We thank Dr. Alc&#225;ntara and Dr. Ortega from UB and the rest of the members in Dr. Acosta&#39;s lab for the insightful discussions. S.A. is Serra-Hunter fellow assistant professor from the Generalitat de Catalunya at Universitat de Barcelona.

The White Leghorn chicken (Gallus gallus) embryos were treated by following the Guide for the Care and Use of Laboratory Animals from the Institute of Laboratory Animals Resources, Commission of Life Sciences, National Research Council, USA, and the experiments were approved by the Council for Care and Use of Experimental Animals from the University of Barcelona.
1. Non-guided brain organoid preparation
<ol>
	<li>Maintain H9 human embryonic stem cells (hESCs) in mTE...

Grafting Human Brain Organoids into the Chorioallantoic Membrane of the Chick Embryo

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+vascularized+brain+organoids+to+study+neurovascular+interactions.">Generation of vascularized brain organoids to study neurovascular interactions.</a> Elife. 11, e76707 (2022).</li><li>Paredes, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte+precursor+cell+specification+is+regulated+by+bidirectional+neural+progenitor-endothelial+cell+crosstalk.">Oligodendrocyte precursor cell specification is regulated by bidirectional neural progenitor-endothelial cell crosstalk.</a> Nat Neurosci. 24 (4), 478-488 (2021).</li><li>Matsui, T. K., Tsuru, Y., Hasegawa, K., Kuwako, K. 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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+vascularized+brain+organoids.">Generation of human vascularized brain organoids.</a> Neuroreport. 29 (7), 588-593 (2018).</li><li>Cakir, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+human+brain+organoids+with+a+functional+vascular-like+system.">Engineering of human brain organoids with a functional vascular-like system.</a> Nat Methods. 16 (11), 1169-1175 (2019).</li><li>Shi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+human+cortical+organoids+(vorganoids)+model+cortical+development+in+vivo.">Vascularized human cortical organoids (vorganoids) model cortical development in vivo.</a> PLoS Biol. 18 (5), e3000705 (2020).</li><li>Mansour, A. 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C., Jaff, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+induce+blood-brain+barrier+properties+in+endothelial+cells.">Astrocytes induce blood-brain barrier properties in endothelial cells.</a> Nature. 325 (6101), 253-257 (1987).</li><li>Janzer, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier:+Cellular+basis.">The blood-brain barrier: Cellular basis.</a> J Inherit Metab Dis. 16 (4), 639-647 (1993).</li><li>Lobrinus, J. A., Juillerat-Jeanneret, L., Darekar, P., Schlosshauer, B., Janzer, R. 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In this study, we describe a detailed protocol with numerous key steps that provide favorable growth and development of human brain organoids upon grafting without perturbing the survival of the chicken embryos. We recommended the use of sterile needles to puncture the air chamber of the egg after 24 h of incubation (day 1). Additionally, we also tried to make the puncture at day 4 (after checking through the eggshell by light to test the development of the vasculature to be sure that we were working only with healthy em...

