Development of a simplified and scalable hydrogel-based method for 3D cell culture

Objective To develop a cost-effective and mechanically robust 3D collagen hydrogel system suitable for pressure-based culture, enabling physiologically relevant in vitro modeling of mechanical stress responses in cells. Methods A rat tail type I collagen-based hydrogel was formulated through optimized component ratios and cast into standard 24-well plates to form uniform gel columns. Endothelial cells was embedded and subjected to 30 mmHg pressure culture for up to 48 h. Gel morphology and fiber architecture were assessed via scanning electron microscopy. Cell viability, proliferation (Ki67 immunostaining), and tube formation ability were evaluated. A custom mechanical compression setup was used to apply and monitor sustained pressure. Results The hydrogel exhibited stable gelation, uniform porosity, and resistance to deformation under mechanical loading. SEM confirmed a consistent nanofiber network, with fiber diameter unaffected by 30 mmHg pressure. After 24-h pressure culture, the gel retained its height and structure. Endothelial cells remained viable but showed reduced proliferation and impaired tube formation under pressure, as indicated by Ki67 staining and angiogenesis assays. Conclusions This 3D collagen hydrogel provides a simple, cost-effective, and scalable alternative to complex bioprinting methods, supporting broader application of 3D cell culture in biomedical research.