Decoupling Bulk Stiffness and Pore Size in 3D Rat Tail Collagen I Matrices.

The interplay between extracellular matrix (ECM) mechanics and the tumor microenvironment is increasingly recognized as a key driver of cancer progression. Three-dimensional (3D) collagen I culture systems provide more physiologically relevant environments than traditional 2D cultures, yet seperately controlling the structural and mechanical features of 3D matrices remains challenging due to their inherent interdependence. Here, we present a collagen-based approach to decouple bulk stiffness and pore size by varying collagen concentration and polymerization temperature. Collagen concentration was varied to modulate bulk storage modulus, while polymerization temperature was adjusted to alter pore dimensons at comparable collagen concentrations. Using this approach, we generated 3D Collagen I matrices with bulk storage moduli of approximately 80, 228, and 360 Pa while maintaining a similar median pore sizes near 2.5 µm. Conversely, 1.5 mg/mL collagen, median pore size varied from approximately 2.5 to 3.1 µm while bulk storage modulus ramined near 80 Pa, whereas at 3.5 mg/mL collagen, pore size varied from approximately 2.0 to 2.4 µm while bulk storage modulus remained near 350 Pa. We further evaluated the responses of MCF-10A epithelial cells and MDA-MB-231 metastatic breast cancer cells to matrices spanning these sturactual and mechanical conditions. Although the changes in pore size within each stiffness range were modest, they were associated with measurable and statistical differences in cell morphology. These findings demonstrate that even modest changes in collagen network architecture can produce measurable differences in cellular behvarior under comparable bulk mechanical conditions, highlighitng the importance of considering matrix structure in addition to stiffness when investigating cell-matrix interactions.