lectin-aided separation of circulating tumor cells and assay of their response to an anticancer drug...

Research Article

Lectin-aided separation of circulating tumorcells and assay of their response to ananticancer drug in an integratedmicrofluidic device

Metastasis caused by the entry of circulating tumor cells (CTCs) into the bloodstream or

lymphatic vessels is a major factor contributing to death in cancer patients. Separation of

CTCs and studies on CTC–drug interactions are very important for prognostic and

therapeutic implications of metastatic cancer. In this study, an integrated microfluidic

device for CTC separation through the combination of lectin and microstructure is

presented. This microfluidic device and lectin concanavalin A were utilized for the

separation of K562 cells in whole blood samples. The results showed that the separation

efficiency can reach 84%, which is much higher than that of an experiment without

concanavalin A treatment. To further demonstrate the feasibility of this microfluidic

device application in sequential studies after target cells were separated, the interactions

of K562 cells and an anticancer drug, cytarabine, were also examined. After 6 h on-chip

treatment with cytarabine, the viabilities of K562 cells were 85.29, 77.05, and 40% for

drug concentration levels of 0.25, 0.5, and 1.0 g/L, respectively. This system can facilitate

the rapid and efficient in vitro investigation of CTC separation and CTC-related studies.

Keywords:

Circulating tumor cell / Lectin / Microfluidic device / Microstructure / SeparationDOI 10.1002/elps.201000139

1 Introduction

Metastasis is the underlying cause of death in cancer

patients [1]. It results from circulating tumor cells (CTCs)

that escape from the primary tumor into the bloodstream or

lymphatic vessels. CTCs may constitute the seeds for the

subsequent growth of additional tumors (metastasis) in

different tissues. Detection and assay of CTCs have

important prognostic and therapeutic implications for

understanding the process of metastasis, disease staging,

prognosis prediction, patient monitoring during therapy,

and improvement in therapy design [2, 3]. However, CTCs

exist in the peripheral blood of cancer patients, who have

very low blood concentration [2], so their separation and

identification are very difficult and time consuming.

The conventional techniques utilized for the large-scale

preparation of CTCs include centrifugation and membrane

filtration. However, although they have been used for many

decades, these two techniques are complicated and have low

efficiency and specificity in sorting rare target cells. More

sophisticated methods such as immunocytochemistry [4],

PCR [5], flow cytometry, immunofluorescence or fluores-

cence-activated cell sorting, and magnetically activated

separation have been established as the standard methods

for high-quality cell separation [6–11]. However, each of

these methods still has its disadvantages. For instance,

immunocytochemistry and immunofluorescence (their

detection rates range from 1 to 62%) are limited mainly by

their inability to retrieve live cells for downstream analysis.

PCR can detect the mutation in the DNA of a cancer cell

from as little as one cell in 1� 106–1� 107 normal cells, but

it cannot differentiate whether the DNA is from living CTCs

or from dead tumor cells. Fluorescence-activated cell sorting

is limited by the specificity required for antibodies, the long

separation time, and the need for skilled technicians and

expensive equipment. Magnetic cell sorting is also limited

by the specificity required for antibodies [12, 13]. Therefore,

Li Li1

Wenming Liu1

Jianchun Wang1

Qin Tu1

Rui Liu1

Jinyi Wang1,2

1College of Animal Medicine andCollege of Science, NorthwestA&F University, Yangling,Shaanxi, P. R. China

2Shaanxi Key Laboratory ofMolecular Biology forAgriculture, Northwest A&FUniversity, Yangling, Shaanxi,P. R. China

Received March 9, 2010Revised June 21, 2010Accepted June 24, 2010

Abbreviations: Con A, concanavalin A; CTCs, circulatingtumor cells; FBS, fetal bovine serum; PI, propidium iodide;

RBCs, red blood cells; WBCs, white blood cells

Correspondence: Professor Jinyi Wang, College of AnimalMedicine, College of Science, and Shaanxi Key Laboratory ofMolecular Biology for Agriculture, Northwest A&F University,Yangling, Shaanxi 712100, P. R. ChinaE-mail: [email protected]: 186-29-8708-2520

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2010, 31, 3159–3166 3159

there is an urgent need for the development of novel and

convenient methods for isolating CTCs.

