Guiding brain organoid vascularization via intravital reverse 3D bioprinting

Brain damage caused by conditions such as tumors, strokes, and traumatic injuries leads to cognitive dysfunctions, with limited self-repair capacity due to the brain’s unfavorable microenvironment. Human brain organoids (hBOs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising alternative for neural regeneration. However, their clinical application is still limited by challenges such as uncontrolled growth, aberrant synaptic connections, and insufficient vascularization, which restricts nutrient supply and cellular viability. This thesis focuses on optimizing vascularization guidance for transplanted hBOs using intravital reverse 3D bioprinting, a technique that enables controlled scaffold formation within living brain tissue. A photosensitive biomaterial based on 7-carboxymethoxy-4-methylcoumarin (CMMC) conjugated to hyaluronic acid (HA) was developed to create a tunable biomaterial scaffold capable of crosslinking and selectively de-crosslinking upon exposure to specific light stimuli. This biomaterial was evaluated for its biocompatibility, biodegradation, printing precision, depth-specific targeting, and vascularization capabilities within a murine model of cortical brain injury. Live/Dead staining assays confirmed that CMMC-HA maintains biocompatibility with neural stem cells and brain organoids, supporting their viability and growth. In vivo implantation studies revealed no induction of neuroinflammation, with a possible trend toward anti-inflammatory effects. The biomaterial exhibited controlled biodegradation, maintaining its structural integrity for at least two weeks—an essential period for organoid integration. Precision assessments both in vitro and in vivo demonstrated high printing accuracy (∼90%) and a resolution of 2 µm, ensuring reliable microchannel formation. The ability to print microchannels at targeted cortical depths (up to 600 µm) was also validated, showing successful microchannel formation at targeted depths. Channels printed at deeper levels remained intact, whereas surface-printed structures exhibited minor swelling. Finally, evaluation of the capability of intravital reverse 3D bioprinting to guide vascularisation demonstrated that 10 µm-wide microchannels successfully guided endothelial cell infiltration and the formation of functional blood vessels, while smaller 1 µm channels remained unoccupied, indicating the ability to selectively direct vascular integration. These findings establish intravital reverse 3D bioprinting as a promising strategy for guiding the integration of hBOs into host brain tissue, addressing vascularization challenges and enabling precise neural circuit reconstruction. This work advances the potential of bioengineered approaches for brain repair and regenerative medicine, offering a foundation for future in vivo applications in cortical injury treatment and neuroengineering.