No abstract

To precisely investigate the mechanobiological responses of valvular endothelial cells, we developed a microfluidic flow profile generator using a pneumatically-actuated micropump consisting of microvalves of various sizes. By controlling the closing pressures and the actuation times of these microvalves, we modulated the magnitude and frequency of the shear stress to mimic mitral and aortic inflow profiles with frequencies in the range of 0.8-2 Hz and shear stresses up to 20 dyn cm-2. To demonstrate this flow profile generator, aortic inflow with an average of 5.9 dyn cm-2 shear stress at a frequency of 1.2 Hz with a Reynolds number of 2.75, a Womersley number of 0.27, and an oscillatory shear index (OSI) value of 0.2 was applied to porcine aortic valvular endothelial cells (PAVECs) for mechanobiological studies. The cell alignment, cell elongation, and alpha-smooth muscle actin (αSMA) expression of PAVECs under perfusion, steady flow, and aortic inflow conditions were analyzed to determine their shear-induced cell migration and trans-differentiation. In this morphological and immunocytochemical study, we found that the PAVECs elongated and aligned themselves perpendicular to the directions of the steady flow and the aortic inflow. In contrast, under perfusion with a fluidic shear stress of 0.47 dyn cm-2, the PAVECs elongated and aligned themselves parallel to the direction of flow. The PAVECs exposed to the aortic inflow upregulated their αSMA-protein expression to a greater degree than those exposed to perfusion and steady flow. By comparing these results to those of previous studies of pulsatile flow, we also found that the ratio of positive to negative shear stress plays an important role in determining PAVECs' trans-differentiation and adaptation to flow. This microfluidic cardiac flow profile generator will enable future valvular mechanobiological studies to determine the roles of magnitude and frequency of shear stresses.

A microfluidic diffusion diluter to create stable chemical gradients across an array of cell cultures was demonstrated. The device enabled concentration based studies to be conducted at 256 different concentrations across individual, low shear cell cultures. A gradient of staurosporine on cells stained with Mitotracker Deep Red (MTDR) showed a concentration-based effect on cell apoptosis across the cell culture array.

A microfluidic diffusion diluter was used to create a stable concentration gradient for dose response studies. The microfluidic diffusion diluter used in this study consisted of 128 culture chambers on each side of the main fluidic channel. A calibration method was used to find unknown concentrations with 12% error. Flow rate dependent studies showed that changing the flow rates generated different gradient patterns. Mathematical simulations using COMSOL Multi-physics were performed to validate the experimental data. The experimental data obtained for the flow rate studies agreed with the simulation results. Cells could be loaded into culture chambers using vacuum actuation and cultured for long times under low shear stress. Decreasing the size of the culture chambers resulted in faster gradient formation (20 min). Mass transport into the side channels of the microfluidic diffusion diluter used in this study is an important factor in creating the gradient using diffusional mixing as a function of the distance. To demonstrate the device's utility, an H2O2 gradient was generated while culturing Ramos cells. Cell viability was assayed in the 256 culture chambers, each at a discrete H2O2 concentration. As expected, the cell viability for the high concentration side channels increased (by injecting H2O2) whereas the cell viability in the low concentration side channels decreased along the chip due to diffusional mixing as a function of distance. COMSOL simulations were used to identify the effective concentration of H2O2 for cell viability in each side chamber at 45 min. The gradient effects were confirmed using traditional H2O2 culture experiments. Viability of cells in the microfluidic device under gradient conditions showed a linear relationship with the viability of the traditional culture experiment. Development of the microfluidic device used in this study could be used to study hundreds of concentrations of a compound in a single experiment.