Development of Optical and Mechanical Techniques to Investigate Rheology and Adhesion in Biological and Biomimetic Systems

Mechanical interactions between cells and their environment have a profound impact on many cellular functions. In our body, the stiffnesses of different types of tissues differ by multiple orders of magnitude. While brain is very soft, other kinds of tissue such as muscle or cartilage is considerably stiffer. Individual cells can exert forces on their environment in order to sense the rigidity of their environment and in turn, adapt their behavior accordingly. By now, it is well established that many cellular processes such as adhesion, migration, and morphogenesis are influenced by the mechanical properties of the cellular environment. A common model system to investigate the mechanical interactions between cells and their surrounding tissue are soft, biomimetic polymer substrates, which are typically polymerized in the form of thin films with different stiffnesses. Experiments, in which cells are allowed to adhere on the surface of such soft substrates have not only revealed that several processes such as cell migration, adhesion, and differentiation are influenced by the rigidity of the substrate. Such substrates can also provide an in-depth view into the dynamics of the mechanical machinery of individual cells, in particular the forces adherent cells exert on their environment. For all of those studies, precise methods to measure the elastic properties of such thin polymer films are necessary. In the first experimental part of this work, a simple technique to measure the elastic modulus and the Poisson ratio of films with a thickness of a tenth of a millimeter is presented. Steel spheres with different radii were placed on such substrates, the indentation was measured with an inverted microscope and the interplay between the elastic properties and finite thickness effects was exploited to determine both elastic parameters in a single experiment. The technique was applied to four different materials, which are commonly used in such studies, namely polyacrylamide, porous polyacrylamide, poly-N-isopropylacrylamide, and polydimethylsiloxane. Furthermore, Monte-Carlo simulations were carried out to optimize the precision of future experiments. Another cellular process, which is inherently governed by mechanical interactions, is the internalization of extracellular objects into cells. As one central part of our innate immune system, macrophages can engulf objects such as bacteria or whole cells in a process called phagocytosis by wrapping their membrane around the target. The protrusion of the membrane is driven by the polymerization of cortical actin, which is a central part of the cytoskeleton of macrophages located directly below the membrane. Thus, for a theoretical understanding of the process, knowledge of the cortical rheology during phagocytosis is necessary. However, there is only very limited data of the changes of the cortical rheology during cup formation available. In the experiments described in the second experimental part of this work, a technique based on blinking holographic optical traps to measure the viscoelastic properties of macrophages during phagocytosis is presented. As a model system, antibody-coated polystyrene particles were used as phagocytic targets and attached to the macrophages. After cell-particle contact, temporally modulated optical forces were exerted on the particles to mechanically probe the cells. The technique was demonstrated to be capable of resolving the viscoelastic properties of the macrophages as well as the temporal evolution of the cell-particle contact radius during the early phagocytic binding phase. This phase takes roughly 1 to 2 minutes and it was established, that cortical remodeling does not occur in a systematic manner during this phase. The setup was extended with a 3-dimensional feedback system to enable constant optical forces before, during, and after phagocytic cup formation. Furthermore, fluorescence microscopy was used to visualize the cortical dynamics and to identify the internalization process. To quantify the cell-particle adhesion strength, which is presumably an important driver of the early phagocytic particle binding mechanism, a microfluidic device was designed and verified in the final experimental part of this work. To quantify the adhesion strength between hundreds of particles and cells simultaneously, the particles were sedimented on the cells and subsequently exposed to a shear flow. The binding and unbinding rates, the fraction of irreversible binding events, and the number of particles remaining after the exertion of a hydrodynamic force of 50 pN were quantified as measures for the adhesion strength. For the study, nominally identical polystyrene particles were purchased from eight different manufacturers and it is demonstrated that the adhesion strength of those particles differed drastically, depending on the zeta-potential of the particles. Additionally, some particles were incubated in salt and freshwater to model environmental exposure of microplastic particles. This exposure goes along with the formation of an eco-corona on the surface of the particles, which also increased the cell-particle adhesion strength. All three techniques developed in this work contribute to the understanding of the mechanical interplay between cells and their environment. In particular, the combination of all three techniques is a powerful tool set to unravel further details about the mechanics of phagocytosis in the near future. Interesting directions for future experiments to investigate the mechanics of phagocytosis are discussed in the final part of this work.