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tools to replicate neuronal differentiation and assess the neuronal subtype composition of specific brain
regions. Moreover, they are widely used to investigate neural migration and establishment of connective
neural circuits reflecting brain development. These processes are essential for neurodevelopmental disorders
including DEEs, which are very challenging to capture and investigate in 2D in vitro models or in in vivo
models, which often times show species-specific differences.

A successful example of the application of 3D cerebral organoids in neurodevelopmental disorders is autism
spectrum disorder, where cerebral organoids have revealed an accelerated cell cycle, increased number
of synapses and an overrepresentation of GABAergic inhibitory neurons caused by overexpression of
[69]
FOXG1 . Another disorder that was modeled with cerebral organoids is Miller-Dieker syndrome (MDS).
MDS is caused by a deletion at 17p13.3 and characterized by severe intellectual disability, intractable epilepsy
and lissencephaly. MDS cerebral organoids revealed a mitotic defect in outer radial glia, which are critical
[70]
for human neocortical expansion . These examples provide evidence that organoids can be very useful
in understanding the pathology behind brain disorders, but the usefulness of organoids in large-scale
drug efficacy tests is still challenging due to the variability of neuronal subtypes, organoid size and limited
penetration of compounds into organoids due to the absence of vascularization.

Currently, some of the most promising research studies combine the fusion of cortical organoids enriched in
glutamatergic neurons and sub-pallium organoids enriched in GABAergic neurons. These fused organoids
show the development of GABAergic neural progenitors and their integration in glutamatergic organoids.
Most importantly, in this system, GABAergic interneurons can functionally integrate with glutamatergic
neurons to form an electrophysiologically active network [71,72] .

A pioneer study in the field of organoids demonstrated that organoids generated from patients with primary
microcephaly showed morphological and cellular defects similar to those observed in the postmortem
brains of patients with microcephaly. These defects included abnormal migration and proliferation of neural
progenitors, which resulted in smaller organoids compared to controls, reflecting the in vivo situation in
[73]
microcephaly . Even though recent advancements in organoid technologies have made it possible to
[74]
standardize organoid growth , there are still size limitations implemented due to the development of a
necrotic core in larger sized organoids. Those necrotic cores are caused by a switch from a proliferative neural
progenitors to terminally differentiated neurons and impaired diffusion of oxygen and nutrients to the core
along with hampered metabolite exchange. Those are now actively addressed via, for example, bioprinting
attempts where endothelial cells are introduced via extrusion or droplet deposition of cells suspended in
biocompatible gel in a patterned manner, sacrificial networks where specific matrices are used to seed neural
progenitors and endothelial cells in the generated tubular structures, or more classical approaches of growing
cerebral organoids in co-culture with endothelial cells, which will cause angiogenic sprouting from the
[75]
endothelial cells and infiltration of the cerebral organoids . Even though these are significantly increasing
the complexity of working with cerebral organoids, vascularization would be a leap forward to generate more
in vivo-like conditions. In the case of epilepsy studies, the functional analysis of changes in excitability is
crucial. Here, major improvements have been made in applying optogenetics and MEA electrophysiology.
Furthermore, next-generation imaging technologies can monitor neural activities in live imaging during the
high throughput drug screening of antiepileptic drugs [24,76] . Despite the mentioned advantages, organoids are
also associated with disadvantages including being time-consuming and having low reproducibility between
experiments, and compound screens are extremely challenging in this system.

Consequently, all in vitro models need to be substantiated by relevant in vivo models to understand complex
pharmacological processes and disease phenotypes. Several preclinical animal models for epilepsy have
been used to develop potential treatment strategies. Unfortunately, these models have failed to translate into
successful clinical trials. This is in part due to the critical differences in gene expression, protein function