Júlia Vallverdú Ginés, PhD
Lead ScientistCerebral organoids have emerged to address the limitations of both 2D cell cultures, which lack cell-cell and cell-extracellular matrix (ECM) interactions, and animal models, which differ substantially from humans in brain organization, gene expression, and cellular composition.
Since Lancaster et al. first described 3D self-organizing brain structures derived from pluripotent stem cells in 2013, the field has advanced rapidly. Today, cerebral organoids can replicate specific regions of the human brain, model genetic and environmental causes of neurological disease, and serve as platforms for drug screening. More recently, the development of assembloids, systems formed by integrating multiple organoids or cell types, has extended what is achievable in vitro, enabling the study of cell migration, axon guidance, and circuit formation across brain regions.
How Cerebral Organoids Are Generated from Stem Cells to Recapitulate Brain Development
The generation of cerebral organoids begins with pluripotent stem cells, either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), reprogrammed from individual patients. iPSCs have become the preferred source because they preserve the donor’s unique genetic background and sidestep ethical constraints associated with embryonic material.
The process broadly mirrors embryonic brain development. Briefly, stem cells form embryoid bodies, which are transferred to neural induction medium and then embedded in Matrigel, a 3D extracellular matrix. Spinning bioreactors or orbital shakers provide mechanical stimulation and improve nutrient delivery. Over weeks to months, neural progenitor cells self-organize and differentiate into structures sharing architectural and gene expression features with the developing human brain.
Two differentiation strategies exist:
- Unguided protocols allow spontaneous differentiation, producing organoids containing multiple brain regions, well-suited for studying inter-regional interactions but limited in reproducibility.
- Guided protocols add specific growth factors and small molecules, directing differentiation toward a defined regional identity, producing cortical, hippocampal, midbrain, or cerebellar organoids with greater consistency.
Generation of cerebral organoids. Source: Luo D, Xu J, Liu F, Gu Z. Advances and Challenges in Cerebral Organoids Research. Adv NanoBiomed Res. 2024;4(5):2300126.
Types of Cerebral Organoids in Brain Research and Their Structural Limitations
Region-specific organoids include three main types:
- Forebrain organoids, the most extensively studied, replicate the structure of the cerebral cortex and have been applied to neurodevelopmental and neurodegenerative disorders, including Alzheimer’s disease, epilepsy, autism spectrum disorder, and Zika virus infection. Their relevance stems partly from the unique evolutionary expansion of the human neocortex and its involvement in the majority of neurological conditions.
- Midbrain organoids feature spatially organized clusters of dopaminergic neurons with active synapses, alongside astrocytes and oligodendrocytes. They are particularly relevant to Parkinson’s disease, where dopaminergic neurodegeneration in the midbrain is the defining pathological feature.
- Cerebellar organoids model the hindbrain region responsible for motor coordination, balance, and cognitive regulation. The most recent protocols have succeeded in producing cerebellar organoids that include functional Purkinje cells and support the long-term culture of all primary cerebellar cell types, enabling the study of conditions such as medulloblastoma.
Despite the advances in organoid culture, there are still some drawbacks holding the field back. Single-region organoids face structural constraints that limit their translational utility. The human brain is a highly interconnected network, and the function of any region depends on inputs and outputs from others that a single-region model cannot provide. In addition, these models lack key non-neuronal cell types, particularly microglia and oligodendrocytes, which play essential roles in immune response, synaptic pruning, and myelination. Oligodendrocytes and microglia are generally absent, as they derive from a different developmental lineage. Blood vessels are lacking, which means organoids larger than roughly 500 µm develop necrotic cores due to inadequate oxygen and nutrient delivery. This absence also limits waste removal, restricts the establishment of physiological gradients (e.g., oxygen, nutrients, and signaling molecules), and prevents critical vascular-neural interactions that influence tissue maturation, metabolic regulation, and functional relevance.
Modeling Human Brain Development: From Single-Region Organoids to Multi-Region Assembloids
In 2017, Sergio Pasca’s group published a landmark study in Nature describing the assembly of human forebrain spheroids and used them to attribute, for the first time, defects in neuronal migration to Timothy syndrome. This rare genetic condition predisposes people to autism, epilepsy, and cardiac malfunction.
An assembloid is a 3D aggregate formed by the fusion and functional integration of different organoids with each other, or with specialized cell types not generated by standard protocols. Formalized through work in Pasca’s laboratory at Stanford, assembloids solve single-region limitations by promoting specific inter-regional connections in vitro. These constructs allow for the study of neural migration, axon projection, and circuit formation.
Example image of an assembloid modeling human forebrain circuits (Pasca Lab, Stanford University)
The biological rationale is straightforward. During human brain development, neurons are not born in their final location. GABAergic interneurons of the cortex, for example, originate in the ventral forebrain and must migrate long distances to reach their final positions in the cerebral cortex, where they integrate into local circuits. This migration, its spatial cues, its cellular mechanics, and its disruption in disease cannot be studied in a cortical organoid alone, because the source population is absent. By fusing a ventral forebrain organoid with a cortical organoid, forebrain assembloids recreate the migratory journey of these interneurons in vitro.
After years of research, Pasca’s assembloid platform has subsequently enabled preclinical testing of an antisense oligonucleotide (ASO) therapeutic strategy that successfully restored normal migration patterns. They are now trying to find patients carrying the genetic defect causing Timothy syndrome to prepare a clinical trial to test the safety and therapeutic potential of the ASO.
Neuronal Circuit Formation and Functional Connectivity in Cerebral Assembloids
Assembloids provide the conditions for circuit formation by bringing together the cell populations that need to interact. Interestingly, functional neural circuits can be modeled in three-component assembloids, for instance, to create the cortico-spinal-muscle pathway modeling neuromuscular diseases, such as amyotrophic lateral sclerosis (ALS). In these assembloids, muscle fibers contract in response to optogenetic stimulation of the cortical component, proving the ability of motor neurons to regulate muscle activity via neuromuscular junctions.
The practical bottleneck is the assembly step itself. Bringing two or more organoids together requires physically transferring intact 3D structures between wells without disrupting their morphology, a task currently performed manually. Physical disruption during handling damages organoids, compromises viability at the fusion interface, and prevents proper integration. At the scale required for high-throughput screens, manual assembly introduces inconsistencies in timing and handling. We need a system capable of performing gentle, controlled plate-to-plate organoid transfer that preserves 3D integrity throughout, while also enabling the selection of high-quality, sufficiently matured organoids prior to assembly to ensure reproducibility and functional integration.
The MO:BOT was designed with exactly these constraints in mind. Its aspiration mechanics allow individual organoids to be picked up and relocated between wells as discrete intact structures. This plate-to-plate transfer capability transforms the assembly step, currently a manual bottleneck requiring skilled operator intervention for each organoid pair, into a reproducible and standardized workflow applicable across an entire plate in a single run. In addition, our quality control module, the MO:CROSCOPE, enables image-based selection of organoids, ensuring the consistent use of structurally uniform organoids for downstream assembly.
For assembloid-based drug screening and disease modeling, where consistent fusion quality directly determines the reliability of downstream biological readouts, this level of standardization should be a prerequisite.
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