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. 2010 Aug 17;107(33):14524-9.
doi: 10.1073/pnas.1001515107. Epub 2010 Aug 2.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

Antoine-Emmanuel Saliba  1 , Laure Saias, Eleni Psychari, Nicolas Minc, Damien Simon, François-Clément Bidard, Claire Mathiot, Jean-Yves Pierga, Vincent Fraisier, Jean Salamero, Véronique Saada, Françoise Farace, Philippe Vielh, Laurent Malaquin, Jean-Louis Viovy
Affiliations

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

Antoine-Emmanuel Saliba et al. Proc Natl Acad Sci U S A. .

Abstract

We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

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Conflict of interest statement

Conflict of interest statement: Part of the methodology described is dependent from CNRS patent WO9823379 and Curie Institute patent application PCT/FR2009/051942.

Figures

Fig. 1.
Fig. 1.
Principle and practical implementation of the Ephesia system. (A) Principle of magnetic self-assembly. A hexagonal array of magnetic ink is patterned at the bottom of a microfluidic channel. Beads coated with an antibody are injected in the channel. Beads are submitted to Brownian motion. The application of an external vertical magnetic field induces the formation of a regular array of bead columns localized on top of the ink dots. (B) Two levels PDMS integrated microchip. Channels were filled with colored water. Delivery and separation channels for the cells appear in yellow. Inlets ports appear in orange. The separation channel is the longer vertical branch. The area bearing magnetic posts is marked by the dotted white box. Channels in the upper PDMS layer, controlling the opening and closing of the inlet channels, appear in blue. The green wire is a thermocouple for in situ control of the temperature in the system. (Scale bar: 0.5 cm.) (C) Magnetically assembled array of columns of 4.5 μm beads coated with anti-CD19 mAb (specifically retaining Raji B-Lymphocytes) (Movie S1). Typical column shapes are shown in the insets. (Scale bar: 80 μm.) (D) Optical micrograph of the columns after the passage of 1,000 Jurkat cells. No cell can be seen (Movie S2). (Scale bar: 80 μm.) (E) After the passage of 400 Raji cells, numerous ones are captured and rosetted on the columns (Movie S3). (Scale bar: 80 μm.)
Fig. 2.
Fig. 2.
Cell capture profile in the channel. x-axis: magnetic columns row, numbered from the capture microchannel entrance. y-axis: Number of cells captured in each row (sum over all the columns in the row). Data from three experiments with different total numbers (N) of cells injected are plotted as triangles (N = 214), circles (N = 320) and squares (N = 400). Theoretical best-fit values for each number of cells are represented by full lines (all derived with a single set of parameters r = 0.18 and L = 25). The last two curves for N = 700 and N = 1,000 are extrapolations for larger numbers of cells, showing a saturation plateau progressing with time from the entrance to the outlet.
Fig. 3.
Fig. 3.
In-situ culture and observation of Raji cells in the magnetic array. Numbers under the frames represent the time elapsed since capture, in hours:minute. The cell with the white arrow undergoes cell division during this time lapse. The cell on the left has extracted a bead from the magnetic column (black arrow), and is moving it around. (Scale bar: 40 μm.)
Fig. 4.
Fig. 4.
Examples of normal and malignant B-cell analysis by cytological staining, flow cytometry, and Ephesia system. Each line represents one subject. Columns represent, from left to right: (i) patient status; (ii) cytological observation (May Grunwald Giensa (MGG) coloration); (iii and iv) flow cytometry gated on lymphoid population data representing the intensity of CD19 versus CD5 and CD10. The color code distinguishes non B (red) and B CD19+ (green) lymphocytes; (v to viii): Ephesia representative confocal images selected from an image library for each patient. Subsequent images are, respectively: vi: equatorial z-plane bright field image, with indication of cell diameter; (vii): equatorial z-plane nucleus image (Hoechst staining); (vii): integration of three z confocal planes centered on the cell’s center, for fluorescent anti-CD10 (yellow); (viii) same as (vii) for anti-CD5 (red); in each image, the dashed circle represents the magnetic bead localization; (ix) a quantification of CD10 versus CD5 fluorescence intensity extracted from confocal images of trapped CD19 population; a red circle points to the data from the cells presented in vignettes (v) to (viii). Note that these datasets are to be compared with the green dots of flow cytometry data (columns (iii) and (iv)) only, because CD19- cells are not captured in Ephesia. A, first line: Healthy subject (identified as n°1 in Table S2) shows, among the CD19+ cells in FC, a majority of CD5-/CD10- cells, and minor populations identified as CD5+ or CD10+. Ephesia reproduces this trend with minor populations as CD10+ and CD5+. The nucleus has a regular outline, both in MGG (col. ii) and Hoechst staining in Ephesia, with a preferred chromatin localization at the periphery of the nucleus. B, line 2: Patient with chronic lymphocytic leukemia (n°3 in Table S2) shows a vast majority of CD5+ and CD10- cells, according to both flow cytometry and Ephesia fluorescence quantification. Ephesia additionally shows CD5 located at the membrane. C, line 3: Cells from patient with Mantle-cell lymphoma (n°7 in Table S2) show a larger diameter than normal B-cell and nucleolus on cytological observation (arrows); CD19+ cells are CD5+ and CD10- in FC data. Ephesia reproduces these results showing intranuclear nucleolus (arrow), no expression of CD10 and a bright membrane expression of CD5. D, line 4: Cells from patient with follicular lymphoma (n°9 in Table S2) show a fragmented nucleus on cytological observation (arrow). FC shows that B-cells even if CD19 are expressed at low levels, present a high expression level of CD10 but not CD5. Ephesia reproduces these results showing cleaved nucleus (arrow), no expression of CD5 and an expression of CD10, localized at the membrane and in the cytoplasm. (Scale bar: 5 μm.)
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References

    1. Hortobagyi GN. Trastuzumab in the treatment of breast cancer. N Engl J Med. 2005;353:1734–1736. - PubMed
    1. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997;3:730–737. - PubMed
    1. Pachmann K, et al. Monitoring the response of circulating epithelial tumor cells to adjuvant chemotherapy in breast cancer allows detection of patients at risk of early relapse. J Clin Oncol. 2008;26:1208–1215. - PubMed
    1. Allard WJ, et al. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res. 2004;10:6897–6904. - PubMed
    1. Jorgensen JL. State of the art symposium: flow cytometry in the diagnosis of lymphoproliferative disorders by fine-needle aspiration. Cancer. 2005;105:443–451. - PubMed

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