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.

Figure 1

CTC-iChip schematic. The CTC-iChip is composed of two separate microfluidic devices that house three different microfluidic components engineered for inline operation: DLD to remove nucleated cells from whole blood by size-based deflection by using a specially designed array of posts performed in CTC-iChip1, inertial focusing to line up cells to prepare for precise magnetic separation and magnetophoresis for sensitive separation of bead-labeled WBCs and unlabeled CTCs, which are performed in CTC-iChip2. PLTs, platelets.

Figure 2

Protocol flowchart. Flowchart of the protocol including references to the steps in the text and related figures.

Figure 3

Structure of the CTC-iChip1. DLD is designed to separate nucleated cells from blood, and it is performed in CTC-iChip1. (a) High-resolution photograph of the fabricated chip. (b) Schematic of CTC-iChip1 (left image shows only two lanes, whereas the device is composed of many). Whole blood and buffer inlets enter from opposite top corners of the post array (right, 1). The post geometry (bottom, egg-shaped with a width • length • height of 24 • 17 • 150 μm) is engineered to deflect particles with a diameter >4 μm. As such, posts deflect nucleated cells (green-labeled lanes) away from smaller RBCs, platelets and plasma (red-labeled lanes) and toward the buffer (blue-labeled lanes). As the device operates in a laminar flow regime (Supplementary Fig. 3), RBCs, platelets (PLTs) and free beads remain in their lateral positions. (c) Electron micrographs of CTC-iChip1. Start and end of post array (top; scale bars, 100 μm) and posts (bottom; scale bar, 20 μm).

Figure 4

Structure of CTC-iChip2. (a–c) CAD drawing (a), high-resolution photograph (b) and electron micrographs (c) of CTC-iChip2. Highlighted are inlet and outlets, filters, focusing regions and magnetic deflection channels (width • length of magnetic deflection stage 1 = 1 mm • 37 mm; width • length of stage 2 = 0.5 mm • 37 mm). Scale bars, 250 μm. W2, waste 2a; W2b, waste 2b; P, product. (d) Schematic of CTC-iChip2. Note that, for simplification, the schematic was linearized; in the actual device, before entering the second stage, fluid flow turns around 180° and thus moves in the opposite direction of the first stage. (e) Fluorescence images of cell streaks formed during CTC-iChip2 operation showing both stages of inertial focusing and magnetophoresis of WBCs with (green) or without (orange) magnetic load. CTC-iChip2 initially separates the flow equally into two parallel and equivalent channels for higher throughput. Cells get tightly focused in the inertial focusing region, and subsequently separated in the magnetophoresis region (dashed lines symbolize channel wall). In the first separation stage, the magnetic force is directed toward the middle of the channel, and thus labeled cells deflect toward the middle and exit through waste 2a (d, Fig. 5). The first magnetophoresis stage is designed to have a relatively lower magnetic gradient, as a result only the portion of the WBC population with more than seven beads is successfully deflected. Channels on each side of waste 2a lead to the second stage, where cells are refocused and deflected with higher sensitivity (cells with more than two beads are deflected). Separated populations exit through their respective outlets (WBC → waste 2b, CTC → product).

Figure 5

Magnetophoresis setup. Deflection channel positioning in the CTC-iChip2 magnetic manifold is crucial in the success of the protocol. (a) zy plane schematic of the magnetophoretic setup for both stage 1 (yellow) and stage 2 (red). (b) The plot on top shows the calculated magnetic field gradient on points across the y axis, where the indicated stages of the deflection channels are. Stage 2 positioning is designed to enable the highest sensitivity separation possible, whereas stage 1 positioning is designed to push the cells toward the middle with a softer magnetic field gradient. (c) CTC-iChip2 design includes a ruler to help align CTC-iChip2 with its magnetic manifold. (d) When aligned, the stage 1 deflection channel is positioned in the middle of two magnets.

Figure 6

Schematic of the production of CTC-iChip2. (a–d) Schematic showing the production steps of CTC-iChip2 in the clean room in device view (top) or inlet/outlet view (bottom panel). Thin PDMS is produced in Steps 1–13 and bonded to a second layer of thick PDMS pieces in Steps 14–16; inlet and outlets are punched in Step 17 and bonded to glass slides in Steps 18–22.

Figure 7

CTC-iChip assembly. (a) CTC-iChip1 manifold bottom piece with inlet and outlet O-rings attached (left) and top piece with screws in place (right). (b) CTC-iChip1 is placed on top of O-rings and fits between the metal guide posts. Fluid reservoirs, O-rings and CTC-iChip1 inlets are automatically aligned in this step. (c) The top piece is screwed into the bottom piece with the chip. (d) CTC-iChip2 manifold bottom piece (left) and top piece (right). CTC-iChip2 manifold houses the permanent magnets oriented to form a quadrupole (Fig. 5). (e,f) CTC-iChip2 is placed on the manifold bottom piece (e), is aligned (as detailed in Fig. 5 and Supplementary Video 2) and the top piece is pushed and screwed in f. When connected with tubing, these assembled devices form the CTC-iChip (Fig. 8).

Figure 8

Running setup. Schematic (left) and picture (right) of CTC-iChip operation. A constant pressure of 140 kPa (20 p.s.i.) is applied to the buffer and blood, which are both connected to CTC-iChip1 inlets via tubing. Blood is rocked with a period of 5 s during the operation. CTC-iChip1 separates WBCs and CTCs from whole blood. Purified nucleated cell suspension exit from the bottom outlet and is transferred to CTC-iChip2. The remaining blood, which gets diluted with buffer, is collected in a 60-ml syringe at a rate of 600 μl/min controlled by using a syringe pump running in withdrawal mode. CTC-iChip2 separates CTCs from WBCs in two stages. The output containing highly bead-labeled WBCs exit from waste 2a. Sparsely bead-labeled cells exit from waste 2b collected at the bottom of the manifold. The product containing CTCs exit from the outermost two outlets and are collected together.

Figure 9

Sequence of steps that are crucial to priming and starting the CTC-iChip. Schematic showing critical clamping sequence during priming (left) and starting (right) the CTC-iChip.

Figure 10

Expected results. (a) Characterization of the CTC-iChip protocol shows high yield of spiked cancer cells isolated from whole blood that is independent of spiked cell type with low number of nucleated cell impurities. Mean yield: 97 ± 2.7% (± s.d.). (b) Logarithmic plot of the number of nucleated cell impurities after application of the CTC-iChip to healthy donor samples and clinical samples from patients of breast and pancreatic cancers. Mean carryover: 1,200 ± 900 (± s.d.) nucleated cells/ml of blood. Box plots were generated using JMP (SAS Institute) with the following settings: boxes represent first and third quartiles, lines represent upper and lower data points and exclude outliers. (c) Fluorescence micrographs of CTCs and impurities from the CTC-iChip product (20× objective, Nikon). Rare cell suspension is immobilized using Spintrap (Supplementary Fig. 1), fixed using 4% (wt/vol) paraformaldehyde and stained for immunofluorescence microscopy using antibodies against cytokeratins (CK; red, CTC marker), CD45 (green, leukocyte marker) and DAPI (nuclear stain, blue). Bright green dots indicate anti-CD45–coated magnetic beads.