Two-photon decrosslinked hydrogel microchannels for guiding cell invasion within brain organoids

Brain organoids are in vitro 3D models created by the aggregation of pluripotent stem cells, which can self-organize under specific conditions. These models can partially resemble interactions and functionalities found in some regions of human brain. Despite their great potential, brain organoids do not show vessel-like structures found in the brain, which are fundamental for oxygen and nutrients supply to cells in the inner tissues. This lack of vessels can bring to necrosis in the inner regions of the organoid. In order to overcome limitations due to the lack of vascularization, brain organoids can be put into contact with blood vessel organoids, which are able to generate endothelial cells, which are typical of blood vessels’ walls. In our project, a hyaluronic acid hydrogel-based scaffold has been used to study endothelial cell invasion within brain organoids tissues. Our hyaluronic acid has been functionalized with coumarin derivatives, functional groups which are able to undergo cycloaddition when exposed to ultraviolet radiation, allowing the polymer cross-linking. The scaffold shows two wells in which a brain organoid and a blood vessel organoid have been placed. Then, these two wells have been put into connection with microchannels, which can be obtained via hydrogel de- crosslinking, which exploits the cycloreversibility of coumarin derivatives’ linkages following multi-photon near-infrared radiation. De-crosslinked microchannels which can be found in literature biomedical applications are microchannels with squared cross-section. De-crosslinked microchannels of our scaffold have a round cross-section, which better resemble in vitro vascular structures found in vivo. Endothelial cells from the blood vessel organoid have invaded microchannels towards the well in which the brain organoid is placed. Our scaffold will be useful to study different cell migration and invasion phenomena in vitro, using different types of organoids.