Collagen is the most abundant extracellular matrix protein. The concentrations, structural arrangement, and directionality of collagen depend on the type of tissue. Thick fibril bundles of collagen are observed in most collagenous tissues, including connective tissues, bones, and tendons, indicating that they play a critical role in many cell functions. In this study, we developed a new method to regulate collagen bundling without altering the protein concentration, temperature, or pH by using sodium sulfate to replicate bundled collagen fibrils found in vivo. Microstructure analysis revealed that both the thickness of the fibril bundles and the pore size of the matrix increased with the amount of sodium sulfate. In contrast, there was no significant change in the bulk mechanical stiffness of the collagen matrix. The modified collagen bundle matrix was used to investigate the responses of human cervical cancer cells by mimicking the extracellular environments of a tumor. Compared to the normal collagen matrix, cells on the collagen bundle matrix exhibited significant changes in morphology, with a reduced cell perimeter and aspect ratio. The cell motility, which was analyzed in terms of the speed of migration and mean squared displacement, decreased for the collagen bundle matrix. Additionally, the critical time taken for the peak turning angle to converge to 90° decreased, indicating that the migration direction was regulated by geometric cues provided by collagen bundles rather than by the intrinsic cell persistence. The experimental results imply that collagen bundles play an important role in determining the magnitude and direction in cancer cell migration. The proposed method of extracellular matrix modification can be applied to investigate various cellular behaviors in both physiological and pathological environments.
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