Though not all 3D systems for neuronal culture could be regarded as brain organoids, methods to stimulate neuronal differentiation from RGCs into 3D neuronal structures have been pursued since the early 1990s. Brain organoids may replicate the brain’s developmental process and reflect the physiological, pathological, and pharmacological characteristics of the brain. They also have many anatomical and cellular similarities with the real brain [8, 9].

The creation of brain organoid technology is based on early research into the two-dimensional induction of neuroectodermal cells and the three-dimensional differentiation of embryoid bodies (EBs). Researchers also started looking at EB differentiation approaches because of the relatively basic cell types in two-dimensional (2D) culture systems, the stark disparities between cell interactions and actual tissue, and the challenge of directly examining human brain tissue. Advances in stem cell technology have made it possible for researchers to employ human induced pluripotent stem cells (hiPSCs) to construct brain-like tissues and organs from a 3D viewpoint. Researchers have also started to investigate neural cell differentiation procedures of PSCs [10, 11].

The creation of innovative procedures for brain organoid formation was made possible by the initial groundbreaking studies of differentiation employing 2D monolayer cultures [12,13,14,15,16] and the groundbreaking work on 3D cultures by the Sasai and Kleber group [17]. Generally speaking, there are two primary methods for creating brain organoids: VSC self-assembly and external sensor inputs. Using neural guiding molecules and extracellular arrays, the research groups of Sasai and Knoblich conducted experiments on 3D brain organoid culture systems [11, 13]. Key features of the fetal brain are mimicked by human brain organoids; nevertheless, the inability to create 3D brain structures that accurately represent late fetal development is due to the drawbacks of existing organoid techniques, including intrinsic hypoxia and cell death [18]. The development of a technique by Gordon et al. to create three-dimensional organoids of the human cerebral cortex with characteristics of the neonatal period is astounding [19] (Fig. 2).

Advances in methodology of brain organoid generation. (A) A simple method using a minimum of medium and extracellular matrix to create self-organized brain organoids. (B) Synthetic materials promote organoid maturation. (C) Organoids with mixed systems, such as neuromuscular organoids, make it possible to study the interaction between organs. (D) Microfluidics develop vasculature in organoids

Brain organoids have progressed from non-directional whole brain organoids to several brain organoids with distinct regional features, including cortex [20, 21], midbrain [22, 23], hippocampus [24], cerebellum [25], and spinal cord [26]. Specifically, realized brain organoid vascularity and regional brain organoids.

Currently, brain organoid cultures are classified into two categories based on whether or not targeted differentiation is carried out. First, stem cell differentiation produces organoid structures that, when grown in cultures, produce multi-brain organoids without the need for external morphogenetic agents. The second approach involves timing the addition of exogenous morphogenetic and neurotrophic substances to cultivate organoids in certain brain areas in accordance with the regulatory systems of the human brain development process (Fig. 3).

Unguided and guided approaches for making brain organoids. Unguided methods take use of hPSCs’ inherent signaling and self-organization abilities to allow them to naturally differentiate into tissues that resemble the growing brain. The resultant brain organoids often have diverse tissues that mimic various parts of the brain. Directed techniques use growth factors and tiny chemicals to create spheres that symbolize a particular tissue type. Organoid techniques specific to brain regions include the early usage of modular variables to influence the destiny of stem cells. Later phases of differentiation subsequently eliminate these components. Moreover, alignment techniques may be used to create two or more spheres or organoids that symbolize the identities of various brain areas. These can then be combined to create “class assemblies” that simulate the interactions of various brain regions

Serum-free culture was primarily used to create neuroectoderm during the early stages of brain organoid differentiation [27], while matrigel and bioreactors were used to accomplish long-term neuronal differentiation culture [11], among other methods. The SFEBq culture method, for example, successfully differentiates embryoid bodies of mouse embryonic stem cells (mESCs) into telencephalic tissue by simultaneously adding inducers for neuronal differentiation (e.g., Wnt antagonists and Nodal antagonists). Sasai’s team [28] produced a serum-free liquid culture of embryoid body-like aggregates with rapid reaggregation in 2005. By blocking the Notch pathway, the team used SFEBq to create human and mouse embryonic stem cells (ESCs) that resemble the retina in terms of composition and cell structure, so mimicking the retina’s developing process to some degree. He established the foundation for the creation of brain organoids [18, 29].

Using the SFEBq technique, Lancaster et al. used hiPSCs for the first time in 2013 to promote differentiation into entire brain organoids [11] (Fig. 2). They stimulated and directed the differentiation of hiPSC embryoid structures with endoderm, mesoderm, and ectoderm. After that, they went through neuroectoderm and neuroepithelium to produce structures that resembled the early embryo’s cerebral cortex, which may have represented the early embryo’s development of the human brain.

