Development Of High-turoughput Inertial Microfluidic Chips For The Separation Of Rare Tumor Cells And Clusters

Separation of tumor cells and clusters from malignant pleural effusions is an important supplement to liquid biopsy technology,and has important clinical significance in early tumor screening,efficacy assessment,and disease progression monitoring.Current separation methods of cell clusters are difficult to balance in terms of throughput,cost,recovery rate,and more.Therefore,there is an urgent need for a new sorting structure or a breakthrough in separation technology.On the basis of in-depth exploration of the basic structure of inertial microfluidic sorting devices and common innovative methods,a sinusoidal microchannel chip was used to achieve efficient sorting of tumor cells in malignant effusion samples.A novel trapezoidal-section spiral chip modified with local convex expansion structure was proposed for focusing microparticles of different sizes in three different positions along the channel width.Furthermore,through the integration of multi-stage sorting units,the synchronous,high-purity,and high-throughput sorting of single tumor cells and cell clusters in malignant pleural effusion can be achieved.The specific research results obtained in this paper are as follows:(1)An inertial sorting chip with a sinusoidal channel structure was designed to achieve high-purity,high-efficiency and stable sorting of tumor cells.The size-based differential focusing maps in the sinusoidal channel were tested and the optimal sorting flow rate of the mixed microparticles was determined to be 1200μL/min.At this time,the recovery rate of the target microparticles was 99.17%and the purity was 99.45%.The separation efficiency of tumor cells at different concentrations was tested.When the concentration of spiked leukocytes was in the order of 10~5 counts/m L,the recovery rate of tumor cells was as high as 93.30±1.29%,and the purity was 91.01±2.08%.The recovery rate was greater than 80%when doped tumor cells were at a very low concentration of 100 counts/m L.For malignant pleural effusion samples,the detection rate of tumor cells was 100%(n=5).(2)A spiral microfluidic chip coupled trapezoidal cross section with partial convex expansion was developed,which can simultaneously separate single tumor cells,cell clusters,and leukocytes.The convex expansion structure was modified on the spiral flow channels with trapezoidal cross section to generate additional local convex expansion induced lift.As a result,leukocytes were focused near the inner wall of the channel and single tumor cells were focused in the center of the channel,while tumor cell clusters were located at the outer wall of the channel.Explore the effects of the width of the convex expansion unit and the height of the outer wall on the focusing behavior of microparticles.In cell sorting experiment,the removal rate of leukocytes in outlet III was 94.0%under the optimal flow rate,and the recovery rate of single tumor cells in outlet II was 93.4%.The chip was further used for the sorting of malignant pleural effusion.At this time,the purity of tumor cell clusters in outlet I was about 16%and the average cell size was approximately 20.07μm.The purity of single tumor cells in oultet II was 12%,and the average cell size was approximately 15.08μm.Malignant cells and clusters were detected in each sample after treatment of fluid accumulation in cancer patients with different cancer types,stages and fluid types.Monitoring cells in the body fluids of cancer patients before and after treatment cycles.The results showed a reduction in the number of tumor cells and clusters.(3)Aiming at the low sorting purity of single-stage spiral chips,a two-stage sorting chip integrating sinusoidal channels and trapezoidal spiral channels was developed to achieve high-throughput,high-purity,high-throughput,and simultaneous analysis of single tumor cells and cell clusters.In view of the excellent performance of sinusoidal microchannel chips in high-concentration cell sorting,a two-level integration scheme was designed for the advantages of multi-layer stacking of thin-film chips.The first-stage structure connected multiple sinusoidal microchannels in parallel to remove leukocytes and collect tumor cells and clusters with extremely high throughput.The second-stage sorting chip coupled trapezoidal cross-section with local convex expansion further removed residual leukocytes and recovered high-purity single tumor cells and clusters at the same time.Draw the focus map of microparticles of multiple sizes in the integrated chip channel;use cell lines to verify the separation performance of the integrated chip.The integrated chip was applied for the treatment of malignant pleural effusion with a throughput up to 8 m L/min.Compared with the single-stage trapezoidal spiral chip,the purity of tumor cell clusters in outlet 2-1 was increased to 35%(19%increase in purity).The purity of single tumor cells in outlet 2-2 was increased to 68%(an increase of about 5.7 times).