Cell Separation And Single-cell Array Using Integrated Microfluidicses

As the development of microfluidic technology,cell separation and single-cell array methods based on different size and deformability has been attracted increasing attention.Microstructure-based microfluidic technology is research hotspot for cell separation and single-cell array,due to their advantages such as simple,non-invasive operation without special labels and more efficient techniques and well maintenance of cell viability and function.For cell separation,compared with other methods,microstructure-based separation techniques have various significant advantages,such as high colloction effieciecy and purity.The main challenge of the microstructure-based method is the potential for clogging when a large number of cells are processed,which can result in irregular flows and low separation efficiency.Specifically,as the pores of microstructure filters were gradually blocked by the trapped cells,the hydrodynamic resistance of the microstructure filters changed unpredictably,resulting in the functional filter reducing and the separation efficiency decreasing.In addition,captured cells cannot be easily extracted from microstructures in some devices,limiting the subsequent research of the captured cells such as genomic and protein analysis.For single-cell array construction,microstructure-based method can construct single-cell array with high throughput.In tumor cellular heterogeneity research,construction of single-cell arrays based on different different size and deformability can be used for studying the effect of cell biomechanical(e.g.,size and deformability)heterogeneity on their biological characteristics(e.g.,drug-resistance and tumor-initiating features).In this study,we design integrated microfluidic devices through combination of microstructure matrices and the microvalve system for physical separation of cells with different biomechanical property,and construction of single-cell arrays and assay of their drug-resistance.Our microfluidic devices provide new methods for physical separation of cells and single-cell arrays according to their different biomechanical properties.The results obtained in the present work are as follows:1.The integrated microfluidic device utilised for cell separation on the basis of size and deformability was fabricated using a multilayer soft lithography method.The integrated microfluidic device was combined the microstructure-constricted filtration and pneumatic microvalves.Using this device,the cell populations sorted by the microstructure matrices canbe easily released in real time for subsequent analysis.Moreover,the periodical sort and release of cells greatly avoided cell accumulation and clogging and improved the selectivity.Numerical simulation of the flow through the microstructure-constricted filtration showed that the device provided a well-controlled hydrodynamic environment for cell capture or passing,avoiding the potential risk of clogging or cell damage caused by the prolonged contact between the cells and the filters.By infusing two different food-dye solutions,the simulation of the whole separation process suggested that the integrated microfluidic device could realize cell infusion,sorting and controllable release in a fast,orderly and efficient manner.2.By optimizing the operating parameters,the device could separate cancer cells(MCF-7,MDA-MB-231 and MDA231-LM2 cells)with different deformability from two kinds of cell mixtures(MCF-7 and MDA-MB-231 cells,and MCF-7 and MDA231-LM2cells)? This device showed high purity(more than 75%)for these different cancer cells,indicating that the device could be used to separate less flexible cells and flexible cells for the study on the cancer metastatic and cancer-initiating cells.3.The separation of cancer cells from blood samples with a cancer cell-to-blood cell ratio of 1/106,suggested that the device could be used to separate cancer cells from the blood samples with more than 90% cell recovery and more than 80% purity.Comparing these results with the current filtration methods,the device possesses the potential to separate rare cells with high collection recovery and purity.4.An integrated microfluidic device was performed for single-cell array construction and drug-resistant analysis.The microfluidic device was combined multi-obstacle architecture-liked filter matrices with the microvalve system.Using this device,high-throughput single-cell arrays could be easily realized according to their biomechanical(size and deformability)heterogeneity.The microfluidic device also allowed tracking of dynamic behavior of hundreds of cells and monitoring temporal changes within single cells.This device provides a new approach for single-cell isolation according to their different biomechanical properties.5.Evaluating the vincristine-resistance of single glioblastoma cells with different biomechanical properties.The results indicated that tumor cell biomechanics was significant implication for their drug-resistance,that is,small and/or more deformable tumor cells had higher drug-resistance than the big and/or less deformable tumor cells.This device possesses practical potential for tumor studies on a single-cell level.