Human brain organoids are an emerging technique that allows us to recapitulate the early development of the human brain in vitro1,2,3. Nevertheless, one of the major limitations of this model is the lack of vascularization, which plays indispensable roles not only in brain homeostasis but also in brain development4. In addition to the delivery of oxygen and nutrients, accumulating evidence suggests that the vascular system of the brain regulates neural differentiation, migration, and synaptogenesis during development5,6. Therefore, there is an urgent need to establish reliable models that can provide the missing vascular signaling and structure to brain organoids, enhancing the complexity of human brain organoid generation7.
Among the proposed methods for vascularization, two main streamlines can be considered: organoid engrafting into a living organism and purely in vitro technologies co-culturing endothelial cells and neural cells8,9,10,11,12. Intracerebral transplantation in mice is costly and time-consuming, making other technologies relevant for simpler models. The chick chorioallantoic membrane (CAM) assay has been used extensively to study angiogenesis13,14,15. In the last decade, several groups have successfully engrafted different types of organoids, including kidney16,17, cardiac18, and tumor organoids19,20, into CAMs. Nevertheless, little is known about the efficacy, toxicity/rejection, physiological effect, and methods to engraft human brain organoids into the CAM. Another interesting and yet unexplored aspect is the formation of a chimeric blood-brain barrier (BBB) between the CAM and the organoid astrocytic interface. Previous pioneering work suggested the putative feasibility of generating a BBB in the CAM by transplanting astrocytes and astrocyte-conditioned medium21,22,23. However, mature astrocytes seem to be unable to achieve this24,25. Thus, the astrocyte-induced formation of the BBB remains debatable, and transplanting human brain organoids would allow us to shed light on this controversy.
This video article describes a protocol for an in ovo human brain organoid transplant into CAM that promotes growth, improvement, and vascularization, resulting in organoids that encompass histologically compatible BBB elements. Here, we present a protocol ensuring the survival of the chicken embryo and report on the permissivity of the CAM to sustain brain organoid growth.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-TUBB3 [Tuj1], mouse&#160;</td>
			<td>BioLegend</td>
			<td>801201</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Anti-GFAP, rabbit</td>
			<td>GeneTex</td>
			<td>GTX108711</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Anti-rabbit AlexaFluor 488, goat.</td>
			<td>Invitrogen</td>
			<td>A-21206</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Anti-mouse AlexaFluor 594, goat</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-585-150</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Fertilized White Leghorn chicken (Gallus gallus) eggs</td>
			<td>Granja Gibert (Cambrils, Spain)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>1:10,000</td>
		</tr>
		<tr>
			<td>DPX</td>
			<td>Sigma</td>
			<td>100579</td>
			<td>xylene-based mounting medium&#160;</td>
		</tr>
		<tr>
			<td>Gentle Dissociation Solution</td>
			<td>CreativeBiolabs</td>
			<td>ITS-0622-YT187</td>
			<td>cell dissociation solution</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 4-88 mounting media</td>
			<td>Merk</td>
			<td>81381</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towel, lab-grade</td>
			<td>Sigma-Aldrich</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y27632</td>
			<td>Millipore</td>
			<td>SCM075</td>
			<td>10 nM</td>
		</tr>
		<tr>
			<td>Sharp-Point Surgical Scissors</td>
			<td>VWR</td>
			<td>470106-340</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Adhesion Microscope Slides</td>
			<td>Epredia</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
	</tbody>
</table>

early unguided human brain organoid neurovascular niche modeling into the permissive chick embryo chorioallantoic membrane

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants.
This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed.
No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.

Author Spotlight: Optimizing the Neurovascular Development of Human Brain Organoid in Chick Embryo

Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane

The key questions I aim to address revolve around the mechanisms and processes involving human brain formation in the context of normal development, as well as various pathological states. This includes investigating the factors influencing brain development, such as genetics, environmental, and epigenetic influences. Obtaining brain organoids that closely resemble the adult human brain, including vascularization, blood brain barrier functionality, the presence of microglia and cellular diversity is challenging.Overcoming this is essential for the development of more physiologically relevant brain organoid models that can efficiently mimic the adult human brain to enhance clinical applications. Through our research, we provide optimal conditions for transplanting human brain organoids into chorioallantoic membrane to ensure good development of the microglia. This clinical improvement is central for forthcoming efforts in the field to generate synthetic vascularized environment for brain organoids.The advantage of our protocol compared to other techniques lies in its high effectiveness and cost efficiency. We have achieved a high success rate in transplanting viable organoids using fertile eggs from farm chickens. In the future, our laboratory will focus on conducting functional assessments of neo vasculature associated with organoids, exploring potential use of this vascular design model for application of intravenous nanomedicine.Additionally, we aim to evaluate the biodistribution of these nanomedicines in the human central nervous system.

Selecting the embryo maturation schedule for the transplant 
The experiment begins at D0 when fertilized eggs are incubated at 38 &#176;C and 60% relative humidity. The chorioallantoic membrane (CAM) is a highly vascularized extraembryonic membrane that develops after egg incubation. It is formed by the fusion of the allantois and chorion. At D1, after 24 h of incubation, the air chamber is punctured to prevent the CAM from attaching to the inner shell membrane. Puncturing the air chamber at D1 impr...

Watch this Scientific Journal Video about Author Spotlight: Optimizing the Neurovascular Development of Human Brain Organoid in Chick Embryo at JoVE.com

Here, we present a protocol to engraft human brain organoids at multiple maturation stages into the chick chorioallantoic membrane (CAM). Brain organoids were grown following unguided standardized protocols.

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has ...

early-unguided-human-brain-organoid-neurovascular-niche-modeling-into