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Isolation of circulating tumor cells under hydrodynamic loading using microuidic technology

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Isolation of circulating tumor cells under hydrodynamic loading using microuidic technology

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Cancer is a leading cause of mortality worldwide causing human deaths. Circulating tumor cells (CTCs) are cells that have detached from a primary tumor and circulate in the bloodstream; they may constitute seeds for subsequent growth of additional tumors (metastasis) in different tissues. The detection of CTCs may have important prognostic and therapeutic implications but, because their number is very small, these cells are not easily detected. Circulating tumor cells are found in the order of 10-100 CTCs per mL of whole blood in patients with metastatic disease. Isolation of tumor cells circulating in the blood stream, by immobilizing them on surfaces functionalized with bio-active coating within microfluidic devices, presents an interdisciplinary challenge requiring expertise in different research areas: cell biology, surface chemistry, fluid mechanics and microsystem technology. We first review the fundamental of cell biology of CTCs and summarize the key microfluidic techniques for isolation of CTCs via cell-ligand interactions, magnetic interactions, filtration; detection and enumeration of CTCs; in vivo CTCs imaging.
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力学进展, 2014 ,44 : 201412
Isolation of circulating tumor cells
under hydrodynamic loading using
microfluidic technology
ZHAO Cong1LEE Yi-Kuen1,2,XU Rui1LIANG Chun3LIU Dayu4
MA Wei2PIYAWATTANAMETHA Wibool5,6ZOHAR Yitshak7
1Division of Biomedical Engineering, HKUST, Hong Kong, China
2Department of Mechanical and Aerospace Engineering, HKUST, Hong Kong, China
3Division of Life Science, HKUST, Hong Kong, China
4Guangzhou First Municipal People’s Hospital, Guangzhou 510180, China
5Department of Electronics, King Mongkut’s Institute of Technology,
Ladkrabang, Bangkok, Thailand
6Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
7Department of Aerospace and Mechanical Engineering,
University of Arizona, Tucson, AZ, USA
Abstract Cancer is a leading cause of mortality worldwide causing human deaths.
Circulating tumor cells (CTCs) are cells that have detached from a primary tumor
and circulate in the bloodstream; they may constitute seeds for subsequent growth
of additional tumors (metastasis) in different tissues. The detection of CTCs may
have important prognostic and therapeutic implications but, because their number
Received: 2014-05-20; accepted: 2014-10-08; online: 2014-10-20
E-mail: meyklee@ust.hk
Cite as: Zhao C, Lee Y-K, Xu R, Liang C, Liu D Y, Ma W, Piyawattanametha W, Zohar Y.
Isolation of circulating tumor cells under hydrodynamic loading using microfluidic tech-
nology. Advances in Mechanics, 2014, 44: 201412
c
2014 Advances in Mechanics.
448 力 学 进 展 44 : 201412
is very small, these cells are not easily detected. Circulating tumor cells are found
in the order of 10–100 CTCs per mL of whole blood in patients with metastatic
disease. Isolation of tumor cells circulating in the blood stream, by immobilizing
them on surfaces functionalized with bio-active coating within microfluidic devices,
presents an interdisciplinary challenge requiring expertise in different research ar-
eas: cell biology, surface chemistry, fluid mechanics and microsystem technology.
We first review the fundamental of cell biology of CTCs and summarize the key
microfluidic techniques for isolation of CTCs via cell-ligand interactions, magnetic
interactions, filtration; detection and enumeration of CTCs; in vivo CTCs imaging.
Keywords circulating tumor cells (CTC), microfluidics, cancer, microsystem,
micro-electro mechanical system (MEMS), epithelial–mesenchymal transitions, metas-
tasis
Classification code: R318 Document code: ADOI: 10.6052/1000-0992-14-038
Zhao Cong et al. : Isolation of circulating tumor cells 449
1 Introduction
Blood is a specialized bodily fluid that delivers necessary substances to every cell in
the body, such as nutrients and oxygen, and transports waste products away from those
same cells. Blood is circulated around the body through vessels by the pumping action
of the heart, and the entire blood volume is re-circulated throughout a human body in
about a minute. Blood contains a wealth of information concerning the condition of all
tissues and organs in the body. Therefore, analysis of blood samples plays a major role
in the diagnosis of many physiologic and pathologic conditions, and extraction of reliable
information for clinical or basic research requires the understanding of the relevant biology
as well as proper technologies (Toner & Irimia 2005). Indeed, knowledge about blood has
expanded with the general progress in biology, while technological advancements enabled
several breakthroughs (Wintrobe 1980). Today, staining procedures along with full blood
count are still two basic yet very informative blood tests performed in hematology (Bauer
1999). Only flow cytometry can rival these techniques in terms of fine details and high
throughput, representing the current state-of-the-art in cell isolation and characterization.
The Clinical Laboratory Improvement Act (CLIA), imposing high-quality standards since
1988, has transformed the field of blood analysis (Kost et al. 1999); in particular, analysis
of cellular components is restricted to highly specialized laboratories. Nevertheless, despite
considerable automation, many blood-handling procedures are still carried out manually
or under conditions that could impact the analysis results even in advanced laboratories.
Reducing errors and processing time while enhancing analysis capabilities are well-known
challenges requiring improved methods.
Microfluidic-based technology is one of the most promising approaches for the next-
generation blood analysis (Toner & Irimia 2005). For clinical applications, realizing ade-
quate labs at the patient bedside for comprehensive blood analysis holds the potential of
revolutionizing the health-care landscape. Portable systems for reliable blood testing at
home or in office will allow accurate and rapid diagnosis of a host of bodily malfunctions
such as cancer. They may also accelerate the transition to personalized medicine in effort to
reduce side effects and improve the therapeutic efficiency. Microdevices inherently require
minute sample volumes for analysis, allowing repetitive sampling while minimizing blood-
drawing adverse effects. A diverse array of microfluidic systems have been developed in the
last decade for immunoassays including cell sorting, detection and counting, lysis, as well as
isolation and amplification of nucleic acids and proteins (Mauk et al. 2007). Microfluidic-
450 力 学 进 展 44 : 201412
based techniques developed for pathogen detection can be extended for the more challenging
task of cancer screening and diagnostics. Automated immunoassays of a single or multiple
markers can be implemented for point-of-care cancer screening. More robust tests can be
realized by quantifying a panel of nucleic acids or proteins to determine a cancer-specific
gene transcription or expression profile. To assess a gene transcription profile, microfluidic
systems need to include components for cell sorting and lysis, nucleic acid isolation and
amplification, as well as multiplex detection and quantification of gene transcripts. The
promising benefits of lab-on-a-chip cancer diagnostics systems include the use of small sam-
ple volumes, automated operation, short processing times, reduced reagent consumption,
reproducibility and consistency, minimal risk of sample contamination, convenient disposal,
and low cost.
This review summarizes recent advancements in the basics of circulating tumor cells
(CTCs) and the application of microfluidics for the isolation of metastatic cancer cells cir-
culating in the blood stream. We first present a general overview of disease progression in
carcinomas, during which tumor cells from primary tumors of epithelial origin enter blood
and lymphatic vessels. This is followed by a brief discussion of the challenges in blood sample
preparation and cell separation techniques. Finally, we detail the recent techniques devel-
oped for isolation of CTCs, including affinity-based method, magnetic method, filteration,
identification and enumeration and imaging method using optical Micro-Electro Mechanical
System (MEMS) technology.
2 Tumor-derived epithelial cells circulating in the blood stream
Metastasis is the spread of a disease from one organ or part to another non-adjacent
organ or part. Only malignant tumor cells and infections have the capacity to metastasize;
although, this is recently reconsidered by new research (Cristofanilli et al. 2004, Kahn et al.
2004, Smerage & Hayes 2005). Cancer cells can leave a primary tumor, enter lymphatic and
blood vessels, circulate through the bloodstream, and settle down to grow within normal
tissues elsewhere in remote sites; and the first observation of circulating tumor cells was
described by Ashworth in a post-mortem breast cancer patient (Ashworth 1869). Metas-
tasis is one of the hallmarks of malignancy, and most tumors can metastasize in varying
degrees (Zieglschmid et al. 2005). When tumor cells metastasize, a new secondary tumor is
formed with its cells resembling those in the original tumor. Viable tumor-derived epithelial
cells (CTCs), present in peripheral blood of cancer patients, are most likely the agents of
Zhao Cong et al. : Isolation of circulating tumor cells 451
metastatic disease (Chang et al. 2005, Du et al. 2007, Park et al. 2007, Toner & Irimia
2005).
Epithelial–mesenchymal transition (EMT) occurs during critical phases of embryonic
development in many animal species (Kalluri & Weinberg 2009). Without such a transi-
tion, in which epithelial are converted into motile cells, multicellular organisms would not
be able to advance beyond the early stages of embryonic development. A similar program
seems to play a key role in tumor progression as well (Thiery 2002). Epithelial-mesenchymal
transition presents a new mechanism for carcinoma progression to more malignant states.
Mesenchymal cells develop a morphology during EMT enabling them to migrate in an ex-
tracellular domain and to settle in regions associated with organ formation. These cells
can be involved also in forming epithelial organs through a reverse mesenchymal–epithelial
transition (MET). Although the existence of epithelial and mesenchymal cells was known
for over a century, EMT has only recently been identified as a distinct process (Greenburg &
Hay 1982). Since then, EMT has become a major research topic (Stoker & Perryman 1985).
