Single-Cell Isolation of Circulating Tumor Cells from Whole Blood by

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Single-cell isolation of circulating tumor cells from whole blood by lateral magnetophoretic microseparation and microfluidic dispensing Jinho Kim, Hyungseok Cho, Song-I Han, and Ki-Ho Han Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00570 • Publication Date (Web): 11 Apr 2016 Downloaded from http://pubs.acs.org on April 16, 2016

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Analytical Chemistry

Single-cell isolation of circulating tumor cells from whole blood by lateral magnetophoretic microseparation and microfluidic dispensing

Jinho Kim, Hyungseok Cho, Song-I Han, and Ki-Ho Han*

Department of Nano Science and Engineering Center for Nano Manufacturing, Inje University Gimhae 621-749, Republic of Korea

*Corresponding Author Address: Department of Nano Science and Engineering, Inje University 607 Obang-dong, Gimhae, Gyongnam 621-749, Republic of Korea Tel: +82-55-320-3715/ Fax: +82-55-320-3631/ E-mail: [email protected]

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Abstract This paper introduces a single-cell isolation technology for circulating tumor cells (CTCs) using a microfluidic device (the ‘SIM-Chip’). The SIM-Chip comprises a lateral magnetophoretic microseparator and a microdispenser as a two-step cascade platform. First, CTCs were enriched from whole blood by the lateral magnetophoretic microseparator based on immunomagnetic nanobeads. Next, the enriched CTCs were electrically identified by single-cell impedance cytometer and isolated as single cells using the microshooter. Using 200 µL of whole blood spiked with 50 MCF7 breast cancer cells, the analysis demonstrated that the single-cell isolation efficiency of the SIM-Chip was 82.4%, and the purity of the isolated MCF7 cells with respect to WBCs was 92.45%. The data also showed that the WBC depletion rate of the SIM-Chip was 2.5×105 (5.4-log). The recovery rates were around 99.78% for spiked MCF7 cells ranging in number from 10 to 90. The isolated single MCF7 cells were intact and could be used for subsequent downstream genetic assays, such as RT-PCR. Singlecell culture evaluation of the proliferation of MCF7 cells isolated by the SIM-Chip showed that 84.1% of cells at least doubled in five days. Consequently, the SIM-Chip could be used for single-cell isolation of rare target cells from whole blood with high purity and recovery without cell damage.

Keywords: cancer, circulating tumor cells, isolation, microfluidics, single-cell

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Introduction Owing to the cellular heterogeneity of tissue and culture samples, molecular analyses using collective cell populations can only provide averaged data, meaning that important information about subpopulations within the collective cells may be obscured.1-4 Even a single tumor, for instance, can consist of numerous subclones because cancer typically develops via a series of mutations, cellular selections, and clonal expansions.5 Therefore, the detection of rare genetic information from bulk cell populations requires a large effort spent on DNA sequencing and bioinformatics.6, 7 Meanwhile, molecular analyses using single cells originating from cellular subpopulations can provide detailed information to understand disease evolution and the behaviors of heterogeneous cell populations, such as cancer cells, stem cells, and neurons.8-10 Single-cell analysis can also be applied in research on drug discovery11, 12 and immunology.13, 14

In particular, genomic analyses of rare single cells, such as circulating tumor cells (CTCs)

against a background of billions of normal blood cells, have had an increasing clinical impact on the prognosis and treatment of cancer.15, 16 Many methods, including mechanical microfilters,17-19 density gradient,20,

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dielectrophoresis,22 and magnetophoresis,23 have been developed for isolating and collecting CTCs from blood of patients with cancer. Size-based filtration and density gradient methods typically suffer from low purity of isolated CTCs and difficulty of collection.24 Dielectrophoresis method also has limitation of low-throughput. For this reason, magnetophoresis technique based on immunomagnetic beads25-27 is the most commonly used for isolating CTCs. Although many methods have been successfully demonstrated for isolating CTCs from blood, these techniques should be integrated with a single-cell isolation method for further downstream single-cell analyses.

