(73c) Magnetized 3D Bioprinting to Fabricate Neural Assembloids

Neural organoids are a promising new platform to study development, evolution, and disorders of the human nervous system. To model circuit formation and cell-cell interactions in the nervous system, organoids can be integrated to form assembloids. However, manual positioning of organoids for assembloid generation within a liquid has limited reproducibility and scalability, and conventional 3D bioprinting technologies for single-cell suspensions (including inkjet, microextrusion, or laser-assisted printing) are not well suited for the millimeter-scale size of organoids. While aspiration-based 3D bioprinting has recently been used to precisely arrange osteogenic and cardiac spheroids, this technique requires relatively small (d ~ 150-600 μm) cell clusters that exhibit a high surface tension (~ 125 mN/m). In contrast, we observed that this approach deforms human neural organoids due to their large diameters (~ 1000-2000 μm) and weak surface tensions (~ 75 mN/m), disrupting their internal cellular organization. To address these critical challenges, we developed a biomaterial-based strategy that enables the controlled patterning of organoids in 3D space through magnetic positioning. We demonstrated the power of this versatile, magnetized 3D bioprinting platform by fabricating assembloids from human brain region-specific organoids. Our 3D bioprinting technique consists of an electromagnetic 3D printer that transfers organoids coated in a magnetic ink into a gel-phase support bath within the same custom-designed chip. For the magnetic ink, iron-oxide particles were encapsulated within a 0.025 wt% cellulose nanofiber (CNF) solution dispensed onto the neural organoids, such that the magnetic particles were evenly dispersed on the outer surface. The presence of the CNFs in the ink delayed magnetic particle sedimentation to ensure homogeneous extrusion. Once coated, the organoids were lifted with an electromagnet (15 V) and deposited within a shear-thinning 0.5 wt% CNF hydrogel support bath, which both stabilized their position and prevented dehydration throughout the assembly process. To select the support bath material and formulation, we screened for biomaterials with the appropriate rheological properties to immobilize organoids while not interfering with the cell migration between adjacent organoids that allows for their integration. The movement and activity of the electromagnet was controlled by a custom-modified 3D printer. We were therefore able to reproducibly localize the organoids in a variety of 3D configurations. The neural organoids fused over four days in the CNF support hydrogel, creating precisely arranged assembloids with high cell viability and maintenance of the cytoarchitecture of individual organoids. To remove the assembloid from the support hydrogel after organoid fusion, the CNF could be degraded on demand by treatment with cellulase. Finally, within our custom-designed chips fabricated with stereolithography 3D printing, we demonstrated the differentiation, printing, and assembly of dorsal and ventral forebrain organoids in a fully enclosed microenvironment. In conclusion, this biomaterials platform represents a significant advance in 3D bioprinting technologies for integrating organoids into neural assembloids.