Single Cell Sepration And Long-Term Culture Using Microfluidic Technologyies

Stem cells have important applications in the fields of regenerative medicine and precision medicine due to their immortal proliferation and the ability to differentiate into various organs in the adult body.However,due to the lack of in-depth understanding of stem cell fate control,there are many applications are not easy to achieve in the above two fields.Tracing back to its origin,as a highly heterogeneous cell,stem cells are comprehensively affected by different chemical stimuli and mechanical stimuli.Therefore,to quantify their division,differentiation behavior and molecular control mechanisms at the single cell level are very critical for stem cells' scientific research and clinical applications.However,the current research is limited by expensive culture conditions,complicated technical equipment and other conditions.It is impossible to continuously cultivate single cells to divide multiple generations and collect them for gene sequencing research.What is more,the gene sequencing research cell samples are either from a single cell in a mixed sample or a single cell in the same clonal line,or two daughter cells divided from the same mother cell.These studies are "end-point determinations",and "end-point determinations" is impossible to explore the influence of the fluctuation of gene expression over time on stem cells' fate at the single-cell level.To solve this problem,it is necessary to use microfuidic chip to isolate single cells at critical time points(for example,before or after predicted cell division),and then using single-cell sequencing to reveal the cell potential molecular composition at different time points.In this paper,we focus on the single cells seperation and long term cultivation,and replace stem cells with human chronic myeloid leukemia cells(K562)to eatablish a microfluidic system for single cell culture and multi-generation daughter cell separation.This paper optimized a microfluidic chip for the separation of multi-generation daughter cells.A microfluidic chip was used as the single cells culture and separation device,and the whole fully automatic inverted fluorescence microscope and a precision syringe pump were used as the control device to achieve the long-term culture of single cells and the separation of multi-generation daughter cells.Based on the microfluidic technologyies,this paper established a numerical simulation of multiphase flow model to simulate the cell flow,separation,capture and other processes,and on this basis,the design parameters of the microfluidic chip were optimized.Through numerical simulation,it was found that when the main capture channel width was 30 ?m,the critical capture distance between the cells and the trap is 3 ?m.Based on the critical capture distance and the 18 ?m average diameter of K562 cells,in order to ensure the separability of daughter cells,the trap depth range was set in the range of 10 ? 18 ?m,and the feasibility of this size is verified by simulation and experiment.A daughter cell separation and long-term culture system with a microfluidic chip as the core was established,which realized the long-term culture of K562 cells and the separation of four consecutive generations of daughter cells.On this basis,the study of cell capture efficiency under different flow rates and the statistics of the survival rate and separation rate of single-cell long-term culture were carried out.Experiments show that the chip can achieve a capture efficiency of 91.65%,an average cell survival rate of 51.7%,and a cell separation rate of 45%.This system can realize the long-term culture and separation of single cells,and provide new methods and ideas for stem cells to quantify their division,differentiation behavior and molecular control mechanisms at the single cell level.