Accurate and efficient separation of tumor cells and clusters from malignant effusions is crucial for cancer diagnosis and prognosis. Tumor cell clusters are particularly significant due to their enhanced metastasis and treatment resistance compared to single cells. Existing methods, such as immune-affinity-based cell sorting, suffer from low efficiency for cluster purification and may damage cells. Label-free microfluidics offers a promising alternative, utilizing the physical properties of cells for separation. While progress has been made in separating single tumor cells based on size, density, and deformability, high-throughput, continuous-flow ternary separation of single cells, clusters, and WBCs remains a challenge. Previous microfluidic devices often suffer from low throughput, require multiple steps, or struggle with purifying clusters from single cells. This research aims to address these limitations by developing a novel spiral-contraction-expansion microfluidic device for efficient and high-throughput ternary separation.

The literature review highlights the clinical importance of identifying tumor cells and clusters in malignant effusions for diagnosis and prognosis. Existing immune-affinity-based methods are inefficient for separating clusters and may compromise cell viability. Label-free microfluidic approaches based on inertial focusing show promise, with various designs focusing on single-cell separation. However, a high-throughput, continuous-flow, single-step method for simultaneously separating single tumor cells, clusters, and WBCs is lacking. Existing methods have limitations including low throughput, multiple steps, and difficulties in isolating clusters from single cells. The authors reviewed examples of previous microfluidic devices and their limitations, highlighting the need for a more efficient and comprehensive approach.

The authors designed a microfluidic device consisting of slanted spiral channels coupled with periodic contraction-expansion arrays. This design utilizes inertial lift force (FL), Dean drag force (FD), and vortex-induced lift force (FV) to achieve size-based separation. The device was fabricated using a chip-on-film technique involving UV laser cutting and polymer film assembly. Polystyrene microparticles of varying sizes (10, 15, 20, and 25 μm) were used to characterize focusing performance. MDA-MB-231 breast cancer cells (including single cells and clusters) and WBCs were used to evaluate cell separation efficiency. Clinical pleural and abdominal effusion samples from six patients were also tested. Immunofluorescence staining was used to identify tumor cells (Pan-CK positive, CD45 negative) and WBCs (CD45 positive, Pan-CK negative) after separation. A high-speed CCD camera and ImageJ software were used for data acquisition and analysis. The experimental setup involved a syringe pump to control flow rate and a cell counter to determine cell concentrations.

The study showed that the spiral-contraction-expansion device effectively focused particles based on size. At an optimal flow rate of 3500 µL/min, 10 μm particles were focused near the outer wall, 15 μm particles near the center, and 20-25 μm particles near the inner wall, demonstrating successful ternary focusing. In cell separation experiments, the device achieved a 93.4% recovery rate for MDA-MB-231 cells and 94.0% removal of WBCs at 3500 µL/min. Importantly, more than 90% of tumor cell clusters were preserved after separation. The device also successfully performed ternary separation of cells from clinical pleural and abdominal effusion samples from six cancer patients, demonstrating its applicability to real-world samples.

The results demonstrate the effectiveness of the spiral-contraction-expansion device for high-throughput, continuous-flow, label-free ternary separation of tumor cells, clusters, and WBCs from malignant effusions. The device's performance surpasses existing methods by achieving high recovery rates for tumor cells and clusters while efficiently removing WBCs. The successful application to clinical samples highlights its potential for use in cancer diagnostics. The combined effect of inertial lift, Dean drag, and vortex-induced lift forces enables size-based separation, simplifying the process and enhancing throughput. This technology could significantly improve the efficiency and accuracy of cytological analysis of malignant effusions, potentially leading to earlier and more accurate cancer diagnosis.

This study successfully demonstrates a novel microfluidic device for high-throughput and simultaneous separation of tumor cells and clusters from malignant effusions. The device's high efficiency, continuous flow, and label-free operation offer significant advantages over existing techniques. Future research could focus on optimizing the device for other types of cells or fluids and integrating it with downstream analysis methods for a fully automated diagnostic workflow.

The study primarily focused on MDA-MB-231 breast cancer cells. Further research is needed to evaluate the device's performance with other cancer cell types and to investigate potential variations in separation efficiency due to differences in cell size, deformability, or clustering characteristics. The current device design may need further optimization to handle highly viscous or heterogeneous clinical samples.