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A precise isolation technology that can obtain single target cells from heterogeneous populations is essential to perform sophisticated single-cell analyses in various fields of biomedical research and clinical applications. Conventionally, four main single-cell isolation technologies have been used, namely, serial dilution, micromanipulation, laser-capture microdissection, and fluorescence-activated cell sorting.2, 7, 28 However, these conventional single-cell isolation technologies suffer from being time consuming and inconvenient, having moderate performance, being potentially deleterious to cell integrity, and requiring expensive instruments.29 In particular, the previous technologies have a critical limitation for application to single-cell isolation of extremely rare subpopulations from within heterogeneous cell populations. Therefore, a new advanced single-cell isolation technology, which has high yield, purity, integrity, automation, and compatibility with established workflows, is required for recent advanced genetic techniques such as next-generation sequencing (NGS) and digital PCR in single-cell analyses. Recently, microfluidic technologies have been developed for single-cell manipulation and analyses in microdevices.30-32 However, most microfluidic devices are still unable to isolate the required specific cell types and transfer single cells directly into conventional standard containers for the established downstream analyses. Therefore, the single-cell samples prepared by microfluidic devices are usually transferred by pipetting steps for subsequent downstream analyses. Here, we introduce a single-cell isolation microfluidic device (SIM-Chip) that enables the isolation of particular single cells directly from a heterogeneous cell admixture. In addition, the SIM-Chip conveniently transfers the singlecell droplets into conventional standard containers for subsequent downstream analyses (see Movie S1).

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Materials and methods Working principle and design The SIM-Chip comprises three functionalities for isolating extremely rare single cells from heterogeneous cell populations, namely, a lateral magnetophoretic microseparator, an electrical impedance cytometer, and a single-cell microshooter, as shown in Fig. 1A. First, CTCs from blood samples are enriched by the lateral magnetophoretic microseparator23 using immunomagnetic nanobeads (MNBs), which are specifically bound to CTCs. Subsequently, the sizes of the enriched cells are electrically measured using the impedance cytometer by the amplitude modulation-sensing method.33 Because CTCs are generally larger than normal blood cells,34, 35 CTCs can be identified by size among normal blood cells within the enriched cells. Once CTCs have been identified, they are individually transferred into single-wells of 96/384-well plates or standard containers by the single-cell microshooter, as shown in Fig. 1B. Kim and co-workers23 previously reported the working principle and performance of the lateral magnetophoretic microseparator using MNBs for separating CTCs from whole blood. In this study, the lateral magnetophoretic microseparator (Fig. 2A) was fabricated using a bottom glass substrate with an inlaid ferromagnetic permalloy wire array (Fig. 2B) and a poly(dimethylsiloxane) (PDMS) mold with a microchannel, as described in section S1.1. The ferromagnetic wire array was placed at an angle of 5.7° to the direction of flow. When a uniform external magnetic field is applied to the ferromagnetic wire array, a highgradient magnetic field is generated at the edge of the inlaid ferromagnetic wire.36 To obtain a high purity of target cells and to reduce the flow rate into the impedance cytometer, channel widths of outlet 1 and outlet 2 were designed to be 200 and 800 µm, respectively. When CTCs labeled with MNBs are flowing through the microchannel of the lateral magnetophoretic microseparator, they experience magnetic force (Fm) and hydrodynamic

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force (Fd) simultaneously when passing over the wires. The lateral magnetic force (Fl) acting on CTCs is generated as the vector sum of the magnetic force and the drag force;37 thereby, CTCs move laterally along the edge of the wire and flow into the impedance cytometer through outlet 1. Meanwhile, normal blood cells and other blood components are discarded through outlet 2. The microdispenser, including the impedance cytometer and the microshooter (Fig. 2A), was fabricated using a glass substrate with patterned gold electrodes and PDMS with a microchannel (Fig. 2C), as described in section S1.2. To achieve the amplitude modulation, two excitation electrodes and a single sensing electrode were placed in parallel and patterned on a bottom glass substrate for simple fabrication. To reduce ensemble noise, an excitation sinusoidal signal of 500 kHz is applied to the two excitation electrodes out of phase with one another, as shown in Fig. 1B. When a cell passes through one of the excitation and sensing electrode pairs, an amplitude-modulated sensing signal is induced on the sensing electrode by the imbalance between the two electrode pairs and restored by demodulation. In this way, the size of cells enriched by the lateral magnetophoretic microseparator can be simultaneously measured by the impedance cytometer. Once a CTC is identified by the amplitude of the sensing signal, the shooting buffer of phosphate-buffered saline (PBS) is exploded by rapid pulsed air pressure using a fluid dispensing controller (SBD-A101N, SEBA Inc.). During the signal processing and buffer shooting, the identified CTC has already arrived at the dispensing microchamber. Therefore, it spouts via the exhaust needle along with the shooting buffer and is transferred into a single well of a 96-well plate. Right after the shooting of a droplet, the 96-well plate under the SIMChip is automatically positioned for another droplet by a custom-made XYZ stage. Meanwhile, the shooting air pressure creates a sudden backflow in the sensing channel. The sudden change in flow at the sensing region creates a noise pulse on the sensing 6


