Three-dimensional (3D) brain organoid cultures

The human brain is the most complex organ in our body, with many aspects of its development and human-specific pathology remaining unknown because of limited accessibility to living human brain tissue. As a result, many approaches and models have been generated in an attempt to recapitulate the human brain in an in vitro system.

Two-dimensional (2D) cell culture systems have historically been used to model various diseases and systems in vitro; however, they are quite limited when it comes to modeling complex systems such as the human brain. 2D-cell culture systems lack the cellular organization that is present in the brain, as they are grown in a monolayer format which limits cell interactions only to their periphery. This results in a lack of proper oxygen and nutrient diffusion as well as waste clearance (Antoni et al. 2015). Furthermore, 2D systems lack the tissue complexity that is present in the brain. As cells are often cultured as a single cell type, they are missing the cell-to-cell interaction between different cell types that is present in vivo. To create a 2D system that is more resemblant to cellular interactions in the human brain, co-cultures of cells can be created, such as co-culturing neurons with other neural cell types such as microglia (Haenseler et al. 2017; Vahsen et al. 2022). Despite these co-culturing techniques, the cells are still limited to peripheral contacts and lack the organization needed to recapitulate the human brain in vitro.

Various neuronal cells, such as primary neurons and neuroblastoma cells, have been used in 2D systems in neuroscience research (Liu et al. 2022). However, obtaining patient-derived brain tissue, or neural stem cells and embryonic stem cells that can differentiate into neuronal cells, is controversial and not always accessible (Gabriel and Gopalakrishnan 2017). The innovation of induced pluripotent stem cell (iPSC) technology eased this problem by opening the doors to being able to generate human neural cell types without the need to isolate cells directly from the human Central Nervous System (CNS) (Gabriel and Gopalakrishnan 2017). The somatic cell-derived iPSCs are a type of stem cell that can differentiate into other cell types in the body (Lyadova and Vasiliev 2022), which makes them an available source for researchers (Karagiannis and Kim 2021). iPSCs were developed by Takahashi and Yamanaka 2006 at Kyoto University in Japan, where they first induced pluripotency in mouse embryonic stem cells (ESCs) and then in adult human fibroblast cells a year later (Takahashi and Yamanaka 2006; Takahashi et al. 2007). Takahashi and Yamanaka generated their iPSCs from fibroblast culture by using retroviral vectors to introduce specific transcription factors such as Oct3/4, Sox2, Klf4, and c-Myc into the skin cells. They noted that their iPSCs display both the morphology and growth properties of ESCs and express ESC-specific marker genes (Takahashi and Yamanaka 2006; Takahashi et al. 2007). Researchers have created techniques for generating iPSCs without using viral vectors, like plasmid-based or episomal reprogramming, that can eliminate the dangers linked to mutations due to viral integration into the genome (Bang et al. 2018). This method involves electroporation, where the electrical shocks introduce plasmid into the genome of the cells. Additionally, CRISPR-based reprogramming allows for the precise and efficient editing of certain genes for successful reprogramming (Liu et al. 2018).

Human iPSCs (hiPSC) prove a powerful tool for easily generating human neural cell types in culture; however, the differentiated cells are still 2D and limited in the same way other 2D cell culture systems are, as listed previously. This led to the need to develop alternative models to study such systems in vitro while mimicking an in vivo environment, such as 3D culture systems. One of the first successful 3D neural systems was that of neurospheres. Neurospheres are 3D cell aggregates of multipotent neural stem cells (NSC) grown in culture, providing a good resource for studying NSCs in vitro (Soares et al. 2021). These clusters of NSCs can then be differentiated into varying cell types, such as neurons and glial cells, all within the same sphere, also known as a neural spheroid (Dingle et al. 2015; Zhou et al. 2016; Pamies et al. 2017). This further allows for a better representation of cell-to-cell interactions in vitro. Although these systems provide a better representation of the brain in vitro than traditional 2D cell culture, they lack complete cellular composition, organization, and complexity of the human brain (Reynolds et al. 1992; Pamies et al. 2017). The human brain has very specific region specificity and cellular organization that is crucial to its function, and this is lacking in neurospheres and neural spheroids as the cells do not organize (Dingle et al. 2015).

Unlike 2D cell culture systems and neurospheres, brain organoids are able to model the human brain at a cellular, structural, and developmental level, allowing researchers to model the human brain and its function in ways that were previously impossible. Brain organoids were first generated by Lancaster et al. in (2013) as a system to study microcephaly. They were able to successfully generate an iPSC-derived 3D cell system, which they dubbed “cerebral organoids” (COs), that displayed discrete brain regions, dorsal cortical organization, functional cortical neurons, and glial cell populations (Lancaster et al. 2013). The development of this system has been a major breakthrough in neural sciences research as it was the first time the human brain was able to be recapitulated in vitro with correct organization and patterning.

