Research ArticleAPPLIED SCIENCES AND ENGINEERING

Detection and isolation of free cancer cells from ascites and peritoneal lavages using optically induced electrokinetics (OEK)

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Science Advances  05 Aug 2020:
Vol. 6, no. 32, eaba9628
DOI: 10.1126/sciadv.aba9628
  • Fig. 1 OEK microfluidic chip developed for detecting peritoneal metastasis and cell membrane capacitance.

    Ascites obtained from patients are placed in the OEK microfluidic chip. The gastric cancer cells can be separated from ascites rapidly and label-free. Meanwhile, the cell membrane capacitance of the separated cells is obtained.

  • Fig. 2 Experimental system and the OEK microfluidic chip.

    (A) Schematic diagram of the OEK system. The system consists of a computer, a projector, a condensed lens, a signal generator, a three-dimensional (3D) motion stage, a charge-coupled device (CCD) camera, and the OEK microfluidic chip. (B) Schematic of extraction device. The plastic hose, which is connected to the syringe pump, is used for collecting separated cells. (C) Structure of the OEK microfluidic chip. The chip consists of three parts: glass coated with indium tin oxide film (ITO glass), double-adhesive tape, which is used for fabrication of the microchannel, and coated hydrogenated amorphous silicon (a-Si:H) ITO glass.

  • Fig. 3 Cell polarization model and cell separation.

    (A) A single layer of shell core model for cells is established in this system. The model is applied to the theoretical basis of cell separation and to the solution of cell membrane capacitance. (B) Radii of SGC-7901 cancer cell line cells and patients’ peritoneal lavage cells (M1, M2, and M3). (C) Different crossover frequencies between SGC-7901 and patients’ peritoneal lavage cells. ***P < 0.001. (D) Cell membrane capacitances of SGC-7901 and patients’ peritoneal lavage cells. (E) Cell separation after mixing SGC-7901 with different patients’ peritoneal lavage cells (M4 and M5) in a ratio of 1:20, 1:50, and 1:100. The AC voltage of 70 KHz 10 Vpp are applied on the OEK microfluidic chip in the illuminated area and some cells are applied by positive dielectrophoresis (pDEP) force and others are moving away from the light pattern by negative dielectrophoresis (nDEP) force. Scale bars, 50 μm. (F) The finite element method for numerical analysis results of electric field magnitudes ∣E∣ and the direction of the ∇E2 distribution.

  • Fig. 4 Process of cell separation and identification of separated cells.

    (A) Process of cell separation in the OEK microfluidic chip. (a) and (b) show the process of cell separation in the cell mixture. (c) and (d) show the process of dragging separated cells next to the plastic hose in the OEK microfluidic chip. (e) shows separated cells collected by the plastic hose. (B) Cell separation and identification of simulated peritoneal lavage. We mixed SGC-7901 cells, which were incubated with CellTracker Green CMFDA with peritoneal lavage cells in different ratios: 1:10, 1:100, and 1:1000. The separated cells were imaged by a fluorescence microscope using a 10× objective. The second panel of (B) shows representative cells after separation stained with Wright-Giemsa. The cells were hyperchromatic with large nuclei, conforming to the characteristics of cancer cells. The third panel of (B) shows the cells before separation stained with Wright-Giemsa. The red arrows in (B) point to gastric cancer cells.

  • Fig. 5 Cell separation of patients’ ascites.

    We separated gastric cancer cells from patients’ ascites (A1 to A5) with appropriate external frequencies. A1, A2, and A3 are patients’ ascites before treatment and A4 and A5 are after treatment. (A) Radii of separated cells. (B) Cell membrane capacitances of separated cells. (C) Process of cell separation in the OEK microfluidic chip. (a) demonstrates separating gastric cancer cells in the cell collection area. (b) is the process of opt dragging separated cells to the plastic hose. (c) shows the collection process by plastic hose. Scale bars, 50 μm. (D) Wright-Giemsa staining of cells. The left panel shows representative cells after separation. The cells were hyperchromatic with large nuclei, conforming to the characteristics of cancer cells. The right panel shows the cells before separation. Scale bars, 50 μm.

  • Fig. 6 Cell separation in different stages of patient A6 and results summary.

    We separated patient A6’s ascites in the different stages; A6-1 is the ascites before the treatment, and A6-2 and A6-3 are the ascites after treatment. (A) Radii of separated cells in the different stages. (B) Cell membrane capacitances of separated cells. (C) Wright-Giemsa staining of cells. The first panel shows the representative cells after separation. The cells were hyperchromatic with large nuclei, conforming to the characteristics of cancer cells. The second panel shows the cells before separation. Scale bars, 50 μm. (D) EpCAM was positively expressed in the isolated cells from sample A2, A3, A4, and A6-3. (E) Cell membrane capacitance summary of clinical samples. The cell membrane capacitances of the ascites separated cells were all significantly higher than that of three peritoneal lavage cells. Separated cells’ membrane capacitances from ascites after treatment are higher than those before treatment. (F) Purity statistics of cell separations in eight ascites samples. (G) The overall purity of the cell separation. This overall purity was calculated through pooled analysis by Stata. The purity was 71% [95% confidence interval (CI), 62 to 80%]. *P < 0.05. **P < 0.01. ***P < 0.001.

Supplementary Materials

  • Supplementary Materials

    Detection and isolation of free cancer cells from ascites and peritoneal lavages using optically induced electrokinetics (OEK)

    Yuzhao Zhang, Junhua Zhao, Haibo Yu, Pan Li, Wenfeng Liang, Zhu Liu, Gwo-Bin Lee, Lianqing Liu, Wen Jung Li, Zhenning Wang

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