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 neovasculature associated with organoids.

Exploring potential use of this vascularized model for application of intravenous nano medicine. Additionally, we aim to evaluate the distribution of these nano medicines in the human central nervous system. To begin, derive brain organoids from H9 embryonic stem cells and obtain newly fertilized chicken eggs.

On day one, clean the eggshell surface with 70%ethanol and let it dry. Place a surgical paper adhesive bandage on top of the egg. Using a lamp, determined the thinnest eggshell surface at the upper tip.

Then with a sterile needle, gently puncture the eggshell to create air chamber hole without disturbing chick CAM. To protect the embryo from the external environment, cover the air chamber hole with a new bandage and incubate the egg at 38 degrees Celsius. On day seven, remove the bandage and wipe the eggshell surface with 70%ethanol.

Then place a new, bigger bandage covering the extension. Gently puncture the eggshell to enlarge the hole into a two centimeter diameter grafting window. Using tweezers, remove the edges of the eggshell window and peel off the bandage to take off the remaining dust and eggshell pieces.

Using a sterile wide gauge tip, collect the organoid in 50 microliters of PBS and carefully transfer it to the CAM opposite to the location of the embryo. Add a seven microliter drop of matrigel around the organoid. Seal the window using parafilm and attach it to the shell with an adhesive bandage.

Incubate the transplant for CAM differentiation. After five days, remove the adhesive bandage. Using scissors and fine tweezers, enlarge the window to facilitate sample harvest.

Fix it with 4%paraform aldehyde for two minutes, and collect a 1.5 square centimeter section of the CAM containing the organoid. To confirm successful grafting, perform hematoxylin and eosin staining of 20 micrometer thick tissue slices of CAM with organoid. Hematoxylin and eosin staining of the graft tissue indicated that the organoids continued to mature next to the CAM vessels without any evidence of infiltration of blood vessels.