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electrode, which may cause spurious results. To reduce the sudden backflow, an air damping reservoir is included in the sensing channel located between the sensing region and the shooting reservoir, as shown in Fig. 1A. The effect of this air damping reservoir can be simulated using a circuit model equivalent to the SIM-Chip, as shown in Fig. S3A. The simulation analysis demonstrated that the sudden flow change can be dramatically reduced by the air damping reservoir, as shown in Fig. S3B. More detail of the instrumental set-up is described in the Supporting Information S3.

Blood collection and sample preparation Peripheral blood was obtained from healthy human donors, collected in a Vacutainer tube containing the anticoagulant EDTA, and processed within 12 h. A human breast cancer cell line, MCF7, was used for evaluation of the SIM-Chip, and fluorescently-labelled MCF7 cells were spiked into 200 µL of whole blood for the sample preparation. In accordance with the manufacturer’s instructions (STEMCELL Technologies), anti-EpCAM antibodies and MNBs were applied in sequence and incubated on ice for 30 min, respectively. Finally, the blood sample was prepared in 1 mL of solution by mixing with 800 µL of PBS containing 0.1% bovine serum albumin (BSA), thereby having physiological conductivity of 1.6 S m−1.

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Figure 1. (A) Working principle of the SIM-Chip for isolating single CTCs from whole blood. (B) Mechanism of single-cell isolation using the impedance cytometer and the microshooter.

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Figure 2. (A) The fabricated SIM-Chip, in which the lateral magnetophoretic microseparator and the microdispenser, containing the impedance cytometer and the microshooter, are connected by capillary tubing. (B) An enlarged view of the microchannel of the lateral magnetophoretic microseparator. (C) Side view of the microdispenser with the exhaust needle.

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Results and discussion Single-cell isolation efficiency The prepared blood sample and PBS containing 0.1% BSA were injected at a flow rate of 2 mL h−1 through sample and buffer inlets of the lateral magnetophoretic microseparator, respectively. Then, a solution containing normal blood cells that had not been isolated in the lateral magnetophoretic microseparator, was withdrawn through outlet 2 at a flow rate of 3.2 mL h−1. Thus, the flow rate of solution, which included MCF7 cells and contaminated persistent blood cells flowing into the impedance cytometer, remained at 0.8 mL h−1, which is nearly the maximum flow rate to detect sensing signal without attenuation, and the CTC isolation process using a blood sample of 1 mL was completed within 30 min. Flow velocity in the sensing channel (W×H: 40×27 µm2) of the microdispenser was 200 mm s−1; therefore the traveling time of cells from the sensing region to the dispensing microchamber of the microdispenser (length of 10 mm) was 50 ms. Once MCF7 cells were identified by the sensing signals (see Fig. S5 and S6), after 50 ms for waiting until the cells arrive at the dispensing microchamber, the fluid dispensing controller applied an air pressure of 0.175 MPa for 0.15 s through the shooting reservoir, and a droplet containing a single CTC was then shot through the exhaust needle. Figure 3A shows fluorescent images of droplets containing single MCF7 cells. The average volume of the droplets was 4.5 µL, which faithfully matches the simulation result using the equivalent circuit model, as shown in Fig. S3C. Single-cell isolation throughput of the microdispenser was 700 ms, as required for shooting the buffer and positioning of the 96well plate. The single-cell isolation experiment on the SIM-Chip using blood samples spiked with 50 MCF7 cells showed that the average proportion of droplets containing a single MCF7 cell was 82.4% (Fig. 3B). Droplets lacking cells might be caused by contamination with 10


