Forming a Precise and Reproducible Hypoxic Tumor Microenvironment in Glioblastoma by High Cell Density Bioprinting

Glioblastoma (GBM) is the most common and malignant brain tumor, characterized by its highly aggressive and rapidly proliferative behavior. In this study, we developed a high throughput GBM model with a cell density modulated hypoxic niche to investigate the important role of hypoxia in shaping GBM progression and therapeutic response. Harnessing the precise control over materials using digital light processing (DLP) bioprinting, we fabricated GBM constructs with tunable cell densities in gelatin methacrylate (GelMA), a photopolymerizable hydrogel that mimics the extracellular matrix. High cell density (HCD) constructs gave rise to a hypoxic microenvironment, allowing us to study natural hypoxia-driven adaptations, including ROS signaling, migration patterns, and altered metabolic pathways. The major hypoxia pathway, hypoxia inducible factor (HIF-1α), was significantly enriched by 15-fold in the HCD condition compared to its base condition. Following this, we explored cellular response to drug treatment using standard-of-care GBM therapies to validate the hypoxic niche. These data show HCD model provides a more robust and Temozolomide-resistant environment compared to spheroids and low density conditions. Our findings demonstrate that DLP bioprinting provides a precise and reproducible platform for modeling GBM physiology and highlight its potential for high throughput drug screening in vitro.