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Table 3. Challenges in metabolic studies of brain organoids
Challenge Description Impact Potential solutions References
Oxygen and Larger organoids → limited diffusion Core cell death/altered states → ↓ Organoid size, ↑ media 7,133
nutrient diffusion → hypoxia and necrosis in core skewed metabolic data optimization, ↑ perfusion systems
Cellular Diverse cell types → varying metabolic Mixed metabolic signals → Isolate cell populations or design 60,138,139
heterogeneity demands difficult interpretation cell type-specific models
Glucose-induced Hyperglycemic media → oxidative Alter metabolic profiles → Refine glucose levels, monitor ROS 7,140
stress stress, ↑ ROS, impaired neuronal inconsistent outcomes to reduce stress
differentiation
Enzyme activity Transcriptomics≠enzyme function → Enzymatic activity unmeasured Combine transcriptomics with 7,60,141
lack of functional metabolic insight → incomplete metabolic enzyme activity assays
understanding
Spatial Imaging mass spectrometry → Poor spatial resolution of Develop improved spatial profiling 132,137
metabolomics technically complex; hard to integrate metabolic data and integration tools.
with transcriptomics
Abbreviation: ROS: Reactive oxygen species.

these hurdles, researchers can enhance the physiological necessitating the development of faster alternatives such as
relevance of brain organoids, improving their utility for voltage imaging.
studying human brain development and neurological Voltage imaging provides real-time monitoring of
diseases. neuronal membrane potential across networks using
4.3. Advanced functional imaging and voltage-sensitive dyes (VSDs) and GEVIs. 154,167 VSDs offer
electrophysiology in brain organoids: Unraveling high-speed, network-wide activity measurements but can
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neural circuit maturation and disease mechanisms be invasive and susceptible to dye bleaching, whereas
GEVIs provide non-invasive, long-term expression in
Brain organoids serve as in vitro models for NDDs, specific neuronal populations but have slow kinetics
replicating key aspects of human brain development. 15,52 and limited spatial resolution. 167,169,170 These approaches
Their functional activity is assessed using patch-clamp enhance electrophysiological studies, dynamic neuronal
electrophysiology, calcium imaging, MEAs, and voltage activity analysis, and network dynamics investigations.
imaging, each offering unique insights into neuronal
maturation and disease mechanisms (Table 4). 7,142 MEAs facilitate high-throughput, long-term monitoring
of extracellular voltage fluctuations, capturing network-
Patch-clamp electrophysiology remains the gold standard wide electrical events in real-time. 2D MEAs are effective
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for single-cell analysis, providing precise control over for studying network dynamics but lack the complexity of
membrane potential and detailed measurements of synaptic 3D systems. Advances in 3D MEA technology enhance
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responses. 17,163 This technique can be applied to whole spatial and temporal resolution, allowing for more
mount, sliced, or dissociated organoids, each with distinct comprehensive analysis of neuronal network formation
advantages and limitations. Whole-mount organoids offer and activity in organoids. Integration with optogenetics
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a comprehensive view of neuronal activity but are limited enables targeted neuronal stimulation, improving the study
to surface neurons, while sliced and dissociated organoids of specific cell types in neural circuits. Traditional MEA
143
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provide access to deeper structures at the expense of 3D systems, initially designed for 2D cultures, face challenges
integrity. Patch-seq, which integrates patch-clamp with in fully capturing 3D organoid complexity, prompting
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scRNA-seq, has further advanced the understanding of the development of specialized platforms for improved
molecular mechanisms underlying neuronal activity. electrode placement and recording.
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However, patch-clamp remains labor-intensive, requires Fluorescence imaging techniques such as whole-
specialized training, and has limited throughput.
organoid and live-cell imaging enable non-invasive, long-
Calcium imaging enables non-invasive, high-throughput term observation of cellular and network activity. 157,158
monitoring of neuronal network activity using genetically Whole-organoid imaging captures morphological and
encoded calcium indicators (GECIs) such as GCaMP. This functional changes but are limited to surface measurements,
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method provides valuable insights into synaptic plasticity while live-cell imaging provides high temporal resolution
and network maturation, particularly when combined with for tracking dynamic processes such as migration and
optogenetics for precise neuronal control. Despite its differentiation, though it requires advanced microscopy
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advantages for long-term studies, calcium imaging has slow and can cause phototoxicity. In vivo imaging, particularly
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kinetics that limit the resolution of rapid neuronal events, two-photon microscopy, allows for high-resolution, deep-

Volume 1 Issue 3 (2025) 9 doi: 10.36922/OR025100010