Q5 - Assembling 3D Functional Brain Tissue from NeuralSpheroid Building Blocks in PCL Scaffolds

Introduction: : Organoids are increasingly used to model the brain in vitro. Brain organoids have been applied to study the formation of connections between neurons, and the development of spontaneous activity patterns that are characteristic of healthy brain function. However, robust modeling of complex activity patterns such as oscillations and traveling waves has been challenging with existing organoid technologies. This is due to the random arrangement of neuron types within the organoid, leading to formation of highly variable circuits. In contrast, the brain is characterized by well-defined cytoarchitecture that consists of precise spatial arrangement of different neuron types. Here, we report a novel technology based on 3D printed scaffolds, for creation of patterned brain organoids. Patterned organoids generated highly-reproducible traveling waves of neural activity, which are similar to waves recorded in the intact cortex.

Materials and

Methods: : Neural spheroids formed by seeding cell suspensions into 3D microwells. Suspensions included dissociated rat cortex (neurons + astrocytes) or hiPSC-derived neurons with rat astrocytes. These spheroids were then plated into the pores of 3D-printed polycaprolactone (PCL) scaffolds to form the desired patterns. The scaffolds were created using a solvent-cast 3D printing process, where polymer ink was extruded layer-by-layer to form the structure. Functional connectivity was assessed using calcium imaging with GCaMP7f and jRGECO1a indicators. Channelrhodopsin (ChR2) was used to evoke activity in the pattern optogenetically and produce traveling waves similar to those in the cortex.

Results, Conclusions, and Discussions:: The scaffold enabled the formation of spatially patterned neural constructs that supported reproducible calcium dynamics. Brightfield and fluorescence imaging confirmed the establishment of desired spatial configurations, such as letter-shaped neural assemblies and multi-population spheroids composed of different neuron types. Correlation matrices demonstrated stable and increasing inter-spheroid connectivity over time. Constructs containing hiPSC-derived neurons similarly exhibited robust activity and structural integrity. Upon optogenetic stimulation, spheroid networks generated propagating calcium waves with measurable velocities, mimicking natural cortical behavior. These findings underscore the potential of 3D-printed scaffold technologies to overcome the limitations of conventional organoid systems by enabling the design of cytoarchitecturally and functionally predictable brain-like tissues.

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