**Abstract**

The brain organoid technology emerged a little over a decade ago. During this short time span, the handling approach has seen tremendous advancements in order to solve current obstacles and enable the development of new applications. Using these methodologies, the fundamental characteristics of the majority of the brain regions may be mimicked in organoids; however, the existing brain organoids cannot be regarded an exact replica of the human brain or its anatomical regions. This chapter will present some of the biological phenomena on which the brain organoid technology relies. Following this, a summary of the gross common structure and timeline of the brain organoid protocols along with their main components and strategies for their improvement is included. A special selection of protocols for each major brain region will be presented with their origin, rationale, and key specifics. Finally, some of the daunting challenges to brain organoid technology will be highlighted.

**Keywords:** brain organoids, cerebral organoids, embryoid bodies, methodology, technology, challenges

## **1. Introduction**

More than a decade has passed since the first brain organoid emerged from the lab of one of the fathers of organoid technology – Yoshiki Sasai. During that time, an increasing number of researchers worldwide got attracted by the potential of this technology and the hope that we may learn how to build our brains *in vitro* and thus further adopted and developed it [1]. A few years before the first brain organoid protocol was published, Zhang et al. developed a protocol for the generation of freefloating embryoid bodies (EB) from dissociated human embryonic stem cells (hESC), which afterward could be directed to differentiate into neural precursors in rosette structures. This technique actually was an offspring of the methods for aggregated suspension culture from dissociated fetal brain cells like the one by Bjerkvig et al. from 1986 [2, 3]. The team used cells from post-neurulation stages, when loss of potency is present and which can affect their self-organization and differentiation capacities, as some recent studies reveal, while Zhang et al. used embryonic cells from a much earlier stage of the embryogenesis and therefore could direct them to form

neural rosettes (resembling early neural tube-like structures). Noteworthy to mention is the contemporary study by Doetschman et al., in which they could observe nerve cells generated spontaneously and stochastically from EBs formed from blastocyst cells using undefined medium. However, they lacked the knowledge of the molecular mechanisms and ways to direct them to a desired cellular fate [3–6]. Zhang et al. succeeded in generating EBs, which were directed to structures recapitulating very early stages of neural tube formation and comprised mostly of neural precursor cells, thanks to the newer discoveries on the neural induction mechanisms [2]. However, the goal of their study was to get transplantable neural precursors and not to recapitulate later-stage brain regions. Therefore, their protocol was much shorter and more straightforward than the ones for organoids.

By the turn of the twentieth century, the recently acquired knowledge of the molecular control of the embryonic brain patterning got consolidated in updated models of neural induction and morphogenesis. It became evident that the brain morphogenesis is spatiotemporally organized and orchestrated by a dynamic network of transient patterning centers (organizers) that secrete a bunch of molecular controllers (morphogens) that can trigger identity change of nearby cells in a concentration−/distance-dependent fashion. These morphogens are activators or inhibitors of a handful of signaling pathways. In addition, more evidence was accumulated that the neural progenitors without extrinsic signals develop anterior neural specification [7]. So, during the first decade of the new millennium, protocols for stem cell differentiation towards identities from the major brain regions were developed based on this framework. Besides, some of the growth and differentiation conditions were also significantly improved; for example, with the popular dual SMAD inhibition strategy for improved neural induction or with the usage of the survival enhancer of dissociated cells -Y-27632 (ROCK inhibitor that diminishes dissociation-induced apoptosis) [8, 9]. This review will present some of the key biological phenomena engaged in brain organoid generation. The structure and the main components of the brain organoid protocols will be analyzed and summarized. The protocols specific to different brain regions will be presented and analyzed to introduce and guide the readers in this growing field. Furthermore, some of the major obstacles related to brain organoid technology and recapitulating the brain and its regions will be discussed.
