Airway smooth muscle cells (ASMCs) exist in a form of helical winding bundles within the bronchial airway wall. Such tubular tissue provides cells with considerable curvature as a physical constraint, which is widely thought as an important determinant of cell behaviors. However, this process is difficult to mimic in the conventional planar cell culture system. Here, we report a method to develop chips with cell-scale tubular (concave and convex) surfaces from fused deposition modeling 3D printing to explore how ASMCs adapt to the cylindrical curvature for morphogenesis and function. Results showed that ASMCs self-organized into two distinctively different patterns of orientation on the concave and convex surfaces, eventually aligning either invariably perpendicular to the cylinder axis on the concave surface or curvature-dependently angled on the convex surface. Such oriented alignments of the ASMCs were maintained even when the cells were in dynamic movement during migration and spreading along the tubular surfaces. Furthermore, the ASMCs underwent a phenotype transition on the tubular (both concave and convex) surfaces, significantly reducing contractility as compared to ASMCs cultured on a flat surface, which was reflected in the changes of proliferation, migration and gene expression of contractile biomarkers. Taken together, our study revealed a curvature-induced pattern formation and functional modulation of ASMCs in vitro, which is not only important to better understanding airway smooth muscle pathophysiology, but may also be useful in the development of new techniques for airway disease diagnosis and therapy such as engineering airway tissues or organoids.

Airway hyperresponsiveness (AHR) is the cardinal character of asthma, which involves the biomechanical properties such as cell stiffness and traction force of airway smooth muscle cells (ASMCs). Therefore, these biomechanical properties comprise logical targets of therapy. β2-adrenergic agonist is currently the mainstream drug to target ASMCs in clinical practice for treating asthma. However, this drug is known for side effects such as desensitization and non-responsiveness in some patients. Therefore, it is desirable to search for new drug agents to be alternative of β2-adrenergic agonist. In this context, sanguinarine, a natural product derived from plants such as bloodroots, that has been reported to relax gut smooth muscle emerges as a potential candidate. So far, it is unknown whether sanguinarine can regulate the biomechanical properties of ASMCs and reactivity of ASMCs to irritants. Thus, we tested the hypothesis that sanguinarine reduce the contractile potentials of ASMCs in culture. To do so, the primary cultured rat ASMCs were first treated with different concentration of sanguinarine. Then, cell stiffness, traction force, fiber distribution, and calcium signaling of the ASMCs were evaluated by optical magnetic twisting cytometry, Fourier transform traction microscopy, atomic force microscopy, and Fluo-4/AM based fluorescence confocal scanning microscopy, respectively. The results indicated that sanguinarine (0.05 and 0.5 µmol/L) significantly decreased cell stiffness and traction force, inhibited reactivity of ASMCs to histamine, and disrupted the fiber structures in ASMCs in dose-dependent manner. These findings establish that sanguinarine can indeed change the biomechanical properties of ASMCs and may be used to treat AHR in asthma.

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