Cells linked to the hippocampus, retina, forebrain, midbrain, hindbrain, and other areas with apico-basal polarity are seen in whole-brain organoids [11]. Additional research has shown that the integration of tissue engineering and three-dimensional culture may enhance the tissue structure of brain organoids and boost the repeatability of differentiation [30]. Researchers used microfilaments of poly (lactide-co-glycolic acid) (PLGA) fibers as scaffolding to generate microfilament-engineered brain organoids (MEOs) in 2017 [31]. The brain organoid scaffolds made from microfilms are part of a system that advances cortical development and encourages the production of neuronal ectoderms [31]. He used air-liquid interface culture of whole brain organoids (ALI-COR) in 2019; this method improves axonal development and neuron survival in whole brain organoids [32].

To put it briefly, the unguided brain organoids created in this work mimic the cellular makeup and anatomical structure of the brain in vivo, can model the brain’s developmental process, and can represent the physiological and pathological characteristics of the brain. These capabilities open up new avenues for research on brain function, disease simulation, drug discovery, and other related topics.

Organoids made out of the whole brain exhibit unique heterogeneity [33]. Brain organoids were developed to mimic specific functional properties of individual brain regions in order to better understand the functions of various brain regions and their interregulation, as well as to look into patterns of neuronal development and the onset and progression of diseases in particular brain regions. Organoids of the brain were created to mimic the properties of several brain areas, including the cerebellum, midbrain, and forebrain (Table 1).

The optic nerve, hippocampus, thalamus, hypothalamus, and other brain areas are included in the forebrain. In 2011, Sasai et al. caused PSCs to spontaneously generate upper hemisphere vesicles [18], inhibiting the WNT pathway and concurrently activating the WNT pathway to establish a proximal-distal axis. The distal portion folds inward to create the optic cup structure, while the proximal section includes retinal features including inner nuclear layer (INL) and retinal ganglion cells (RGCs). The research produced organoids called optic cups that contained several kinds of retinal cells. After that, the scientists constructed a 3D culture system that resembled cortical growth [21, 22], setting the stage for the production of organoids of the brain that are particular to a certain area. 2015 Sasai and colleagues optimized cortical organoids and produced hippocampal organoids by inhibiting SMAD pathways and activating WNT and BMP pathways [25]. In 2016, Qian et al. created a hypothalamus organoid by inhibiting the SMAD route and activating the WNT and SHH signaling pathways [34]. In order to create neuroectodermal organization and increase BMP7, Xiang et al. blocked the SMAD pathway in 2019 [35]. This resulted in the development of thalamic organoids.

In the pathophysiology and therapy of Parkinson’s disease (PD), the midbrain—which regulates information transmission, motor control, and sensory processing between the forebrain and spinal cord—has drawn a lot of interest. Jo et al. produced midbrain organoids in 2016 by adding SHH/FGF8 to induce ceiling structure [24], activating the WNT pathway, and inhibiting the SMADs pathway. By identifying dopamine production, they opened up a new avenue for research on Parkinson’s and other illnesses. Furthermore, in 2015, MONZEL et al. stimulated neuroepithelial stem cells to produce organoids in the midbrain using SHH inhibitors and GSK3 inhibitors [23].

The brain’s motor control center, the cerebellum, has the capacity to develop into distinct neuronal groupings. To form cerebellar organoids, Muguruma et al. created a boundary structure between the midbrain and cerebellum by blocking the SMADs pathway, adding FGF2 and insulin to promote caudation of cerebellar organoids, and then adding FGF19 and SDF1 to induce cells to promote neuroepithelial formation in the cerebellar lamina [26]. Because of the diverse cell types and fragile structure of the cerebellum area, the cerebellar organoid culture system still needs a long-term culture system.

In addition to neurons, microgila also play a pivotal role in the brain’s functionality. Currently, the lack of microglia with the ability to reshape neuronal networks and phagocytose apoptotic cells and debris is a major shortcoming of the midbrain organoid system. Moreover, modeling of diabetes-related neurological complications is not possible in the absence of microglia. By co-culturating hiPSCs-derived mesodermal progenitor cells (Brachyury+) with neurospheres, Worsdorfer et al. renewably generated vascularized neuroorganoids that included vasculoid structures (CD31+) and microglia-like cells [36]. This study provided a model for studying angiogenesis and neurodevelopment, but did not investigate the function of microglia in the organoids. In the another study, Fagerlund et al. reported that hiPSCs-derived eythro-myeloid progenitors (CD41+) migrated into human brain organoids [37]. Differentiated into microglia-like cells, and interacted with synaptic material. Whole-cell patch-clamp and multi-electrode array recording showed that microglia within organoids promoted the maturation of neural networks. A recent study that co-cultured human midbrain organoids with hiPSCs-derived macrophage progenitor cells also reported that microglia integration let to increased nerve maturity and function [38]. Whole-cell patch-clamp and multi-electrode array recordings showed that lower action potential generation thresholds and shorter peak-to-peak intervals were observed in midbrain organoids with microglia integration, suggesting that microglia integration improve neural maturation.

