Anisotropic microarchitecture owing to extracellular matrix (ECM) fiber orientation is ubiquitous in mammalian tissues. The ECM fiber orientation leads to physical anisotropy, i.e., direction-dependent variation in physical properties (stiffness, topography). Cells respond to ECM anisotropy in 2D by changes in cell shape, cell protrusions, reorganization of actin filaments, and focal adhesions. Cells in vivo often exist in a 3D milieu and are physically confined by ECM (collagen) alignment. Therefore, studying the cell mechanical characteristics under 3D anisotropic confinement will provide vital insights into cellular processes like cell migration and signaling.
In this context, we created a confined, anisotropic collagen-I 3D matrix and investigated the link between matrix anisotropy and cell membrane anisotropy. An array of microchannels (~50 µm channel width) was formed by unidirectionally freezing silk fibroin. A collagen or collagen/cell mixture was polymerized within the silk microchannels, resulting in a confined, anisotropic collagen matrix. MCF-7 cells embedded in these collagen matrices displayed elongated morphology with anisotropic migration. The anisotropic viscoelasticity of collagen was then demonstrated using optical tweezer (OT) microrheology of collagen-bound beads. Notably, the elastic modulus of the collagen matrix is greater along the confinement direction. The OT microrheology of cell membrane-bound beads was performed to determine the cell membrane viscoelasticity. The cell membrane was effectively decoupled from the surrounding collagen matrix using a quenching approach. Under confinement, the cell membrane viscoelasticity was found to be anisotropic, and the cell membrane was softer despite the five times stiffer local environment. Further, OT measurements of actin-depolymerized cells show that actin disruption has less effect on the cell membrane viscoelasticity under confinement. These results indicate that ECM viscoelastic anisotropy correlates with cell migration and cell membrane viscoelastic anisotropy.