However, it still took a long time to recognize EMT as an important mechanism driving
carcinoma progression. A key reason for this late recognition is due to the inability to follow
EMT in space and time in human tumors. The diverse cellular arrangements observed in
tumors makes it extremely difficult to perceive EMT with clarity. Nonetheless, some cancer
cell lines undergo EMT in vitro as illustrated in Fig. 1, and pathologists have suggested
several criteria for distinguishing between carcinomas and sarcomas (tumors of epithelial
and mesenchymal origin, respectively). The mechanisms leading to EMT are now studied
extensively; a great similarity between EMT in embryonic development and in tumor pro-
gression has been observed, and a few common signaling pathways have been discovered
in both EMTs. Thus, the EMT concept provides a new framework for identifying genes
important for the cancer progression towards more malignant states.
Loss of E-cadherin expression has been implicated in EMT and, therefore, E-cadherin
is proposed as a characteristic of the epithelial phenotype (Holcomb et al. 2002, Yoshida et
al. 2001). Renewal expression of E-cadherin in normal and mesenchymal cells can lead to
stable contacts among cells resulting in adherens junctions. Initial contacts in epithelial cells
are also mediated by E-cadherins that evolve into small complexes to form stable adherens
junctions (Adams & Nelson 1998, Garrod et al. 1996, Kowalczyk et al. 1998). While
E-cadherin is required for the maintenance of stable junctions, E-cadherin antibodies can
break these contacts and induce a mesenchymal phenotype (Imhof et al. 1983). The loss of
E-cadherin expression at EMT sites as well as E-cadherin expression at MET sites has been
452 力 学 进 展 44 : 201412
Normal epithelium
Localized carcinoma Invasive carcinoma
Basement membrane
Blood vessel
Extravasation Intravasation
Metastasis
MET
EMT
EMT
CTCs
Endothelial
cell
Fig. 1
The epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET)
in cancer progression (modified from Thiery 2002)
documented (Kuure et al. 2000). It has been reported that N-cadherin is expressed in some
cancer cells that lost E-cadherin (Frixen et al. 1991). E-cadherin expression is maintained
in most differentiated tumors, but E-cadherin levels seem to be inversely correlated with
either cancer grade or patient survival (Birchmeier & Behrens 1994, Hirohashi 1998). While
contributing to the differentiation-program maintenance, E-cadherin could also important
in regulating cell proliferation. Hence, loss of E-cadherin is central to EMT in normal
development as well as in cancer cells. The models used for studying EMT in vitro suffer
from a few drawbacks. EMT is either incomplete or slow in several models, taking several
days for completion. Only few cancer cell types of epithelial phenotype can complete EMT
in vitro. On the other hand, in vivo, the molecular mechanisms of EMT have not yet been
discerned, although promising results have been obtained in some model systems; EMT
in tumors is particularly difficult to study because it can take such a long time. Thus,
an important aim of continuing research is to elucidate the precise stages of EMT, both
in embryos and in mouse models of carcinogenesis. It might be possible to prevent EMT
altogether by understanding the conditions that lead to it. This therapeutic concept could
potentially block metastasis and, perhaps, prevent recurrence since micrometastases often
survive conventional therapeutic modalities (De Boer et al. 2009).
Zhao Cong et al. : Isolation of circulating tumor cells 453
It is well known that cancer cells can be released from primary tumors and, in animal
models, about 106cells/g of tumor (109cells) are estimated to reach the bloodstream
each day (Butler & Gullino 1975, Chang et al. 2000). The existence of cancer cells in
areas surrounding primary tumors is an indicator of metastasis, and these cells are routinely
investigated in various carcinoma types. Circulating tumor cells have also been found in
blood of patients having primary tumors, while bone micrometastases have been observed
in a large number of patients with tumors smaller than 2 cm in size (Pantel et al. 1999,
Thiery 2002). Detection of metastatic tumor cells serves as an independent prognostic
indicator for recurrence and survival (Braun et al. 2000, Naume et al. 2001), and could be
more significant than lymph-node metastasis in some carcinomas (Braun et al. 2000).
Circulating tumor cells are rare, as few as one cell per 109haematologic cells in the
blood of patients with metastatic cancer; hence, their isolation presents a tremendous tech-
nical challenge (Cristofanilli et al. 2004, Pantel & Alix-Panabies 2010). However, although
extremely rare, CTCs represent a potential alternative to invasive biopsies as a source of
tumor tissue for the detection, characterization and monitoring of non-haematologic cancers
(Paterlini-Brechot & Benali 2007, Lianidou & Markou 2011). Intensive research effort in
this field is now geared towards identifying specific markers (Nelson 2010), and determining
the aggressive nature of these cells (Mishra & Verma 2010, Nelson 2010). It is still unknown
whether CTCs share similar phenotypic characteristics with cancer cells in either the pri-
mary tumor or the lymph nodes, how do they home to specific organs, and how do they
flourish in a seemingly hostile environment? Clearly, techniques for the rapid isolation of
rare cells from blood would aid in not only diagnostic but also therapeutic applications (Lee
et al. 2009).
3 Challenges in cell sorting of blood sample
Blood is composed of two primary constituents: plasma and a variety of cells performing
multitude of tasks. The cell mixture in blood is not only very complex but also continuously
changing in response to various biochemical and biophysical stimuli; thus, collecting infor-
mation from blood cells is a major technical challenge. Depending on the required clinical
or research information, only a sub-population of cells is relevant and therefore has to be
isolated from the blood sample. The other blood cells have to be filtered out as they may
interfere with the subsequent analysis of the target cells. However, the sheer number and
vast diversity of cell types dramatically complicate the task of selectively isolating specific
454 力 学 进 展 44 : 201412
cells. In every microliter of blood, there are millions of cells including red and white blood
cells (RBCs & WBCs); and, for each WBC, there are about one thousand RBCs in blood.
White blood cells (leukocytes) are themselves quite diverse, and their classification into five
groups is insufficient for many applications (Table 1); a considerable number of subgroups
and sub-subgroups that be identified only by the presence of specific proteins, which are
either expressed on the surface of or secreted by the white blood cells have been discovered
(Goldsby et al. 2003). Several markers are typically needed to identify cells and elucidate
their function in the body. This immense cell diversity presents formidable obstacles for
obtaining the final desired purity adequate for further analysis. Even if accurately identi-
fied, careful extraction of target cells from a complex mixture such as a blood sample is a
greater challenge since most WBCs respond rapidly to environmental changes; hence, they
can be modified by various procedures during extraction. It has been demonstrated that the
exposure of cells to various insults when processing blood samples could alter the original
characteristics of the separated cells (Fukuda & Schmid-Schonbein 2002, Hallden et al. 1999,
Lundahl et al. 1995); in such a case, further analysis would not be able to recover the in
vivo state of the target cells.
Lab-on-a-chip technologies offer a promising alternative for overcoming the obstacles
associated with the diversity of blood cells and their susceptibility to varying conditions.
The new blood processing techniques can be more than just scaling down current assays.
Table 1 Absolute and relative number of cell populations and subpopulations in
normal blood (Wintrobe 1980)
Cell type Average/µL Percent of WBC Size
Erythrocytes (RBC) 5 000 000 6-8 and 2 µm
Reticulocytes 30 000–70 000
Platelets 200 000–500 000
All leukocytes (total) 5 000–10 000 100%
Neutrophils 4 000–8 000 40%–66% 10–12
Monocytes 200–800 4%–8% 14–17
Eosinophils 50–300 1%–3% 10–12
Basophils 0–100 0%–1% 12–15
Lymphocytes (total) 1 000–4 000 20%–40% 7–8
CD4 + T Cell 400–1 600 15%–20% 7–8
CD8 + T Cell 200–800 7%–10% 7–8
B-Cell 200–800 8%–12% 7–8
NK 100–500 4%–6% 7–8
Zhao Cong et al. : Isolation of circulating tumor cells 455
Taking advantage of demonstrated concepts for microenvironment as well as single-cell ma-
nipulation (Vilkner et al. 2004, Voldman et al. 1999), new techniques have been proposed
to overcome the technical obstacles in the analysis of blood. In microfluidic systems, it is
possible to efficiently recapitulate the in vivo micro-environment of each cell in blood and,
thus, minimizing the risk of modifying the original state of the isolated target cells. Novel
cell separation principles coupled with their amenability to on-chip integration with other
cell-analysis procedures are key elements in the efforts to meet the strict requirements for
unbiased analysis of isolated blood cells.
4 Microfluidic-based cell sorting techniques
4.1 Cell-separation microdevices based on mechanical principles
Variations in cell size and shape can readily be observed when probing cell mixtures
under a microscope (Wintrobe 1980). These geometrical differences between various cells in
blood samples can be utilized in microfluidic devices designed for separation of cells based
on mechanically trapping a sub-population of flowing cells. While it is relatively easy to
construct such systems using standard micromachining techniques; in general, they do not
offer the flexibility to accommodate variations in cell properties within the tested samples.
The separation of a selective cell sub-population of a different size would normally require
either a different design or a different device with similar design but different dimensions.
Microdevices designed based on size-dependent separation frequently also suffer from low
efficiency and poor cell purity.
Arrays of pillars and weir-type structures have been employed separating leukocytes
from whole blood samples (Wilding et al. 1998). Successive arrays of passages continuously
decreasing in size have also been used in separating target tumor cells suspended in normal
blood samples (Mohamed et al. 2004). The common feature was based on trapping target
and other larger cells at the array, while, allowing smaller cells to pass through some nar-
row spaces. However, despite their larger size, target cells can also “squeeze” through the
same mechanical constrictions compromising the capture efficiency (Wilding et al. 1998).
Furthermore, trapping target cells in designated structures can significantly alter the flow
pattern within the microdevice; this is a critical aspect when processing blood samples that
contain millions of cells. Another interesting phenomenon was observed blood cells were
forced to pass through a device featuring an array of coated posts (Carlson et al. 1997).
Among the trapped leukocytes, various cells were stopped at different distances along the
456 力 学 进 展 44 : 201412
array. This self-fractionation of cells was attributed to a combination of differences in viscos-
ity and nonspecific adhesion between the various cell types as they interact with the posts
during the separation process. The limited achievements of these early efforts highlight the
gaps in our understanding and difficulty in taking advantage of the cell-surface interaction
to isolate a selective group of cells.
