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by necrotic regions, which confound volumetric analyses and cellular composition. This intrinsic variability limits
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and cell counting. Addressing these issues requires the robustness of disease modeling and the predictive value
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advances in computational image analysis, such as machine of drug screening efforts.
learning-based segmentation tools, to automate cell type Furthermore, organoids often lack key components of
and structural feature identification. Standardized labeling the in vivo brain microenvironment, including vasculature,
protocols and tissue segmentation methodologies must the blood–brain barrier (BBB), and resident immune cells
also be developed to improve reproducibility and ensure such as perivascular macrophages. 201-203 The absence of these
consistent data interpretation across studies.
features compromises nutrient and oxygen diffusion, limits
Integrating robust cellular markers, standardized the maturation of certain cell types, and fails to recapitulate
imaging techniques, and computational tools will neurovascular interactions critical to brain physiology and
facilitate reproducible and precise brain organoid pathology. Although recent bioengineering approaches aim
analysis. Refinement of these methodologies is essential to incorporate vascular networks and BBB-like structures,
for leveraging organoids as reliable models of human these systems are still in early development and are not yet
brain development and disease modeling. Techniques widely adopted.
such as tissue dehydration and expansion offer detailed Ethical considerations also continue to evolve as brain
imaging at high resolutions and are particularly suitable organoid models become more complex. 204,205 Questions
for various types of organoids and cultured cells. regarding the potential for sentience, the extent of functional
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CUBIC stands out for its minimal distortion and high connectivity, and the use of human-derived stem cells pose
transparency, enabling effective imaging of organoids while unique bioethical challenges. 206,207 As organoids approach
preserving morphology. In addition, iDISCO combines higher levels of structural and functional sophistication, the
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immunolabeling with clearing, allowing for the retention scientific community must establish clear ethical guidelines
of antigenicity, which is vital for developmental biology and to ensure responsible use of this technology.
immunological studies involving organoids. 196,197 However,
the complexity of protocols, particularly for methods like Addressing these challenges will require a concerted
3DISCO, which facilitates the visualization of entire organs, effort to standardize protocols, develop quality control
can pose challenges in both cost and implementation. 198 benchmarks, and integrate missing physiological elements.
Continued dialogue around the ethical implications
To optimize the use of these techniques, careful
consideration of their limitations is necessary. For example, is equally critical for the responsible advancement of
organoid-based neuroscience.
organic solvent-based methods are effective for dense or
lipid-rich tissues but may involve toxicity and extended 6. Engineering advances in brain organoid
processing times. Meanwhile, PARS (enzyme-assisted culture: Innovations in static, microfluidic,
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clearing), although preserving proteins and nucleic
acids, is limited to specific tissue types. Therefore, and vascularized systems
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a comprehensive understanding of each technique’s The development of human brain organoid culture
capabilities and constraints is essential for advancing techniques has made significant strides through the
research in brain organoid studies. integration of various engineering approaches, such as static
culture, microfluidic systems, and vascularization strategies
5.3. Current limitations in brain organoid models: (Table 6). These advancements have reduced structural
Reproducibility, microenvironment, and ethical heterogeneity, enhanced neuronal tissue maturation, and
challenges improved functional outcomes. Recent methodologies
Despite significant methodological advances, brain in brain organoid culture offer notable advantages and
organoid systems face a range of persistent challenges that limitations, with promising future applications.
hinder their full translational and experimental utility. Long-term static culture systems maintain cells
One of the most pressing concerns is the evaluation of under physiological conditions without active nutrient
reproducibility across different laboratories. Variability replenishment or waste removal. Neural-inducing
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in protocols, cell lines, culture conditions, and analysis molecules drive self-organization, leading to the formation
techniques often results in divergent phenotypes and of spheroids that differentiate into cortical neurons
outcomes, complicating the comparison and integration of and non-reactive astrocytes. Low-adsorption culture
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findings across studies. 51,79 plates and U- or V-bottom perforated plates promote
Batch-to-batch variability is another major issue, even 3D self-assembly. Embedding organoids in Matrigel and
when using the same hPSC line. Subtle differences in media transferring them to rotating bioreactors further enhances
7
composition, passage number, or even incubator humidity oxygen diffusion and nutrient distribution. However,
can lead to significant variations in organoid morphology challenges such as necrotic core formation due to inadequate

Volume 1 Issue 3 (2025) 13 doi: 10.36922/OR025100010