Hybrid microfluidic sorting of rare cells based on high throughput inertial focusing and high accuracy acoustic manipulation

Abstract

The ability to isolate rare circulating tumor cells (CTCs) from blood samples is essential to perform liquid biopsy as a routine diagnostic and prognostic test. Both label-free and surface biomarker-based cell sorting technologies have been developed to address the demand in high-integrity isolation of rare CTCs for cancer research. Label-free cell sorting mainly relies on the size difference between CTCs and blood cells; thus, it lacks sufficient sorting specificity. Surface biomarker-based cell sorting is highly specific; however, it requires expensive, labor-intensive, and time-consuming labeling due to the use of multiple sets of surface biomarkers. Because of the complex nature and high heterogeneity of tumorigenesis, it is difficult to rely on a single sorting process for high-integrity rare cell isolation. In this study, for the first time, we present a hybrid microfluidic cell sorting method combining high throughput size-dependent inertial focusing for size-based pre-enrichment and high accuracy fluorescence activated acoustic sorting for single cell isolation. After one single hybrid sorting process, we have demonstrated at least 2500-fold purity enrichment of MCF-7 breast cancer cells spiked in diluted whole blood samples with cell viability maintained at 91 ± 1% (viability before sorting was 94 ± 2%). This developed hybrid microfluidic cell sorting technique provides a promising solution for rare cell isolation needed in a variety of biological research and clinical applications.

Fig. 1. Hybrid sorting method combining inertial focusing and acoustic manipulation for rare cell isolation. (a) Schematic of the size-based inertial cell sorting device (one reverse wavy channel unit near the trifurcated section). Cancer cells and blood cells are expected to have differential focusing positions because of the size difference. (b) Schematic of the fluorescence-activated acoustic sorting at the single cell level. Sandwiched by two asymmetric sheath flows, the cell sample suspension flows through the fluorescence interrogation region where the fluorescent label on target cancer cells is excited. Upon the detection of single cell fluorescence emission, the focused interdigital transducer (FIDT) generates a focused acoustic wave beam to laterally transport the detected target cell toward the target outlet, while the non-target cells follow the original streamline to the waste outlet. (c) Schematic of the cell content after each enrichment cycle. Size-based inertial sorting and fluorescence activated acoustic sorting can both enrich the target cells 50–100 fold, resulting in an overall 2500–10 000 fold enrichment that is typically needed for rare cell isolation.

Fig. 2. Schematic of the fluorescence-activated acoustic cell sorting system, which is composed of three main parts: (i) fluorescence excitation and detection subsystem, (ii) acoustic cell sorting subsystem and (iii) observation stage.

Fig. 3. Inertial focusing behavior of differently sized microspheres in the inertial sorting device. (a) 15 μm fluorescent microsphere is used to represent the minimum size of cancer cells. (b) 10 μm fluorescent microsphere is used to mimic white blood cells. (c) 7 μm fluorescent microsphere is used to mimic red blood cells. (d) 3 μm fluorescent microsphere is used to mimic platelets. Scale bar is 125 μm.

Fig. 4. Time-lapsed microscopic image showing the process of sorting one single fluorescently labeled MCF-7 cancer cell from blood cells to the upper target outlet. Unlabeled non-target blood cells flow into the lower waste outlet. The image was obtained by overlapping 12 frames recorded every 2 ms. The scale bar is 50 μm.

Fig. 5. Representative microscopic photographs of cell samples collected from (a) input inlet of inertial sorting device, (b) target outlet of inertial sorting device, and (c) target outlet of acoustic sorting device. A representative cancer cell in each sample is indicated by a blue dashed circle. Scale bar is 100 μm. (d) Experiments 1–3 refer to the three repeated experiments under the same sorting conditions. Purity 1 and viability 1 refer to the purity and viability of cancer cells from sample (a). Purity 2 and viability 2 refer to the purity and viability of cancer cells from sample (b). Purity 3 and viability 3 refer to the purity and viability of cancer cells from sample (c). (e) and (f) show proliferation of cancer cells collected from the final sorting output.