Induction of inverted morphology in brain organoids by vertical-mixing bioreactors

Organoid technology provides an opportunity to generate brain-like structures by recapitulating developmental steps in the manner of self-organization. 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. 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. Overall design: We conducted single-cell RNA-sequencing to dissect transcriptional traits of the cells in brain organoids.