Scalable preparation of macroporous collagen microgels by air bubble-induced breakup and ice templating

Collagen I, the most abundant protein of the extracellular matrix, has found widespread use in three-dimensional cell culture, and increasingly also in bioprinting and biofabrication applications. However, several limitations remain, such as the capacity to locally recapitulate the multiscale organization of collagen in native tissues. Bioprinting cellular collagen structures with high feature fidelity so far either requires a more rapidly gelling biopolymer to be added or an acellular collagen structure to be defined before the delivery of cells. Here, we report the flow synthesis of macroporous collagen microgels (MCMs) that serve as building blocks for granular bioinks. Obtained bioinks offer excellent printability, provide an avenue to faithfully recapitulate the multiscale collagen organization of native tissues, and overcome the aforementioned limitations. Viscous collagen solutions with concentrations as high as 10 mg ml-1 are consistently converted into droplets using a parallelized microfluidic device via air bubble induced droplet breakup into a continuous oil phase. MCMs are obtained by inducing gelation, oil removal, and washing, and incorporating internal pores of tunable size via ice templating at freezing rates between 0.1 and 10 degrees C min-1. Independent control over the MCM diameter (175-250 mu m) and porosity (58-76%) allows the extracellular matrix structure to be tailored to different tissue engineering applications. The wall structures within MCMs share similarities with the highly compacted and recapitulated collagen porosity in native tissues. This approach in the future can be used to 3D print more complicated biomimetic structures that require cell positioning during printing. Collagen I, the most abundant protein of the extracellular matrix, has found widespread use in three-dimensional cell culture, and increasingly also in bioprinting and biofabrication applications.