Design of a cone-and-plate device for controlled realistic shear stress stimulation on endothelial cell monolayers

Abstract

Endothelial cells are constantly exposed to blood flow and the resulting frictional force, the wall shear stress, varies in magnitude and direction with time, depending on vasculature geometry. Previous studies have shown that the structure and function of endothelial cells, and ultimately of the vessel wall, are deeply affected by the nature of wall shear stress waveforms. To investigate the in vitro effects of these stimuli, we developed a compact, programmable, realtime operated system based on cone-and-plate geometry, that can be used within a standard cell incubator. To verify the capability to replicate realistic shear stress waveforms, we calculated both analytically and numerically to what extent the system is able to correctly deliver the stimuli defined by the user at plate level. Our results indicate that for radii greater than 25 mm, the shear stress is almost uniform and directly proportional to cone rotation velocity. We further established that using a threshold of 10 Hz of wall shear stress waveform frequency components, oscillating flow conditions can be reproduced on cell monolayer surface. Finally, we verified the capability of the system to perform long-term flow exposure experiments ensuring sterility and cell culture viability on human umbilical vein endothelial cells exposed to unidirectional and oscillating shear stress. In conclusion, the system we developed is a highly dynamic, easy to handle, and able to generate pulsatile and unsteady oscillating wall shear stress waveforms. This system can be used to investigate the effects of realistic stimulations on endothelial cells, similar to those exerted in vivo by blood flow.

A microfluidic chip with gravity‐induced unidirectional flow for perfusion cell culture

“…As a control experiment, we also cultured HUVECs in a chip without the bypass channel, thus giving bidirectional flow with recirculation. Our results show that unidirectional flow was achieved as intended, thus improving the orientation and directionality of the cells, consistent with previous studies . However, there was some degree of orientation observed for cells cultured in the static condition as well.…”

“…Our results show that unidirectional flow was achieved as intended, thus improving the orientation and directionality of the cells, consistent with previous studies. 13,23,35 However, there was some degree of orientation observed for cells cultured in the static condition as well. We speculate that this was because even though we intended a static condition, there was inevitably some degree of natural convection due to several external factors, such as manual handling, vibration of the cell culture incubator, natural convection due to the temperature fluctuation, which could have been facilitated with the channel geometry of the cell culture substrate.…”

“…Exposing cells to athero‐prone waveform, which is inconsistent flow pattern with fluctuations led to increased circularity with cobblestone‐like shapes. On the other hand, exposing cells to athero‐protective waveform, which is more unidirectional, uniform flow pattern, cells showed increased elongation with low circularity . By creating unidirectional and bidirectional flow inside our chip, we effectively recreated athero‐protective and athero‐prone waveform inside our chip, without any complex fluidic design or external device for pumping.…”

“…In particular, vascular endothelial cells are exposed to shear stress from the blood flow, which can change depending on the blood flow direction and consequently affect cell orientation . For example, cells residing at locations with low shear stress or disturbed flow can show weak levels of alignment or elongation . This abnormal alignment or elongation of vascular endothelial cells could be a cause of diseases such as atherosclerosis or intimal hyperplasia .…”

“…23,24 This abnormal alignment or elongation of vascular endothelial cells could be a cause of diseases such as atherosclerosis or intimal hyperplasia. 23 Changes in the morphology of endothelial cells in response to fluidic stimulus have been reported in in vitro systems in several studies, 22,23 and it has been observed that the direction of flow relative to the cytoskeletal axis affect the expression of inflammatory signaling molecules such as nuclear factor-κB and endothelial nitric oxide synthase. 25 Also, various fluidic devices with an aim of creating flow with physiological patterns have been developed, including parallel plate, 26 cone-end plates, 23 and microfluidic chips.…”