To generate organoids, specific conditions, like extracellular matrix (ECM), small molecules, and growth factors, are provided to iPSCs or tissue-derived cells (TDCs) (Zhao et al. 2022). Thus, this environment will differentiate iPSCs or TDCs into the tissue of interest, such as the lung, heart, and cerebral cortex (Zhao et al. 2022). Researchers use stem cells such as iPSCs to generate brain organoids due to their availability (Gabriel and Gopalakrishnan 2017). This method involves differentiating single-cell iPSCs into embryoid bodies (EBs) and then NSCs by using small molecules and growth factors (Hong et al. 2022). Neuroepithelium cells form during the induction phase of EBs (Hong et al. 2022). The expansion phase involves embedding the EBs in ECM such as Matrigel, which results in a budding morphology and promotes further differentiation into several cell types present in COs, such as NSCs, neurons, and glial cells (Agboola et al. 2021). The expanded EBs are cultured in suspension on an orbital shaker (Lancaster and Knoblich 2014) or in a spinning bioreactor (Qian et al. 2016) during and after the maturation phase, where they become self-organized COs.

The organoids generated using these protocols, known as “unguided organoids,” as they are allowed to freely organize themselves into forebrain, midbrain, and hindbrain regions (Lancaster et al. 2013; Qian et al. 2016). This allows for a recapitulation of the entire brain in vitro which is an extremely useful tool; however, some diseases affect specific regions of the brain. As a result, it is necessary to be able to model specifically the forebrain, midbrain, or hindbrain alone, as well as specific structures in the brain as organoids.

Many groups of researchers have worked to develop guided protocols using extrinsic factors to generate brain region-specific organoids, which contain more accurate cell populations and organization of specific brain regions and structures. A commonly used guided organoid is a cortical organoid, which is representative of the cerebral cortex. Cortical organoids have been used to study a variety of neural disorders, such as Zika virus (ZIKV) infection (Qian et al. 2016), Japanese encephalitis virus (Zhang et al. 2018), Alzheimer’s disease (AD) (Raja et al. 2016), and several other neural degenerative disorders. Beyond cortical organoids, many other brain regions and structures have successfully been generated using guide protocols. These include forebrain and midbrain organoids (Raja et al. 2016; Jo et al. 2017; Liu et al. 2019; Krenn et al. 2021). In conclusion, brain organoids possess a superior in vitro model to study ZIKV infection, allowing researchers to better understand the viral pathophysiology and utilize them as a platform to screen therapeutic interventions.

Another neurotropic virus that has been associated with microcephaly is the human Cytomegalovirus (CMV). CMV belongs to the Herpesviridae family and is a double-stranded DNA virus that can infect people of all ages. Although the majority of CMV infections are asymptomatic, newborns with congenital CMV infection may present neurologic anomalies such as lethargy and microcephaly (Boppana et al. 2013). Several groups have reported CMV infection modeling in brain organoids using unguided protocols. CMV infection in brain organoids disrupts organoid morphology, probably due to the induced cell death leading to size reduction (Sun et al. 2020). In addition, infection with CMV interferes with neurogenesis and the formation of neural rosette formation from NPCs (Sison et al. 2019). Furthermore, neural signaling, as well as networks important for neurodevelopment, has been shown to be downregulated following CMV infection in brain organoids (O’Brien et al. 2022). Moreover, brain organoids were also modeled for testing the therapeutic potential of neutralizing antibodies for the treatment of newborns with congenital CMV infection (Sun et al. 2020).

In the past three years, the COVID-19 pandemic has been a major health problem worldwide. Although the major target of SARS-CoV-2 is the respiratory system, the viral tropism involves multi-organ systems (Liu et al. 2021). Neurological manifestations in COVID-19 patients have been reported, although the neuropathology associated with SARS-CoV-2 is yet to be elucidated (Mao et al. 2020; Douaud et al. 2022; Ng et al. 2023). In recent years, several brain organoid models have been developed as a tool to gain insight into SARS-CoV-2 neuroinvasion in the CNS, using guided and unguided protocols. Yi et al. reported persistent ACE2 expression during the development of dorsal forebrain organoids from hESCs and showed susceptibility to infection using a SARS-CoV-2-pseudovirus (Yi et al. 2020). Zhang et al. also reported that ACE2, TMPRSS2, and coronavirus entry-associated proteases (cathepsin L, and furin) are readily available in hNPCs and brain organoids generated from hiPSCs and showed that the brain organoids support SARS-CoV-2 infection and replication (Zhang et al. 2020). Jacob et al. have further investigated SARS-CoV-2 infection in region-specific hiPSCs-derived brain organoids. Using cortical, hippocampal, hypothalamic, and midbrain organoids, they reported limited infection in neurons and astrocytes, but robust infection in choroid plexus (ChP) epithelial cells. Moreover, they developed ChP organoids from hiPSCS and reported productive SARS-CoV-2 infection associated with cell death (Jacob et al. 2020). Several other groups also reported SARS-CoV-2 infection modeling in dorsal cortical organoids or cortical organoids. Those studies revealed that SARS-CoV-2 can infect glial cells (McMahon et al. 2021; Andrews et al. 2022), ChP cells (McMahon et al. 2021), and neuronal cells (Zhang et al. 2020; Song et al. 2021). Mesci et al. showed that infections in neurons promote cell death with the loss of excitatory synapses. Moreover, the same group showed that the antiviral drug Sofosbuvir inhibits SARS-CoV-2 replication in a model of cortical organoids (Mesci et al. 2022). Hou et al. also utilized forebrain and midbrain organoids to study replication efficiency and neurotropism of different variants of the virus (SARS-CoV-2 WT, Delta, Omicron BA.1 and Omicron BA.2) and demonstrated a higher replication efficiency of variant Omicron BA.2 with positive viral infection in dopaminergic neurons in midbrain organoids and cortical neurons in forebrain organoids (Hou et al. 2022). Unguided brain organoids have also been developed to mainly demonstrate the virus’s cellular tropism. In line with findings using guided protocols, various neural cell types were shown to be susceptible to infection with SARS-CoV-2 (Bullen 2020; Ramani et al. 2020; Tiwari et al. 2021). Another group showed that SARS-CoV-2 infection in neurons was boosted by the presence of astrocytes in brain organoids which also led to synaptic loss and neuronal toxicity (Wang et al. 2021). Infection in astrocytes was also linked to the promotion of neuronal death (Kong et al. 2022). Pellegrini et al. reported SARS-CoV-2 infection in ChP with limited neuronal infection, probably due to the higher expression levels of ACE2 in this cell type (Pellegrini et al. 2020). Moreover, by using brain organoid models, Samudyata et al. showed SARS-CoV-2 nucleocapsid protein expression in PAX6, MAP2, GFAP, SOX10, OLIG2, and Iba1 positive cells, and revealed a role of microglia in infected organoids in increasing the engulfment of postsynaptic termini with increased phagocytosis and synapse elimination (Samudyata et al. 2022). In summary, brain organoid models have provided a superior platform to investigate neuronal susceptibility, disease mechanisms, and treatment strategies for SARS-CoV-2 infection.

