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for advancing research in brain development, disease scaffold biocompatibility and optimize fluid dynamics
modeling, and drug discovery. within organoid cultures. Using hydrogel-based bioinks,
complex multicellular structures, including precisely
3.3.2. Application of three-dimensional printing in the positioned ECs and pericytes, can be printed. This approach
vascularization of BOs supports the formation of perfusable vascular networks that

Three-dimensional printing technology has integrated integrate with organoids and promote tissue maturation.
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advanced bioengineering techniques with the self- Salmon et al. demonstrated the potential of 3D-printed
organizing properties of cells to construct bioengineered microfluidic chips, which allowed for the formation
organoids with enhanced reproducibility and improved of organized and perfusable vascular networks within
structural fidelity. This approach leverages the ability to organoids. These networks supported essential nutrient flow
precisely control the spatial arrangement of multiple cell and oxygenation, thereby promoting organoid maturation
types, biomaterials, and bioactive molecules, enabling the and functionality. The 3D-printed chips are customizable,
fabrication of highly complex tissue models, including allowing organoid-specific designs that facilitate
organoids, engineered tissues, and even functional organs. By vascular growth and integration, overcoming limitations
employing 3D bioprinting, it is possible to accurately define associated with conventional polydimethylsiloxane-based
the external and internal geometries, spatial organization, methods. By enhancing nutrient supply, waste removal,
and cellular orientation of tissues, thereby effectively and mechanical fluid flow, these chips support the self-
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mimicking the structural and functional characteristics of organization and development of BOs. The integration of
their in vivo counterparts. This ensures the interconnectivity 3D printing with microfluidics has enabled more efficient
of different regions within the organoids and supports modeling of physiological processes, offering a better
adequate perfusion, which is crucial for tissue development, platform for disease modeling, drug screening, and the
maturation, and repair. Recent applications of 3D printing study of neurodegenerative disorders such as AD and PD.
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in BOs’ research have included the use of printed scaffolds 3.4. In vivo vascularization of BOs
to support organoid formation and maturation. 3D-printed
scaffolds have been used to generate planar BOs that exhibit In vivo vascularization of BOs has emerged as a critical
gyrification. In particular, fibrous scaffolds fabricated using strategy for overcoming the limitations posed by the lack
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electrospinning or cryogen techniques have been shown to of perfusion and nutrient supply in organoid cultures.
enhance neuronal cultures by providing structural support Early efforts to vascularize BOs involved transplantation
as well as improving oxygenation and nutrient delivery to the into host animals, particularly immunodeficient rodents,
tissue. 155,156 In addition, by employing PLGA microfilaments where the host’s vascular system supported the engraftment
as scaffolds, researchers have successfully induced the and integration of the transplanted tissue. Daviaud
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formation of elongated embryoid bodies, leading to well- et al. transplanted hESC-derived COs into the cortex of
defined neuroectoderm and cortical development, with immunodeficient mice, creating an in vivo environment
organized cortical plates and radial structures. 157 that promoted neuronal maturation and vascularization
(Figure 8A). Mansour et al. developed a vascularized
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Three-dimensional bioprinting has also significantly
enhanced the development of vascular networks within model by grafting hESC-derived BOs into the retrosplenial
cortex of immune-deficient, non-obese diabetic-severe
BOs by creating intricate, biocompatible vascular structures combined immunodeficiency mice. Within a week, the
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that closely resemble native vasculature. Xu et al. used mouse blood vessels migrated toward the grafts, and
two-photon polymerization 3D printing to create high- vascular networks were confirmed by dextran dye injection.
resolution meshed vessels with micropores that facilitated Notably, 85.4% of the grafts were vascularized, while non-
the diffusion of nutrients and oxygen, thereby reducing vascularized organoids failed to survive. This observation
hypoxia and apoptosis in the core of BOs (Figure 7B). suggests that blood flow was essential for delivering oxygen
Such vascularized organoids exhibited enhanced growth, and nutrients. Vascularized organoids were larger, exhibited
maturation, and functional integration of different brain reduced apoptosis, and had more mature Fox-3, Rbfox3,
regions when assembled into multi-regional structures, or hexaribonucleotide binding protein-3-positive neurons
supporting complex neurovascular interactions. compared to those cultured in vitro. Subsequently, Revah et
Meanwhile, vascular structures within organoids also al. transplanted COs into the retrosplenial cortex of adult
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promoted dimensional breaking growth and enabled mice. By integrating transparent microelectrodes with two-
the co-culture of various neural regions, such as cortical, photon microscopy, they monitored the organoids over
striatal, and medial ganglionic eminence regions, fostering time. Their results demonstrate the successful integration
cellular migration, projection, and signaling pathways. of the organoids into the mouse brain, with vascularization
In addition to direct organoid fabrication, 3D printing and functional responses to visual stimuli confirmed by
can also be applied to create microfluidic chips that enhance electrophysiological recordings and imaging.

Volume 1 Issue 2 (2025) 17 doi: 10.36922/or.8162