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

This study investigates the influence of geometrical variations in volumetrically printed (Vol3DP) structures on the attachment, survival, and organization of cancer cells (143b) and human umbilical vein endothelial cells (HUVECs). We adapted a gelatin methacryloyl (GelMA)–poly(ethylene glycol) diacrylate (Gel-PEG) resin for volumetric bioprinting. Compared to GelMA, Gel-PEG improved printing fidelity and resolution, superior mechanical properties, and reduced swelling. We fabricated disc-like constructs and channel geometries, including straight channels and angular designs of 60°, 90°, and 110° and cultured human umbilical vein endothelial cells (HUVECs) and 143b human osteosarcoma cells, a highly metastatic cell line, for up to 14 days. Using label-free holographic microscopy, we visualized cellular protrusions, important for adhesion and mechanosensing, in real-time and without staining, an advantage for long-term, live-cell analysis in 3D constructs. HUVECs adhered well, expressed CD31, and showed preferential spreading in channels with specific geometrical angles, indicating geometry-sensitive behavior. This is physiologically relevant, as it reflects the native mechanosensitive and alignment behavior of endothelial cells during vascular formation. In contrast, osteosarcoma cells spread uniformly throughout the constructs, formed dense, geometry-independent agglomerates, and exhibited enhanced growth and spreading within the Gel-PEG matrix compared to GelMA. This behavior is consistent with the aggressive and geometry-insensitive nature of metastatic tumor cells. These findings highlight Gel-PEG’s utility for generating stable, biomimetic 3D environments and demonstrate the application of holographic microscopy for assessing cell– material interactions within volumetrically bioprinted constructs, underscoring the potential of this approach for developing vascularized models and studying mechanobiological responses in engineered tissues.