High-throughput and simultaneous inertial separation of tumor cells and clusters from malignant effusions using spiral-contraction-expansion channels

Exfoliated tumor cells and clusters present in pleural or abdominal effusions are associated with malignancy, metastatic spread, and poor prognosis. Accurate cytological diagnosis, including enumeration of tumor cell clusters, is clinically important because clusters have markedly higher metastatic potential than single cells. Conventional immune-affinity cell sorting methods effectively detect individual tumor cells but are inefficient for further purification of clusters and do not readily yield viable cells for downstream analyses such as culture, drug testing, and sequencing. Label-free microfluidic approaches leveraging physical properties (density, size, deformability) or external fields (dielectric, magnetic, optical) have advanced single tumor cell separation, yet cluster separation has received less attention until recently. Prior devices (e.g., Cluster-Chip, pillar arrays, Labyrinth) demonstrated enrichment of clusters but faced limitations including low throughput, potential damage or release steps, and difficulty further purifying clusters from single tumor cells due to their rarity. There remains a need for high-throughput, continuous-flow, single-step, label-free devices that can simultaneously separate single tumor cells, tumor cell clusters, and blood cells. In this work, the authors developed a spiral-contraction-expansion microfluidic device that couples slanted spiral channels with periodic contraction–expansion arrays to exploit the combined inertial lift, Dean drag, and local vortex-induced lift forces for ternary, size-based separation. They characterized particle focusing versus size and flow rate, optimized operation, quantified recovery and purity for tumor cells and clusters, and demonstrated ternary separation from clinical malignant effusions.

Conceptual design and principle: The device integrates slanted spiral microchannels with periodic contraction–expansion arrays to achieve continuous, high-throughput, size-based ternary separation. In the slanted spiral section, the inertial lift force (FL) drives larger cells toward the inner wall, while the Dean drag force (FD) drives smaller cells toward the outer wall, producing binary pre-separation (large tumor cells vs. small WBCs). In the final loop, periodic expansion structures at the outer wall generate local vortices that introduce an additional vortex-induced lift force (FV) toward the outer wall. The balance among FL, FD, and FV produces three distinct equilibrium positions: largest clusters near the inner wall, single tumor cells near the channel centerline, and WBCs trapped near the outer wall within the Dean vortex, enabling ternary separation. Device design and fabrication: A chip-on-film method was used. Four polymer films were UV laser-cut (TH-UV200A) and assembled. The inner wall was formed by a 70 µm film and the outer wall by a 160 µm film, yielding slanted (trapezoidal) cross-sections after bonding to a cover plate. The device comprises a 4-loop slanted spiral channel with 28 periodic expansion structures along the outer wall. Channel geometry: trapezoidal cross-section with 70 µm inner-wall height, 160 µm outer-wall height, and 500 µm width. Expansion structures are spaced every 5 degrees along the circumference and each has 500 µm width. Geometric optimization details are provided in supplementary figures and table. Sample preparation: Polystyrene particles (10, 15, 20, 25 µm; Thermo Fisher Scientific) were suspended in PBS (0.01 M) with 1% Pluronic F-127 to 1×10^6 particles/mL for focusing characterization. Human breast tumor cells (MDA-MB-231) were cultured in DMEM with 10% FBS and 1% penicillin–streptomycin at 37 °C, 5% CO2. Clusters were generated by trypsinizing adherent cells (0.05% trypsin-EDTA), low-speed centrifugation, and gentle resuspension in PBS. WBCs were prepared by lysing donor blood with ACK buffer and diluting in PBS. Test mixtures contained ~10^5 WBCs/mL and ~10^3 MDA-MB-231 cells/mL, with ~154±5 clusters per experiment. Clinical pleural/abdominal effusions (20 mL each) from 6 metastatic cancer patients were processed by centrifugation (1000 rpm, 5 min), and cell pellets were resuspended in the original volume of PBS to minimize viscoelastic effects prior to separation. Immunofluorescence staining and identification: Post-separation, cells from all outlets were centrifuged and adhered to Polysine slides, fixed with −20 °C methanol (5 min), blocked with 10% normal goat serum, stained with FITC-conjugated Pan-CK (tumor cells) and APC-conjugated CD45 (WBCs) at 4 °C overnight, and mounted with DAPI. Pan-CK+/DAPI+/CD45− cells were classified as tumor cells; CD45+/DAPI+/Pan-CK− cells as WBCs. Experimental setup and data analysis: The device was clamped in a transparent fixture on a microscope (Olympus IX71) with a high-speed CCD (Retiga EXi) to capture trajectories. ImageJ was used to stack time-series images and extract focusing maps and profiles. A syringe pump (Legato 270) controlled flow rates. Cell concentrations were measured by an automated counter (Countess II FL). Operational flow rates were scanned from 500 to 4000 µL/min; an optimal flow rate near 3500 µL/min was identified for ternary focusing and separation.

