Geometrical Designs in Volumetric Bioprinting to Study Cellular Behaviors in Engineered Constructs

This study investigates how geometrical variations in volumetrically printed (Vol3DP) structures influence the attachment, survival, and organization of human umbilical vein endothelial cells (HUVECs) and osteosarcoma cells (143b). A gelatin methacryloyl-poly(ethylene glycol) diacrylate (GelMA-PEGDA) resin was optimized for volumetric bioprinting. Compared to GelMA, Gel-PEG enhanced printing fidelity, mechanical properties, and dimensional stability. Disc-like constructs and channels with straight or angled geometries (60°, 90°, 110°) were fabricated and cultured with both cell types for up to 14 days. Label-free holographic microscopy allowed real-time visualization of cellular protrusions, critical for adhesion and mechanosensing, without staining, enabling long-term live-cell analysis in 3D constructs. HUVECs adhered, expressed CD31, and exhibited geometry-dependent spreading, reflecting their native mechanosensitivity and alignment during vascular morphogenesis. In contrast, 143b cells spread uniformly, formed dense, geometry-independent aggregates, and showed enhanced growth in Gel-PEG compared to GelMA, consistent with their aggressive, metastatic behavior. These findings demonstrate that Gel-PEG provides a stable, biomimetic matrix suitable for high-resolution Vol3DP and that holographic microscopy enables dynamic assessment of cell-material interactions. Together, they underscore the potential of this approach for engineering vascularized tissue models and for studying mechanobiological responses in both endothelial and cancer cell systems.