In an alternative approach, the characteristic motion of particles in a laminar flow has
been investigated for separation of different particles. Discrimination of particles varying
in size has been demonstrated in microfabricated devices. Particles were separated as a
result of a differential lateral movement due to the asymmetry of laminar flow around me-
chanical obstructions with a length scale on the order of the particles size (Huang et al.
2004). Continuous separation of suspended particles in liquid was also demonstrated by
forcing the suspension through a narrow passage with an opening comparable to the parti-
cle size. A stream with and a stream without suspended particles were merged together in
a pinched microchannel; as a results, the particles were aligned along the sidewall such that
smaller particles were closer to the wall, while larger particles were closer to the channel
center. Thus, after passing the narrow constriction, initially mixed particles were re-aligned
along distinct streamlines depending on their size (Yamada et al. 2004). The flow pattern
around a microstep structure was also used for the separation of micro-particles and bacteria
(Vankrunkelsven et al. 2004).
The compliance of biological cells varying in size typically compromises the efficiency
of size-dependent cell separation; however, some devices were designed to separate cells
based on both their size and stiffness. Microchannels simulating blood vessels were used
to separate infected and more rigid red blood cells from normal, uninfected ones (Shelby
et al. 2003). The higher rigidity of infected cells hinders their passage, resulting in their
aggregation at the channel entrance. Separation of plasma and other cellular components
from blood samples in microfluidic devices has also been reported. Plasma was collected by
driving whole blood through microchannel arrays (Yang et al. 2004, Blattert et al. 2004),
or by sample centrifugation in lab-on-a-disk devices (Brenner et al. 2004, Kang et al. 2004).
4.2 Dielectrophoresis-based microdevices for cell separation
In dielectrophoresis (DEP), cells are electrically polarized in a non-uniform electric field
to induce affect their dynamic response. In mammalian cells, the induced polarization is
determined by numerous physiological conditions (Gascoyne & Vykoukal 2004). The cell
response to the applied electric field is sufficiently sensitive to distinguish between different
Zhao Cong et al. : Isolation of circulating tumor cells 457
cell types (Griffith & Cooper 1998). In microdevices, it is rather easy to generate intense
electrical fields under low voltages. Moreover, the electric fields can be fine-tuned at a
length scale on the order of the cell size by using integrated micro-components. Due to these
advantages, many DEP microdevices have been presented for manipulating suspended cells
or particles. Separating cells from mixtures in microfluidic devices, using dielectric fields, has
been discussed in several reviews (Gascoyne & Vykoukal 2002, Gascoyne & Vykoukal 2004,
Hughes 2002), and various strategies have been proposed for dielectrophoretic separation
depending on the sample volume.
DEP-based cell trapping is based on creating energy traps with a characteristic scale
on the order of the target cell size. Trapped cells are in stable equilibrium; they can be
released by turning off or perturbations of the electric field (Voldman et al. 2002, Lapizco-
Encinas et al. 2004). The efficiency of capturing target cells can be improved by setting the
electric-field frequency such that the non-target cells are driven away (Huang et al. 2002).
Under no-flow conditions, the efficiency can be enhanced by dielectric levitation of target
cells above electrode arrays (Gascoyne et al. 2002).
Target cells in large samples can be isolated using hyper-layer techniques, in which
DEP forces are exerted on cell suspensions driven through microchannels utilizing electrode
arrays. The forces acting on each cell include the gravitational force proportional to its
density, hydrodynamic drag force proportional to fluid velocity, and DEP force exponentially
decaying with its distance from the electrodes. Different cells attain equilibrium states at
different channel heights as a result of these three forces. Each cell type then moves with
different speed due to the non-uniform velocity profile across the channel. Thus, cells in a
flowing mixture were separated along a channel and collected at its outlet at different time
intervals (Gascoyne & Vykoukal 2004). The hyper-layer technique was used to separate
between normal and cancer cells in whole blood samples (Becker et al. 1995). Interdigitated
electrodes were placed at the bottom of thin flow chambers were used to levitate normal
and cancerous cells at different heights, depending on their dielectric properties (Yang et al.
1999). However, the separation efficiency has to be improved for clinical applications such
as the detection of cancerous cells in clinical blood samples.
DEP forces were also exploited to divert cells into distinct streams, dissociate cell
clusters, and separate cells along a particular stream for the isolation of individual cells
(Fiedler et al. 1998). Following passage over an interdigitated-electrode array, the suspension
main flow was branched into three streams (Holmes et al. 2003); the separation efficiency
can be enhanced by forcing the cells into a narrower stream above the electrodes (Holmes
458 力 学 进 展 44 : 201412
& Morgan 2003). In a different approach, tumor cell lines in mixtures were also separated
using DEP forces in devices with integrated microelectrode arrays (Muller et al. 2000,
Pethig 1996). Typically, DEP microdevices are simple and do not require pre-treatment of
cells; therefore, they are still considered to be prominent candidates for on-chip applications
(Yang et al. 2000).
4.3 Microscale optical techniques for cell separation
Optical techniques for separation of cells from complex suspension are very appealing
because they do not require intimate contact between the target cells and solid surfaces
minimizing the risk of cell activation. Using laser-tweezers in microdevices, single cells can
optically be trapped and manipulated. However, although suspended cells can be controlled
very precisely, only one cell at a time can be handled (Ozkan et al. 2003). Using such
microsystems, manipulation of millions of cells in parallel, as is expected for blood samples
in clinical applications, would be extremely challenging. Size-dependent cell separation has
been demonstrated using a diode laser bar in a microdevice. Size-based separation of cells
can be accomplished as larger cells deviate from their path when crossing a laser beam
while smaller cells continue un-affected (Applegate et al. 2004). An adaptive optical lattice,
formed as an interference pattern, was utilized for separating certain particles and cells
from mixed suspensions based on differences in their size and refractive index (MacDonald
et al. 2003). More recently, tunable optical lattices were applied for separating RBCs
from WBCs with about 95% efficiency (MacDonald et al. 2004). Such microdevices can be
reconfigured easily by adjusting the interference pattern; furthermore, since narrow passages
are not required, they are less susceptible to clogging during operation. The wide spread
availability coupled and continuous cost reduction of coherent light sources are additional
incentives to utilize optical techniques for separating target cells from complex mixtures
such as blood samples.
4.4 Magnetic techniques for microscale cell separation
Hemoglobin, the iron-bearing oxygen-transport protein contained in red blood cells
only, could provide a clear difference in magnetic characteristics between these and all other
blood cells. However, despite the iron content, measurements have shown that RBCs ex-
hibit magnetic behavior similar to that of WBCs, at least in oxygenated blood; a weak
paramagnetic behavior was reported for RBCs in deoxygenated blood (Paul et al. 1981). In
the presence of high magnetic fields, small differences in paramagnetic characteristics could
Zhao Cong et al. : Isolation of circulating tumor cells 459
be exploited for isolating WBCs from RBCs. Capture of RBCs from whole blood samples
was first demonstrated utilizing a metal mesh embedded in a strong magnetic field. The
generation of intense magnetic fields still requires very large magnets, which are difficult to
miniaturize; nevertheless, it is easier to obtain high magnetic-field gradients in microdevices,
and magnetapheresis has been demonstrated experimentally (Fuh et al. 2004, Zborowski
et al. 2003). Both particles and cells varying in magnetic characteristics were separated
from thin layers of suspensions flowing under an externally applied magnetic field (Fuh et
al. 2004).
The performance of magnetic-based cell separation can dramatically be enhanced by
binding magnetic beads decorated with ligands to membrane receptors on the surface of the
target cells. However, in this approach, the cell identification is based on protein: protein
specific interaction rather than on differences in cellular magnetic properties. Therefore, mi-
crodevices employing this principle are considered to be part of the affinity-based separation
class.
4.5 Biochemical interactions for cell separation in microfluidic devices
Subtle biochemical variations between cell populations can be exploited effectively for
manipulation of selective cell sub-populations by exposing the entire cell suspension to partic-
ular environmental conditions. For example, WBCs are much less vulnerable to ammonium
chloride than RBCs which, under exposure to such a solution, are lysed within a very short
time period (Szilard 1923). In standard protocols, blood samples are mixed with the lysing-
agent solutions for time intervals sufficiently long to allow RBC lysis. However, WBCs are
also exposed at the same time to the same agent and, therefore, could be affected to a certain
degree by the lysing solution. As a result, it is highly desirable to shorten the exposure time
in order to minimize the risk of damaging the WBCs (Kouoh et al. 2000). Experiments and
simulations suggest that the diffusion of the lysing agent within the cell suspension is the
limiting factor in trying to speed up the reaction; hence, shortening the distance required
for diffusion would provide a significant advantage. In microfluidic devices, short diffusion
length and time scales are inherent system characteristics. Indeed, lysis of RBCs has been
completed within one minute of sample exposure to an isotonic lysis buffer with recovery
of almost all the WBCs. In hypotonic deionized water, lysis of RBCs was completed even
faster while the WBCs remained intact (Sethu et al. 2004). Cell lysis in microfluidic devices
clearly results in improved WBC yield and viability due to the shorter processing time. Fur-
thermore, all cells in the sample are subjected to the same treatment; hence, by eliminating
460 力 学 进 展 44 : 201412
another source of variability, subsequent analysis could be more reliable.
