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

Abstract

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 requires a more rapidly gelling biopolymer to be added or an acellular collagen structure to already be defined before cells are delivered. Here, we report the flow synthesis of macroporous collagen microgels (MCMs) that serve as building blocks for granular bioinks. Viscous collagen solutions with concentrations as high as 10 mg ml-1 are consistently converted into droplets using a parallized microfluidic device via air bubble induced droplet breakup within a continuous oil phase. MCMs are obtained via gelation, oil removal, washing, and incorporation of size-tunable internal pores via ice templating while freezing at rates between 0.1 and 10 °C min-1. Independent control over the MCM diameter (175-250 µm) and porosity (58-76%) allows tailoring the extracellular matrix structure for different tissue engineering applications, making MCM internal walls similarly compact as collagen fibers in native tissues. Obtained granular bioinks allow for the delivery of cells, offer excellent printability and provide an avenue to faithfully recapitulate the multiscale collagen organization of native tissues. The presented approach may in the future enable extrusion 3D bioprinting of intricate cellular structures.