The methodologies for constructing region-specific brain organoids are also examined. The development of these organoids continues to advance, integrating developmental inducers, biomaterials, and bioreactor systems. It is anticipated that more precise realization of region-specific brain organoids can be achieved. The aim is to replicate the maturation processes of various brain regions and establish relevant disease models. These may be used to research the control of neurodevelopment and the beginning of illness in certain brain areas. To more accurately mimic the physiological structure of the brain, however, further work has to be done on the precision of the generated brain areas and the repeatability of the cells.

Although brain organoids may be utilized to model many interacting brain areas, their sizes and spatial configurations are very varied and unpredictable. Researchers have attempted to mimic the structure and environment of the genuine human brain by integrating several brain organoid areas in an effort to create a more realistic brain organoid design that can replicate the development of different brain regions and model disorders. 2017, Team Pasca performed neural induction of PSC by SFEBq to induce the formation of dorsal and ventral telencephalic organoids by regulating WNT and SHH signalling [39], spontaneously fused ventral and dorsal telencephalic organoids and observed irregular migration of interneurons in the cortical tissue. Interneurons in fused organoids from Timothy syndrome patients migrated abnormally. Bagley et al. fused the ventral telencephalon and whole brain organoids to form brain organoids fused to the dorsoventral axis [40]. Based on this, Xiang et al. studied the migration of CXC chemokine receptor 4 (CXCR4) dependent interneurons from ventral to dorsal migration [41]. Medial ganglionic neurite (MGE) organoids were constructed, and then fused with cortical organoids to examine CXC chemokine receptor (CXCR4) dependent interneurons.

To better imitate the mutual projection of the thalamus and cortex, XIANG et al. produced thalamocortical fusion organoids by physically combining thalamic and cortical organoids [35]. Studies of neurological conditions including schizophrenia and autism spectrum disorders may be conducted using the biaxial projection between the thalamus and cortex, which replicates synaptic connections in the body. Subsequently, MIURA et al. employing fusion of striatal and cortical organoids, showed that cortical neurons project axons to striatal organoids and make synaptic connections with neutral invertebrate neurons, showing enhanced electrical features and calcium activity [42]. By combining the two kinds of organoids, functional integration was accomplished in these four investigations. In an in vitro three-dimensional culture media, they replicated the tangential movement of human endoneurons [39,40,41,42].

Brain organoid fusion methods provide a potent platform for investigating the relationships between various brain regions/tissues, including the impact of tissue growth centers on brain organoid development and the investigation of cell-cell interactions in vitro. Nevertheless, in order to create functioning circuits and provide helpful instruments for researching brain function, current fusion organoid manufacturing techniques need to be further refined to represent particular brain space projections and physiological reactions.

Organoids in the brain still differ significantly from the genuine human brain. Lack of a circulatory system is one of the main obstacles. Gas penetration, nutrition delivery, neuron differentiation, and other processes are all impacted by vascular function [43, 44]. Necrotic regions will form in the organoid center as a result of inadequate oxygen and nutrient penetration, which will interfere with the proper growth of brain organoids and the neuronal migration route [45]. Thus, the development of a vascular network is a critical requirement for the optimization of brain organoids. As of right now, there are primarily two methods for vascularizing brain organoids: creating blood vessels in the organoids by in vivo transplantation and creating blood vessels in vitro.

In 2018, researchers transplanted brain organoids into the cerebral cortex of NOD-SCID immunodeficient mice, and the blood vessels of mice infiltrated into the implanted brain organoids within 14 days after transplantation; Compared with non-vascularized brain organoids in vitro, the in vivo development environment improves cell maturation and survival in brain organoids [46]. For the purpose of achieving the functional link between human axons and neurons in the mouse brain, brain organoids that have been transplanted may produce a lot of new neurons and live for over 200 days [46]. To create human-mouse vascular tissue linkages in the grafts, human vascularized organoids (vOrganoids) co-cultured with human umbilical vein endothelial cells were inserted into the mouse S1 cortex. Compared to non-vascularized brain organoids, vascularized brain organoid transplantation increases blood vessel development and cell survival [47]. As a result, a series of transplantation experiments have demonstrated the importance of vascularization in the maturation of brain organoids.

In terms of in vitro vascularization, in 2019, Cakir et al. co-differentiated human embryonic stem cells expressing ETV2 (ETS variant 2) with wild-type embryonic stem cells to achieve directional induction of vascular endothelial cell differentiation in cortical organoids, based on which vascularization cortical organoids were constructed [48]. vascularized human cortical organoids (vhCOs) form perfusable blood vessels; Compared with control cortical organoids, the cell survival rate in vascularized cortical organoids was significantly improved [48]. In addition, by co-culturing with venous endothelial cells, researchers were able to establish vascularized brain organoids in vitro, in which venous endothelial cells can form well-developed reticular or tubular vascular systems, as confirmed by single-cell RNA sequencing. This vascularized brain organoid system has similar molecular properties and cell types to the human fetal telencephalon [47]. The integration of brain organoids and vascular system under in vitro culture will help to improve the phenomenon of central necrosis during the long-term cultivation of brain organoids.