5 Antibody-mediated isolation of CTCs
Cellular adhesion is the binding of a cell to another cell or to a surface or matrix, and it
is regulated by specific cell adhesion molecules that interact with molecules on the opposing
cell or surface. Such adhesion molecules are also termed “receptors” and the molecules
they recognize are termed “ligands” (or “counter-receptors”). Cells are not often found
in isolation, rather they tend to stick to other cells or non-cellular components of their
environment. Therefore, a major research effort has been devoted to discover what makes
cells sticky. Cell adhesion molecules (CAMs) are proteins located on the cell surface involved
in binding with other cells or with extracellular matrix (ECM). These proteins are typically
transmembrane receptors composed of three domains: an intracellular domain that interacts
with the cytoskeleton, a transmembrane domain, and an extracellular domain that interacts
either with other CAMs of the same kind (homophilic binding) or with other CAMs or the
extracellular matrix (heterophilic binding). Most of the CAMs are classified in 4 protein
families: Ig (immunoglobulin) superfamily (IgSF CAMs), integrins, cadherins, and selectins.
Immunoglobulin superfamily CAMs (IgSF CAMs) are either homophilic or heterophilic
and bind to integrins or different IgSF CAMs. Some molecules included in this family are:
NCAMs Neural Cell Adhesion Molecules, ICAM-1 Intercellular Cell Adhesion Molecule,
VCAM-1 Vascular Cell Adhesion Molecule, PECAM-1 Platelet-endothelial Cell Adhesion
Molecule, L1, CHL1, MAG, nectins and nectin-like molecules. Integrins are a family of het-
erophilic CAMs that bind to IgSF CAMs or the extracellular matrix. They are heterodimers,
consisting of two noncovalently-linked subunits, called alpha and beta. Twenty-four differ-
ent alpha subunits that can link in many different combinations with the 9 different beta
subunits are known; however, not all combinations are observed. Cadherins are a family
of Ca2+-dependent homophilic CAMs. The most important members of this family are
E-cadherins (epithelial), P-cadherins (placental), and N-cadherins (neural). Selectins are
a family of calcium-dependent heterophilic CAMs that bind to fucosylated carbohydrates,
e.g., mucins. The three family members are E-selectin (endothelial), L-selectin (leukocyte),
and P-selectin (platelet). The best-characterized ligand for the three selectins is P-selectin
glycoprotein ligand-1 (PSGL-1), which is a mucin-type glycoprotein expressed on all white
blood cells.
Zhao Cong et al. : Isolation of circulating tumor cells 461
6 Microfluidic devices for the isolation of CTCs
The analysis of circulating tumor cells (CTCs) includes isolation, characterization and
enumeration of CTCs. CTCs are very rare (as low as few CTCs per 10 mL of blood), the
isolation of CTCs is therefore technologically challenging. Ideally, a platform for capturing
CTCs should have the following characteristics: (i) high sensitivity, i.e., detection of every
single target CTC (no false negative); (ii) high specificity, i.e., removal of all non-target
cells (no false positives); (iii) high purity, i.e., isolation of all target CTCs with no contami-
nating non-target cells; (iv) preservation of CTC viability, morphology, protein and nucleic
acids; rapid and minimal processing of blood could improve the quality of CTC material for
molecular characterization studies; and (v) economical, i.e., low cost, high-throughput and
minimal operation time.
In the absence of any gold standard with which to evaluate various technologies, defining
their absolute accuracy, sensitivity, and specificity in detecting CTCs remains a challenge.
The ultimate goal, i.e., to efficiently isolate this rare sub-population of cells in a viable and
intact state and with high purity from the vast number of surrounding blood cells, presents
a daunting technological challenge. In recent years, substantial progress has been made to
improve and automate the isolation and characterization of CTCs, increasing the detection
sensitivity while decreasing the cost. These innovative CTC analytical technologies hold the
potential to serve as highly efficient and low-cost diagnostic tools for cancer. In general,
there are two basic formats that can be used for CTC isolation: macroscale and microscale.
Commonly used macroscale systems for CTCs detection include: (a) magnetic tag-
ging utilizing ferromagnetic micro-beads functionalized with homing ligands, classified as
immunomagnetic-assisted cell sorting , such as CellSearchTM (Allard et al. 2004); (b) size-
based separations that use porous membranes, such as ISET (Paterlini-Brechot & Benali
2007, Vona et al. 2000), ScreenCellTM (Vona et al. 2000); (c) fluorescence-activated cell
sorting (FACS) (Wang et al. 2012); and (d) reverse-transcription polymerase chain reaction
(RT-PCR) of mRNAs (AdnaTest BreastCancerTM) (Lankiewicz et al. 2006), which are used
as surrogates for cell identification. In general, commercial systems allow the collection of
rare cells from large sample volumes; however, their operation involves overcoming serious
obstacles that include labor-intensive sample preparation, sample loss due to transfer be-
tween instruments, and high cost associated with the completed device as well as the large
quantity of reagent consumption.
In comparison, microfluidic systems enable the application of processing techniques
462 力 学 进 展 44 : 201412
which are difficult to utilize in macrosystems (Chen et al. 2012, Das & Chakraborty 2013,
Dong et al. 2013, Sequist et al. 2009). In the microscale domain, the behavior of the fluid is
more predictable. Therefore, it is possible to take advantage of micro and nanostructures to
improve the capture efficiency of viable rare cells. Particularly, microsystems allow handling
individual cells without loss, which is not easy to duplicate with conventional systems.
Microsystem technologies are also attractive due to the smaller physical dimensions, reduced
power requirements, lower reagent consumption and high-throughput automation potential
in comparison with macroscale alternatives. Furthermore, CTC characterization following
capture can also be integrated into a microsystem to provide a fully integrated analysis.
A comparison of the fabrication technology, blood sample requirements, capture efficiency,
target cancer cells, cell viability, clinical assessment, required processing time of various
micro/nano CTC chips is summarized in Table 2. In addition, the detailed performance
of the systems for the detection of CTC developed by private companies is summarized in
Table 3.
6.1 CTC isolation via cell-ligand interactions
Affinity-based isolation of CTCs is frequently utilized in microfluidic systems. This
method relies on the specific interaction between a ligand — an antibody (Nagrath et al.
2007, Santana et al. 2011), aptamer (Dharmasiri et al. 2009, Fan et al. 2009), or lectin
(Wang et al. 2010), and a particular receptor on the surface of the target cell. Affinity-based
cell isolation can be achieved by either positive or negative approach. Negative method
aims to remove all non-target cells with minimal capture of target cells; this method is
attractive when the target-cell biomarkers are not well known. Positive method aims to
capture directly the target cells by exploiting surface markers unique to a certain cell sub-
population. Typically, cell selection requires a fluidic conduit bounded by a solid surface
on which the homing ligands are immobilized, and the CTC-containing sample is forced
through this conduit to enable the selective binding interaction.
Essential factors to achieve high-performance CTC isolation are: (a) frequency of con-
tact between target cells and the chemically modified solid phase, (b) adhesion strength
between target cell and recognition ligands, (c) specificity of cell receptor-surface ligand
interactions, (d) adequate flow rate to maintain the stability of cell-ligand binding, and
(e) high throughput. In microfluidic devices, microchannels may include additional micro
or nano features designed to enhance the cell-surface interactions (Isselbacher et al. 2010,
Wang et al. 2011). By applying properly configured topography of the solid phase (Talasaz
Zhao Cong et al. : Isolation of circulating tumor cells 463
Table 2 Summary of various micro/nano CTC chips
References Device
name
Operation
principle
Sample Test Clinical Assessment Time
Consumption
Fabrication
complexitya
Overall
scoreb
Cell Lines; Medium;
Volume/mL; Optimum
throughput/(mL·h1)
Capture
efficiency;
Purity;
viability
Cancer type;
Sample vol-
ume/mL
Sensitivity;
Speci-
ficity
Sample pro-
cessing;
Turnaround
/min
Nagrath et al. 2007 Micropost
chip
Antibody
coated microp-
osts
PC3-9, SkBr-3, T-24,
NCI-H1650; PBS, WB;
NA; 1–2
>65%;
50%;
99%
Lung,
Prostate,
Pancreas,
Breast,
Colon; 2.7
99%;
100%
81–162;
398–472
2 3
Zheng S et al. 2007
Lin H K et al. 2010
Microfilter Size based fil-
tering
LNCaP; PBS, WB; 1; 6 >90%;
NA;
>90%
Prostate,
Breast,
Colon, Blad-
der; 7.5
96% 16% <10; 150 1 4
Adams et al. 2008
Soper et al. 2009
Soper et al. 2011
HTMSU
chip
Antibody/ ap-
tamer coated
curvilinear
microchannels
MCF-7, LNCaP,
SW620, HT29; WRB,
WB; 1; 2
90%–
97%;
100%;
NA
NA NA 29–40;
279–290
2 2
Fan et al. 2009
Sheng et al. 2012
Micropillar
chip
Aptamer
coated
micropillar
DLD-1, HCT 116,
CCRF-CEM; PBS,
WB; 1; 2.16
90%;
81%;
93%
NA NA 28; 250 2 3
Lim C T et al. 2009
Tan et al. 2010
Micro cres-
cent well
chip
Size and
deformability
based filtering
MCF-7, MDA-MB-
231, HT-29; PBS, WB;
1–3; 0.7
>80%;
>83%;
NA
NA NA 86; 450 1 2
Talasaz et al. 2009 MagSweeper Antibody
coated
magnetic beads
MCF-7; PBS, WB; 9; 9 >50%;
51%–
100%;
94%
Breast; 9 100%;
NA
60; >130 1 4
464 力 学 进 展 44 : 201412
Table 2 Summary of various micro/nano CTC chips (continued)
References Device
name
Operation
principle
Sample Test Clinical Assessment Time
Consumption
Fabrication
complexitya
Overall
scoreb
Cell Lines; Medium;
Volume/mL; Optimum
throughput/(mL·h1)
Capture
efficiency;
Purity;
viability
Cancer type;
Sample vol-
ume/mL
Sensitivity;
Speci-
ficity
Sample pro-
cessing;
Turnaround
/min
Wang S et al. 2009 SiNP chip Antibody
coated SiNP
MCF-7, PC3, HeLa,
Daudi; PBS, WRB; 1;
NA
45%–
65%; NA;
84%–91%
NA NA 45; 205 2 2
Gleghorn et al.