During the 1990s, when microelectromechanical

systems technology became widely available to researchers,

and PDMS microchannels were introduced [14], micro-

fluidics became an increasingly useful tool for cell biologists

due to its capability to control, monitor, and manipulate

cellular microenvironments precisely [15]. To date, the

applications of microfluidics in cell biology have rapidly

expanded. For example, several biological studies use

microfluidics fabricated with PDMS as a platform for

miniature immunoassays, separation of proteins and DNA,

sorting and manipulation of cells, and microscale bio-

reactors [16–23]. Additionally, the introduction of integrated

microfluidics facilitates more complex biological manipula-

tions in one single device [22]. All these advancements have

allowed the miniaturization of filtration devices and the

realization of precise channel geometries, leading to the

more sophisticated separation of cells in microfluidics, such

as the isolation of red blood cells (RBCs) and leukocytes

[24–26], embryos [27], and cancer cells [28, 29].

Generally, the straightforward methods utilized for cell

separation in microfluidics are based on size-scale discri-

mination using filtration sieves. In the past few years,

several groups have developed microfiltration chips with

different-sized microsieves integrated in microfluidic chan-

nels to fractionate cell mixture [30, 31] and trapped RBCs,

white blood cells (WBCs), and spiked neuroblastoma [29].

Previous studies have proved that the sizes of RBCs and

WBCs are smaller than those of tumor cells [32, 33].

Moreover, tumor cells not only have a larger volume and

cellular karyon but also low deformability [28]. Therefore,

the separation of tumor cells from a cell mixture is realiz-

able. For example, Chen et al. have successfully sorted

tumor cells (SPC-A-1) from a cell mixture using a pool-dam

structure filter [28]. Hisham Mohameda et al. have separated

eight cancer lines one by one in one microdevice using a

multicolumn microstructure [29]. However, all devices used

in these studies had a simple function (only for cell sorting).

Additionally, they are mainly dependent on the physical

structure size (smaller cells need a smaller gap space

between two adjacent microstructures). These issues

complicate the fabrication of microfluidics, especially for the

separation of rare and small cells, as well as sequential

studies on cell identification, culture, and response to

drugs. Therefore, new methods for special cell sorting and

the development of a multifunctional microdevice are

indispensable.

Tumor cells have been reported to alter carbohydrate

expression pattern frequently compared with healthy cells,

and can be agglomerated by lectin to form a large mass [33].

Therefore, based on our study on the preparation of anti-

bodies and integrated microfluidics [34, 35], a new method

for CTC isolation is presented by combining lectin with

integrated microfluidics. Also, the sequential on-chip iden-

tification, culture, and interaction of CTCs with an antic-

ancer drug were investigated.

2 Materials and methods

2.1 Materials

RTV 615 PDMS pre-polymer and a curing agent were

purchased from GE Silicones (Minato-ku, Tokyo); surface-

oxidized silicon wafers from Shanghai Xiangjing Electronic

Technology (Shanghai, China); AZ 50XT photoresist and

developer from AZ Electronic Materials (Somerville, NJ,

USA); SU-8 2025 photoresist and developer from Micro-

chem (Newton, MA, USA); Goat antimouse IgG-FITC

from Boster (Beijing, China); Cytarabine, Concanavalin A

(Con A), and propidium iodide (PI) from Sigma-Aldrich

(MO, USA); and cell culture medium and fetal bovine

serum (FBS) from Gibco Invitrogen (CA, USA). Mouse

ascites polyclonal anti-K562 cell surface membrane antigen

antibody was prepared and identified following the method

reported previously [34]. All solvents and other chemicals

were purchased from local commercial suppliers and were

of analytical reagent grade, unless otherwise stated. All

solutions were prepared using ultra-purified water supplied

by a Milli-Q system (Millipores).

