Construction Of A High-throughput Single-cell Extracellular Vesicle Research Platform And Its Application On The Study Of Oral Squamous Cell Carcinoma

Objectives:To construct a high-throughput single-cell capture and secretion platform for Extracellular Vesicles（EVs）,and to achieve EVs research at the level of single-cell origin in Oral squamous cell carcinoma（OSCC）on this platform.Materials and Methods:In this experiment,a silicon wafer template was prepared by soft lithography,and polydimethylsiloxane（PDMS）was poured to prepare a single cell capture chip and a microchannel chip.The cell suspension was evenly dispensed onto the single cell capture chip and then covered with an antibody barcode array slide.The formation process of the antibody barcode array slide is as follows:First,the microchannel chip is bonded to the poly-L-lysine slide,and then the antibodies are coated by the flow patterning method to form an antibody barcode array slide.Both the slide and the single cell capture chip after the addition of the cell suspension were clamped together,and incubated in a cell culture incubator.EVs secreted by single cells were captured by the capture antibodies in the corresponding region above.The slide was removed after18h and the biotinylated antibody（Biotin-CD63）and Streptavidin-APC or Streptavidin-PE were sequentially added to detect EVs.The slide was then scanned using scanner（GenePix 4300A）to obtain an image.Finally,a series of analysis softwares were used to statistically analyze the fluorescence signals of the experimental results,showing the heterogeneity of EVs secretion at the single cell level.Results:We constructed a high-throughput single-cell capture and detection platform for secreted EVs.The platform consists of two main components:a single cell capture chip and an antibody barcode array slide.The cells showed a Poisson distribution on the slide.The average number of single cell capture per experiment was 1,318,and the average capture rate was 20.5%.In order to verify the survival rate of the cells on the chip,the cell suspension droplets stained by the cell tracker were applied to a single cell capture chip,and after 18 hours,the cell survival rate was 91.5%.The 9 channel entrances of the microchannel chip were all added with FITC-BSA,and then the antibodies were coated onto the poly-L-lysine slide by Flow Patterning,and finally scanned by scanner.The result showed that the antibody was distributed very evenly on the slide.This study uses immunoaffinity to capture EVs.The fluorescence images of the experimental result was observed under an atomic force microscope,The size（82.031 nm）of an individual vesicle was randomly measured,which was in accordance with the size range of EVs currently reported internationally.After constructing the platform and identifying that the vesicles captured by the immunoaffinity method are EVs,we applied the platform to the OSCC cell lines UM-SCC6,SCC25,and OSCC primary cells,respectively.The results demonstrate the heterogeneity of OSCC single cells in the secretion of EVs.We can simultaneously capture and detect 5 kinds of EVs and 3 kinds of cytokines secreted by single cell,as the capture antibodies used in this experiment includes not only EVs capture antibodies,but also cytokine antibodies.ViSNE software analysis showed that cells from each patient or cell line gave rise to three structured clusters.Group 1 is mainly distinguished with EVs secretion,like ^CD9+CD63+EVs and ^CD63+EVs;Group 3 dominated proteins secretion,mainly for IL-8;while Group 2 accommodates both EVs secretion and proteins secretion,but with much attenuated frequency.Demonstrating the functional architecture of population cells is relatively stable across both cell lines and primary cells.In addition,the primary OSCC single-cell data obtained in this experiment were analyzed by cluster software,and the clinical samples of lymph node metastasis and non-metastasis were distinguished.Conclusions:1.Based on microfluidic technology,we constructed a high-throughput single-cell EVs detection platform.The platform was used to verify the significant heterogeneity of the ability and species of OSCC single cells to secrete EVs.2.The single cells secreting EVs and cytokines have different divisions in the same cell population.3.The platform differentiated clinical samples of lymph node metastasis and non-metastasis at the single cell level.