Tailoring agarose fluid gels for use in suspension bath bioprinting and culture of spheroid-based bioinks

Suspension bath bioprinting, whereby bioinks are extruded into a yield stress bath with rapid recovery from shearing, has enabled the printing of low viscosity bioinks into constructs with high geometric complexity. Previous studies have often relied upon external stabilisation of the suspension bath (e.g. collagen) in order to culture soft materials without loss of printed structure. Here, we report a systematic investigation of suspension bath properties that support the printing, fusion, and culture of spheroid-based bioinks without added stabilisation. Specifically, agarose fluid gels of varied polymer concentrations and dilutions were produced and characterised morphologically and rheologically. Juvenile bovine chondrocytes or mesenchymal stromal cells (MSCs) were formed into spheroids of ∼150µm in diameter and investigated within agarose suspension baths either for their fusion in hanging drop cultures or as jammed bioinks. MSC spheroids were also printed when mixed with hydrogel microparticles to demonstrate additional versatility to the approach. Suspension baths of lower polymer concentrations and increased dilution enabled faster spheroid fusion; however, the most heavily diluted suspension bath was unable to maintain print fidelity. Other formulations supported the printing, fusion, and culture of spheroid-based inks, either as simple lines or more complex patterns. These findings help to inform the design of suspension baths for bioprinting and culture.