Controlling the physical microenvironment of cells with microfluidics for studying mechanically regulated cellular behaviors
Mechanical cues are one of the most important biophysical regulators of cellular machinery under both physiological and pathophysiological conditions. Advancements in the microtechnology in the last two decades allowed researchers to develop automated, high-throughput, and multifunctional experimental tools based on microfluidic techniques in order to facilitate conventional mechanobiological experiments and enable novel experimental paradigms that would not be possible otherwise. In this work, we developed various microfluidic devices and techniques that can be used to generate different mechanical microenvironments for the understanding of the role of mechanical cues at the cellular level.
Suspended mammalian cells such as lymphocytes constantly migrate within the tissues following the complex patterns of chemokine gradients in order to carry out diverse immune system functions. The in vitro analysis of the immune cell migration under precisely controlled microenvironments have a potential to enable in-depth understanding of the mechanism that regulate immune cell motility behaviors. However, in order to unveil the cell migration through cellular locomotion suspended immune cells should be isolated from external mechanical forces such as shear stress dependent drag forces. Therefore, we generated a microfluidic flow-free gradient generator system and used this approach with Jurkat cells to analyze their motion patterns within various CXCL12 gradients and extracellular matrix configuration. Using this system, we found that the strength of the chemotactic response of Jurkat cells to CXCL12 gradient was reduced by increasing surface fibronectin in a dose-dependent manner. Moreover, we the observed that the chemotaxis of Jurkat cells was governed not only by the CXCL12 gradient but also by the average CXCL12 concentration. Moreover, we developed a framework where the distinct migratory behaviors in response to chemokine gradients in different contexts might be physiologically relevant for shaping the host immune response and may serve to optimize the targeting and accumulation of immune cells to the inflammation site. Lastly, we used this system with primary murine CD8+ T cells obtained from either healthy mice or B16 mouse tumor model in order investigate effect of tumor microenvironment in T cell motility.
Another example of the biological phenomena where mechanics plays an important role is development. Drosophila melanogaster (fruit fly) is a well-established model organism which has been used in developmental biology studies for over a century. Although most of the Drosophila research largely focused on biochemistry and genetics as the regulatory mechanisms for development, recent studies showed that both internally and externally generated mechanical signals also play an important role during development. In this regard, we developed a novel microfluidic system for automatically aligning and loading hundreds of Drosophila embryos into microchannels where they can be simultaneously compressed to desired levels using pneumatically actuated deformable sidewalls. Using this microsystem, we demonstrated the effect of different levels of acute and chronic compression on the developmental progression and viability of the Drosophila embryos. Furthermore, we quantitatively characterized dose- and time-dependent induction of the ectopic expression of Twist —a crucial transcription factor that governs gastrulation process— upon mechanical compression.
One of most mechanically sensitive cell type in mammalian body is the endothelial cells —monolayer of which constitutes the inner wall of the vasculature— as they are constantly subjected to mechanical cues in the form of blood shear stress. We further enhanced the scope of this work by developing a novel microfluidic system that can generate various physiologically relevant shear stress modalities such as different levels of shear stress and shear stress gradients for the investigation of endothelial cell polarity and orientation in response to flow which is considered to be a marker for endothelial dysfunction. In this microfluidic device, human umbilical vein endothelial cells (HUVECs) exhibited a rapid and robust response to shear stress, with the relative positioning of the Golgi and nucleus transitioning from non-polarized to polarized in a shear stress magnitude- and gradient-independent manner. By contrast, polarized HUVECs oriented their Golgi and nucleus polarity to the flow vector in a shear stress magnitude-dependent manner, with positive shear stress gradients inhibiting and negative shear stress gradients promoting this upstream orientation.
Lastly, we developed a new microfabrication technique called Polycarbonate Heat Molding (PCH molding) that can facilitate the fabrication of microfluidic devices. We tested this technique with master molds fabricated through photolithography, mechanical micromilling as well as 3D printing. Using this technique, we were able to successfully copy microstructures with submicron feature sizes and high aspect ratios. We characterized the copying fidelity of this technique and tested mechanically active microfluidic devices fabricated via PCH molding. We also used this approach to combine different master molds with up to 19 unique geometries into a single monolithic copy mold in a single step displaying the effectiveness of the copying technique over a large footprint area to scale up the microfabrication. This novel microfabrication technique can be performed outside the cleanroom without using any sophisticated equipment, suggesting a simple way for high-throughput rigid monolithic mold fabrication that can be used in mechanobiological studies.
- Doctor of Philosophy (PhD)