2.2 Fabrication of PDMS microfluidic devices

The microfluidic device utilized for this study was fabricated

using the multilayer soft lithography method [35–38]. Two

different molds were first produced by photolithographic

processes to create the fluidic components (channel width:

200 mm; channel height: 35 mm; chamber diameter: 300 mm;

height: 35 mm; length: 1900 mm) and the control channels

(channel width: 25–100 mm; channel height: 25 mm)

embedded in the respective layers of the PDMS matrix. To

prepare the mold utilized for the fabrication of the fluidic

components, a 35 mm-thick positive photoresist (AZ 50XT)

was spin-coated onto a silicon wafer. After UV exposure and

development, the wafer was heated above the glass

transition temperature of the positive photoresist. As a

result, the surface profile of the patterned positive photo-

resist was transformed into a round profile. The mode for

control channels was made by introducing a 25 mm-thin

negative photoresist (SU8-2025) pattern on a silicon wafer.

To achieve the reliable performance of each valve, the widths

of the control channels were set to 150–200 mm in sections

where the valve modules are located.

Before fabricating the microfluidic device, both the

fluidic and control molds were exposed to trimethyl-

chlorosilane vapor for 2–3 min. A well-mixed PDMS pre-

polymer (RTV 615 A and B in 5:1 ratio) was poured onto the

fluidic mold placed in a Petri dish to yield a 3 mm-thick

fluidic layer. Another portion of PDMS pre-polymer (RTV

615 A and B in 20:1 ratio) was spin-coated onto the control

mold (1600 rpm, 60 s, ramp 15 s) to obtain the thin control

layer. The thick fluidic layer and the thin control layer were

cured in an 801C oven for 50 min. After incubation, the

thick fluidic layer was peeled off the mold, and holes were

Electrophoresis 2010, 31, 3159–31663160 L. Li et al.


introduced into the fluidic layer for cell and nutrient supply

access, chamber purging, and waste exclusion. The fluidic

layer was then trimmed, cleaned, and aligned onto the thin

control layer. After baking at 801C for 60 min, the assembled

layers were peeled off the control mold, and another set of

holes was punched for the access of control channels. These

assembled layers were then placed on top of a glass slide

coated (3000 rpm, 60 s, ramp 15 s) with PDMS pre-polymer

(GE RTV 615 A and B in 10:1 ratio) that had been cured for

15 min in an oven set at 801C. The microfluidic device was

ready for use after baking at 801C for 48 h.

2.3 Control interface

The control setup consisted of eight sets of eight-channel

manifolds (Ningbo Lida Pneumatic, Ningbo, China)

controlled through a NI-PCI-6513 controller board (National

Instruments, Austin, TX, USA) connected to a computer

through a USB port. Nitrogen gas provided pressure (30 psi)

to the manifolds. Twenty-one control channels in the

microfluidic device were first filled with water and

individually connected to the corresponding channels on

the manifolds with metal pins (23 Gauge, Jinke Wei, China)

using polyethylene microbore tubing. When a regulator on

the manifold was activated, nitrogen gas entered the

respective control line connected with the regulator,

providing pressure to the closed valves in the microfluidic

device. The control interface was created using LabVIEW

program (Version 8.0, National Instrument) on a personal

computer, allowing for the manual control of individual

valves and for the automation of our microfluidic system.

2.4 Blood collection

Fresh blood (10 mL) was collected from healthy mice. After

the addition of anticoagulant, 100 mL 150 unit/mL Heparin,

and gentle shaking in a shaking incubator for 1 min, whole

blood was maintained at room temperature prior to use. An

isotonic saline solution of PBS (pH 7.4) was used for the

dilution of whole blood when required.