Future advancements

As far as COs have come in recent years, there is still much improvement to be done. One of the biggest hindrances in CO models is the fact that they are size-restricted due to the lack of nutrient and oxygen diffusion to their centers. An improvement in this area would allow for the generation of larger, more complex, and more stable COs and organoids in general. This area is currently being explored in a variety of ways, such as bioengineering special devices to increase diffusion and organoid size (i.e., spinning bioreactors and microfluidic chips) (Lancaster et al. 2013; Kim et al. 2015; Qian et al. 2016; Karzbrun et al. 2018). Another method for improving the diffusion of nutrients and oxygen in COs is to establish vascularization within the organoids. Not only would vascularization increase nutrient diffusion throughout the CO, but it would allow for the addition of a BBB component, which is extremely important in modeling neurovirology and is lacking in brain COs (Miller et al. 2012). As mentioned previously, several attempts at vascularization in organoids have been made through various means and with various degrees of success. Methods such as endothelial cell co-culture and assembloids between vascular organoids and COs have been used with relative levels of success (Pham et al. 2018; Sun et al. 2022). However, these protocols are tedious and complicated and are not yet a reliable and feasible way to generate vascularized COs. Thus, creating reliable and reproducible methods to generate vascularized COs is an area that needs to be explored to generate more accurate models of neuroviral infections, and of the human brain modeling in general. There are also reported variations between COs due to a variety of factors. Donor cell variability due to genetic background and sex differences needs to be further characterized, as they can lead to large differences between different lines of iPSCs (Burrows et al. 2016; Volpato and Webber 2020). Additionally, there can be quite a large batch variability between COs generated from the same line of iPSCs. Batch variability from the same iPSC lines may be a result of handling, source of growth factors, media, equipment, and differences in protocols. Further improvements in protocols and techniques are needed to improve the reliability, reproducibility, and batch variations. Furthermore, new methods for measuring the electrophysiology of COs may be explored through means such as an optimized multi-electrode array. The CO models of the human brain is still a new era of modeling neurotropic viral infections and there is an endless number of possibilities that can and should be explored to improve these systems.

Conclusions

3D brain organoid models are clearly a superior in vitro platform over 2D-cell cultures with their tissue-cell complexities comparable to in vivo conditions. Human brain organoids are able to represent the human brain at the cellular, structural, and developmental levels, allowing researchers to model neurotropic viral infections in ways that were previously not possible. Brain organoids are developed by “unguided protocols,” allowing them to freely organize into the forebrain, midbrain, and hindbrain or “guided protocols” for generating organoids representing specific brain regions of interest (Fig. 1). Over the last decade, many groups of researchers have developed various types of brain organoids and modeled major neurotropic viral infections (Table 1). As we reviewed above, brain organoid models have provided invaluable knowledge towards a better understanding of molecular regulation of neurotropic viral infections and cellular responses. Although they are proven to be a significantly better platform for modeling the brain in vitro, brain organoids also possess some limitations. They are limited in size and prone to cell death at their center due to the limited diffusion of nutrients and oxygen. Improvements with vascularization attempts are in progress with promising outcomes and the biotechnology for 3D modeling of brain organoids is a fast-growing research era. Nonetheless, 3D brain organoids have shown their potential to take the in vitro culture systems to a new level and have allowed for better modeling of neurotropic viral infections.

Table 1 Types of brain organoids used to study various neurotropic viruses
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