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Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics

Nature Protocols volume 11, pages 134–148 (2016)Cite this article

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Abstract

Circulating tumor cells (CTCs) are rare cancer cells that are shed from primary or metastatic tumors into the peripheral blood circulation. Phenotypic and genetic characterization of these rare cells can provide important information to guide cancer staging and treatment, and thus further research into their characteristics and properties is an area of considerable interest. In this protocol, we describe detailed procedures for the production and use of a label-free spiral microfluidic device to allow size-based isolation of viable CTCs using hydrodynamic forces that are present in curvilinear microchannels. This spiral system enables us to achieve ≥85% recovery of spiked cells across multiple cancer cell lines and 99.99% depletion of white blood cells in whole blood. The described spiral microfluidic devices can be produced at an extremely low cost using standard microfabrication and soft lithography techniques (2–3 d), and they can be operated using two syringe pumps for lysed blood samples (7.5 ml in 12.5 min for a three-layered multiplexed chip). The fast processing time and the ability to collect CTCs from a large patient blood volume allows this technique to be used experimentally in a broad range of potential genomic and transcriptomic applications.

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Figure 1: Overview of CTC isolation using spiral microfluidics.
Figure 2: Schematic illustration and working principle of particle or cell focusing in straight and curvilinear microchannels.
Figure 3: Illustration of different steps for fabrication of spiral microfluidic chips.
Figure 4: Characterization of the spiral biochips for CTC isolation.
Figure 5: Immunostaining of enriched CTCs from clinical patient blood samples.
Figure 6: Validation of the spiral inertial biochip for clinical analysis.

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Acknowledgements

We express our sincere gratitude to all patients and healthy volunteers who participated in this trial and donated blood samples for characterization of our device. Financial support by the Singapore-MIT Alliance for Research and Technology (SMART) Center (BioSyM IRG) is gratefully acknowledged. This work is also supported by the use of NTU's Micro-Machine Center (MMC) facilities for wafer fabrication and the lab facilities at the Mechanobiology Institute (MBI) and the Nano Biomechanics Laboratory at the National University of Singapore. The clinical sample and data collection was supported by the Singapore National Medical Research Council grant no. NMRC 1225/2009.

Author information

Author notes

  1. Majid Ebrahimi Warkiani and Bee Luan Khoo: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Mechanical and Manufacturing Engineering, Australian Center for NanoMedicine, University of New South Wales, Sydney, New South Wales, Australia

    Majid Ebrahimi Warkiani

  2. Medical Oncology Group, Ingham Institute for Applied Medical Research, Liverpool, New South Wales, Australia

    Majid Ebrahimi Warkiani

  3. BioSystems and Micromechanics (BioSyM), Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore, Singapore

    Majid Ebrahimi Warkiani, Bee Luan Khoo, Andy Kah Ping Tay, Jongyoon Han & Chwee Teck Lim

  4. Mechanobiology Institute, National University of Singapore, Singapore, Singapore

    Bee Luan Khoo & Chwee Teck Lim

  5. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Lidan Wu & Jongyoon Han

  6. Department of Biomedical Engineering, National University of Singapore, Singapore, Singapore

    Andy Kah Ping Tay & Chwee Teck Lim

  7. Clearbridge BioMedics Pte Ltd, Singapore, Singapore

    Ali Asgar S Bhagat

  8. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Jongyoon Han

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Contributions

M.E.W., B.L.K., L.W., A.K.P.T., A.A.S.B. and C.T.L. contributed to the design of the spiral biochips. M.E.W., B.L.K., L.W., A.K.P.T. and A.A.S.B. prepared the manuscript and J.H. and C.T.L. commented on the manuscript.

Corresponding authors

Correspondence to Majid Ebrahimi Warkiani, Jongyoon Han or Chwee Teck Lim.

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Competing interests

A.A.S.B. works for Clearbridge Biomedics Pte Ltd, which is commercializing the spiral biochips for cancer cell enrichment.

Integrated supplementary information

Supplementary Figure 1 Recovery efficiency of spiral biochip device.

(a) Histogram plot indicating a high separation efficiency of ~ 90% for different cancer cell lines tested. All error bars represent standard deviation (SD) of triplicates. (b) Diagram showing the recovery of MCF-7 cells spiked into lysed blood at clinically relevant concentrations.

Supplementary Figure 2 Optical image of a metallic mold made by conventional micromilling in stainless steel.

Supplementary Figure 3 The workstation setup for CTC separation from lysed blood.

The lysed blood and one sheath flow are pumped through the single (or multiplexed) spiral biochip using 2 different syringe pumps where CTCs are separated from other blood components rapidly and efficiently.

Supplementary Figure 4 Schematic diagram illustrating the correct assembly of single (or multiplexed) spiral biochip for CTC isolation from lysed blood.

For the multiplexed biochip (i.e., 3 spirals), the flow rate of sample and sheath flow must be multiplied by three.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1–3 (PDF 808 kb)

Supplementary Data 1

Spiral CAD design for manufacture of a biochip. (ZIP 946 kb)

High-speed video illustrating the complete focusing of WBCs, platelets and RBC residuals after lysis to the outer wall of the channel from a healthy blood sample.

WBCs are clearly distinguished from platelets and RBC residuals based on size/morphology and phase contrast. (AVI 588 kb)

41596_2016_BFnprot2016003_MOESM283_ESM.avi

High-speed video illustrating the complete isolation of MDA-MB-231 cells from WBCs (lysed blood) at the device outlet using a single spiral biochip. (AVI 706 kb)

High-speed video captured at the outlet of a spiral biochip showing isolation of a CTC cluster from the peripheral blood of a patient with advanced metastatic lung cancer.

This movie clearly demonstrates the performance of our device for efficient enrichment of CTCs and micro-clusters from blood samples. (AVI 375 kb)

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Warkiani, M., Khoo, B., Wu, L. et al. Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics. Nat Protoc 11, 134–148 (2016). https://doi.org/10.1038/nprot.2016.003

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