Development Of A Microfluidic Platform For High-throughput Dynamic Single Cell Screening And Recovery

Cells are highly heterogeneous in terms of morphology,proliferation,and response to external stimuli.Single cell analysis is the study of hundreds or thousands of cells at a single cell level,and it is impossible to obtain important data from traditional studies based on large cell averages.Therefore,this technology had been voted as one of the ten technical breakthroughs by MIT technical review in the 2007.Single cell clonal analysis can study the growth dynamics mechanism,division or differentiation mechanism of cells at the single cell level.Isolation of individual cells from a mixed cell population is a prerequisite for single cell cloning.Microfluidic chips are now widely used for single cell capture and analysis due to their unique single cell handling capabilities and the potential for precise control of the cellular microenvironment.However,the survival rate and clonal colony formation rate of single cells in existing microfluidic devices are very low.Moreover,the cells on the chip are difficult to recover independently of each other for subsequent analysis using conventional equipment.Therefore,inoculation of a large number of single cells into separate culture chambers is one of the key issues in single cell clonal amplification studies.This paper introduced an innovative microfluidic chip which can be integrated with the standard well plate.The chip utilized an integrated adjustable valve and corresponding through-holes to screen and recover individual microspheres of the required size onto a standard 384-well plate,ensuring that one microsphere per well is achieved.The device had three independent layers(gas layer,membrane layer and fluid layer)and two operating modes(high throughput capturing single cells or size-based cells dynamic screening and addressable recovery).Firstly,this paper systematically established the hydrodynamic model for single microsphere screening and recovery using theoretical calculations,numerical simulations and statistical analysis.Among them,the deformation of the membrane in the microchannel of the chip,the variation of flow resistance and flow rate generated by the membrane were studied.Secondly,this paper studied the design and fabrication process of the chip in detail,from the design of the channel,to the separate processing of each layer of the PDMS chip,and then to the final chip bonding.Based on the microfluidic chip fabrication experiments,this paper summarized the important parameters of multi-layer PDMS chip fabrication to ensurethe repeatability of the device and experiments.Finally,the microspheres were used to model spherical suspension cells,and the feasibility of single-cell screening was tested by experimental methods.Experiments results showed that the microfluidic chip developed in this paper can be used for dynamic screening and recovery of single cell.This device solved the requirements for dynamic isolation,screening and vaccination of single cells,and was of great significance for clonal analysis of single cells.