- Particle focusing and ternary separation: The coupling of slanted spiral channels with periodic contraction–expansion arrays shifted equilibrium positions and enhanced focusing. At 3500–4000 µL/min, ternary focusing was achieved: 10 µm particles migrated to the outer wall under dominant Dean drag; 15 µm particles focused at the channel centerline due to the added vortex-induced lift; 20–25 µm particles focused near the inner wall under dominant inertial lift. Quantitatively at 3500 µL/min, equilibrium positions were approximately: 10 µm at 373–456 µm from the inner wall; 15 µm at the centerline (~253 µm); 20–25 µm near the inner wall (~83 µm). - Cell separation performance: Using lysed blood spiked with MDA-MB-231 cells and clusters at 3500 µL/min, the device removed 94.0% of WBCs and recovered more than 97% of MDA-MB-231 tumor cells, while preserving more than 90% of tumor cell clusters. Reported outlet analysis noted 93.4% of MDA-MB-231 cells collected at outlet II (additional distribution not fully shown), consistent with high overall recovery. - Clinical applicability: The device successfully performed one-step, label-free ternary separation of single tumor cells, tumor cell clusters, and WBCs from pleural or abdominal effusions from 6 metastatic cancer patients, demonstrating feasibility as a high-throughput sample pretreatment tool.

The study addresses the need for simultaneous enrichment of tumor cell clusters and single tumor cells from complex effusion samples. By harnessing the interplay among inertial lift, Dean drag, and vortex-induced lift forces within a spiral-contraction–expansion architecture, the device generates three distinct equilibrium positions, enabling one-step ternary separation at high volumetric throughput (up to 3500–4000 µL/min). This overcomes the binary limitation of conventional spiral inertial devices and avoids low-throughput or potentially damaging capture-and-release strategies. The measured high WBC removal (94%), high tumor cell recovery (>97%), and high cluster preservation (>90%) indicate effective purification with minimal loss, which is essential for downstream analyses requiring viable and intact cells and clusters. Successful application to clinical pleural/abdominal effusions underscores translational relevance, suggesting utility as a preparatory step for cytological diagnosis, enumeration of clusters for prognostic evaluation, and potential downstream molecular or functional assays.

The authors introduced a chip-on-film spiral-contraction–expansion microfluidic device that achieves high-throughput, continuous, label-free ternary separation of WBCs, single tumor cells, and tumor cell clusters. By integrating periodic contraction–expansion arrays into slanted spiral channels, the device leverages combined hydrodynamic forces to produce size-dependent equilibrium positions, validated with particles, cultured cells, and clinical effusions. Key outcomes include 94% WBC removal, >97% tumor cell recovery, and >90% cluster preservation at 3500 µL/min, demonstrating suitability as a sample pretreatment platform for malignant effusions. Future work could include broader clinical validation across cancer types and effusion viscosities, assessment of post-separation cell/cluster viability and function over time, integration with downstream analytical modules (culture, drug testing, genomics), and further optimization of channel geometry for even higher throughput and robustness.