2010
Kirby et al. 2012
GEDI chip Antibody
coated stag-
gered obstacle
arrays
LNCaP; PBS, WB; 1;1 85%–
97%;
62%–
68%;
NA
Prostate; 1 94%; NA 60; >520 2 3
Hosokawa et al.
2010
Hosokawa et al.
2013a
Hosokawa et al.
2013b
Microcavity
array
Size based fil-
tering
NCI-H358, MCF-7,
SW620, AGS, SNU-1,
Hs578T, NCI-H69,
NCI-H82, NCI-H358,
NCIH441, HCC827,
DMS79, A549, PC-14;
PBS, WB; 1; 12
80%–
99%;
33%;
98%;
Lung; 4 95.7%;
20%
20; 120–180 1 4
Isselbacher et al.
2010
Herringbone-
chip
Antibody and
herringbone
mixer
PC3; PBS, WB; NA;
2–3
92%;
14%; 95%
Prostate; 4 93%; NA 100–160;
300–400
2 3
Lu B et al. 2010
Xu et al. 2010
Microslot
filter
Size based fil-
tering
PC3, DU145; WB; 1;
12
90%; NA;
90%;
Prostate; 7.5 NA; 93% 38; NA 1 4
Wang J Y et al.
2010
Lectin-
aided chip
Microcolumn
and lectin
K562; WMB; NA; 0.06 84%;
99%; 94%
NA NA NA; >168 2 3
Zhao Cong et al. : Isolation of circulating tumor cells 465
Table 2 Summary of various micro/nano CTC chips (continued)
References Device
name
Operation
principle
Sample Test Clinical Assessment Time
Consumption
Fabrication
complexitya
Overall
scoreb
Cell Lines; Medium;
Volume/mL; Optimum
throughput/(mL·h1)
Capture
efficiency;
Purity;
viability
Cancer type;
Sample vol-
ume/mL
Sensitivity;
Speci-
ficity
Sample pro-
cessing;
Turnaround
/min
Di Carlo et al.
2010
Inertial mi-
crofluidics
Deformability
induced
inertial force
Hela, MCF7; WB; 1;
0.17
97%; NA;
>91%
NA NA 360; >380 1 2
Alazzam et al.
2011
Comb-like
DEP chip
DEP MDA231; WB; NA; 0.1 96%; NA;
NA
NA NA <60; NA 1 1
Jen et al. 2011
Jen et al. 2012
DEP-FFF
chip
DEP Hela; WB; NA; NA 76%; NA;
100%
NA NA 3; NA 1 1
Di Carlo et al.
2011
Hur et al. 2011
Microvortice
chip
Size induced
inertial force
Hela, MCF7; WB; 1
and 10; 300
10%–
20%;
85%; 90%
NA NA <3; <63 1 3
Duraiswamy et al.
2011
SiNP chip Antibody
coated SiNP
with micromix-
ers
MCF7; PBS, WB; 1; 1 >95%;
NA; NA
Prostate; 1 75%; 22% 60; >550 3 2
Hoshino et al. 2011 Immuno-
magnetic
chip
Magnetic
nanoparticles
conjugated to
antibody
COLO205, SKBR3;
WB; 3.5; 10
90%; NA;
NA
NA NA 21; >75 1 2
Jung et al. 2011 MOFF-
DEP chip
Combine
multi-orifice
flow fractiona-
tion (MOFF)
with DEP
MCF7; WB; 2; 7.56 99%;
16%; NA
NA NA 16; NA 1 2
466 力 学 进 展 44 : 201412
Table 2 Summary of various micro/nano CTC chips (continued)
References Device
name
Operation
principle
Sample Test Clinical Assessment Time
Consumption
Fabrication
complexitya
Overall
scoreb
Cell Lines; Medium;
Volume/mL; Optimum
throughput/(mL·h1)
Capture
efficiency;
Purity;
viability
Cancer type;
Sample vol-
ume/mL
Sensitivity;
Speci-
ficity
Sample pro-
cessing;
Turnaround
/min
Bhagat et al. 2011 Pinched
flow cou-
pled shear-
modulated
inertial mi-
crofluidics
Size based iner-
tial force
MCF-7, MDA-MB-
231; PBS, WB; 1;
24
>80%;
NA; NA
NA NA 50; >145 1 2
Thierry et al. 2010
Thierry et al. 2011
MicroPDMS
chip
Antibody
coated PDMS
microposts
SK-BR-3, NCI-H69;
PBS, WB; –0.5; 1
>70%;
NA; NA
NA NA 30; >1500 2 1
Wo et al. 2011
Chen et al. 2012
Microdisk
chip
Magnetic and
centrifugal
force
MCF-7; PBS; 0.2; –1.7 80%; NA;
90%
NA NA 7; >85 2 3
Zheng S et al. 2011 3D micro-
filter
Size based fil-
tering with 2
layers of mem-
branes
LNCaP, MCF-7; WB;
10; 120
86%;
NA;
>85%
NA NA 3–5; 50 2 3
Zheng X et al.
2011
High-
performance
microsys-
tem
Antibody
coated mi-
crochannels
MDA-MB-231; PBS;
0.02; 0.03
95%;
85%; NA
NA NA 40; >70 2 3
Augustsson et al.
2012
Acoustopho-
resis chip
Ultrasonic res-
onances force
DU145, PC3, LNCaP;
PBS, WB; 1; 4.2
93.9%;
99.7%;
100%
NA NA 14; >60 1 4
Zhao Cong et al. : Isolation of circulating tumor cells 467
Table 2 Summary of various micro/nano CTC chips (continued)
References Device
name
Operation
principle
Sample Test Clinical Assessment Time
Consumption
Fabrication
complexitya
Overall
scoreb
Cell Lines; Medium;
Volume/mL; Optimum
throughput/(mL·h1)
Capture
efficiency;
Purity;
viability
Cancer type;
Sample vol-
ume/mL
Sensitivity;
Speci-
ficity
Sample pro-
cessing;
Turnaround
/min
Bichsel et al. 2012 CTC cap-
ture and
3D culture
platform
Antibody
coated
micropillars
PC3; WB; NA; 1 8%–42%;
NA; 52%
NA NA NA 2 0
Issadore et al. 2012 µHD chip Antibody
coated mag-
netic nanopar-
ticles and
micro-Hall
detector
A431, MDAMB-
468, MDA-MB-453,
SkMG3; NA; NA; 107
cells/min
NA Ovarian; 7.5 91%;
100%
120; 150 3 2
Kang et al. 2012 Microfluidic-
magnetic
chip
Antibody
coated mag-
netic beads
M6C; WMB; 0.1; 1.2 87%;
NA; 90%
NA NA 5; >45 2 3
Schiro et al. 2012 MicroeDAR
chip
Fluorescent an-
tibodies
SKBr-3, MCF-7; WB;
1; 3
93%;
10%–
50%;
NA
Breast; 1–2 100%; 0 20; >230 1 3
Lim L S et al. 2012 Microsieve
chip
Size based fil-
tering
HepG2, MCF-7,
BT474; WB; 1; 60
>80%;–1;
NA
Breast; 3 NA 3; <90 1 3
Sun et al. 2012 Double
microspiral
chip
Size-dependent
hydrodynamic
forces
Hela, MCF7; WB; 1;
20
97%; NA;
NA
NA NA 3; NA 1 2
468 力 学 进 展 44 : 201412
Table 2 Summary of various micro/nano CTC chips (continued)
References Device
name
Operation
principle
Sample Test Clinical Assessment Time
Consumption
Fabrication
complexitya
Overall
scoreb
Cell Lines; Medium;
Volume/mL; Optimum
throughput/(mL·h1)
Capture
efficiency;
Purity;
viability
Cancer type;
Sample vol-
ume/mL
Sensitivity;
Speci-
ficity
Sample pro-
cessing;
Turnaround
/min
Loutherback et al.
2012
MicroDLD
array
Size-dependent
determinis-
tic lateral
displacement
MCF10A, MDA-MB-
231; WB; 5; 600
>85%;
NA;
>95%
NA NA 0.5; NA 1 3
Hou et al. 2013 DFF chip Size-dependent
centrifugal
forces
MCF-7; WB; 1; 3 85%;
10%;
>98%
Lung; 7 100%;
NA
120; >150 1 4
Lin M X et al. 2013 Microvortex
chip
Antibody
coated mag-
netic beads
and size based
filtering
MCF-7, MDA-MB-
231; WB; 7.5; 36
91.3%;
NA; NA
NA NA 11.25; >60 2 2
Hyun et al. 2013 p-MOFF
chip
Size-dependent
inertial forces
MCF-7, MDA-MB-
231; WB; 10; 36
>91%;
NA; NA
Breast; 7.5 19 out
of 24
patients
<30; <60 1 3
Lu Y T et al. 2013 NanoVelcro
chip
Antibody
coated SiNW
and PDMS
chaotic mixer
PC3, LNCaP, C4-2;
PBS, WB; 1; 0.5
>80%;
NA; NA
Prostate; 1 NA 2; >240 3 2
Huang et al. 2013 ODEP chip Optically
induced-DEP
PC3, OEC-M1; WB;
0.001; 0.006
61%;
64%; 92%
NA NA 10; NA 2 2
Zhao Cong et al. : Isolation of circulating tumor cells 469
Table 2 Summary of various micro/nano CTC chips (continued)
References Device
name
Operation
principle
Sample Test Clinical Assessment Time
Consumption
Fabrication
complexitya
Overall
scoreb
Cell Lines; Medium;
Volume/mL; Optimum
throughput/(mL·h1)
Capture
efficiency;
Purity;
viability
Cancer type;
Sample vol-
ume/mL
Sensitivity;
Speci-
ficity
Sample pro-
cessing;
Turnaround
/min
Warkiani et al.
