Development Of Novel Microfluidic Device For Cell Migration And Chemotaxis Research

Immune cell migration and chemotaxis plays a key role in immune response.It is of great significance to study the migration and chemotaxis of immune cells.The microfluidic device can produce precisely controlled concentration gradient,making it an ideal tool for chemotaxis research.However,the use of microfluidic device in the absence of professional equipment and technical personnel is very difficult,which hinders the promotion of microfluidic device in the field of biological and medical research.In this paper,we have developed several microfluidic devices for addressing a series of problems existing in the field of cell migration and chemotaxis research.The final purpose of this thesis is to provide some easy-to-use and practical solutions for cell migration and chemotaxis research.First,a microfluidic device integrated whole blood cell separation and precalibration was developed,which was beneficial to improve the experimental efficiency and analytical accuracy.The whole blood separation method and microfluidic device were validated by testing neutrophil chemotatic to chemokine(N-formylmethionyl-leucyl-phenyl-alanine,fMLP)and chronic obstructive pulmonary disease(COPD)sputum samples.On this basis,a dual-docking device integrating multi-zone cell calibration and gradient generation functions was designed for performing neutrophil chemotaxis and memory effect research.The results supported using bias-random-walk to characterize neutrophil chemotaxis on a simple two-dimensional plane and demonstrated that the chemotactic memory effect was not only related to the concentration of chemokine but also to the 'past and present gradient' the cells experienced.In order to improve detection efficiency,we developed a microfluidic device with three parallel gradient channels that allowed three parallel exepriments under a single microscope field of view(10X objective)on a single microfluidic device simultaneously,which was beneficial to save time,reduce labor intensity,and ensure the authenticity and reliability of the experiment.The microfluidic device was used to study the biomarker of fibroblast growth factor 23(FGF23)associated with chronic kidney disease(CKD).We for the first time found FGF23 affected the chemotaxis of neutrophil,and disclosed the significance of studing FGF23-mediated neutrophil chemotaxis for further studying the pathogenesis of disease and formulating treatment regimens.Finally,a real-time imaging system for cell migration and chemotaxis research by integrativing microfluidic device and smartphone was developed for addressing some shortcomings met in current cell chemotaxis research community,such as using expensive microscope,and high time and experiment cost.The system integrated microfluidic device,live cell imaging,experimental environment control and data analysis.The system can quickly collect the experimental data in real time and realize the automation analysis,which greatly improves the efficiency of cell migration experiment.In conclusion,this thesis provides the detail design and experimental basis for the development of cell chemotaxis research equipment based on microfluidic device.The microfluidic devices are expected to be widely used in cell migration and chemotaxis research community.