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Meanwhile, other researchers have pre-vascularized translatability of such models to human physiology.
BOs using various co-culture techniques with ECs or Species-specific differences between murine and human
endothelial-like cells to induce vascularization before vasculature can lead to inconsistencies in nutrient delivery,
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transplantation. For example, Shi et al. co-cultured BOs signaling pathways, and organoid maturation, potentially
with HUVECs for several weeks before transplantation compromising the physiological relevance of the model.
164
into immunodeficient mice (Figure 8B). This pre-culturing Furthermore, while the integration of host vasculature can
resulted in enhanced vascular integration and improved support basic organoid functions, the differences between
perfusion within the organoids. 3D reconstruction human and rodent vascular cells may affect organoid
revealed blood vessel structures in the V-Organoid grafts, development, limiting the applicability of such models
with human ECs from the V-Organoids and mouse- for studying human-specific neurological conditions. In
derived ECs from the host forming a cohesive vascular addition, immune compatibility challenges persist even in
network. Functional blood flow was observed in the immunodeficient mouse models, where residual immune
grafts using live two-photon microscopy after injecting responses may affect organoid survival and function.
Alexa Fluor 594-labeled dextran, indicating integration Ethical concerns and biological variability associated with
between the graft and host vascular systems. Compared animal models further complicate data interpretation and
to non-vascularized organoids, V-Organoids exhibited limit the translational applicability of findings to human
earlier angiogenesis after implantation. Similarly, Pham systems. 165
et al. used iPSC-derived ECs to pre-vascularize BOs, These challenges underscore the need for further
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which has led to higher survival rates and more efficient refinement of vascularization strategies to generate fully
vascularization post-transplantation. These approaches humanized vascular systems within BOs. To address
highlight the potential of co-culture systems to promote these issues, we discuss alternative strategies, including
vascularization and improve the physiological relevance of humanized animal models, in which murine hosts
organoids. are engineered to express human vascular endothelial
Advances in genetic engineering have also enabled markers, thereby improving compatibility between the
the prevascularization of BOs. Cakir et al. developed a host and transplanted organoids. Moreover, advances in
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novel approach by overexpressing the transcription factor in vitro vascularization techniques, such as microfluidic
ETV2 in iPSCs, which enabled the generation of functional perfusion systems and co-culture approaches with human
ECs within the BOs. These pre-vascularized organoids, ECs, provide promising alternatives to reduce reliance
when transplanted into mice, integrated with the host’s on murine models. In addition, 3D bioprinting and
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vascular system, supporting long-term organoid survival synthetic scaffolds used to pre-vascularize organoids
and neural development. The V-Organoids showed clear before transplantation show the potential to enhance
MRI contrast and evidence of vascularization compared vascular network formation and reduce species-related
to controls, which exhibited limited host vessel invasion. discrepancies. These advancements aim to provide a
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Dynamic contrast enhancement imaging indicates that more comprehensive understanding of the limitations of
the V-Organoids were vascularized, though the structures murine vasculature in human-specific organoid research
displayed lower permeability than the adjacent muscle and offer methods to improve the translational relevance
tissue. Immunostaining with human-specific CD31 and of such models. 168
fluorescein isothiocyanate, following dextran perfusion, In summary, the development of vascularized BOs
identified functional blood vessels within the V-Organoids, represents a significant advancement in recapitulating
which were absent in the control organoids. In addition, the complex structure and function of the human brain
the V-Organoids contained significantly more hCD31 in vitro. Through innovative engineering strategies such
+
and fluorescein isothiocyanate-dextran-filled vessels, as microfluidic technologies, 3D bioprinting, and in vivo
highlighting the importance of in vitro-generated vascular transplantation, researchers have made considerable
networks in establishing blood flow and functional progress in overcoming the limitations posed by the
integration with the host vasculature. This method lack of vascularization in BOs’ cultures. Microfluidic
circumvents the need for complex in vitro vascularization platforms have facilitated continuous nutrient and oxygen
protocols, offering more precise control over vascular supply, improving organoid viability and maturation,
network formation and improving the overall function of while 3D bioprinting has enabled the construction of
the organoids.
intricate tissue models with functional vascular networks.
Despite these advancements, in vivo vascularization In vivo transplantation into rodent models has further
of BOs remains associated with several limitations. demonstrated the critical role of host vasculature in
One significant challenge is that the vascular networks supporting long-term organoid survival and functionality.
formed are often of murine origin, which limits the Nevertheless, challenges remain, particularly regarding the

Volume 1 Issue 2 (2025) 19 doi: 10.36922/or.8162