Fig. 3. Fibrin brain-mimicking 3D-bioink integrated with 3D engineered printed perfusable vascular network.

(A) Schematic illustration of the 3D-bioprinting model multistage process. (B) 3D-printed Pluronic-based vascular bioink (in cyan) on top of 3D-printed layers of fibrin 3D GB-stroma bioink (in white). (C) 3D-bioprinted vascularized GB model sealed in a metal frame showing the complete perfusion chip. (D) The vascularized 3D-bioprinted GB model is connected to a peristaltic pump through a tubing system, placed in a designated incubator. (E) Tiled Z-stack confocal microscopy images of the 3D-printed penta-culture vascularized GB model. Blood vessels are lined with iRFP-labeled hPericytes (in cyan) together with mCherry-labeled HUVEC (in red) (10⁷ cells/ml; 4:1 ratio) and surrounded by azurite-labeled PD-GB4 (in blue), GFP-labeled hAstro (in green), and nonlabeled hMG (2.1 × 10⁶ cells/ml; 1:1:0.1 ratio). The dashed box represents a coronal cross-sectional plane of the vessel. (F) Fluorescence microscopy images of the 3D-bioprinted vascularized GB model before (top) and after (bottom) perfusion of 70-kDa dextran-FITC. The 3D-bioprinted model is composed of a fluorescently labeled vascular network (mCherry-labeled HUVEC and iRFP-labeled hPericytes) surrounded by nonlabeled GB-bioink (hAstro, PD-GB4, and hMG).