**2. Brain organoid protocols: general timeline, stages, strategies to improve outcome and classification**

Most of the current brain organoid protocols use the dissociation-re-aggregation method to form 3D aggregates from the stem cells. After some period of recovery and establishment of cell–cell contacts and maturation, these aggregates become EBs, which are used as a starting point for organoid formation and development. The EBs are not formed from every stem cell aggregate. So far, the studies have revealed a set of important variables [10, 11]. Firstly, the critical cell mass, i.e., a sufficiently large number of cells, is needed to form EB. Secondly, size and uniformity have a huge effect on the homogeneity and structure of the EBs and may impact the fate choice. Studies found that the addition of Y-27632 is beneficial for these results. To overcome these issues, geometric confinement approaches are often used. Thirdly, the time for aggregation also plays a critical role. These and other variables secure the establishment of an appropriate intra-EB microenvironment to trigger the intrinsic embryo developmental programs. The EBs are multicellular aggregates of stem cells, which resemble
