Microfluidic systems offer controlled microenvironments for cell-to-cell and cell-to-stroma interactions, which have precise physiological, biochemical, and mechanical features. The opti- mization of their conditions to best resemble tumor microenvironments constitutes an experimental modeling challenge, particularly regarding carcinogenesis in the central nervous system (CNS), given the specific features of the blood–brain barrier (BBB). Gel-free 3D microfluidic cell culture systems (gel-free 3D-mFCCSs), including features such as self-production of extracellular matrices, provide significant benefits, including promoting cell–cell communication, interaction, and cell polarity. The proposed microfluidic system consisted of a gel-free culture device inoculated with human brain microvascular endothelial cells (HBEC5i), glioblastoma multiforme cells (U87MG), and astrocytes (ScienCell 1800). The gel-free 3D-mFCCS showed a diffusion coefficient of 4.06 × 10 − 9 m 2 · s − 1 , and it reconstructed several features and functional properties that occur at the BBB, such as the vasculo- genic ability of HBEC5i and the high duplication rate of U87MG. The optimized conditions of the gel-free 3D-mFCCS allowed for the determination of cellular proliferation, invasion, and migration, with evidence of both physical and biochemical cellular interactions, as well as the production of pro-inflammatory cytokines. In conclusion, the proposed gel-free 3D-mFCCSs represent a versatile and suitable alternative to microfluidic systems, replicating several features that occur within tumor microenvironments in the CNS. This research contributes to the characterization of microfluidic approaches and could lead to a better understanding of tumor biology and the eventual development of personalized therapies. (PDF) A Versatile Microfluidic Device System that Lacks a Synthetic Extracellular Matrix Recapitulates the Blood-Brain Barrier and Dynamic Tumor Cell Interaction. Available from: https://www.researchgate.net/publication/384802118_A_Versatile_Microfluidic_Device_System_that_Lacks_a_Synthetic_Extracellular_Matrix_Recapitulates_the_Blood-Brain_Barrier_and_Dynamic_Tumor_Cell_Interaction#fullTextFileContent [accessed Oct 10 2024].