2.5 Cell culture

K562 cells were obtained from the Chinese Academy of

Sciences (Shanghai, China; a type of immortalized myeloge-

nous leukemia line. The cells are nonadherent and rounded,

and grow in suspension). They were routinely cultured using

DMEM (Invitrogen, Grand Island, NY) supplemented

with 10% FBS (Invitrogen), 100 units/mL penicillin, and

100 mg/mL streptomycin in a humidified atmosphere of 5%

CO2 at 371C. To maintain their exponential growth phase, the

cells were normally passaged at a ratio of 1:2 every two days

through dilution. Before use, they were harvested after

centrifuging at a rotational speed of 800 rpm for 3 min. The

cells were then resuspended in healthy whole blood. The

densities of RBCs and K562 cells in the blood mixture were

2� 106 and 2� 105 cells/mL, respectively, determined

through the hemocytometer method.

2.6 Lectin concentration optimization

A series of Con A solutions with various concentrations

(10.00, 1.00, 1.0� 10�1, 1.0� 10�2, 5.0� 10�3, 2.5� 10�3,

1.25� 10�3, and 6.25� 10�4 g/L) was, respectively, added to

eight parallel 100 mL fresh blood samples (cell density of

RBCs was 2� 106 cells/mL) and eight parallel 100 mL K562

cell suspensions (2.0� 105 cells/mL). After gently shaking

for 3 min at 371C, these were continuously incubated at

371C for 30 min. The agglomerated state of cells was

monitored every 10 min.

2.7 On-chip CTC sorting and viability assay

Before whole blood with K562 cell conglomeration was

loaded into the microfluidic device for K562 cell isolation,

the PDMS device was sterilized with UV light for 1 h. After

rinsing with PBS and drying with N2, fresh blood mixtures

were loaded into the microdevice through a microtubing

connected to the inlet of the device under the coordination

of microvalve groups. The blood flow velocity was adjusted

using an automated pump. The separation efficiency was

the ratio of sorted cells and tumor cells. Sorted cells were

quantified by counting the number of trapped cells using

Software Image-Pro Plus 6.0 (Media Cybernetics, Silver

Spring, MD, USA). Tumor cells were quantified by cell

concentration, flow velocity, and loading time.

Assay of trapped K562 cell viability was performed using

PI staining assay [39, 40]. Briefly, after rising with PBS thrice,

1.0 mmol/L PI was introduced into the cell trapping cham-

bers. After incubation for 10 min, cell viability was quantified

by counting the live (unstained) and the dead (red) cells.

2.8 Identification of the trapped CTC cells

To identify the trapped cells, after washing with PBS thrice,

they were first fixed with 4% paraformaldehyde and

sequentially permeabilized with 0.2% glutaraldehyde at

room temperature for 15 min, respectively. Then the

trapped K562 cells were incubated with Mouse anti-K562

cell ascites polyclonal antibody and secondary goat anti-

mouse IgG-FITC antibody in PBS for 2 h one after another,

followed by washing with PBS thrice.

2.9 Assay of CTC responses to an anticancer drug

After the K562 cells were separated and trapped in the

device, they were washed with normal culture medium

Electrophoresis 2010, 31, 3159–3166 Microfluidics and Miniaturization 3161


thrice. Then culture medium DMEM supplemented with

10% FBS and 1% penicillin/streptomycin was introduced

into the cell culture chambers and cultured in an incubator

at 371C with 5% CO2 for 24 h. The on-chip growth status of

the cells was monitored every 6 h, and viability was analyzed

using fluorescence dye PI assay following the procedures

described above.

For the assay of K562 cell responses to anticancer drugs,

three devices with the trapped K562 cells were first cultured

for 24 h following the same method above. Then the normal

culture medium was, respectively, replaced with 0.25, 0.5,

and 1.0 g/L cytarabine dissolved in normal culture medium.

Later, the devices were placed in a plastic Petri dish, and the

entire experiment setup was kept in an incubator at 371C

with 5% CO2 for 6 h. K562 cell response to cytarabine was

evaluated through assay of the changes in cell viability after

cytarabine treatment.

