A 3D-Printed Pulsatile Shear Stress Platform for Studying Endothelial Cell Mechanobiology.

The tools for subjecting cells to high-magnitude shear stress within conventional cell culture dishes and well plates on an orbital shaking platform have remained unchanged for the past 30 years. Here, we develop a pipeline for creating custom cell culture dishes and well plates of arbitrary size and complexity using 3D printing technology. We describe two methods: direct 3D printing and reversible bonding of ACLAR film to 3D-printed structures. We show that the custom chambers support alignment of human aortic endothelial cells cultured under flow while capturing robust activation of extracellular signal-regulated kinase in a shear stress- and time-dependent manner. We show that shear stress regulates the post-translational modification of the shear-stress-sensitive mechanosensitive ion channel PIEZO1, resulting in an increase in N-linked glycosylation that may be relevant to the channel's ability to sense and respond to shear stress. We also developed the first scanning and transmission electron microscopy protocol compatible with cells mechanically stimulated on an orbital shaker and demonstrated another approach for customizing conventional labware. The simplicity of fabrication, cost-effectiveness of this pipeline, and the ability to process a large number of cells simultaneously for multiple downstream experimental end points mean that the methodology developed here is likely to be of broad utility in the