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Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device

Nature Protocols volume 13, pages 34–58 (2018)Cite this article

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Abstract

The development of personalized cancer therapy depends on a robust system to monitor the patient's individual response to anticancer treatment. Anticancer drug efficacy has been tested on circulating tumor cells (CTCs) derived from patient blood samples after ex vivo expansion into CTC clusters. Current attempts to culture these primary cancer cells focus on long-term maintenance under growth factor supplements into cell lines, which usually takes >6 months and results in a CTC expansion efficiency of <20%. We recently developed a simple but unique microfluidics-based culture approach that requires minimal preprocessing (∼30 min) and does not require prior enrichment of CTCs or depend on the use of growth factor supplements. The approach capitalizes on co-culture of immune cells from the same patient blood sample within specially designed microwells that promote CTC cluster formation within 2 weeks, with an overall cluster formation success rate of ∼50%. Drug screening is facilitated by the incorporation of a gradient generator for parallel exposure to two or more drugs at various concentrations. Owing to the cost-effectiveness and less-invasive nature of this procedure, routine monitoring of disease progression can be achieved. The described microfluidics system can be operated with a single syringe pump to introduce drug compounds (which takes ∼6 min), followed by incubation of the CTC clusters for 48 h before analysis. In addition to its applications in biomedical research, the rapid readout of our platform will enable clinicians to assess or predict a patient's response to various therapeutic strategies, so as to enable personalized or precision therapy.

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Figure 1: Steps 72–82: Schematic illustration and working principle of CTC cluster formation.
Figure 2: Steps 83–99: Overview of CTC cluster formation.
Figure 3: Applications of the CTC cluster assay.
Figure 4: Clinical relevance of CTC cluster formation.
Figure 5: Working principle of back-diffuser exposure.
Figure 6: Steps 50–52: Assembly of PDMS layers.
Figure 7: Preparation of device and concentration gradient characterization.

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Acknowledgements

We express our sincere gratitude to all volunteers who participated in this trial and donated blood samples for characterization of our device. The clinical samples and data collection were supported by a Singapore National Medical Research Council (NMRC) grant. This work was also supported by the Mechanobiology Institute and the Singapore–MIT Alliance for Research and Technology (SMART) BioSystems and Micromechanics (BioSyM) Interdisciplinary Research Groups (IRGs), which are funded by the National Research Foundation, Prime Minister's Office, Singapore, under CREATE.

Author information

Author notes

  1. Gianluca Grenci and Ying Bena Lim: These authors contributed equally to this work.

Authors and Affiliations

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

    Bee Luan Khoo, Jongyoon Han & Chwee Teck Lim

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

    Gianluca Grenci & Chwee Teck Lim

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

    Gianluca Grenci, Ying Bena Lim & Chwee Teck Lim

  4. Department of Hematology–Oncology, National University Cancer Institute, National University Hospital, Singapore, Singapore

    Soo Chin Lee

  5. Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore

    Soo Chin Lee

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

    Jongyoon Han

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

    Jongyoon Han

  8. Department of Mechanical Engineering, National University of Singapore, Singapore, Singapore

    Chwee Teck Lim

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  1. Bee Luan Khoo
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Contributions

B.L.K. and G.G. conceived and designed the experiments. B.L.K., G.G. and Y.B.L. performed the experiments. C.T.L., S.C.L. and J.H. contributed by providing reagents, materials and analysis tools. B.L.K., G.G., Y.B.L., S.C.L., J.H. and C.T.L. wrote the manuscript.

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Correspondence to Bee Luan Khoo, Jongyoon Han or Chwee Teck Lim.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Optimization of microwell fabrication.

(A) Schematics illustrating the formation of a microwell. The distance of the laser from the substrate, the speed of laser ablation as well as laser power affects the depth and width of microwell formed. The higher the laser power, the deeper and wider the resultant microwell becomes. The layer of the recast is an artifact generated by the laser process, which becomes prominent with stronger laser power. (B) Phase contrast images of the smallest (left; average inner diameter 50 μm; Scale bar is 50 μm) and largest possible microwell that can be fabricated (middle; average inner diameter 341.5 μm; Scale bar is 100 μm.) with laser-ablation. (Right) Magnified view of the array of microwells with optimal parameters. (C) Simulation results of flow velocity at the entrance of the channel. (left) Cross-sectional slices of the velocity magnitude in the XZ-plane. Coloured scale bar represents the velocity magnitude in mm/s. Red arrows depict the direction of the flow and have lengths that are proportional to the velocity magnitude. (right) Zoomed in view of the panel a where the microwells are. The uniform dark blue colour within the microwells indicate that there is no flow in that region i.e. cells in culture do not experience shear stresses from the flow of fluid entering the channel from the gradient generator.

Supplementary Figure 2 Optimization of microwell parameters.

Phase contrast images of cancer cells seeded into microwells of different average diameters (∼107 μm, 187.5 μm and 341.5 μm from left to right). Images were obtained with a 20X objective lens. Scale bar is 50 μm.

Supplementary Figure 3 Custom tapered microwells for CTC cluster formation.

Clinical samples do not form clusters in conventional round bottom wells (left) but are able to develop clusters consistently in our tapered microwell assay. These clusters can be formed in microwells generated by either laser-ablation (middle) or microfabrication (right).

Supplementary Figure 4 Step 79: Enumeration of cell counts in a fixed area reveal cell packing density.

