PhD Defense Agnese Ravetto, MSc.
- 07 April
- 16:00 - 18:00
Monocytes play a key role in the development of inflammatory diseases, such as atherosclerosis. During this pathological process they undergo several modifications of their cytoskeletal and membrane structure, which result in changes in cell shape and cellular mechanical and/or adhesive properties. The alteration in cell mechanical properties can significantly influence vascular flow and might lead to vascular complications. Given this shift in cell structure and mechanical properties, we hypothesized that mechanical screening of circulating cells may become an important additional approach for detecting inflammatory diseases. The objective of this research was to develop and evaluate microfluidic devices to investigate and interrogate monocytic cells with respect to their mechanical properties as carriers of biomarkers suitable for discriminating patients with an increased risk of atherosclerosis.
After reviewing the existing microfluidic devices, we characterized the mechanical properties of monocytic cells, both in the healthy and in the activated state, by using a range of experimental setups and devices. We investigated the response to deformation and the effect of cystoskeletal changes on cell mechanical behavior. First, we developed a system for compressing cells by deflection of a flexible PDMS membrane. By measuring the viscoelastic response of cells, we were able to distinguish between circulating and adherent cells, being monocytic and fibroblastic cells. In order to characterize the full elastic behavior of quiescent and activated monocytic cells, we used a technique named Capillary Micromechanics. This technique is based on cell deformation within a tapered glass capillary by the application of external fluid pressure. The changes in the mechanics of monocytic cells upon cell activation were studied for the entire physiologically relevant range of deformations, allowing us to obtain both the compressive and the shear modulus of a cell from a single experiment. From both studies, it appeared that the mechanical properties of monocytic cells are mainly driven by actin content and organization. Furthermore, it was shown that the mechanical behavior of the monocytes strongly depends on the activation of the cells. The cytoskeletal modifications due to activation cause an increase in the compressive modulus, but a decrease in the shear modulus. This effect was especially pronounced at high strains, such as those occurring during diapedesis through the vascular wall.
Taken together, these discoveries suggest that mechanical characterization can indeed reflect the activation state of monocytic cells. Therefore, as a next step, we developed a microfluidic device to subject cells to deformation in a constriction to differentiate between cells on the basis of changes in cell structure and mechanical properties upon actin disrupting and pro-inflammatory stimuli. It was proven that the device is sufficiently sensitive to detect differences in cell stiffness as evidenced by observed changes in entry time of the cells in the constriction and in velocity of the cells in the channel.
A major benefit of using the microfluidic technology is that system designs can be easily adapted to improve sensitivity and to adapt the setup to multiple applications. Thus, the principle of the constriction channel was used as a basis for a novel microfluidic device that allows chemical and mechanical manipulation of single cells within the same device. The effect on cell mechanical properties of an actin-disrupting drug and a clinical anti-inflammatory drug were tested. The chemicals were diffused through a custom-made porous membrane while cell mechanical properties were characterized before and after stimulation in a series of constrictions. The evaluation of this system demonstrated its potential for investigating changes in cell mechanical properties upon drug stimulation within a single microfluidic device. The live imaging of cells and the controlled delivery of chemicals within the device provide powerful options to test cellular mechano-response to various drug concentrations in real time and in physiologically relevant microenviroments.
In conclusion, we demonstrated that microfluidics is an appropriate technology to study and characterize monocytic cells to obtain mechanical markers for diseases, such as atherosclerosis. In future work, our microfluidic concepts can be applied to further investigate the mechanobiology of circulating cells in health and disease and to test the effect on mechanical properties of drugs for treating circulating cells, such as in inflammatory diseases like atherosclerosis.