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From 2D to 3D In Vitro Cultures

Traditionally, adult and embryonic neurons have been cultured using 2D tissue culture techniques. This reductionist approach has enabled scientists to identify key mechanistic pathways associated with relatively simple phenomena. Using simplified 2D neural cultures has enabled researchers to gain important insights into fundamental aspects of cell biology. However, because animal tissues, and the brain in particular, are extremely complex 3D arrangements of cells and extracellular matrices, 2D cell cultures are unable to fully represent complete tissues. Therefore, certain research questions, such as those relating to the structural development of neural tissues, cannot be addressed using a 2D cell culture system. In order to better model the human brain in vitro, researchers have developed 3D brain models that recapitulate the brain’s structural arrangement. It’s important to note that compared to their established 2D counterparts, 3D cell culture systems are much more heterogeneous and complex, making them more challenging to master and analyze. Often a combination of 2D and 3D cell culture models can be used in tandem to answer research questions.

3D neural culture systems span a continuum of complexity and cellular composition, from relatively simple aggregates to highly organized organoid models, as described in the sections below:

Neurospheres: One of the earliest 3D neural culture systems developed was the neurosphere assay.¹ Since its introduction, it has been widely used as a method to identify neural stem cells (NSCs) from the central nervous system (CNS).^2-5 In the neurosphere assay, NSCs and neural progenitor cells (NPCs) are cultured in the absence of an adherent substrate. Single cells proliferate to form small clusters of cells, known as neurospheres, which grow in suspension. The multipotent cells that make up the neurospheres retain the capacity to differentiate into all of the main neural cell types of the CNS.¹

Neural Aggregates: Neural aggregates These represent an intermediate 3D culture format that bridges pluripotent stem cell differentiation and more complex neural tissue models. Neurosphere-like neural aggregates are formed using pluripotent stem cells, usually by first forming an embryoid body (EB).⁶ EBs are 3D aggregates of pluripotent stem cells that represent an early stage of embryonic development. Forming EBs from human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC) colonies is now a very common starting point in both directed and spontaneous differentiation protocols. In neural differentiation, CNS-type NPCs are generated from hESC/hiPSC cells using EB-based neural induction; however, monolayer-based neural induction protocols are becoming increasingly popular.⁷ The NPCs produced are then usually transitioned to 2D culture for further differentiation into neurons and glial cells. As such, neural aggregates are commonly used to study early neural induction, lineage specification, and cell–cell interactions under controlled 3D conditions.

Neurospheres and neural aggregates are useful 3D cell culture systems that can answer simple research questions; however, these systems are often enriched for neural stem and progenitor cells and typically lack the cellular diversity and structural organization of more advanced neural tissue models. In some applications, neural aggregates are intentionally generated with defined or mixed cell populations, enabling controlled studies of specific cell-type interactions and scalable assay development. To take the 3D culture system further, it is important to not only develop systems that contain the relevant neural cell types in the brain, but also to have them structured in a manner that represents the brain’s in vivo cortical layering. Cortical spheroids (often referred to as regionally patterned neural aggregates) and neural organoids are 3D brain models derived from hPSCs that have been shown to produce structures organized in a similar manner to the brain itself. The complexity of cellular organization and composition is modulated by either omitting or adding exogenous patterning factors. Adding patterning factors results in models of a specific brain region (for example, midbrain or forebrain). Omitting patterning factors results in a heterogeneous structure with areas representing multiple brain regions within one model, known as an unguided organoid.

Unguided Organoids: Up until the publication of Lancaster and Knoblich’s cerebral organoid model,⁸ no one had generated single neural organoids that represented several different brain regions. Unguided organoids, sometimes referred to as cerebral organoids, are cultured under conditions that promote self-organization and self-patterning. The aggregates are embedded in an extracellular matrix (ECM), Corning® Matrigel®, that improves the polarization of the neural progenitors and supports the outgrowth of large neuroepithelial buds. The buds then spontaneously develop into various brain regions in the absence of exogenous patterning factors. These regions contain “‘layers”’ of neurons that resemble the organization of the developing human brain. Culturing cerebral organoids in smaller, individually maintained volumes allows greater control over organoid number and size, reducing variability compared to traditional large-volume bioreactor systems, and improving reproducibility.

Guided Organoids: Sometimes referred to as regionalized or region-specific neural organoids, guided organoids are generated by directing pluripotent stem cells toward a defined neural identity using precisely timed combinations of morphogens and small-molecule inhibitors.¹¹ Unlike unguided organoids, which rely primarily on intrinsic self-organization to produce mixed brain-region identities, guided organoids use patterning cues to restrict fate and promote the emergence of a single, targeted brain region. Early work from Paşca and colleagues established robust, chemically defined protocols for generating cortical spheroids and later cortical organoids that display consistent dorsal forebrain identity and contain progenitor zones, expanding radial glia, and glutamatergic neurons organized in laminar-like arrangements.¹¹ Pașca’s group also developed protocols for generating other region-specific organoids, such as ventral forebrain,^9,11 midbrain,^10,11,12 and spinal cord organoids.^12,13 These guided organoids exhibit predictable developmental trajectories and more mature electrophysiological properties compared to unguided organoids and earlier 3D neural culture models. Importantly, Paşca’s work further demonstrated that these region-specific organoids can be combined to form assembloids, enabling the study of neuro-immune interactions, neuronal migration, synaptic integration, and disease-relevant circuit-level interactions.¹⁴

Neurodevelopmental and Neuropsychiatric Disorders

Neural organoids have been particularly impactful in modeling neurodevelopmental and neuropsychiatric disorders for which disease onset occurs during early brain development and human-specific mechanisms play a central role. In the first paper that described unguided development of cerebral organoids, the researchers used the system to study microcephaly.⁸ The organoids developed from patient-derived iPS cells were smaller, reminiscent of the reduced brain sizes observed in patients with microcephaly. Because of the potential link between the Zika virus and infants with microcephaly,²⁷ research into Zika has increased significantly using neural organoids as a useful tool to study the virus’ effect on brain development. This work established neural organoids as a powerful system for linking genetic mutations to developmental phenotypes.