(a) Cell counts in a fixed area (e.g. 50 μm by 50 μm) are obtained from images of cultures with WBCs from healthy volunteer only and spiked cultures with WBC and cancer cell lines. (b) Spiked cultures start to form aggregates of higher cell density (> 8 cells per 2500 μm2) after only 3 days in culture.

Supplementary Figure 5 Substrate adherence requirement.

(a) Surfactant treatment of polystyrene surfaces is temporary, and spheroids of cell lines cultured in laser-ablated microwells dispersed after three days in culture. N=3. Scale bar is 50 μm. (b) Clinical samples can form loose clusters with microwells of 187.5 μm average diameter in the absence of surfactant treatment. N=3. Scale bar is 50 μm.

Supplementary Figure 6 Step 79: Cluster identification.

(a) Morphological phenotyping of a cluster in laser-ablated microwells using a phase-contrast microscope. Scale bar is 100 μm. (b) Cluster stained in situ with live dye for visualization and quantification of cell density. Scale bar is 100 μm. (c) Clusters demonstrate a region of lower gray scale values, while regions without cells reflect most of the light, leading to high gray scale values. (d) Microfabricated microwells with sparse blood cell monolayer (top) or cluster (bottom) respectively at 10X magnification. Corresponding scatter plots of grey values reflect cell density within the microwell. Microwells with sparse cell density have high grey values within the microwell region, while those with high cell density reflect lower grey values < 50% of peak. Scale bar is 100 μm.

Supplementary Figure 7 Panel of healthy sample cultures.

Cultures of healthy blood samples demonstrate either cell debris (25%) or monolayer of residual blood cells (75%) (n=16). Scale bar is 50 μm.

Supplementary Figure 8 Step 6: Fabrication of the primary mold with elliptical pillars.

(a) The 4” soda lime mask with the elliptical opening patterned in the Cr layer, already coated with the 500-nm thick PMGI sacrificial layer (step 28). (b) SU-8 spin coating of the mask plate. SU-8 3050 is poured carefully on the plate while this is placed on the vacuum chuck of the spin coater (steps 29 and 31). (c) Soft baking of the SU-8 (steps 30 and 32). (d) Loading of the mask (face down) in the UV exposure system. A blank mask plate is placed face up to prevent reflection of the UV light from the bottom, after passing through the SU-8 layer. (e) The opal diffuser is placed on top of the mask (step 33). (f) Development of the mask. When all the unexposed SU-8 has been removed, the plate is cleaned with spraying SU-8 developer first and IPA later, and then dried with gentle nitrogen flow (step 35).

Supplementary Figure 9 Step 34: Soft-lithographic replica of the primary mold.

(a) The mask plate with the SU-8 dome-shaped pillars is placed in a petri dish face up (Step 39). (b) PDMS is poured on the mask (step 40). (c) De-gassing of the PDMS to remove all trapped air. (d) After curing, the PDMS is cut along the mask border with a razor blade (step 43). (e) Using a pair of tweezer, the cured PDMS is gently peeled off from the mask. (f) PDMS replica and original mold after the peeling-off step is completed.

Supplementary Figure 10 Step 37: The soft-lithographic replica of the secondary mold.

(a) Surface activation of the PDMS Secondary mold using oxygen plasma (step 45). (b) The activated PDMS secondary mold is placed in a vacuum jar along with a small quantity of silane for its silanization process (step 45). (c) After the silanization is completed, the Secondary Mold is placed in a petri dish and fresh uncured PDMS is poured (same as in previous image and step 39-40 and 48). Then, after curing the PDMS is cut along the Secondary Mold border with a razor blade (as in step 48) and the replica is gently peeled off. (d) Secondary Mold (right) and its PDMS replica (left)

Supplementary Figure 11 Step 48: Release of PDMS from the mold after curing.

(a) Carefully cut out the PDMS without cutting the SU-8 pattern of the gradient generator. (b) Slide the blade along the inner wall of the aluminum mold for the barrier layer. (c) Cut along the outline of the PDMS replica mold of the microwell layer drawn in Step 50. (d-f) Use the blade to lift the corners of the PDMS from each of the three molds. (g-i) Peel out the gradient generator, barrier and microwell layers from their respective molds.

Supplementary Figure 12 Steps 49 and 50: Preparation of each PDMS layer prior to assembly.

(a) Trim the sides of the gradient generator and punch two vertical holes at the inlets. (b) Cover the top surface of the barrier layer with tape. (c) Flip the barrier layer upside down and cut through the thin layer of PDMS along the length on both sides of each channel. (d) Peel off the tape and use a pair of tweezers to tear out the thin strips of PDMS. (e, f) Place the microwell layer under the barrier layer and align it with the channels in the barrier layer. Trim off the sides of the microwell layer using the edges of the barrier layer as a guide.

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1–12 and The Supplementary Tutorial. (PDF 5929 kb)

Supplementary Table

Supplementary Table 1 (XLSX 13 kb)

Supplementary Data 1

Design of photolithography mask for gradient generator layer. (ZIP 7230 kb)

Supplementary Data 2

Design of photolithography mask for microwell layer. (ZIP 18 kb)

Supplementary Data 3

CAD file for aluminum mold for barrier layer. (ZIP 96 kb)

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Khoo, B., Grenci, G., Lim, Y. et al. Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device. Nat Protoc 13, 34–58 (2018). https://doi.org/10.1038/nprot.2017.125

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