2014
Slanted
microspiral
chip
Size-dependent
inertial lift
forces
MCF-7, T24, MDA-
MB-231; WB; 7.5; 102
85%;
4log
depletion
of WBCs;
>90%
Breast,
Lung; 7.5
10 out
of 10
patients
<8; 60 1 5
Abbreviations: PBS, phosphate buffered saline; WB, whole (human) blood; WMB, whole mouse blood; WRB, whole rabbit blood; DEP, dielectrophoresis;
MOFF, multi-orifice flow fractionation; FFF, field flow fractionation; DLD, deterministic lateral displacement; DFF, dean flow fractionation; SiNP, silicon
nanopillar; SiNW, silicon nanowire; NA, not available. aThe score of fabrication complexity +1 for (1) every mask layer, (2) the special structure in the
chamber (with another mask) and (3) antibody coating. bThe overall score (05) +1 if the device has a (1) relatively high capture efficiency of >80%, (2)
purity of >50%, (3) viability of >90%, (4) throughput of >5 mL/hr as well as an acceptable turnaround time of <3 hr, and (6) clinical assessment that shows
its potential in clinical use.
470 力 学 进 展 44 : 201412
Table 3 Summary of the CTC system developed by the companies in the world
Platform Company Operation principle
Turnaround
time/min;
Blood
sample/mL
Pros Cons Clinical applications References
CellSearchTM Veridex, LLC,
Raritan, NJ,
USA
Immunomagnetic
enrichment and im-
munofluorescence
90;
7.5
FDA approved;
Validated
in multiple
clinical trails
High complex-
ity; Captured
CTCs are no
longer alive
Metastatic breast,
prostate and colorectal
cancer
Cristofanilli et al.
2004
AdnaGenTM
CTC Detection
Adnagen AG,
Langenhagen,
Germany
Immunomagnetic en-
richment and RT-PCR
480;
5
CE marked;
Providing
genetic profiles
Low auto-
maticity;
without CTC
counting
Metastatic breast, and
colorectal cancer
Fehm et al. 2009
CTCscopeTM Advanced Cell
Diagnostic,
Hayward, CA,
USA
RNA in situ hybridiza-
tion (ISH) and im-
munofluorescence
>300;
5
Without the
need for spe-
cial CTC
enrichment or
purification
Can’t capture
the apoptotic
or dead CTCs
Simultaneous identifi-
cation and molecular
characterization of
CTCs
Payne et al. 2012
OncoCEE-
BRTM
Biocept Inc.,
San Diego, CA,
USA
Immunocytochemical
microfluidic device
with HER2 FISH
420–540;
16
Capture CTCs
undergoing
EMT
Large volume
blood sample
required
CTC enumeration with
CK detection or HER2
FISH
Mayer et al. 2011;
Pecot et al. 2011
HD- CTCTM Epic Sciences,
La Jolla, CA,
USA
Immunofluorescence
and morphological
identification
NA;
NA
Without the
need for CTC
isolation
With CTC
cluster prob-
lem
Advanced prostate,
breast, lung, and
pancreatic cancer.
Wendel et al. 2012
Halloysite
Nanotubes
(HNTTM )
NaturalNano
Inc.,
Rochester,
NY, USA
Immunocytochemical
microtube
>240;
10
Straightforward
technique us-
ing off-the-shelf
materials
Low auto-
maticity
CTC isolation Hughes et al. 2012
Zhao Cong et al. : Isolation of circulating tumor cells 471
Table 3 Summary of the CTC system developed by the companies in the world (continued)
Platform Company Operation principle
Turnaround
time/min;
Blood
sample/mL
Pros Cons Clinical applications References
Vita-CapTM,
Vita-AssayTM
Vitatex Inc.,
Stony Brook,
NY, USA
Cell Adhesion Ma-
trix(CAM)
NA;
NA
Not biased to
capture by par-
ticular physical
or antibody
markers
Only capture
invasive CTCs
iCTC capture and cul-
ture
www.vitatex.com
Rarecells R
System:
Patented ISET
RareCells SAS,
Paris, France.
CTC size 120;
10
CE-IVD la-
beled; Without
a previous
immune-based
selection
Low purity and
loss of CTCs
smaller than
8µm size
CTC isolation (except
Chronic Lymphocytic
Leukemia, CLL)
www.rarecells.com;
Farace et al. 2011
ApoStreamTM ApoCell, Hous-
ton, TX, USA
Dielectrophoresis field
flow fractionation
(DEP-FFF).
NA;
0.05–10
Antibody-
independent;
High auto-
maticity
Limited sample
suspension col-
lection volume
CTC isolation www.apocell.com
Canopus’ CTC
Platform
Canopus
Bioscience
ltd., Toronto,
Canada
Viscoelastic properties NA;
NA
Free from filter
clogging
With a first
stage removes
red blood cells
CTC isolation http://canopus-
bioscience.com/
CellSieveTM Creatv Mi-
croTech, Inc.
MD, USA
CTC size NA;
10
Rapid sam-
ple filtering
(<2 min)
With CTC
cluster prob-
lem
CTC isolation, enu-
meration, and culture
www.creatvmicro-
tech.com
ClearCell R
FX
System,
CTChip R
FR
Clearbridge
BioMedics Pte
Ltd., Singapore
Inertial focusing princi-
ple
>150;
6
Entirely label-
free
Low purity CTC isolation www.clearbridge-
biomedics.com;
Hou et al. 2013
472 力 学 进 展 44 : 201412
Table 3 Summary of the CTC system developed by the companies in the world (continued)
Platform Company Operation principle
Turnaround
time/min;
Blood
sample/mL
Pros Cons Clinical applications References
ClearCell R
CX,
CTChip R
CS,
CTChip R
CR.
Clearbridge
BioMedics Pte
Ltd., Singapore
CTC size and deforma-
bility
450;
3
Label-free iso-
lation
High complex
system
Integrated system for
CTC enumeration,
staining and retrieval
www.clearbridge-
biomedics.com;
Lim C T et al.
2009
sureCELL R
CellSievo Pte
Ltd., Singapore
CTC size 90;
3
Label-free iso-
lation
Low purity CTC isolation and enu-
meration
Lim L S et al. 2012
Cynvenio’s
Platform
Cynvenio
Biosystems,
Westlake Vil-
lage, CA,
USA
Immunomagnetic
beads
>150;
5
Isolation via
ultrahigh mag-
netic gradient
Only cap-
ture Ep-
CAM+/CK+
CTCs
CTC isolation www.cynvenio.com
OncoQuick R
Greiner Bio-
One GmbH,
Frickenhausen,
Germany
Buoyant density >45;
15–30
Fast, antibody-
independent
Large volume
blood sample
required
CTC isolation www.greinerb-
ioone.com
ScreenCell R
ScreenCell,
Paris, France
CTC size 3;
1
Independent
of any equip-
ment; CE-IVD
labeled
Low purity CTC isolation www.screencell.com
Desitter et al. 2011
Zhao Cong et al. : Isolation of circulating tumor cells 473
et al. 2006, Wang et al. 2011, Wang et al. 2009), it is possible to achieve high adhesion
strength between target cells and functionalized surfaces. Highly specific recognition ligands,
such as monoclonal antibodies and aptamers, are immobilized on the capture surface with
a proper density to result in stable and selective binding.
Nagrath et al. (2007) developed a microfluidic system for removing CTCs from whole
blood samples (Fig. 2(a)). The CTCs originated from various solid tumors and were
targeted using epithelial cellular adhesion molecule (EpCAM) monoclonal antibodies im-
mobilized on the surfaces of a microchannel and micropillars. The device contained 78 000
microposts that were 100 µm tall and 100 µm wide with a total surface area of 970 mm2.
The EpCAM antibodies provided the selectivity for the capture of CTCs from blood sam-
ples since EpCAM receptors are expected to be overexpressed in epithelial originated cancers
(Baeuerle & Gires 2007, Went et al. 2004). Under a 1 mL/h flow rate, 65% of the target
CTCs were recovered, while 98% of the captured cells were viable. Enrichments from blood
samples yielded about 50% purity. A similar system was also used for isolating rare lung
cancer cells from blood samples (Maheswaran et al. 2008). Utilizing the CTC chip, CTCs
were detected in 100% of early-stage prostate cancer patients. The application of the chip
for monitoring patient reaction to anticancer therapy was also tested. In a group of patients
with metastatic cancer undergoing systemic treatment, variations in the number of CTCs
were found to correlate with the disease clinical course (Maheswaran et al. 2008, Nagrath
et al. 2007).
High CTC capture efficiency is expected due to the enhanced interaction between the
target CTC receptors and the immobilized surface ligands. However, fluid flow in microchan-
nels is laminar in nature and, consequently, cells follow streamlines and display minimal
species diffusion across flow channels. This lack of mixing results in a limited number of
receptor-ligand encounters, which is critical for target cell capture. Stotta et al. (Isselbacher
et al. 2010) developed a CTC capture microdevice (Fig. 2(b)), using an alternative strategy
that involves the use of surface ridges or herringbones in the wall of the device to disrupt
streamlines, maximizing contact between target cells and the antibody-functionalized walls.
The herringbone-chip (HB-Chip) provided an enhanced platform for CTC isolation. Effi-
cient cell capture was validated using defined number of cancer cells spiked into a control
blood sample, and clinical utility was demonstrated in specimens from patients with prostate
cancer. CTCs were detected in 93% of the (14/15) patients with metastatic disease. The
HB-Chip proved to be highly efficient; at a flow rate of 1.2 mL/h, the capture efficiency of
PC3 cells was 79%.
