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The fusion of human organoids holds promising potential in modeling physiological and pathological processes of tissue genesis and organogenesis. However, current fused organoid models face challenges of high heterogeneity and variable reproducibility, which may stem from the random fusion of heterogeneous organoids. Thus, we developed a simple and versatile acoustofluidic method to improve the standardization of fused organoid models via a controllable spatial arrangement of organoids. By regulating dynamic acoustic fields within a hexagonal acoustofluidic device, we can rotate, transport, and fuse one organoid with another in a contact-free, label-free, and minimal-impact manner. As a proof-of-concept to model the development of the human midbrain-to-forebrain mesocortical pathway, we acoustically fused human forebrain organoids (hFOs) and human midbrain organoids (hMOs) with the controllable alignment of neuroepithelial buds. We found that post-assembly, hMO can successfully project tyrosine hydroxylase neurons towards hFO, accompanied by an increase of firing rates and synchrony of excitatory neurons. Moreover, we found that our controllable fusion method can regulate neuron projection (e.g., range, length, and density), projection maturation (e.g., higher firing rate and synchrony), and neural progenitor cell (NPC) division in the assembloids via the initial spatial control. Thus, our acoustofluidic method may serve as a label-free, contact-free, and highly biocompatible tool to effectively assemble organoids and facilitate the standardization and robustness of organoid-based disease models and tissue engineering.

Controllable fusion of human brain organoids using acoustofluidics