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

Area of Science:

• Biomedical Engineering
• Cell Biology
• Mechanobiology

Background:

• Conventional cell culture tools for applying shear stress have not evolved in 30 years.
• Studying endothelial cells under shear stress is crucial for understanding cardiovascular health.

Purpose of the Study:

• To develop a novel pipeline for creating custom cell culture dishes using 3D printing.
• To investigate the effects of shear stress on human aortic endothelial cells and PIEZO1 channel.
• To establish new microscopy protocols for mechanically stimulated cells.

Main Methods:

• Utilized 3D printing technology to fabricate custom cell culture dishes and well plates.
• Employed direct 3D printing and reversible bonding of ACLAR film.
• Cultured human aortic endothelial cells under flow conditions on custom-designed platforms.
• Analyzed cellular responses including extracellular signal-regulated kinase activation and PIEZO1 N-linked glycosylation.
• Developed scanning and transmission electron microscopy protocols for mechanically stimulated cells.

Main Results:

• Custom 3D-printed chambers successfully aligned human aortic endothelial cells under flow.
• Demonstrated shear stress- and time-dependent activation of extracellular signal-regulated kinase.
• Showed shear stress regulates PIEZO1 mechanosensitive ion channel's post-translational modification (N-linked glycosylation).
• Established the first electron microscopy protocol for cells stimulated on an orbital shaker.

Conclusions:

• The 3D printing pipeline offers a versatile, cost-effective method for creating custom labware for cell culture.
• This methodology enhances the study of endothelial mechanobiology by enabling high-throughput analysis of cellular responses to shear stress.
• The findings provide new insights into PIEZO1 channel regulation by shear stress, relevant for cardiovascular research.