Embedded 3D printing in self-healing annealable composites for functional patterning of human neural constructs

Human in vitro models of neural tissue with controllable cellular composition, tunable microenvironment, and defined spatial patterning are needed to facilitate studies of brain development and disease. Towards this end, bioprinting has emerged as a promising strategy. However, precise and programmable printing of extremely soft and compliant materials that are permissive for stem cell differentiation and functional neuronal growth has been a major challenge. Therefore, solutions for engineering structurally and functionally defined human neural constructs remain scarce. Here, we present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. The approach combines rheological benefits of granular soft microgel supports with the versatile biomimicry of bulk hydrogels to simultaneously enable precise freeform patterning of stem cells, and consequent generation and long-term maintenance of subtype-specific neurons within engineered networks that extend into the bulk of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular channels. Due to its modularity, SHAPE biomanufacturing toolbox not only offers a solution for functional modeling of mechanically sensitive neural constructs, but also has potential to be applied to a wide range of biomaterials with different crosslinking mechanisms to model tissues and diseases where recapitulation of complex architectural features and topological cues is essential. ### Competing Interest Statement Tom Kamperman is the CTO of IamFluidics BV, which provided one of the control samples used in Fig. S3.