2.10 Microscopic imaging and data analysis

An inverted microscope (Olympus, CKX41) with a CCD

camera (QIMAGING, Micropublisher 5.0 RTV) and a

mercury lamp (Olympus, U-RFLT50) were used to acquire

phase contrast and fluorescent images. Software Image-

Pros Plus 6.0 (Media Cybernetics) and SPSS 12.0 (SPSS)

were employed to perform image analysis and data

statistical analysis, respectively.

3 Results and discussion

3.1 Microfluidic fabrication and control interface

The integrated microfluidic device utilized for this study was

fabricated using the multilayer soft lithography method

[35–38]. Figure 1 shows the composition and functions of

each part of this microdevice. Generally, the device consists

of two functional regions (Fig. 1A): (i) the sample mixing

region with two dentation structures for sample mixing [41]

and (ii) the cell trapping region composed of six short

columns for CTC trapping, culture, and cell–drug interac-

tions. Figure 1B shows the optical image of the actual device

in which the channels were loaded with various food-dye

solutions to help visualize the different components of the

microfluidic chip. Red and green lines, respectively, indicate

the control and fluidic channels. The red squares located in

the middle and terminal of the control channels represent

the valves that can regulate intentionally the opening and

closing of channels in the fluidic layer to allow delivery and

localization of samples, and exchange of culture medium.

Each channel in the control layer is independently

controlled by an external solenoid valve that can be

modulated manually or automatically through the control

interface (Supporting Information, Fig. S1). The consecu-

tively arranged dentation on the side of the fluidic channels

in the mixing region, as the SEM image shows in Fig. 1C

(top and engaged part of Fig. 1A), can accomplish reagent

mixing in the fluidic channels based on the design

developed by Sheen et al. [41]. The long assuasive channels

connecting the dentation structures are utilized for sample

incubation before being loaded into the cell trapping region.

Inside these trapping compartments, many microdam

structures are arranged alternately (Fig. 1C, bottom and

engaged part of Fig. 1A) for cell sorting. The width of the

dam gap is of the same order as the cell size, with a large

ostium on top (45 mm) and a small one at the bottom

(15 mm). Tumor cells and their aggregation are larger than

the width of the gap, facilitating cell trapping for subsequent

study. Furthermore, each compartment can be isolated

during sample solution loading and waste exclusion, and

each has enough room for cell culture and proliferation.

A LabVIEW-controlled interface was used to automate

the operation of the integrated microfluidic system. Its

applied feasibility and flexibility were demonstrated through

the injection of different food-dye solutions to simulate the

manipulation process of sample mixing and delivery of

targeted samples to the cell sorting chamber (Supporting

Information, Movie S1), as well as the exchange of culture

medium and drug (Supporting Information, Movie S2).

3.2 Cell sorting

In this study, Con A was the key for the combination of

tumor cell characteristic and microstructure for K562

separation. Con A is a lectin isolated from the seeds of jack

beans (Canavalia ensiformis), which can bind to cell

membrane glycoprotein and glycolipid of many cell types,

such as RBCs, hepatocytes, and transformed and nontrans-

formed cell lines, especially many kinds of tumor cells

[42, 43]. In determining the possible mechanism for Con A

derivational cellular interactions, cell member asialofetuin, a

glycoprotein possessing several branched oligosaccharide

side chains with terminal nonreducing galactosyl residues is

recognized to bind to the membrane of tumor cells and to

induce homotypic aggregation, presumably by serving as a

cross-linking bridge between adjacent cells [44]. Compared

with healthy cells, tumor cells excessively express glycopro-

tein, resulting in a remarkable tumor aggregate when Con A

is added. Therefore, the separation of CTCs from whole

blood without healthy cell aggregation is possible when an

appropriate Con A concentration is used.

To optimize the Con A concentration for K562 cell

agglomeration, a series of Con A solutions with different

concentrations was, respectively, added into parallel whole

blood samples and K562 cell suspensions. The results

(Fig. 2, and Supporting Information Fig. S2) show that

RBCs coagulated into an irregular conglomeration and

formed virgulate cells when the Con A concentration

was 10.0, 1.0, 0.1, and 0.01 mg/L, but no aggregates were

formed when the Con A concentration was less than

0.005 mg/L. The K562 cells all coagulated when the Con

A concentration ranged from 10.0 to 1.25� 10�3 mg/L.



