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Microfluidic, marker-free isolation of circulating tumor cells from blood samples

Abstract

The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen–independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 107 cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2–5 d.

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Figure 1: CTC-iChip schematic.
Figure 2: Protocol flowchart.
Figure 3: Structure of the CTC-iChip1.
Figure 4: Structure of CTC-iChip2.
Figure 5: Magnetophoresis setup.
Figure 6: Schematic of the production of CTC-iChip2.
Figure 7: CTC-iChip assembly.
Figure 8: Running setup.
Figure 9: Sequence of steps that are crucial to priming and starting the CTC-iChip.
Figure 10: Expected results.

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Acknowledgements

We express our gratitude to all patients and healthy volunteers who participated in this study and contributed blood samples. We thank L. Libby, O. Hurtado and A.J. Aranyosi for coordination of the research laboratories; D.M. Lewis and B. Hamza for expertise in device fabrication; and L. Nieman and J. Walsh for expertise in microscopy. This work was supported by the US National Institutes of Health (NIH) P41 Resource Center (M.T.); a NIH National Institute of Biomedical Imaging and Bioengineering Quantum Grant (M.T. and D.A.H.); Stand Up to Cancer (D.A.H., M.T. and S.M.); the Howard Hughes Medical Institute (D.A.H.); the Prostate Cancer Foundation and the Charles Evans Foundation (D.A.H. and M.T.); and Johnson and Johnson (M.T. and S.M.).

Author information

Authors and Affiliations

Authors

Contributions

All authors contributed to design of the CTC-iChip. N.M.K., P.S.S., F.F., V.P., E.O., N.K., K.S. and M.T. prepared the manuscript. E.J.L., J.M.M., P.C., J.Y., H.H., B.M., J.T., T.A.B., S.L.S., S.M., R.K. and D.A.H. commented on the manuscript.

Corresponding author

Correspondence to Mehmet Toner.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Spintrap design and characterization.

(a, b) We designed and custom-made a closed centrifugation system, called Spintrap, for large volume, high throughput, and thin-layer preparation of CTCs. Due to its low fluidic resistance, the design allowed inline use with CTC-iChip with a processing time of 15 minutes for up to a fluid volume of 23 mL. The Spintrap consistently prevented air-drying and thus conserved native cell morphology. Using the Spintrap protocol (c, steps 88-92), and LBX1 cell line with cytoplasmic GFP label, we characterized cell attachment strength, yield, and resulting cellular morphology. (d) We performed automated fluorescence microscopy for quantifying efficiency of immobilization, and found the cell yield to be >95% at optimized conditions. (e, f) Confocal and epifluorescence microscopy revealed native cellular morphology at a relative centrifugal force of 50 g. A slight increase in cellular radius at 500 g and significant pancaking at 2,000 g was found, while cell circularity remained similar to a non-centrifuged control. We found centrifugation for 5 minutes at 50 g to be optimal for cell immobilization with high yield and without morphological disturbance. Cells collected with the Spintrap method showed diagnostic quality on cytological evaluation. Using Spintrap, we characterized CTCs from cancer patients isolated using CTC-iChip58.

Supplementary Figure 2 Spintrap fluidic simulation results.

Spintrap fluidic simulations show gradual deceleration and soft landing of cells to glass surface under 50g gravitational field.

Supplementary Figure 3 Calculated fluidic streamlines in CTC-iChip1.

a) Design of CTC-iChip1 posts used in fluidic simulations. b) 2D view of streamlines in the middle of the microfluidic channel, where the flow rate is highest. c) 3D view of streamlines.

Supplementary Figure 4 CTC-iChip1 assembly.

Detailed assembly of CTC-iChip1 with its manifold and tubing.

Supplementary Figure 5 Buffer and syringe cap schematics.

Schematic showing snorkel buffer cap and syringe cap. These parts help apply air pressure to blood in a syringe and buffer in a bottle using air-tight connections, and enable constant pressure operation of CTC-iChip

Supplementary information

Supplementary Figure 1

Spintrap design and characterization. (PDF 4349 kb)

Supplementary Figure 2

Spintrap fluidic simulation results. (PDF 1523 kb)

Supplementary Figure 3

Calculated fluidic streamlines in CTC-iChip1. (PDF 3409 kb)

Supplementary Figure 4

CTC-iChip1 assembly. (PDF 2067 kb)

Supplementary Figure 5

Buffer and syringe cap schematics. (PDF 641 kb)

Numerical simulation of the movement of a 10μm-diameter deformable cell surrounded by fluid in the Spintrap system centrifuged at 50g.

The applied centrifugal acceleration of 50g (direction: left to right) moves the cell toward the right wall where the glass slide is located. The custom-built finite-element software package PAK [ M. Kojic, R. Slavkovic, M. Zivkovic, N. Grujovic and N. Filipovic, PAK Finite Element Program for solid and fluid mechanics, mass and heat transfer, coupled problems and biomechanics., Mech. Eng. Dept. University of Kragujevac and R&D Center for Bioengineering Kragujevac, Serbia (2010)] was modified to solve for the fluid velocity field as well as the deformations of the cell, during a constant 50g acceleration. The video shows the resulting fluid velocity field (color scale bar indicates speed in μm/s) and depicts the gradual decrease in the cell speed as it approaches the glass slide (see also Supplementary Figure 2). The resulting minimal cell deformation is in good agreement with the experimentally observed cell morphology for 50g (see Supplementary Figure 1e). (AVI 1952 kb)

High-speed video of Deterministic Lateral Displacement of a WBC in blood.

A moving WBC is tracked within CTC-iChip2. WBC is deflected due to DLD whereas RBCs are following fluid streamlines. By the 15th second, the WBC is almost completely separated from the RBC population. The video was captured at 10× magnification while the stage was moving to enable tracking of the WBC. The video is played at 200× slower speed than the particles are moving in the channel. (AVI 16779 kb)

High-speed video of magnetophoretically separated cells entering outlets of CTC-iChip2.

PC3-9 cells being enriched by deflection of WBCs. The video is played at 200× slower than real-time. Note that this video was taken from a slightly different design, which has the product outlet in the middle and magnetophoresis setup pulling the WBCs to the sidewall. (AVI 5177 kb)

CTC-iChip2 Assembly.

Assembly of CTC-iChip2 with its magnetic manifold and their alignment is demonstrated. (MOV 43125 kb)

Supplementary Data

CTC-iChip2 Design CAD. (ZIP 354 kb)

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Karabacak, N., Spuhler, P., Fachin, F. et al. Microfluidic, marker-free isolation of circulating tumor cells from blood samples. Nat Protoc 9, 694–710 (2014). https://doi.org/10.1038/nprot.2014.044

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