Study On The Migration Phenotype Of Neutrophils In Diabetic Patients Based On Microfluidic Concentration Gradient Chip

Background and purpose In recent years,as a metabolic disorder,the prevalence of type II diabetes has been increasing year by year.Due to metabolic disorders,diabetic patients often lead to low immune system function,which is the key factor that causes diabetic patients to be prone to concurrent infection.Neutrophil is a key cell in innate immune system and its chemotaxis plays an important role in anti-infection immune response.Therefore,the direct study of the whole process of neutrophil chemotaxis is of great scientific significance for monitoring the immune function of diabetic patients and evaluating the therapeutic effect.However,most traditional chemotactic methods are inefficient and difficult to observe the whole process of cell migration in real time.Methods To investigate the migration ability of neutrophils in patients with type II diabetes,a six-channel concentration-gradient microfluidic chip was designed.In order to cooperate with the microfluidic chip,a neutrophil separation device was designed and coupled with a commercial neutrophil separation kit,which was used to extract neutrophils from six tubes of 50 μL whole blood at one time.Firstly,the finite element analysis software Comsol Multiphysics 5.5 was used to simulate the generation of concentration gradient in migration pipeline.To verify the practicability of microfluidic chips,chemotactic agent of different concentrations and neutrophil attractants of the same concentration and different types were used to screen chemotactic agent and the optimal concentration gradient of the selected chemotactic agent in the chip.Subsequently,we compared neutrophil migration data between patients with type 2 diabetes mellitus and healthy subjects,and conducted Pearson correlation analysis between migration data of patients and some physiological parameters.In order to determine the influence of different glucose concentrations on the migration function of neutrophils,neutrophils were incubated with glucose solutions of different concentrations(0 mmol/L,5 mmol/L,10 mmol/L,20 mmol/L)to observe the migration of cells in microfluidic chips.Then,in order to further investigate the reason why the migration function of neutrophils was inhibited under high blood glucose concentration,neutrophils were incubated with mannitol and glucose solutions of the same concentration(20 mmol/L)and their mixed solutions to observe the migration status of the cells.At the same time,the neutrophils were incubated with pravastatin and metformin hydrochloride,and the effects of both on neutrophils migration were detected by microfluidic chip.Finally,the transepithelial/endothelial cell assay of neutrophil granulocyte and MD-MB-231 cell migration assay of breast cancer were used to further verify the practicality of microfluidic chips.Results The microfluidic chip designed in this study relied on laminar flow and diffusion mechanisms,and was simulated by the finite element analysis software Comsol Multiphysics 5.5.It was proved that it could simultaneously and independently generate stable concentration gradients in six groups of main cell migration channels in a short time,with a stable duration of more than 75 min.Chemotactic screening experiments showed that in the concentrate-gradient chip designed in this study,the attraction ability of f MLP to neutrophils was better than that of IL-8 at the same concentration,and the optimal concentration of f MLP was 100 nmol/L.The results of neutrophil migration experiment between diabetic patients and healthy subjects based on microfluidic chip showed that the neutrophil migration response of diabetic patients was lower than that of healthy subjects.Glucose gradient experiment showed that high glucose could inhibit neutrophil migration.Through the experiments of incubation of neutrophils with mannitol,glucose and the mixture of the two drugs,it was suggested that the reason why the migration function of neutrophils was inhibited in high concentration glucose solution might be because high concentration glucose damaged the cells themselves.The diabetes drug experiment showed that pravastatin reduced neutrophil migration function to some extent,but metformin hydrochloride did not show significant effect on neutrophil migration function.In addition,the migration of neutrophils across epithelial/endothelial cells and breast cancer MD-MB-231 cells can be achieved on the microfluidic chip designed in this study.Conclusion Neutrophil migration function is impaired in diabetic patients,which may be due to motor dysfunction caused by the interaction between high concentration of glucose and neutrophils.Moreover,the proposed six-flux microfluidic concentration gradient chip can quickly and selectively detect the migration characteristics of cells at the single cell level,which can be used as a new tool for cell migration research.