Independent Tuning of Stiffness and Pore Size in 3D Rat Tail Collagen I Matrices

The interplay between the extracellular matrix (ECM) mechanical properties and the tumor microenvironment is increasingly recognized as a critical factor in cancer progression. Three-dimensional (3D) culture systems have emerged as essential platforms for in-vitro cell-based applications, offering microenvironments that are more physiologically relevant compared to traditional two-dimensional (2D) cultures. However, independently controlling the topological and mechanical features of 3D matrices remains challenging due to the interdependence of these parameters. In this study, we demonstrate a method for independently tuning pore size and stiffness in collagen I (Coll I) networks and examine their effects on breast cancer and epithelial cell morphology and cluster formation. Collagen concentration was used to modulate bulk stiffness, while polymerization temperature was adjusted to control pore size. Using this approach, we developed a 3D Coll I matrix with tuned stiffnesses from 80, 228 and 360 Pa while simultaneously holding pore size constant (2.5 μm). Similarly, we developed a low- (1.5 mg/mL) and high- (3.5 mg/mL) concentration collagen hydrogel with varying pore sizes from 2.5 μm to 3.1 μm and 2.0 μm to 2.4 μm, respectively, without altering stiffness (80 Pa and 350 Pa). Integrating a breast epithelial cell line, MCF-10A, and metastatic breast cancer cell line, MDA-MB-231, we demonstrate matrix stiffness and pore size independently and differentially regulate cell morphology and cluster formation. Our results establish a robust method for decoupling stiffness and pore size in Coll I matrices enabling more precise investigations into how ECM mechanical properties influence metastatic and epithelial cell behavior.