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debris during the sample preparation, and in cases with two or three MCF7 cells (Fig. S7) in a droplet, it could be assumed that they were initially bound together. Thus, the single-cell isolation efficiency of the SIM-Chip might be further improved through careful sample preparation. The average number of total MCF7 cells isolated by the SIM-Chip was 49, and the numbers of contaminated white blood cells (WBCs) and red blood cells (RBCs) were 4.0 and 6.6, respectively, as shown in Fig. 3C. These results indicate that the purity of MCF7 cells among the isolated nucleated cells, including MCF7 cells and WBCs, was 92.45%. They also demonstrate that the WBC depletion rate of the lateral magnetophoretic microseparator was about 2.5×105 (5.4-log; mean, 4 WBCs per 200 µL of whole blood). In general, the size ranges of CTCs and WBCs overlap somewhat.24 Therefore, in spite of the high WBC depletion rate of the lateral magnetophoretic microseparator, there is still a remote possibility that the impedance cytometer can misjudge WBCs as CTCs. The misidentified WBCs, however, will be transferred into a single container and filtered out by subsequent downstream analyses. In addition, owing to the size difference between WBCs and CTCs, CTCs among cells enriched by the lateral magnetophoretic microseparator can be further purified by simple positioning control of the XYZ stage, thereby further improving the WBC depletion rate of the SIM-Chip. The recovery rate of the SIM-Chip was measured for various numbers (from 10 to 90) of MCF7 cells spiked into 200 µL of whole blood. The results (Fig. 3D) showed that the recovery rate was consistently around 99.78% for various numbers of spiked MCF7 cells. This high recovery rate demonstrates that neither the lateral magnetophoretic microseparator nor the microdispenser loses MCF7 cells. According to the measured recovery rate of the SIM-Chip, the recovery rate of the microdispenser was also almost 100%, owing to the size difference between MCF7 cells and WBCs, as shown in Fig. S6. 11



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The sample-loading procedure of the SIM-Chip is very simple and consists of the following steps: (i) buffer injection only, (ii) sample and buffer injection at the same flow rate, and (iii) a second injection of buffer only. In addition, the SIM-Chip does not involve an expensive fabrication process, as explained in section S1, and has no complex structures in the microchannel. For this reason, no clogging problems occur when a blood sample passes through the microchannel when isolating single CTCs.

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B

82.4%

Percentage (%)

80 60 40 20

9.7% 0

7.5% 0.4%

0

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2

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The number of isolated MCF7 cells in a single-wells 60

C

50

Number of cells

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49

40 30 20 10

4 0

MCF7

WBCs

6.6 RBCs

Type of cells separated into a 96-well plate 13


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D

Isolated number of MCF7 cells


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100

80

60

y=0.9978x r=0.99753

40

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0

20

40

60

80

100

Spiked number of MCF7 cells Figure 3. (A) Photographs of fluorescence-probed single MCF7 cells. (B) Relative proportion of the number of MCF7 cells isolated into single wells. (C) Number of MCF7 cells, WBCs, and RBCs isolated from 200 µL of whole blood spiked with 50 MCF7 cells. (D) Regression analysis of the recovery rate for various numbers of MCF7 cells spiked into 200

µL of blood.

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Viability and downstream genetic analyses To measure the viability of single MCF7 cells isolated by the SIM-Chip, isolated cells were

examined

using

a

fluorescence-based

viability

assay

(Live/Dead®

Viability/Cytotoxicity Kit for mammalian cells; Invitrogen), as shown in Fig. 4A. The viability of the isolated single MCF7 cells was 88.53%, compared with 88.98% (not shown here), for innate MCF7 cells harvested from a culture in a Petri dish before single-cell isolation.38 These results suggest that the fluidic shooting for making a droplet containing a single cell does not affect the viability of MCF7 cells. Isolation of the intact CTCs could facilitate more precise CTC-based downstream analyses. In addition, through the culture of isolated CTCs, more sensitive genetic analyses of cancer and drug screening may be possible. To determine whether isolated single MCF7 cells are suitable for subsequent genetic analyses, we examined the expression of cytokeratin-19 (KRT19), an epithelial cell-specific marker.39 Figure 4B shows that the CTC-specific gene can be detected using isolated single MCF7 cells based on reverse-transcription PCR (RT-PCR). The detailed RT-PCR protocol is provided in section S6. This result also demonstrates that single CTCs isolated by the SIMChip can be used for genetic analyses of cancer.