No aggregation occurred when the Con A concentration was

6.25� 10�4 mg/L. Therefore, the optimal Con A concentra-

tion utilized in this study for K562 cell separation was

2.50� 10�3 mg/L. In addition, the use of K562 cell density

(2� 105 cells/mL) in blood was according to the criterion of

diagnosis definition – patients with markedly elevated WBC

counts (>1� 108 cells/mL) in the blood [45]. The K562 cell

concentration (2� 105 cells/mL) utilized in this study was

much lower than that in patient blood.

When fresh blood mixture with K562 cells was loaded

into the microdevice for K562 cell sorting, small cells such

as RBCs and WBCs went through the dam structure,

whereas agglomerated K562 cells were boxed up in the dam

structure (Supporting Information Fig. S3 and Movie S3).

The width of the dam gap is the most essential factor for

tumor cell sorting. In static state, RBCs assume a biconcave

discoid shape with a diameter of 8 mm and a thickness

of 2 mm. Although lymphocytes are small cells, mostly

6–15 mm in diameter, most WBCs, mainly including

60–75% neutrophils, 20–45% lymphocytes, and 2–10%

monocytes, are spherical with a diameter of more than

10 mm [24]. Therefore, when blood samples containing K562

cells flowed through the cell trapping region, all RBCs and

WBCs smoothly passed the dam structures, whereas K562

cell conglomerations were trapped. As shown in Supporting

Information Fig. S3 and Movie S3, the agglomerated K562

cells with a diameter of more than 40 mm (two cell

agglomerate diameter), as well as individual K562 cells, were

boxed up in the dam structures. This result was in accor-

dance with the original intention of the dam dimension

design. Further, the results indicated that the utilization of

Con A greatly enhanced separation efficiency. Taking into

account the impact of flow velocity of the blood samples on

separation efficiency, the relationship of separation effi-

ciency and flow velocity was also investigated. The results

(Fig. 3) indicate that an increase in flow velocity can

decrease separation velocity. When the flow velocity was less

than 1.0 mL/min, a higher efficiency, more than 80%, was

obtained. In fact, the flow velocity of 1.0 mL/min was utilized

in the subsequent study.

3.3 Cell identification

After K562 cell sorting in the microfluidic device and the

subsequent deposition of individual cells, the identification

of K562 cells was performed through immunofluorescence

assay [46]. For this purpose, the primary antibody, mouse

ascites polyclonal anti-K562 cell surface membrane antigen

antibody, was first loaded into the cell trapping region. After

incubation for 1 h and rinsing with PBS, FITC-labeled

diagnostic polyclonal antibody, goat antimouse IgG-FITC

Figure 1. Configuration of the integratedmicrofluidics. (A) Schematic representationof the two functional regions in the device,i.e. sample mixing region and cell trappingregion. The consecutive, arranged denta-tion micromixer in the sample mixingregion and the microdam in the cell trap-ping region were designed, respectively, forquick sample mixing and tumor cell trap-ping (enlarged images of the square indotted lines). (B) Optical image of the actualdevice. (C) SEM images of the micromixer(top) and microdam (bottom), which wererecorded on a scanning electron micro-scope (SEM, JSM-6701F, Japan).

Figure 2. Mean diameter of the aggregated RBCs and K562 cellsunder different concentrations of Con A treatment, quantifiedusing Software Image-Pro Plus 6.0 (Media Cybernetics).



antibody, was injected into the same region for the labeling

of target cells. The results (Fig. 4A) showed that almost 99%

trapped cells are target cells. On-chip cell viability assay

showed that the viability of K562 cells was 94.4% (Fig. 4B).

Few cell deaths were due to natural cell apoptosis.

