Macromolecular crowding-based biofabrication utilizing unmodified extracellular matrix bioinks

The extracellular matrix (ECM) is the body's natural cell-scaffolding material, and its structure and content are often imitated for applications in tissue engineering and regenerative medicine to promote biocompatibility. One approach toward biomimicking natural ECMs is to utilize decellularized extracellular matrices (dECMs), which involve removing cellular components from native tissues to preserve natural components. Solubilizing dECMs to produce bioinks therefore holds high potential for 3D biofabrication and bioprinting of bioactive scaffolds and tissues. However, solubilized ECMs have low printability owing to their slow gelation times, which necessitates additional artificial modifications (e.g. crosslinking) to facilitate biofabrication applications. In this study, we demonstrate a method utilizing macromolecular crowding (MMC) to confer printability, via rapid gelation, to solubilized unmodified dECMs from a variety of tissue types - heart, muscle, liver, small intestine, and large intestine. We show cell spreading and contractility in cell-laden dECM gels fabricated through MMC, highlighting biocompatibility with our method. Finally, we demonstrate successful extrusion bioprinting of complex 3D structures using unmodified dECM solutions as bioinks, revealing the potential of our MMC-based fabrication method for layer-by-layer building of user-designed bioinks made from wide-ranging fully physiological tissues. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrix (dECM) bioinks are among the most promising materials for simulating native organ-specific extracellular matrices. However, standard methods for gelling solubilized dECMs are slow and result in poor mechanical and structural characteristics, reducing printability. dECM solutions are typically supplemented with additional crosslinkers for the formation of robust hydrogels. The crosslinkers may be toxic to cells, and they often need UV light for activation. Here, we present a method that allows wide-ranging dECMs to be easily patternable and 3D printable in their unmodified forms. We demonstrate cell spreading and contractility in cell-laden unmodified dECM gels created demonstrating cell viability and bioactivity. We also demonstrated successful extrusion bioprinting of complex 3D structures utilizing low concentration unmodified dECM bioinks and normal healthy lung fibroblasts.