with highest aspect ratio (diameter, and height of 3 µm, and 35 µm) can detect the smallest traction force of 38 pN/µm. The micropost arrays with different geometries were used to study VSMCs. We have also found that the traction force exerted by VSM cell increases as the stiffness of the micropost increases. It demonstrates that VSM cell tends to adjust its traction force to adapt to its physical environment.;VSMCs with integrin-linked kinase enzyme (ILKE), referred to as CK 4 cell, and without ILK module, referred as ILK cell, were also studied using PDMS micropost array. Both cells were able to deflect or pull the microposts towards their physical center. They were also studied in the presence of Angiotensin II (ANGII), a potent VSM contractile agent, and Cytochalasin-D (CYTO), an actin depolymerizing agent. The results demonstrate that the addition of ANGII protein leads to contraction of CK4 cell while the ILK cells fail to evoke the contractile response. The VSMC's contraction disappeared by 6-10 minutes after the ANGII treatment and the cell recovered its original morphology. CK4 cells showed significant relaxation within 15 minutes after adding CYTO to cell bath. Similarly, the ILK cells relaxed in the presence of ANGII and CYTO. No contraction behavior was found out from ILK cells during the experiment. It has also been found that both CK4 and ILK cells tend to exert larger traction force when they grow on stiffer micropost array. Moreover, the traction forces generated by CK4 cells are more than 80% larger than that generated by ILK cells when the same size of micropost arrays were used. This study indicates that the function of ILK molecule is involved to the VSMC contraction and the control its traction force.;Finally, high temporal resolution analysis of CK4 cells was performed on PDMS micropost array. The VSMCs demonstrated that traction sites in CK4 cells attached to individual microposts 'oscillate' when cells were in the resting states. Such behavior becomes noticeable by observing the movement of micropost's free end in real time. The analysis showed that the oscillation of VSMC has different constant periods. The oscillation was also observed when external stimulation of ANGII and CYTO were added. This analysis showed that the amplitude and the period of oscillation are changeable under the external stimulus. Further work needed to understand the mechanism and significance of the oscillating behavior.