Establishment Of Single Cell Analysis Platform Based On Microfluidics And Its Application

Single-cell analysis is an interdisciplinary frontier field derived from the osmotic development between analytical chemistry,biology and medicine.Cell is the basic unit of morphological structure and life activities of the organism.To explore the basic process of life activities and the essence of life,we must explore in great depth of life activities such as the cell growth and differentiation,metabolism and reproduction,movement and contact,aging and death,genetic and evolutionary process and so on based on the study of cells.For a long time,because of the limitation of detection sensitivity and the lack of single-cell separation methods,we can only obtain the statistical average results that cover the differences between the individual cells by group cell analysis and lead to the limitation of the development of many fields such as biology and medicine.Whereas the single-cell level research could provide more abundant and accurate individual information,avoiding the difference of single cell to be covered by a large number of background signals.As people come to realize that single cell research is very important to the reorganization of human itself and disease,the scientists' research on single cell is becoming more and more detailed,and the birth of single cell sequencing technology is timely.In the clinical application of diagnosis and treatment of cancer,cancerous tissue using single cell sequencing analysis the changes of genetic material can be more accurate diagnoses the cause of the cancer,provide more accurate target information for treatment.Meanwhile,using single-cell sequencing technology to analyze the changes of rare CTC genetic information in blood can effectively monitor tumor changes.The single-cell sequencing has been widely used in microbiology,neurology,human development,immune,cancer,research in the field of assisted reproduction and so on,including cancer and assisted reproductive studies in clinical.With the proposed of Human Cell Atlas Project,a large international collaboration in 2016,single-cell research is being push to a new climax.Research of single cell research such as single cell growth consistency,and SCP group,single-cell transcriptome analysis,single cell genome analysis,etc.,and its correlation with cell mapping,the key lies in how to separate single cells from a large number of cell samples.Due to the advantages of microfluidics such as small volume,low consumption,high throughput,highly flexible manipulation,good biocompatibility,able to integrate with various biochemical reactions,microfluidic chip is especially suitable for and has been widely used in high-throughput single cell research.Although microfluidics has demonstrated good development prospect and application potential in single-cell research,such high throughput single cell sequence platform(Dropseq)of 10 x Genomic and ClTM Single-Cell mRNA Seq system of Fluidigm,still cannot analysis rare cell with high efficiency.To settle the aforementioned problems,the major works of this thesis are list as following:1.Microfluidic based high-throughput single capture and 3D cultureAs research on single cells gradually being extensive and ingoing,there are more and more single-cell study based on microfluidic platform,but the existing single-cell analysis platform is based on two main principles.One is to realize the isolation and study of single cell by using the limiting dilution method.The distribution of cells conforms to the Poisson distribution,and the cell loss is too large to achieve the separation and analysis of rare cells.Another is based on hydro-mechanical single-cell separation and capture.According to articles that have published,this method is unable to provide independent microenvironment for separated single cells.Herein,we designed a microfluidic chip based on fluid dynamics,which can capture single cell with very high efficiency and isolated separated single cells in uniform droplets quickly.We inject different number of K562 cells as models into the chip and count the cell number captured by chip.The capture efficiency of different cell number is all over 80%,proving that the chip has very high capture efficiency.At the same time,we achieved efficient capture and separation of five different kinds of cells respectively,which proved that the chip has a good applicability to the capture of different single cells.Combination with low temperature agarose,we successfully carried out single-cell 3D long-term culture with high-throughput.The establishment of the platform and stability for subsequent single-cell analysis platform provides a strong theoretical and experimental basis,and is expected to be applied to single cell epigenetics research,drug screening for clinical medicine,and clinical treatment research for treatment guidance.2.A new type microfluidic based single cell manipulation platformThe application of droplet microfluidics and microarray is the two main platforms for single cell research at present,with some advantages and disadvantages.Compared with the microarray,the droplet microfluidics can control droplets micro operation such as generation,fusion,mixing,dividing,storage,etc.very easily just by the flow velocity and the external force field,such as electric field,magnetic field,sound wave,pressure and so on.However,due to the random dispersion of liquid droplets in the oil phase,random floating and movement can occur,which cannot achieve long-term dynamic observation,washing and liquid exchange.Compared with droplet microfluidics,microarray produce much more stable micro-reactors(wells or reservoirs)for easier dynamic real-time analysis,avoiding the random droplet floating happens to microfluidic droplet,but lacks the flexible operational.In the thesis,we developed a novel microchannel design,which not only can capture single particle rapidly and efficiently,but also can produce droplet fast for in situ observation.What more,by design of a two-layer chip containing the pump and valve,We can realize the fusion,mixing,replacement of reaction fluid of paired droplets and other functions through the micro manipulation of the external forces.We carried out the one cell one cell paired experiment that provide a theoretical basis for cell co-culture research with this platform.We also realized one cell one bead pairing and micromanipulation such as long time incubation,fluid replacement,and controlled single cell lysis and so on,which provide theoretical basis for subsequent single cell detection,single cell drug research,monoclonal screening system,secreted proteins and intracellular protein detection and the establishment of a single cell sequencing platform.3.Establishment of high throughput single cell mRNA sequencing platformWith the rapid development of high-throughput sequencing technology,people have been able to carry out the genetic mutation and gene expression of tumor cells at single cell level.In particular,the emergence of Single Cell Sequencing technology(SCS)has made it possible to carry out comprehensive and precise research on the genomics,transcriptology and epigenomics of tumor single cells.The first challenge to prepare a single cell-sequencing library is to obtain a single cell with high quality.Existing single-cell separation microfluidic platforms for preparation of sequencing library are unable to realize the automation of rare and precious cell sample,and can only rely on time-consuming micromanipulator.In order to solve the problems,we developed a microfluidic chip platform for high-throughput single cell mRNA sequencing sample preparation,which can match one cell one barcode bead with very high efficiency and control single cell lysis on chip.The barcode bead can capture mRNA released by paired single cell.After bead recovery and reverse translation,encoded the transcriptional information of thousands of cells just by one experiment and then carried out transcriptome analysis.By this platform,we can prepare a large number of single cell transcriptome sequencing samples automatic and rapidly with the advantage of less reagent consumption,low cost,High Throughput,strong maneuverability and rare cells analyzable.