3.4 Assay of CTC responses to an anticancer drug

Tumor cell sensitivity to anticancer drugs varies in certain

situations. Cell viability is very important for an effective

cancer therapy [47]. In order to integrate cell-related studies

after CTC separation, the integrated microfluidic device was

applied to the study of K562 cell responses to an anticancer

drug. As mentioned above, the cell sorting chambers can

also be applied for cell culture. They have a steady and

homogeneous culture environment, and are feasible for a

precise cell–drug testing. To demonstrate this feasibility,

the damnification effect of K562 cells after treatment

with cytarabine was examined. Cytarabine, a preferred

chemotherapy drug for the treatment of leukemia [48], can

inhibit DNA and RNA polymerases, as well as the

nucleotide reductase enzymes needed for cell DNA synth-

esis, resulting in tumor cell damage or death [49, 50].

The K562 cells cultured in the microdevice were treated

with various concentrations (0.25, 0.5, and 1.0 g/L) of

cytarabine for 6 h. Cell viability was optically observed and

evaluated using PI staining assay following the methods

reported previously [39, 40]. After the treatment with

cytarabine, the dead cells were observed to be wizened,

anomalistic on the cell silhouette, gray in the micrograph,

and stained as red dots in the fluorescent image (Supporting

Information Fig. S4). On the contrary, the living cells were

round, glazed on the cell silhouette, not stained (black) in

the fluorescent images, and had better brightness in the

micrograph. In addition, the number of dead cells increased

with an increase in the concentration of cytarabine. The

quantitative relationship of cell viability and administered

cytarabine concentration is shown in Fig. 5. After 6 h

treatment, the cancer cells had a viability rate of 85.29, 77.05,

and 40% for drug concentration levels of 0.25, 0.5, and

1.0 g/L, respectively. Further testing is needed to determine

the proper regime of dosing and treatment time for effective

Figure 3. Separation efficiency of the K562 cells under differentflow velocities.

Figure 4. (A) Immunofluorescence assayfor K562 cell identification. Top: opticalimage of the K562 cells; Bottom: thecorresponding fluorescent image. (B)Bottom: analysis of K562 cell viabilityusing PI assay. The K562 cell viabilitywas 94.4% by counting dead cells fromthe fluorescent image. Top: correspond-ing optical image of the fluorescentimage.



chemotherapeutic results. Overall, the testing was

meant to demonstrate the feasibility of using the

proposed platform for an in vitro evaluation of chemo-

sensitivity. Further experiments on the applications of

this microdevice in cellular studies, such as other CTC

separations, more complex blood sample use, as well as

targeted cell responses to other drugs, are underway in our

laboratory.

4 Concluding remarks

The combination of lectin and microstructure was demon-

strated to have the capability to improve CTC separation

substantially. When the flow velocity of the blood samples

was 1.0 mL/min, and the initial K562 cell density was

2.0� 105 cells/mL, the separation efficiency of K562 cells

can reach 84%. Also, integration merged with this micro-

device facilitated in vitro operation for cell separation and cell-

related studies in a rapid and efficient manner. Therefore, the

use of this model is proposed as one of the practical tools for

improving the separation efficiency of targeted cells, as well

as for cell-related studies in an integrated microfluidic device.

Additionally, dam structure sorting has potential in high flux

cell isolation, and it can play an important role in both the

diagnosis and therapy of various diseases. For example, the

device can be employed to capture metastatic cells in patient

peripheral circulation for drug screening, molecular diag-

nosis, and purging of cancer cells prior to transplantation.

This work was supported by the National Natural ScienceFoundation of China (nos. 209 750 82; 207 750 59), Ministryof Education of the People’s Republic of China (NCET-08-0464),State Forestry Administration of the People’s Republic of China(No. 200904004), Scientific Research Foundation for ReturnedOverseas Chinese Scholars, State Education Ministry, andNorthwest A&F University grants.

The authors have declared no conflict of interest.

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Figure 5. Comparison of cell viability at various concentrationsof cytarabine. Cell viability is evaluated based on counting theliving and dead cells from the microscopic images usingSoftware Image-Pro Plus 6.0 (Media Cybernetics). Data arepresented as mean7SD of three repeated experiments.



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