474 力 学 进 展 44 : 201412
ab
c
d
CTCs captured against posts on CTC-chip
CTCs captured against posts on CTC-chip
Output Input
Pt
Electrode
Capture Bed
Pt
Electrode
Fig. 2
Representative micro/nano devices for CTC isolation via cell-ligand interactions. (a) Sche-
matic showing CTCs captured by anti-EpCAM coated microfluidic channel and posts. (Na-
grath et al. 2007). (b) Cartoon illustrating the cell-surface interactions in the Herringbone-
Chip (Isselbacher et al. 2010). (c) Schematic of a microfluidic device featuring an anti-
EpCAM coated silicon nanopillar substrate and herringbone-patterned ceiling to constantly
bring CTCs into contact with the substrate. (Wang et al. 2011). (d) Schematic of a microflu-
idic device with integrated systems for CTC capture, enumeration, and electro-manipulation.
(Dharmasiri et al. 2009)
An anti-EpCAM-functionalized, nanostructured substrate was developed for the iso-
lation of CTCs from whole-blood samples, Fig. 2(c) (Wang et al. 2009, 2011). The
uniqueness of this approach lies in the use of 3D nanostructured substrates—specifically,
a silicon-nanopillar (SiNP) array—which allow for locally enhanced interactions between
the SiNP substrates and nanoscale components of the cellular surface (e.g., microvilli and
filopodia) leading to vastly improved cell-capture affinity compared to unstructured flat sub-
strates (Wang et al. 2009). This work showed that the number of captured EpCAM-positive
cells increased with increasing SiNP lengths, but relatively minor changes were observed for
Zhao Cong et al. : Isolation of circulating tumor cells 475
EpCAM-negative cells. Using these nanostructured substrates, cancer cells can be reliably
captured from artificial CTC blood samples, and cell viability with this platform is as high
as 84%–91%. In effort to augment this performance, the SiNP substrate was combined with
the herringbone structured channel (Wang et al. 2011). When driving a blood sample con-
taining CTCs through the device, the embedded herringbone micropatterns on the channel
roof induce vertical flow in the microchannel. Consequently, the contact frequency between
CTCs and the SiNP substrate increases, resulting in significantly enhanced CTC capture
efficiency compared to the static setting. With an optimal flow rate (1.0 mLh1), a superb
cell-capture efficiency (>95%) was reported using this microdevice. The optimal conditions
were employed to capture and count CTCs in blood samples collected from prostate cancer
patients with different degrees of tumor spread and different sensitivity to treatments. The
results obtained by the devices were compared with those observed by CellSearchTM using
immunomagnetic enrichment demonstrating overall superiority over the commercial instru-
ment. In a different approach, a polymer-based device was fabricated consisting of an array
of 51 high-aspect ratio curvilinear-shaped channels (Adams et al. 2008, Dharmasiri et al.
2009, Dharmasiri et al. 2011) (Fig. 2(d)). CTC recognition molecules, including EpCAM
antibodies (Adams et al. 2008), PSA antibodies and anti-PSMA aptamers (Dharmasiri et
al. 2009) were tethered to the capture surface. The curvilinear-shaped channels improved
both the cell capture efficiency and the device throughput by using parallel channels with
high-aspect ratio. Using a linear velocity profile for optimal cell capture, a recovery >90%
was achieved.
Drawbacks associated with affinity-based CTC isolation include limitation in capture ef-
ficiency and low throughput. However, the most severe challenge remains a clear biomarker.
The most widely used CTC binding ligand, EpCAM, has been found to be expressed in
70% tumors with different histologic type (Went et al. 2004). Similarly, cytokeratin-
negative tumor cells were also found in the blood of a breast cancer patient (Fehm et al.
2002). Furthermore, CK8, CK18 and CK19 were absent in cell lines derived from dissem-
inated tumor cells (Willipinski-Stapelfeldt et al. 2005). The absence of cytokeratins (CK)
an EMT indicator, has been verified in many samples of breast cancer considered to be of a
higher grade (Willipinski-Stapelfeldt et al. 2005). Thus, during EMT, the most malignant
CTCs seem to cease expression of epithelial antigens; this suggests that assays designed
for detecting epithelial cells in blood are prone to fail in discerning the aggressive tumor
cells. Isolating CTCs requires the processing of relatively large sample volumes. Channels
in microfluidic devices are typically within the range of tens to hundreds of microns. These
476 力 学 进 展 44 : 201412
small sized channels are ideal for enhanced frequency of cell-surface interaction. However,
the limited channel dimensions are also a bottleneck in sampling blood of large volumes
(7.5 mL). Higher flow rate facilitates improved throughput but decreases the cell-ligand
interaction time. In addition, significantly increased shearing force due to higher flow rate
may even prevent cell adhesion while compromising the viability of captured cells.
6.2 CTC isolation via magnetic interactions
Magnetic particles are also widely used for selective isolation of CTC. CTC isolation
using magnetic interactions is different from conventional affinity-based isolation in that the
homing ligands are immobilized on the surface of microbeads, rather than on a microchannel
surface. In this type of assay, silica-coated magnetic particles are functionalized with highly
selective ligands. CTCs are then specifically recognized by the ligands immobilized on
the magnetic beads, while the CTC isolation or enrichment is accomplished by utilizing a
magnetic field (Neurauter et al. 2007, Haukanes & Kvam 1993, ˇ
Safaˇr´ıK & ˇ
Safaˇr´ıKov´a 1999).
A method of microchip-based immunomagnetic CTC detection has been reported com-
bining the benefits of both immunomagnetic assay and microfluidic technology (Fig. 3(a))
(Hoshino et al. 2011). A polydimethylsiloxane (PDMS)-based microchannel bonded to
a glass coverslip was used to screen blood samples. As the blood sample flows through
the microchannel closely above an array of magnets, cancer cells decorated with magnetic
nanoparticles are separated from the blood flow and deposited onto the glass coverslip bot-
tom surface, which enables direct observation of the captured cells using a fluorescence
microscope. The dimensions of the microchannel combined with the sharp magnetic field
gradient, in the vicinity of the magnet array with alternate polarities, lead to an effec-
tive capture of target cells. Customized Fe3O4magnetic nanoparticles functionalized with
EpCAM antibodies were introduced into the blood samples for binding to the target can-
cer cells. The blood sample was then driven through the microchip device to isolate the
decorated target cells. Compared to the commercially available Veridex CellSearchTM sys-
tem (www.cellsearchctc.com) (which has been approved by United States Food and Drug
Administration US FDA, Ref No. K031588, for monitoring the patients with metastatic
breast, colorectal and prostate cancer since 2004, and has been approved by China FDA
for metastatic breast cancer patients in 2012), fewer (25%) magnetic particles were required
to achieve a comparable capture efficiency, while the screening speed reached 10 mL/hr.
Using this method, rare cancer cells (from 1 000 cells down to 5 cells per mL) were success-
fully detected, and recovery rates of 90% and 86% were demonstrated for COLO205 and
Zhao Cong et al. : Isolation of circulating tumor cells 477
a
b
cd
500 mm
150 mm
Inlet Outlet
PDMS microchannel
blood cell
Cancer cell
Magnetic force
Cover slip
1
Magnetic beads injection
in a microfluidic channel
Magnetic
pattern
AMagnetic field OFF Magnetic field ON
B
2
3
4
Magnets
Current (IDC)
Electric field
z
Fig. 3
Representative micro/nano devices for CTC isolation via magnetic interactions. (a) Schem-
atic of microfluidic device isolating magnetically-labeled CTCs by magnetic field (Hoshino et
al. 2011). (b) Captured magnetic beads on a ferromagnetic material encapsulated micropil-
lar, Scale bar is 20 µm. (Xia et al. 2011). (c) Schematic of cell-particle complexes isolated
and transported by a traveling magnetic field. (Stakenborg et al. 2010). (d) Principle of
magnetic bead self-assembly in microfluidic channel. (Saliba et al. 2010)
SKBR3 cells, respectively. A micro magnetic activated cell sorting (microMACS) chip based
on ferromagnetic material encapsulated micropillars was reported (Fig. 3(b)) (Xia et al.
2011). This method is capable of simultaneously producing magnetic microstructure arrays
dispersed among channels that are susceptible to magnetic force, providing a uniquely se-
lective feature. Simulations predicted that the target capture would occur precisely at the
two opposing endpoints of micropillars, based on their perpendicular positioning to the flow
direction and their property of maximum magnetic force. To determine the capability of
the microMACS chip in capturing CTCs, SW620 human colon cancer cells were used in an
in vitro flow model system and the capture efficiency was found to be 72.8%.
478 力 学 进 展 44 : 201412
An automated modular microsystem has been developed to facilitate the detection and
subsequent clinical evaluation of CTCs directly from blood (Fig. 3(c)) (Stakenborg et al.
2010). The proposed microsystem integrates three modules: (1) sample preparation and
immunomagnetic-based cell enrichment, separation and counting; (2) reverse transcription
of messenger ribonucleic acid (mRNA) to complementary deoxyribonucleic acid (cDNA) and
multiplex amplification of specific cancer markers; and (3) detection of the amplified DNA.
More specifically, an automated mixing chamber for immunomagnetic cell separation is
introduced as a first module. The main chamber is designed for efficient mixing to enhance
specific magnetic CTC isolation while retaining its viability. Following isolation of the
immunomagnetic-captured CTCs, the sample volume is reduced and directly transported
to a microfluidic chip via a macro- to-micro-interface. On chip, the cells are subsequently
actuated and separated from unlabeled beads on the basis of differences in magnetic mobility.
This process enables direct counting of the magnetically decorated cells in-flow. Following
isolation and detection, the CTCs are transported to a second module for on-chip PCR.