Subsequent studies leveraged both unguided and guided forebrain organoids to investigate Zika virus-induced microcephaly, revealing preferential infection of neural progenitor cells, impaired neurogenesis, and reduced tissue growth.¹⁰ These findings provided mechanistic insights that would have been difficult to obtain using animal models alone and demonstrate the utility of neural organoids for studying neurotropic infections.

Neuropsychiatric diseases, such as autism spectrum disorder (ASD), schizophrenia, and bipolar disorder, have been notoriously hard to model since they affect cognitive and behavioral traits that are uniquely human.²⁸ Using a 3D cell culture model, guided forebrain organoids were generated from patients with idiopathic ASD.²¹ When the organoids were compared with organoids generated from a patient’s unaffected relatives, the ASD-derived organoids showed an accelerated cell cycle, an increase in synapse numbers, and an overproduction of GABAergic neurons due to an overexpression of FOXG1. The authors speculated that the dysregulated gene expression of FOXG1 could be used as a potential biomarker for ASD in the future.

More broadly, guided organoids and assembloids have enabled investigation of processes such as cell migration and circuit assembly,¹⁴ features that are challenging to study in vivo and in 2D systems. These approaches are increasingly being combined with electrophysiology, single-cell and spatial transcriptomics, and functional imaging to link molecular phenotypes with circuit-level dysfunction.²⁹

Neurodegenerative Diseases

Although neural organoids most closely resemble early developmental stages in the brain, advances in long-term culture, metabolic maturation,³⁰ and cellular diversity have expanded their use in neurodegenerative disease research.³¹

Guided midbrain organoids are now widely used to model Parkinson’s disease,³² recapitulating key features such as dopaminergic neuron vulnerability, α-synuclein pathology, and mitochondrial dysfunction. Similarly, neural organoids and assembloids incorporating astrocytes and microglia have been applied to study Alzheimer’s disease,^33,34 enabling investigation of amyloid-β accumulation, tau pathology, neuroinflammation, and neuron-glia interactions in a human-relevant context. Patient-specific iPSC-derived organoids allow researchers to examine genetic risk factors and disease heterogeneity, supporting mechanistic studies and target validation.³⁴

While challenges remain, including achieving adult-like maturation, neural organoids are increasingly viewed as a complementary platform that bridges the gap between 2D cultures, animal models, and human clinical studies.

Infectious Diseases

Neural organoids are also being explored as human-relevant platforms for the study of infectious diseases. hPSC-derived unguided cerebral organoids have been used to model central nervous system infection by Nipah virus, a highly pathogenic, neurotropic virus. Importantly, this work extends the application of neural organoids beyond traditional neurodevelopmental and disease modeling, demonstrating that these systems can be applied in high-containment settings to address complex, human-specific questions.³⁵ This study highlights the potential of neural organoids as components of new approach methodologies (NAMs) to support medical countermeasure development and reduce reliance on animal testing in high-containment research settings.

Assessing Neurotoxicity and Seizure Liability

Neural organoids have been shown to respond in a dose-dependent manner to both pro-convulsant and anti-epileptic compounds, producing electrophysiological signatures that resemble in vivo brain activity. For example, treatment with seizure-inducing compounds results in sustained, high-frequency network oscillations, while treatment with anti-epileptic drugs suppresses (or alters) these activity patterns in a mechanism-dependent manner. These responses support the use of neural organoids as predictive in vitro systems for evaluating seizure liability and neurotoxicity of putative therapeutics.³⁶

Mechanistic Insights and Molecular Profiling

Beyond functional readouts, neural organoids can also be assayed with transcriptomic and molecular approaches to uncover mechanisms of drug-induced neurotoxicity. Exposure to neurotoxic compounds has been shown to induce widespread changes in gene expression related to ion channel function, synaptic signaling, and neuronal survival pathways. These molecular insights complement electrophysiological data and help link drug-induced functional changes to underlying biological mechanisms, supporting both target validation and safety assessment.³⁷

Advancing Drug Discovery with New Approach Methodologies

Building on these functional and molecular insights, neural organoids are increasingly being used to develop potential drug discovery workflows. Recent work using patient-derived cortical organoids and forebrain assembloids demonstrated that antisense oligonucleotide (ASO) modalities could rescue disease-relevant electrophysiological, molecular, and cellular phenotypes associated with Timothy syndrome, a severe neurodevelopmental disorder. Importantly, this study integrated in vitro organoid assays with in vivo validation using transplanted human organoids, illustrating how organoid-based platforms can support therapeutic discovery in contexts where traditional animal models do not fully capture human disease biology.³⁸