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Figure 4. (A) Fluorescence-based live/dead assay for MCF7 cells isolated by the SIM-Chip. (B) RT-PCR amplification of β-actin (244 bp) and KRT19 (211 bp) transcripts using collective MCF7 cells and single MCF7 cells isolated by the SIM-Chip. Lanes 1 and 10, 100bp DNA size markers; lanes 2 and 3, β-actin and KRT19 using collective MCF7 cells as a positive control (PC); lanes 4−8, KRT19 using single MCF7 cells; lane 9, negative control (NC) without cells.

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Isolated single-cell culture Single MCF7 cells isolated using a regular pipette and the SIM-Chip were separately cultured for cloning analysis. MCF7 cells cultured in a Petri dish were manually isolated as single cells using a regular pipette and seeded into commercial 96-well culture plates filled with 100 µL of Dulbecco’s Modified Eagle’s Medium (DMEM) solution containing 10% fetal bovine serum (FBS) in advance. MCF7 cells, spiked into 200 µL of whole blood, were also isolated by the SIM-Chip and seeded into 96-well culture plates. They were then incubated at 37°C in an environment containing 5% CO2 and 95% humidity for five days without refreshing the culture medium, as shown in Fig. 5A and 5B. Proliferation of the seeded single MCF7 cells was monitored by counting them every 24 h. Interestingly, in terms of their proliferation, 81.6% of the single MCF7 cells isolated using a regular pipette at least doubled, while 18.4% did not divide during this period or died, as shown in Fig. 5C. Meanwhile, 84.1% of the single MCF7 cells isolated by the SIM-Chip at least doubled, as shown in Fig. 5D. These two results on the proliferation of single MCF7 cells showed that they exhibit very similar trends and that the single MCF7 cells isolated by the SIM-Chip appear to be slightly more animated than those isolated using a pipette. Thus, the results explain that the isolation of single MCF7 cells by the SIM-Chip does not affect their proliferation. The fact that 84.1% of single MCF7 cells isolated by the SIM-Chip proliferated is comparable to their viability of 88.53% right after single-cell isolation.

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Figure 5. Proliferation of single MCF7 cells seeded into 96-well plates for five days. Single MCF7 cells isolated using (A) a regular pipette and (B) the SIM-chip. Black scale bars: 50 µm. Number of cells cultured from a single MCF7 cell isolated using (C) a regular pipette and (D) the SIM-Chip.

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Conclusions This study introduced a single-cell isolation microfluidic device with the advantages of its simple operation, high performance, the possibility of isolating intact cells, and no requirement for expensive instruments. One of the most important advantages of the proposed SIM-Chip is that it can isolate extremely rare target cells against a background of several hundred million cells as well as transfer single target cells directly into 96/384-well plates or other standard containers for established downstream analyses. In this way, the isolated single target cells can be directly used for recently advanced single-cell analyses. The presented electrical method for detecting target cells can be replaced by other techniques, such as optical or magnetic sensing, for distinguishing specific cell types from heterogeneous cell populations, thereby expanding the range of applications of the SIM-Chip. Furthermore, a workstation for the SIM-Chip could be simply derived and automated. Consequently, the presented single-CTC isolation technology could be used as a tool to open up a new window for understanding the genomic diversity of individual patients with cancer, allowing us to follow the evolution of cancers and establish individually tailored cancer treatment.

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Acknowledgments This work (NRF-2012R1A2A2A03045174) was supported by the Midcareer Researcher Program through an NRF grant funded by the MEST.

Associated content Supporting Information: Detailed descriptions of (a) fabrication process, (b) equivalent circuit model, (c) instrument setup, (d) relationship between the peak output voltage and the volume of cell, and (e) RTPCR protocol.

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