Fabrication of biomimetic topological micropattern arrays to induce anisotropic/isotropic orientation of fibroblast.

During fibroblast activation, fibroblasts exhibit remarkable heterogeneity characterized by distinct subtypes with unique molecular markers and specialized functions. In cancer and fibrosis, fibroblasts drive extracellular matrix (ECM) remodeling and actin stress fiber contractility, thereby inducing a dysregulated ECM mechanical microenvironment, which in turn sustains their persistent activation. ECM topology is a key characteristic of the mechanical microenvironment. ECM topology refers to ECM fiber orientation, which interacts reciprocally with cellular orientation. In cancer, ECM and cellular orientations shift from random (isotropic) to aligned (anisotropic), whereas in fibrosis, these orientation transitions exhibit distinct patterns across different organs and tissues. Thus, understanding the relationship between fibroblast orientation and its activation and heterogeneity in cancer and fibrosis may facilitate identifying novel therapeutic targets. This protocol is established to fabricate biomimetic topological micropattern array chips with simple operation and cost effectiveness to induce anisotropic/isotropic fibroblast. A method is established to fabricate rectangular (300 × 50 μm) and square (300 × 300 μm) micropattern array chips via microcontact printing, utilizing decellularized ECM as the bioink. Micropattern array chips induce anisotropic/isotropic fibroblasts, with fibroblast orientation analyzed by F-actin staining and Fast Fourier transform analysis. These chips enable in situ immunofluorescence staining and imaging.