The Studies Of High-throughput Cell Migration Methodology Based On Microfluidics Technology

[Objective]The migration of vascular smooth muscle cells(VSMCs)is closely related to the pathological process of arterial stenosis and restenosis caused by atherosclerosis after endovascular treatment.Therefore,it is particularly important for the study of VSMC migration.However,traditional methods of cellular migration have great limitations,such as low throughput,poor experimental consistency and poor reproducibility,which restrict the migration characterization of VSMCs and reduce the level of credible evidence of VSMC migration studies.Therefore,based on the microfluidic technology,we designed three kinds of high-throughput cellular scratch migration chip to study the new method of cell migration to improve the experimental throughput,which overcome the shortcomings of poor experimental consistency and poor experimental controllability,verify the feasibility and seek accurate experimental method of VSMC migration.In conclusion,our studies provide reliable experimental data for the migration of VSMC and new ideas and programs for the prevention and treatment of VSMC migration-related diseases.[Methods]1.To Design three different high-throughput cell migration methods based on microfluidics technology,which are the migration method of the two-dimensional cell with electrosurgical thiol,the migration method of the three-dimensional cell(thiol covered with collagen)and the migration method of the two-dimensional cell(thiol covered with collagen);for different methods,design the appropriate type of migration chip.2.Verify the viability of the chip to accurately characterize cell migration.Tumor cells are inoculated into the chip:human lung cancer cell(A549,H1299)and human cervical cancer cells(HeLa).The inoculation,growth and migration of tumor cells in the chip are observed and different cytokines are administered:fetal bovine serum(FBS)and CXCL12 chemokine.3.Using microscope and laser confocal microscope to photograph the cells before and after migration,image processing in Image Pro Plus 6.0,and cell counting in Lab View,the experimental results are expressed as mean ± standard deviation,between groups ANOVA test is used for multiple-group comparisons where values of P<0.05(*)and P<0.01(**)are considered statistical significance and high statistical significance respectively.Migration curve is used to characterize the migration.4.Through the analysis of the advantages and disadvantages of different chips,to further improve.5.A preliminary study of the migration of VSMCs.[Results]1.Three kinds of migration chip production process are definite and mature.2.Tumor cells and VSMCs are all covered with migration chips after inoculation in high-throughput migration chips,and grow well in migration chips.They can be migrated in the chip.3.The results of three different migration methods show that:1)In the migration method of the two-dimensional cell with electrosurgical thiol,tumor cells can be migrated.However,due to the complex structure of the chip and the poor stability of the power supply,there is some difficulty in the cell culture and removal of the mercaptan,and the consistency of the migration is poor.2)In the migration method of the three-dimensional cell(thiol covered with collagen),three-dimensional state takes a long time,the ability of the thiol to adhere to the gold surface decreases,the modification effect decreases,and the cell remains on the gold surface with some errors in subsequent migration.3)In the migration method of the two-dimensional cell(thiol covered with collagen),consistency of the migration is good,the results is reliable,tumor cells and VSMC can migrate.4.In the migration method of the two-dimensional cell(thiol covered with collagen),the results show that:1)H1299 cells can show different migration status in FBS and CXCL12 chemokine.And compare with H1299,HaLe cells in FBS show different migration status;2)Different FBS percentage and different concentrations of CXCL12 have different stimulating effects on H1299 migration,and different FBS percentage have different stimulating effects on HaLe cells.3)In this kind of chip,the consistency of VSMC migration is good.[Conclusion]1.High-throughput cell migration chip design is reasonable,the process is stable.2.The consistency of method of high-throughput cell migration experiment is good,the experimental results are highly credible.3.All three kinds of chips can be used for cell culture,growth and migration.It has been verified that migration of two-dimensional cell(thiol covered with collagen)is less affected by changes of other experimental conditions,and the stability and the experimental consistency are good.4.Migration method of the two-dimensional cell(thiol covered with collagen)is more suitable for VSMC migration studies.