*The Brain Organoid Technology: Diversity of Protocols and Challenges DOI: http://dx.doi.org/10.5772/intechopen.105733*

the inner cell mass of the pre-gastrulation embryo. These cells have the potential to develop into cells from all three germ layers [12]. Afterward, the cells from the EBs were then committed to neural development through medium change and addition in the medium of various signaling molecules. Initially, during this process of differentiation EBs are directed to neuroectoderm formation. The neuroectoderm then gets transformed to neuroepithelium, which depending on the culturing conditions (as dynamic or static culture) attains the form of large neuroepithelium bulges or small neural rosettes, thus recapitulating the neural tube. These structures further differentiate into more specific brain regions according to the applied patterning scheme [13–16].

The summarized workflow of the procedures and timelines can be divided schematically into the following stages (**Figure 1**), (note that the steps and timeline are exemplary and can vary depending on the organism, used patterning strategies, goals, and other factors) [13–16]:


#### **Figure 1.**

*A schematic timeline of generalized brain organoid protocol: A. stages of the treatments; B. timeline of the 3D culture development and growth; C. timeline of the neural differentiation. After [14–17].*

vii.Organoid growth − Maturation medium is commonly used without patterning molecules but instead with growth factors (such as BDNF, NT-3), antioxidants (such as ascorbic acid), and other additives, which enhance the organoid maturation and long-term survival.

**Patterning agents**: Currently, the available protocols for brain organoids try to mimic the molecular control exercised by the embryonal network of brain patterning centers. The major signaling pathways elaborated or triggered by them are the pathways of fibroblast growth factor (FGF), sonic hedgehog (Shh), Wnt, bone morphogenic protein (BMP), and nodal/activin. This patterning network with its morphogen gradients can be presented in a 3D coordinate system where at each spatial coordinate of the brain primordium corresponds a combination of morphogens (which usually are the first messengers of these pathways) with specific concentrations (**Figure 2**) [19, 20]. Based on this model, the researchers approximate the morphogen combination patterning the region of interest and determine the appropriate concentrations afterward. Although in the brain organoid protocols usually are used as morphogens the first messengers of these pathways, in some scenarios are used secondary messengers or their analogs instead – for example, when it is cost-efficient; or it is difficult to work with them; or is needed partial activation of the pathway or for another reason [21]. In general, the used patterning agents can be divided by their ability to caudalize or ventralize the cell populations regarding the most anterior and dorsal end of the central nervous system primordium. As caudalizing agents, the first messengers from the Wnt and BMP families are often employed, while Shh and Wnt are used as ventralizing agents.

**Oxygenation and metabolite exchange:** To cope with the lack of vascularization and the hindered oxygen and metabolite exchange, several strategies are developed:


**Figure 2.**

*The morphogen gradients in the developing embryonal CNS. T – Telencephalon; D – Diencephalon; MB – Midbrain; R1-R8 – Rhombomeres of the rhombencephalon; SC – Spinal cord. After [18].*

*The Brain Organoid Technology: Diversity of Protocols and Challenges DOI: http://dx.doi.org/10.5772/intechopen.105733*

v.Transplantation in animal brain – the maturing organoids are transplanted into the brain of the host animal after one and half months of *in vitro* culture. After a few weeks, the organoids get vascularized and continue to grow in the host [26].

**Mediums and Supplements:** They are the fundamental components of the organoid microenvironment; however, there is no strict rule on which formulation to use. It requires experimenting with a few recipe/brand choices and their combinations to achieve optimum output. Some commonly used mediums are DMEM/F12; Neurobasal, GlasgowMEM, BrainPhys, mTESR1, Essential medium, etc. [13–16, 27, 28].

**As supplements are commonly used:** KSR (knockout serum replacement), N2, B27, Gem21 Neuroplex, etc. [13–16, 27, 28].

**Stage-dependent medium composition specifics**: The biological transformation of the EBs and consequently organoids require adjustment of the microenvironment according to the dynamic transient necessities of the culture. During the initial stages are used DMEM/F12, essential medium supplied with KSR, etc. The medium is often changed during the differentiation stages to Neurobasal, DMEM/F12 with N2, b27(−A). For the maturation stage, often the choice is for Neurobasal, DMEM/F12, or their combination with added supplements [13–17, 27, 28].

**Culture dishes and substrates:** Dishes with U or V-shaped wells with ultra-low attachment characteristics or hanging drop culture are suggested for the initial stages. At later stages, there is more freedom of dish choice – petri dish, microwells, and microfluidics [13–17, 27, 28]. So far, if a substrate is used at all in the protocols, the most commonly used is Matrigel (basement membrane matrix produced from mouse sarcoma cells); it is used either in differentiation or maturation stages or both [13, 28].

**Classification:** The current brain organoid protocols can be classified in several ways. One of the commonest is based on the approaches for differentiation and can be divided into two groups: 1. Guided (use external agents to direct the patterning) and 2. Unguided (accentuate on the intrinsic developmental programs) (**Figure 3**) [13–17, 28]. Another way is based on the anatomical region in which the organoids recapitulate – telencephalic, diencephalic, mesencephalic, and rhombencephalic [14–17, 27, 28]. Here will be used in a nested fashion.

#### **Figure 3.**

*Approaches for generation of brain organoids. (a). Unguided - without use of patterning agents; (B). And (C). Guided – Utilizing patterning agents to direct the patterning process. (B). Generating region-specific organoids. (C). Generation of fussed organoids from separately grown guided region-specific organoids.*
