Abstract

Organoid technology provides an opportunity to generate brain-like structures by recapitulating developmental steps in the manner of self-organization. Here we examined the vertical-mixing effect on brain organoid structures using bioreactors and established inverted brain organoids. The organoids generated by vertical mixing showed neurons that migrated from the outer periphery to the inner core of organoids, in contrast to orbital mixing. Computational analysis of flow dynamics clarified that, by comparison with orbital mixing, vertical mixing maintained the high turbulent energy around organoids, and continuously kept inter-organoid distances by dispersing and adding uniform rheological force on organoids. To uncover the mechanisms of the inverted structure, we investigated the direction of primary cilia, a cellular mechanosensor. Primary cilia of neural progenitors by vertical mixing were aligned in a multidirectional manner, and those by orbital mixing in a bidirectional manner. Single-cell RNA sequencing revealed that neurons of inverted brain organoids presented a GABAergic character of the ventral forebrain. These results suggest that controlling fluid dynamics by biomechanical engineering can direct stem cell differentiation of brain organoids, and that inverted brain organoids will be applicable for studying human brain development and disorders in the future.

Highlights

  • Organoid technology provides an opportunity to generate brain-like structures by recapitulating developmental steps in the manner of self-organization

  • Brain organoids are a self-organization of three-dimensional aggregates resembling brain structures generated from human induced pluripotent stem cells (iPSCs)

  • The fluid dynamics of the organoid and direction of a cellular mechanosensor, primary cilia, in the outside layer of neural progenitor cells were different between vertical and orbital mixing. scRNA-seq analysis revealed that the inverted brain organoids contained a neuronal area of GABAergic neurons, and it was applicable to the analysis of neurological diseases

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Summary

Results

Generation of inverted brain organoids by vertical mixing. We generated brain organoids using orbital shakers for orbital mixing or reciprocal vertical bioreactors for vertical mixing (Fig. 1a, Supplementary Fig. 1). Cell clusters were defined using K-means clustering on principal component analysis (PCA) space, and the number of clusters was decided using the elbow method We evaluated the generation of excitatory neurons and GABAergic neurons along the time axis in orbital mixing and vertical mixing, and found that vertical mixing might facilitate the promotion of GABAergic neuronal differentiation (Supplementary Fig. 5) These data suggested that the inverted brain organoid is a characteristic brain organoid that harbors a unique structure and special cell composition. After 2 months of culture in vertical mixing, brain organoids showed the inverted structure with the expression of hippocampal marker PROX1, consistent with a single-cell RNA-seq (Fig. 8, Supplementary Fig. 4b). Aβ oligomer accumulation was observed in MAP2positive neurons of AD brain organoids (Fig. 8), suggesting that the inverted brain organoids could be a disease model in vitro

Discussion
Methods
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