Aspiration-assisted Freeform Bioprinting of Mesenchymal Stem Cell Spheroids Within Alginate Microgels

Aspiration-assisted freeform bioprinting (AAfB) has emerged as a promising technique for precise placement of tissue spheroids in three-dimensional (3D) space enabling tissue fabrication. To achieve success in embedded bioprinting using AAfB, an ideal support bath should possess shear-thinning behavior and yield-stress to facilitate tight fusion and assembly of bioprinted spheroids forming tissues. Several studies have demonstrated support baths for embedded bioprinting in the past few years, yet a majority of these materials poses challenges due to their low biocompatibility, opaqueness, complex and prolonged preparation procedures, and limited spheroid fusion efficacy. In this study, to circumvent the aforementioned limitations, we present the feasibility of AAfB of human mesenchymal stem cell (hMSC) spheroids in alginate microgels as a support bath. Alginate microgels were first prepared with different particle sizes modulated by blending time and concentration, followed by determination of the optimal bioprinting conditions by the assessment of rheological properties, bioprintability, and spheroid fusion efficiency. The bioprinted and consequently self-assembled tissue structures made of hMSC spheroids were osteogenically induced for bone tissue formation. Alongside, we investigated the effects of peripheral blood monocyte-derived osteoclast incorporation into the hMSC spheroids in heterotypic bone tissue formation. We demonstrated that alginate microgels enabled unprecedented positional accuracy (∼5%), transparency for visualization, and improved fusion efficiency (∼97%) of bioprinted hMSC spheroids for bone fabrication. This study demonstrates the potential of using alginate microgels as a support bath for many different applications including but not limited to freeform bioprinting of spheroids, cell-laden hydrogels, and fugitive inks to form viable tissue constructs.