Research On Devices For Μulti-Stage Sorting Of Tumor Cells Based On Interfacial Viscoelastic Microfluidics

Circulating tumor cells refer to all types of tumor cells that are detached from the primary tumor lesion and follow the peripheral blood circulation,which can metastasize and spread in cancer patients and eventually lead to deterioration of the disease.Therefore,the study of circulating tumor cells is of great significance for early cancer prevention,diagnosis and treatment,and drug development.However,the number of circulating tumor cells is scarce,containing only a few dozens per milliliter of patient blood,and traditional laboratory sorting methods are often difficult to achieve efficient sorting.Based on microfluidic technology,this paper focuses on the interface viscoelastic sorting technique,which is easy to operate and can guarantee high sorting accuracy,based on the summary of existing microfluidic cell sorting techniques.This paper explores and improves the interfacial viscoelastic sorting technology and integrates it with symmetric contraction-expansion-type channel to retain the advantages of interfacial viscoelasticity and improve the sorting performance.Finally,the magnetic sorting technology is introduced to further improve the sorting accuracy,and the multilayer film stacking process of the research group is combined to achieve several times the throughput of the integrated chip.The specific research results achieved in this paper are as follows:(1)The processing process of the existing interface viscoelastic sorting chip was improved and the throughput of the chip was increased,and the optimization chip was used to successfully isolate a variety of tumor cells from diluted whole blood and clinical samples.In this chapter,we first optimized the design of the interfacial viscoelastic sorter chip,greatly accelerated the chip fabrication by changing the processing process,and selected a lower viscosity viscoelastic solution as the sheath solution to achieve a significant increase in throughput.Different sizes of polystyrene microspheres were used to simulate erythrocytes,leukocytes and tumor cells in blood to verify the sorting performance of the optimized chip and explore the optimal sorting conditions.The recovery rate of the optimized chip was higher than99.99% and the purity was 81.77% for the target particles.The optimal experimental conditions obtained from the particle experiments were applied to the cell sorting experiments of diluted blood doped with high/very low concentrations of multiple tumor cells and clinical samples(pleural and abdominal effusions).The average recovery rate of the chip for the three tumor cells was 94.08% and the average purity was 40.58% when the higher concentration of tumor cells was doped.When doped with very low concentration of tumor cells,the average recovery of the three tumor cells reached 99.99%,and the average purity was reduced to 13.43%.For clinical pleural and abdominal effusions samples,the chips were able to detect tumor cells with an average purity of 6.65%.(2)In response to the insufficient sorting accuracy of the interfacial viscoelastic chip,a two-stage passive sorting chip integrating interfacial viscoelasticity and symmetric contraction-expansion-type channel was developed to complete the separation of tumor cells from diluted whole blood and clinical samples with higher accuracy.Given the significant improvement in sorting efficiency of the optimized interfacial viscoelastic chip,this chapter integrates the interfacial viscoelasticity with the symmetric contraction-expansion-type channel to further improve the sorting accuracy using a two-stage sorting technique.For the new symmetric contraction-expansion-type channel,independent structures were designed and their sorting performance was verified using polystyrene microspheres,blood cells and tumor cells.After the integration scheme was determined to be feasible,the sorting performance of the two-stage integrated chip was verified using different sizes of polystyrene microspheres.The two-stage chip was tested to achieve 100% recovery for the target particles with an average purity of 96.29%.The two-stage chip was then applied to cell sorting experiments on diluted blood doped with high/very low concentrations of multiple tumor cells and clinical samples(pleural and abdominal effusions).The average recovery rate of the three tumor cells was 95.74%and the average purity was 61.45% in the high doping tumor cell sorting experiment.In the low-doping tumor cell sorting experiment,the average recovery of the three tumor cells was greater than 99.99%,and the average purity was 33.77%.For clinical pleural and abdominal effusions samples,the average purity of tumor cells recovered by the two-stage chip was24.62%.Compared with the single-stage interfacial viscoelastic chip,the sorting performance of the two-stage chip was significantly improved.(3)To further improve the throughput and sorting accuracy,an active and passive multi-stage sorting chip integrating interfacial viscoelasticity,symmetric contractionexpansion-type channel and magnetic sorting technologies was developed.In view of the limitations of passive sorting based on size alone,this chapter introduces a size-independent magnetic sorting technique and further improves the sorting accuracy by sequentially removing small-sized blood cells and reverse-labeled magnetic beads leukocytes through interfacial viscoelasticity,symmetric contraction-expansion-type channel and magnetic sorting.Taking advantage of the stacking of thin film chips,the integrated chips are stacked in three layers to improve the sorting throughput.For the new magnetic sorting module,a focusing unit and a magnetic sorting unit are designed and tested separately with magnetic/nonmagnetic particles and cells.After determining the feasibility of the integrated solution,the sorting performance of the integrated chip was verified using magnetic/nonmagnetic particles.The recovery rate and purity of the multi-stage integrated chip for the target particles were tested to be 100%.The multi-stage integrated chip was applied to sort tumor cells in diluted blood.The average recovery of the three tumor cells was 97.06% and the average purity reached 82.26% when doped with higher concentration of tumor cells.The average recovery of the three tumor cells was 100% and the average purity reached 66.17% when doping with very low concentration of tumor cells.For clinical pleural and abdominal effusions samples,the average purity of tumor cells recovered by the multi-stage integrated chip was 55.07%.Compared with the two-stage sorting chip,the throughput of the multi-stage integrated chip was improved by 5 times and the sorting accuracy was significantly improved.