Cascading inertial microfluidics for high-throughput, multi-scale enrichment of tumor cells and intact clusters towards enhanced malignancy diagnosis.

The presence and abundance of malignant tumor cells (MTCs), particularly cell clusters (MTCCs) in clinical effusion samples serve as established correlative indicators for aggressive malignancy, elevated metastatic potential, and adverse clinical prognosis. While tumor cell-based liquid biopsy enables the detection of single MTCs, but clinical utility is limited by cellular damage and attrition of MTCCs. To overcome these limitations, we developed a cascaded inertial microfluidic device for high-throughput and multi-scale enrichment of both MTCs and intact MTCCs through two-stage inertial sorting. The parallelized serpentine microfluidics were employed as the first-stage sorter which depleted majority of background blood cell in an ultra-high throughput. The second-stage spiral microfluidics enabled further multi-scale enrichment of single MTCs and intact MTCCs from residual blood cells without cellular damage. After exploring the effects of particle size and flow rate on two-stage inertial sorting, cell enrichment performances using simulated samples were characterized. Quantitative results indicated a high recovery ratio of 81.7% and a high purity of 76.1% for enriched A549 cells, along with a high purity of 79.4% for A549 cell clusters. Using the cascaded microfluidic device, 50 mL pleural effusion samples were processed within 6.5 min, achieving a high purity of 68% for single MTCs and a high purity of 35% for intact MTCCs. Our cascaded inertial microfluidic device may provide a new tool for accelerating clinical cancer diagnosis and enhance malignancy assessment capability.