P26.08.B LASER-ASSISTED FABRICATION OF AN IN VITRO 3D-ENGINEERED BLOOD-BRAIN/TUMOR BARRIER MODEL

Abstract BACKGROUND Efficient drug delivery to brain tumors is greatly hindered by the blood-brain barrier (BBB). Although different in vitro BBB models have been developed, they do not capture the capillary dimensions, incorporate multiple cell types, feature biomimetic flows, and/or provide sufficient 3D cell-to-cell contact. We aimed to develop a model for the blood-brain/tumor barrier (BBTB) that incorporates these characteristics simultaneously. MATERIALS AND METHODS 3D micro-porous capillary-like structures (µPCs) that mimic the brain capillary dimensions were fabricated using two-photon polymerization (2PP), a 3D light-assisted printing technique that enables precise microscale fabrication. These structures serve as a scaffold for culturing and organizing various BBTB cell types. µPCs are fabricated at the intersection of two channels within a microfluidic slide, facilitating controlled fluid flow inside and outside the µPCs using a pressure-driven pump. Human umbilical vein endothelial cells (HUVECs) and the U87 glioblastoma cell line are utilized. Immunofluorescence staining (using CD31, tubulin, actin, and DAPI) and scanning electron microscopy are performed to assess cell colonization. RESULTS Initially, we cultured endothelial cells on µPCs under static conditions (i.e. without fluid flow). We optimized µPC designs by modifying channel and rod diameters, alignment, and pore sizes to improve structure stability, confocal microscopy visibility, and cell colonization. These experiments resulted in a design that achieved complete endothelial cell coverage. We also confirmed that the µPCs setup allows co-cultures of glioblastoma and endothelial cells. Next, we 3D printed the 2PP-fabricated µPCs within single-channel microfluidic slides that allowed control of internal fluid flow within the µPCs. When endothelial cells were perfused through the µPCs under biomimetic flow rates, after three days of culture, they uniformly covered the surface of µPCs. We also developed a protocol for precise printing of µPCs in 2-channel slides. The first channel enables internal fluid flow within the µPCs, where endothelial cells are cultured via physiological microfluidic flows. The second channel controls the external flow surrounding the µPCs, where astrocytes, pericytes, and glioma cells can be cultured in static conditions. We will use this setup to soon perform co-cultures of endothelial cells, gliomas and pericytes. CONCLUSION We designed a representative model