A Y-Shaped Microfluidic Device to Study the Combined Effect of Wall Shear Stress and ATP Signals on Intracellular Calcium Dynamics in Vascular Endothelial Cells

Abstract

The intracellular calcium dynamics in vascular endothelial cells (VECs) in response to wall shear stress (WSS) and/or adenosine triphosphate (ATP) have been commonly regarded as an important factor in regulating VEC function and behavior including proliferation, migration and apoptosis. However, the effects of time-varying ATP signals have been usually neglected in the past investigations in the field of VEC mechanobiology. In order to investigate the combined effects of WSS and dynamic ATP signals on the intracellular calcium dynamic in VECs, a Y-shaped microfluidic device, which can provide the cultured cells on the bottom of its mixing micro-channel with stimuli of WSS signal alone and different combinations of WSS and ATP signals in one single micro-channel, is proposed. Both numerical simulation and experimental studies verify the feasibility of its application. Cellular experimental results also suggest that a combination of WSS and ATP signals rather than a WSS signal alone might play a more significant role in VEC Ca²⁺ signal transduction induced by blood flow.

Keywords: Y-shaped microfluidic device; adenosine triphosphate (ATP) signal; calcium dynamics; combined effect; vascular endothelial cells; wall shear stress.

Figure 1

Schematic diagram of a Y-shaped microfluidic device. (a) Polydimethylsiloxane (PDMS)-glass structure; (b) coordinate system; (c) the integrated experimental system.

Figure 1

Schematic diagram of a Y-shaped microfluidic device. (a) Polydimethylsiloxane (PDMS)-glass structure; (b) coordinate system; (c) the integrated experimental system.

Figure 2

An actual Y-shaped microfluidic chip. (a) PDMS-glass microfluidic chip; (b) generator of dynamic biochemical signals; (c) the actual experimental setup.

Figure 3

An input static fluorescent signal and its spatiotemporal concentration profile in a dynamic flow in the mixing micro-channel C. (a) The static fluorescent signal ${\overline{\mathsf{\varphi }}}_{\mathrm{A}}\left(t\right)$, the volume flow rates Q_A, Q_B(t), Q(t) and wall shear stress (WSS); (b) numerically simulated concentration profile at t = 15 s and t = 45 s in x-z plane, respectively; (c) experimental concentration profiles at t = 15 s and t = 45 s in x-z plane, respectively. Scale bar is 100 μm.

Figure 4

An input dynamic fluorescent signal and its spatiotemporal concentration profile in steady flow in the mixing micro-channel C. (a) The dynamic fluorescent signal ${\overline{\mathsf{\varphi }}}_{\mathrm{A}}\left(t\right)$ and the volume flow rates Q_A, Q_B(t), Q( t )and WSS; (b) numerically simulated spatiotemporal concentration profiles at z = 2 cm in x-t plane under steady flow; (c) experimental spatiotemporal concentration profiles at the region around x = 2 cm in x-z plane under steady flow. Scale bar is 100 μm.

Figure 5

Comparison between simulation results and experimental data of combined WSS and fluorescent signals at different locations at z = 2 cm in the mixing micro-channel C. (a) Static fluorescent signal under dynamic flow; (b) dynamic fluorescent signal under steady flow. All the signals are normalized to a constant reference value.

Figure 6

The intracellular Ca²⁺ response in the human umbilical vein endothelial cells (HUVECs) culture on the bottom of the mixing micro-channel C at z = 2 cm. (a) HUVECs cultured for 1d, 2d and 4d, respectively; (b) the intracellular Ca²⁺ intensity at t = 3 s, 24 s, 69 s, and 111 s, respectively. Scale bar is 100 μm.

Figure 7

The intracellular Ca²⁺ dynamics in HUVECs in response to WSS signal alone and combined WSS and adenosine triphosphate (ATP) signals. (a) static WSS signal alone and (b) static WSS signal with dynamic ATP signal at the religion of x = W/8 or x = W7/8 in mixing micro-channel C under the condition that volume flow rate is constant, respectively; (c) dynamic WSS signal alone and (d) dynamic WSS signal with static ATP signal at the religion of x = W/8 or x = W7/8 in mixing micro-channel C under the condition that volume flow rate is dynamic changing, respectively. Experimental data in (a,b) are measured from one group of HUVECs while those in (c,d) are measure from another group of HUVECs. The average Ca²⁺ intensity is the average value of 20 single cells. All the data are normalized to a constant reference value.