Engineered microenvironments along with robust quantitative models of cell shape metrology that can decouple the effect of various well-defined cues on a stem cell’s phenotypic response would serve as an illuminating tool for testing mechanistic hypotheses on how stem cell fate is fundamentally regulated. As an experimental testbed to probe the effect of geometrical confinement on cell morphology, poly(ε-caprolactone) (PCL) layered fibrous meshes are fabricated with an in-house melt electrospinning writing system. Gradual confinement states of fibroblasts are demonstrated by seeding primary fibroblasts on defined substrates, including a classical two-dimensional (2D) petri dish and porous 3D fibrous substrates with microarchitectures tunable within a tight cellular dimensional scale window (1–50 μm). To characterize fibroblast confinement, a quantitative 3D confocal fluorescence imaging workflow for 3D cell shape representation is presented. The methodology advanced allows the extraction of cellular and subcellular morphometric features including the number, location, and 3D distance distribution metrics of the shape-bearing focal adhesion proteins.

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