Formation and culture of layered cortico-striatal assembloids in acoustic levitation

The need to cultivate and analyze three-dimensional multicellular tissues, such as spheroids and organoids, in a reproducible manner has led to the development of new tools and methods to control how they self-organize and differentiate. Here, we present a novel approach based on acoustic levitation for structuring and cultivating layered neurospheres. First, we demonstrate that viable and well-differentiated neuronal spheroids derived from mouse primary neurons can be cultivated under constant acoustic trapping in bulk acoustic wave resonators. We showed that both striatal and cortical cell aggregates formed in acoustic levitation could self-organize into spheroids within 24 hours and remain viable up to 10 days under these particular culture conditions without medium renewal. These spheroids also demonstrated healthy development with typical phenotypic distributions and synaptic maturation. Second, in order to model the cortico-striatal pathway, we succesfully structured and cultivated concentric cortico-striatal assembloids using optimized acoustofluidic chips and specific protocols. These results confirm that acoustic levitation can be used as a new scaffold-free technique for structuring and cultivating complex 3D multicellular objects.