Generation of Extracellular Matrix Protein-Based Microcapsules by Using Droplet-Based Microfluidics

Interactions of cells with the extracellular matrix (ECM) are involved in many cellular responses <i>in vivo</i>, and consequently activate multiple signaling pathways that initiate, drive and regulate nearly all motions of a cell. Therefore, cell-ECM interactions also underscore their central physiological roles, as well as their involvement in a wide variety of infectious diseases. Consequently, engineering 3D ECM niche systems for controlled observation and manipulation of bacteria <i>in vitro</i> has become an important strategy, particularly in medical applications. In this research, we have established a novel droplet-based microfluidic approach for the controlled assembly of ECM-based protein microcapsules loaded with <i>Escherichia coli (E.coli)</i>. Towards this end, water-in-oil emulsion droplets consisting of negatively charged block-copolymer surfactants are used as a template for the charge-mediated formation of a laminin- or laminin/collagen-based continuous layer on the inner droplet periphery. A double-inlet microfluidic flow-focusing device is implemented to encapsulate <i>E. coli</i>, proteins and divalent ions. The positively charged ions are attracted to the negatively charged block-copolymer surfactant layer at the inner periphery of the droplet wall. This in turn attracts the negatively charged proteins. After incubating the droplets at 37°C for at least 1h, the polymerized protein microcapsules are sequentially released into a physiological environment. Here, the droplets are covered with a layer of the release medium of choice and perfluoro-octanol is added dropwise from above. Perfluoro-octanol acts as a destablizing surfactant, which allows the fusion of the inside aqueous phase to the outer release medium, placed above the droplets. Incubating this solution for approximately 20min at room temperature releases the established protein microcapsules into the layer of release medium. Collecting the medium layer containing the microcapsules and seeding them on a glass coverslip allows for the analysis of bacterial behavior inside the capsules over longer time periods. We discovered two distinct behaviors of the same bacterial strain, depending on the type of protein used for generating the microcapsules. In the case of laminin-111 based microcapsules, bacteria colonies grow inside the microcapsules and simultaneously expand the microcapsule walls. The microcapsules can only withstand this growth up to a certain point, after that, the capsules break and release a great number of bacteria at once. This system can be taken to hand to e.g., conduct infection studies <i>in vivo</i>. By injecting the protein microcapsules at a point of interest, the encapsulated bacteria grow in confinement until the microcapsules burst and hence generate a local source of infection. In contrast to the outburst, E.coli incubated inside laminin-111/collagenIV-mixed microcapsules undergo filamentation. Here, the E.coli turn into long filamentous bacteria and start shedding small bacteria from the end of the filaments. This behavior is widely distributed in uropathogenic infections. This model can be used to conduct antibiotic studies on filamentous bacteria.<br/>These are just two examples to show the purpose of this system. With our new method it is possible to generate different kind of protein microcapsules and further the content is also not limited. Exchanging the aqueous phase allows a wide variety of proteins, organisms and/or molecules being encapsulated inside water-in-oil droplets. Using microfluidics also allows us to tune the size of the microcapsules, which further increases the applicability of this system.