Study On Single Cell 3D Tumor Microspheres Based On Microfluidic Chip

In organisms,cells are usually in a complex microenvironment.Cell-cell and cell-matrix interactions play an important role in maintaining the normal function of cells.Therefore,it is of great significance for the further development of cell research to realize the accurate manipulation of cells,simulate the microenvironment of cells,and develop cell analysis methods with high sensitivity,high selectivity and low sample consumption.Extracellular vesicles are involved in inflammatory immune response,intercellular signal communication,cell survival and apoptosis,and play an extremely important role in maintaining physiological state and disease development.Abnormal secretion of proteins is often accompanied by diseases such as infection,tumor,transplantation rejection,autoimmunity and so on,therefore,the analysis of secreted proteins is of great significance in cell mechanism research,disease diagnosis,treatment and curative effect evaluation.In this study,we have developed a high-throughput PDMS micro pore array chip,which has 4400 micro pores.The diameter of micro pores is 200 μm for large pores and 30 μ m for small pores.With the advantages of high retention rate of the perforation membrane,the three-dimensional tumor microsphere culture of MCF-7 was carried out on the chip,and the MCF-7 single cell was cultured into tumor microsphere in three-dimensional environment.In addition,the effects of pore size,F68 concentration,substrate selection and cell type on the microsphere culture were also investigated.Finally,a single cell culture method of tumor microsphere in three-dimensional environment was successfully obtained.The successful implementation of this culture method provides ideas and possibilities for subsequent detection and immune analysis.In this paper,the microsphere culture method was obtained through the study.The microsphere was cultured in DMEM/F12 complete culture medium containing 0.04%F68 and 1 ng/ml rhEGF in 200 μm～30 μm diameter double-layer PDMS microarray.In addition,the secretion of extracellular vesicles and secreted proteins of human breast cancer cell lines MCF-7 and MDA-MB-231 were also studied at the population level.The results showed that the signals of CD9+EVs and IL-8 were the strongest in the population cell analysis of MCF-7,which provided a reference for the follow-up immunoassay of MCF-7 three-dimensional tumor microspheres.In the follow-up study,we detected the secreted proteins and exosomes of MCF-7 tumor microspheres cultured by this method within 24 hours.Although the results showed that the secretion of IL-8 and CD9+EVs in MCF-7 tumor microsphere was not very high,it established a new target for our research in this direction.