Bacteria in a 3D Transparent Biphasic Solid Matrix

While most of our understanding of the basic principles in microbiology has been realised by studying bacteria on a 2D surface with homogenous material properties and uninterrupted nutrient availability, microbes in vivo experience a much more complex, three-dimensional disordered microenvironment where the local microstructure directly affects both their motility and nutrient transport via diffusion. Recent attempts have been made to study bacteria in 3D by embedding them inside a jammed packing of micron-sized hydrogel particles directly soaked in liquid cell growth media. Although these jammed microgel systems give interesting insights into their motility in 3D disordered media at the single cell level, the porous microstructure of these 3D media does not impede the transport of small molecules such as nutrients and oxygen. Hence, an unmet need remained to mimic certain microenvironments like adipose tissue, soil, and aquifers where microbial motility, nutrient diffusion, etc are affected. Although such porous media have been prepared by stacking glass beads for studying flow through porous media problems, they remained particularly unuseful for imaging living organisms; a higher refractive index of glass beads precludes optical imaging of microbial behaviour while index matching of the cell growth media to glass refractive index is detrimental to cell viability. Here, we have solved this long-standing challenge by introducing a jammed packing of oil-in-water emulsion droplets where the oil phase matches the refractive index of water allowing us to culture bacterium between the pores of the jammed emulsion system without changing any composition of the cell growth media. A similar refractive index helps us to directly probe cellular behaviour due to the transparent nature of the system. While the inner droplet pores are accessible for bacterial motility and nutrient mobility, we establish that these platforms can serve as a 3D cell growth media for growing bacteria. Our porous biomaterial has tuneable stiffness and allows 3D bioprinting as well as the flow of water or media through it, increasing its biological applicability.