A Microfluidic Study On Intracellular Calcium Dynamics Of Vascular Endothelial Cells In Response To Spatiotemporal Wall Shear Stress And ATP Signals

Vascular endothelial cells(VECs)in vivo are exposed to blood flow.environment and are stimulated by a blood-soluble biochemical factor,adenosine triphosphate(ATP),and wall shear stress(WSS)generated by blood flow.The concentration of ATP and the magnitude of WSS that can cause cellular responses often appear in dynamic form over time.VECs can recognize these dynamic signals and transmit them to the interior of cells,resulting in the dynamic response of intracellular calcium ion(Ca2+)concentration.The Ca2+ dynamic response is closely related to cellular behaviors and functions including migration,proliferation,growth,differentiation and apoptosis.Due to various interference factors in VECs' microenvironment,it is hard to directly study the Ca2+ dynamic response to dynamic WSS and ATP signals in vivo.Thus it is particularly important to establish a method to study the Ca2+ dynamic response to WSS and/or ATP signals in vitro.Nevertheless,traditional methods such as cell culture dish and rectangular parallel-plate flow chamber(PPFC)are not easy to be adopted to load spatiotemporal WSS and ATP signals to cells.Microfluidic technology,serving as a new technology which can precisely control the stimuli of biomechanical and biomechanical factors in cellular microenvironment,has been widely applied in recent years.While in existing studies about VECs' Ca2+ dynamic response,the researchers have just focused on the effects of constant biochemical stimuli over time or WSS stimulus.In this study,we conduct theoretical analysis,experimental observation and numerical simulation to build a methodology that can generate dynamic WSS and ATP signals using microfluidic technology,and investigate the intracellular Ca2+ dynamic response of VECs under different stimulating conditions.This study consists of following parts:First,based on the principle of stagnation point flow and mass convection-diffusion equation,a novel microfluidic chip and its peripheral dynamic signal loading system are designed for generating the spatiotemporal WSS and ATP signals.The distributions of spatiotemporal WSS and ATP signals are obtained by fliud dynamic analysis.The ability of this novel microfluidic chip system to generate WSS signal alone,ATP signal alone and the combinations of WSS and ATP signals is further validated.Second,the actual microfluidic chip and its peripheral system are constructed according to the previous design.The transmission characteristics of biochemical signals in steady and pulsatile flow are analyzed in combination with numerical simulations and fluorescent solution experiments.The microfluidic channel is verified to be a low pass filter,and the influence of the frequency,flow rate and transmission distance of biochemical signals on the filtering effect are examined.The nonlinear modulation of pulsatile flows on the biochemical signals,and the dependence on the biochemical signal frequency,flow signal frequency as well as signal transporting distance are further investigated.Third,the spatiotemporal distribution of biochemical signals which can avoid filtering and nonlinear modulation are designed and verified by carefully considering the factors that cause filtering effect and the nonlinear amplitude modulation as well as the sensitivity of cellular response to stimuli.Then,using the peripheral dynamic signal loading system,the spatiotemporal WSS and ATP signals are provided on the human umbilical vein endothelial cells(HUVECs)that are cultured on the bottom of microfluidic channel,and the real-time intracellular Ca2+ dynamic response is observed and analyzed.The intracellular Ca2+ signal dynamics of HUVECs in response to different combinations of WSS and/or ATP signals is acquired.It is found that the synergistic effect of the WSS and ATP signals,but not a WSS signal or ATP signal alone,plays a more significant role in the signal transduction of HUVECs.In particular,under the combined stimuli of WSS and ATP signals with different amplitudes and frequencies,the amplitudes and frequencies of the intracellular Ca2+ dynamic signals is suggested to be closely related to the amplitudes and frequencies of WSS or ATP signals.In summary,the present study design and construct a microfluidic device system being able to provide adherent VECs with spatiotemporal WSS or ATP signals alone or their combinations,and investigate the synergistic effect of WSS and ATP signals on the intracellular Ca2+ dynamic response.The proposed microfluidic device can also be used to study the cellular dynamics of other adherent cells in response to synergistic stimuli of spatiotemporal WSS and/or ATP signals and underlying molecular mechanisms.