(C-90) Understanding Vascular Endothelial Cell Behavior Using a Mechanical Strain Gradient Generated by an Electromagnetic Stretching Device

Introduction:: Mechanical stretch plays a critical role in the human body with its importance to the function and homeostasis of cells, tissues, and organs. For example, endothelial cells lining the vascular system undergo physiological cyclic mechanical deformation due to pulsatile blood pressure and circulating blood flow. Deviation from physiological mechanical stretch can alter the endothelial cell phenotype and function, which can lead to pathological changes and result in cardiovascular diseases such as atherosclerosis and myocardial infarction.¹ It is thus essential to understand the role of mechanical stretch in the normal function of cells, tissue, and organs, and human disease progression. However, most of current mechanical stretch platforms are hindered by a fixed strain and frequency, non-uniform strain, or require direct physical contact with the cells, all unfit for the observation of cell behavior in the pathophysiological range. Therefore, there is a need for pathophysiologically relevant, effective in vitro models to study the role of mechanical stretch in cell regulation. We have developed a simple, cost-effective, electromagnetically driven 1-D stretching device which can provide strain gradients of pathophysiological relevance to PDMS membrane. Human cerebral microvascular endothelial cells (hCMECs) grown on the membrane in the device responded differently to the various strains along the gradient.


Materials and Methods:: The stretching device consisted of a stretching part and an electromagnetic actuator. The stretching part was comprised of several components, including a track, clamps, a container, and multiple dowels (Figure 1). All these components were 3-D printed using a Form 3 3-D printer (Formlabs, Somerville, MA, USA). The stretchable membrane was made of PDMS (Sylgard 184, Dow Corning, Midland, MI, USA). After spin-coating a mixture of PDMS resin and curing agent (10:1.05, w/w) at 300 rpm for 1 minute on a plastic film, a glass coverslip was placed on the PDMS membrane with one end touching the plastic film and another end elevated by two identical glass coverslips. After curing at 75 °C for 1 hour, the PDMS membrane with thickness gradient was cut and mounted onto the stretching device between the fixed and moving components.

The hCMEC cell line (hCMEC/D3; Cat#: CLU-512, Cedarlane, Burlington, NC, USA) were seeded on the PDMS membrane and cultured under the static condition for 4 hours, and then subjected to the mechanical stretch at an overall strain of 15% and a frequency of 1 Hz continuously for 16 hours. The cells were then fixed for immunofluorescence staining with Alexa Fluor 488 phalloidin (Life Technologies, Carlsbad, CA, USA). The nuclei were stained with DAPI (Life Technologies). The fluorescent images were taken by using a Nikon Ti Eclipse Fluorescence Microscope (Nikon, Melville, NY, USA). The orientation and elongation of the nuclei were analyzed using ImageJ.


Results, Conclusions, and Discussions:: Results

The electromagnetically driven 1-D stretching device encompassed a stretching part (moving component, fixed component, membrane, and track), a container, and an electromagnetic actuator as shown in Figure 1. The moving and fixed components were both connected to the track. The fixed component was connected to the base while the moving component was slid onto the track, with the ability to freely move with little friction acting upon it. The PDMS membrane was secured on the device between the upper and lower pieces of the fixed and moving components. A rectangular piece of metal was attached onto the moving component as it was attracted by the electromagnet and therefore pulling the moving component towards the magnet with the disturbing force and being pulled back to the original position by the membrane’s restoring force, creating uniform oscillations.

Upon mechanical stretching, the strain rate ranging from 2% to 25% from the thick end to the thin end, covered both physiological and pathological vascular stretch ranges in vivo. At a smaller strain in region 1, nuclear orientation angles were mostly in the 30 - 90° range with a majority of cells in the 30 - 45° range. As the strain increased with a decrease in the membrane thickness in regions 2 and 3, the cells displayed a stronger nuclear orientation perpendicular to the stretch direction. However, the trend became less significant in regions 4 and 5. It was speculated that regions 4 and 5 were overstretched, likely entering the pathogenic range of mechanical stretch (e.g., hypertension). At these regions, the cell and nuclear orientations became more randomly distributed, similar to the situation in region 1 (Figure 2).

Conclusion

This novel electromagnetically driven 1-D stretching device provides a simple, versatile, cost-effective, pathophysiologically relevant platform that enables us to study the effects of mechanical strain, both normal and abnormal, on cells and tissues.


Acknowledgements (Optional): :

References (Optional): : 1. Jufri, N. F.; Mohamedali, A.; Avolio, A.; Baker, M. S., Mechanical stretch: physiological and pathological implications for human vascular endothelial cells. Vasc Cell 2015, 7, 8.