The Reconstitution of Eukaryotic Architecture and Motility via Microfluidic Technology

The investigation into how life evolved and cellular complexity developed is an ongoing and highly researched pursuit in science. The central goal of my thesis was to develop a bio-inspired synthetic cell model in order to address this important topic. Towards this end, polymer-stabilized water-in-oil droplets were used as cell-sized compartments for the assembly and testing of specific sets of bioactive components. Moreover, an automated, droplet-based microfluidic technology was implemented for high throughput production of these cell-like compartments and their subsequent manipulation. To resemble the eukaryotic cell architecture, I established a highly-tunable method for the on-demand creation of synthetic cell systems in the form of 3D-supported lipid bilayers, multicompartment systems or free-standing giant unilamellar vesicles (GUVs). To accomplish this, small unilamellar vesicles were encapsulated into polymer-stabilized water-in-oil droplets. By tuning the charge of the inner droplet interface, adsorption of lipids can either be inhibited, leading to multicompartment systems, or induced, leading to the formation of droplet-stabilized giant unilamellar vesicles (dsGUVs) or a combination of multicompartment systems and dsGUVs. Following assembly, the successful release of free-standing GUVs from the polymer shell and the oil phase into physiological conditions was demonstrated. This paves the way towards future applications in which synthetic cell interactions with a physiological environment are crucial. Another significant achievement in this thesis was the assembly of a cellular motility module by the reconstitution of actin cytoskeletal networks and adhesion membrane receptors within droplet-based synthetic cells. These disparate cytoskeletal and adhesive elements were united in the lipid membrane structure of dsGUVs. I showed that this module was able to recapitulate key mechanisms in cell migration – namely cytoskeletal pushing and contractile forces. To date, such synthetic cells containing both cytoskeletal and adhesion-associated functional modules capable of self-propulsion has never been demonstrated. Remarkably, this minimal but functional set of building blocks allowed for the generation of autonomous motion in synthetic cells, and led to the analysis of the mechanism behind this self-propulsion. The powerful microfluidic technology and synthetic cell-like compartments presented in this thesis have the potential for widespread and diverse employment in synthetic biology as they allow for the use of varied sets of both synthetic and natural building blocks. I believe that the scientific achievements presented in this thesis will be of great interest to researchers in fundamental biology, bioengineering and biochemistry