After lysis, the mRNA is transcribed to cDNA to enable simultaneous amplification of over
20 gene fragments by means of multiplex ligation-dependent probe amplification (MLPA). In
the final module, the amplified DNA fragments are detected using an array of electrochemical
sensors.
A unique CTC isolation method has been proposed utilizing columns of bio-functionalized
super-paramagnetic beads self-assembled in a microfluidic channel onto an array of magnetic
traps formed by micro-contact printing (Fig. 3(d)) (Saliba et al. 2010). Taking cancer cell
lines as examples, a capture yield better than 94% was demonstrated together with the
potential to cultivate the captured cells in situ. Clinical samples, including blood, pleural
effusion, and fine needle aspirates were also tested using this method. The immunopheno-
type and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber,
and compared with conventional flow cytometry and visual cytology data, in a blind test.
Immunophenotyping results were fully consistent with those obtained by flow cytometry.
This method provides a powerful approach to cell capture and characterization allowing
fully automated, high resolution and quantitative immunophenotyping and morphological
analysis. It requires at least 10 times smaller sample volume and fewer cells than cytometry,
potentially increasing the range of indications and the success rate of microbiopsy-based
diagnosis while reducing analysis time and cost.
Zhao Cong et al. : Isolation of circulating tumor cells 479
6.3 CTC isolation via filtration
CTCs are different from leucocyte in cell size, density, shape and deformability. These
parameters can be exploited in microsystems to isolate CTCs on the basis of mechani-
cal restriction (Mohamed et al. 2004). In comparison with affinity-based CTC isolation,
filtration-based methods use a more active fluid control strategy. In these devices, CTCs in
the blood are forced to pass through the filtration mesh. These methods are usually label-
free and CTC trapping, using these systems, is usually simple and cost effective. In this
approach, the flow rate is typically >1 mL/min; hence, it promises much higher throughput
than that of an affinity-based assay. Furthermore, filtration-based microdevices usually re-
quire less capture area and therefore guarantee parallel sample processing in a miniaturized
device.
Effective CTC isolation has been demonstrated using a microfluidic design based on
the unique differences in size and deformability between cancer cells and other blood cells
(Fig. 4(a)) (Tan et al. 2009, Tan et al. 2010). Placing physical structures along the path
of blood samples in a microchannel, CTCs which are generally larger and stiffer are retained
while most blood constituents are removed. The isolation efficiency is more than 80% for
tests performed on breast and colon cancer cells. The operational conditions for processing
blood are straightforward and permit multiplexing of the microdevices to handle several
different samples in parallel. The microfluidic device is optically transparent, which makes it
simple to be integrated to existing laboratory microscopes, and immunofluorescence staining
can be done in situ to distinguish cancer cells from hematopoietic cells. Viable isolated cells
are obtained on the microdevice, enabling further analysis through on-chip phenotypic and
genotypic tests.
Microfilter devices for CTC detection featuring parylene membranes have also been
proposed (Fig. 4(b)) (Zheng et al. 2011). Using a constant low-pressure delivery system,
the microfilter platform was capable of cell capture from 1 mL of whole blood in less than
5 minutes, achieving 90% capture efficiency, 90% cell viability, and 200-fold sample enrich-
ment. The captured cells retained normal morphology, as confirmed by scanning electron
microscopy, and could readily be manipulated, further analyzed, or expanded on- or off-
filter. CTCs were identified in 51 of 57 patients using the microdevice, compared with only
26 patients with the CellSearchTM method. When CTCs were detected by both methods,
greater numbers were recovered by the microfilter device in all but five patients. These
filter-based microdevices are both capture and analysis platforms, capable of multiplexed
480 力 学 进 展 44 : 201412
ab
d
c
MCF-7 breast
cancer cells
OUTLET
INLET
FLOW
Healthy cells
Flowing freely
Retained
Cancer cells
mm
 mm
 mm
 mm
 mm
Fig. 4
Representative microfluidic devices for CTC isolation via filtration. (a) Crescent-shaped
traps in a microfluidic device. Scale bar is 20 µm. (Tan et al. 2010). (b) Parylene membrane
filter with LNCaP cells captured. (Zheng et al. 2011). (c) Nickel microcavity array with
MCF-7 cells trapped. The microcavities are 9 µm in size, with a 60 µm pitch. (Hosokawa et
al. 2010). (d) Array of microfluidic traps with varying geometrical restrictions. (Mohamed
et al. 2009)
imaging and genetic analysis.
In another approach, a size-selective microcavity array for rapid and efficient detection
of CTCs has been reported (Fig. 4(c)) (Hosokawa et al. 2010). The microfluidic device
contains a nickel plate with a size-selective microcavity array, which can specifically separate
tumor cells from whole blood on the basis of differences in the size and deformability be-
tween tumor and hematologic cells. The arrayed cells were then processed for image-based
immunophenotypic analysis using a fluorescence microscope. This microfluidic device suc-
Zhao Cong et al. : Isolation of circulating tumor cells 481
cessfully detected about 97% of lung carcinoma NCI-H358 cells in 1 mL whole blood spiked
with 10–100 cancer cells. In addition, breast, gastric, and colon tumor cells lines that include
EpCAM-negative tumor cells, which cannot be isolated by conventional immunomagnetic
separation, were successfully recovered on the microcavity array with high efficiency (more
than 80%). On the average, around 98% of recovered cells were viable.
A micromachined device was designed and tested to fractionate whole blood using
physical attributes for enrichment and/or isolation of rare cells from peripheral circulation
(Fig. 4(d)) (Mohamed et al. 2009). This device has arrays of four successively narrower
channels, each consisting of a two-dimensional array of columns, decreasing in spacing from
20 µm to 5 µm. The first 20 µm wide segment disperses the cell suspension and creates an
evenly distributed flow over the entire device, whereas the others were designed to retain
increasingly smaller cells. When cancer cells were loaded into the device, all cancerous cells
were isolated depending on cell size and deformation characteristics. The results demon-
strated that the device is capable of retaining any cancer cell that is larger and/or less
deformable than blood cells. Furthermore, intact cells can be removed from the device by
reversing the flow after retaining the target cancer cells while all blood cells are flushed out
of the device.
In order to achieve high-throughput CTC analysis in microfluidic systems, one impor-
tant issue is the potential hydrodynamic damage of cells flowing inside microfluidic channels.
Ma and the co-workers (Ma et al. 2013) has developed a high-throughput circular multi-
channel microfiltration (CMCM) device integrated with a polycarbonate (PC) membrane
to investigate the hydrodynamic lysing of cancer cells during the filtration. An empirical
formula was proposed to predict the cell viability of the CMCM as a function of shear stress
or Reynolds number. In addition, based on silicon-on-insulator (SOI) technology, a new
micro-filtration chip with an array of high-density regularly spaced pores (Ma et al. 2014)
was developed. Compared to the previous CMCM device with PC filter, the SOI-based
system shows much better performance of CTC isolation and enumeration.
6.4 Identification and enumeration of CTCs
Since all the CTC isolation techniques cannot yield 100% specificity, it is therefore
necessary to characterize the selected cells to allow reliable CTC enumeration. Methods
for CTC characterization include immune-staining and quantitative reverse transcriptase–
polymerase chain reaction (qRT-PCR). The criteria used in the CellSearchTM as well as
most microfluidic-based CTC detection assays include: round to oval morphology, a visible
482 力 学 进 展 44 : 201412
nucleus (4’, 6-diamidino-2-phenylindole or DAPI positive), positive staining for cytokeratins
and negative staining for cluster or differentiation 45 or CD45 (Allard et al. 2004). The
combination of morphology, nucleus staining and surface markers can differentiate CTCs
from blood cells.
Another approach employed for the characterization of CTCs is real-time polymerase
chain reaction (RT-PCR), where mRNAs are the biomarkers indicating the presence of
cancer cells. The sensitivity of this approach is considered to be higher than that of immune-
mediated detection and immunocytochemistry (Saliba et al. 2010). RT-PCR involves several
steps: (a) CTC collection, (b) RNA extraction, (c) complementary DNA (cDNA) synthesis,
(d) target sequence amplification, and (e) PCR product analysis (by gel electrophoresis or
real-time amplification signal monitoring). PCR methods can identify one target cell out of
106–107normal cells which corresponds approximately to one cell in 0.1 mL–1 mL of blood.
A qPCR-TRAP assay has been developed to amplify the telomerase activity signal
from CTCs captured on micro filters (Xu et al. 2010). Using this method, the detection
of telomerase activity from as few as 25 cancer cells suspended in a 7.5 mL of whole blood
sample has been demonstrated. A factor which might affect telomerase activity measurement
is potential cell variability in various clinical states such as inflammatory conditions or
chemotherapy.
A significant disadvantage of RT-PCR is the destruction of CTCs, eliminating the
possibility of counting or analyze them. Another drawback is that the selection of the
marker RNAs, the transcripts indicating tumor cells in blood is not straightforward. TA
good biomarker should be a transcript expressed in all target cancer cells from one particular
tumor, and not expressed at all in all non-target cells, not even by illegitimate transcription
(low level, non-specific transcription of certain genes) (Chelly et al. 1989). Finally, while RT-
PCR has high sensitivity, it suffers from poor specificity which may result in false positives.
These assays are also known to exhibit high inconsistency among different laboratories (Helo
et al. 2009). Indeed, if the marker is a gene typically expressed in epithelial cells, a false-
positive result will be obtained if the patient happened to have non-tumorous epithelial
cells circulating in blood. Cytokeratin 19 (CK19) mRNA was detected in blood of some
healthy donors, in samples of patients with haematological malignancies, and in some control
subjects. Detection of Cytokeratin 19 in healthy donors has been proposed to result from
the gene illegitimate transcription or enhanced secretion of cytokines that can upregulate
transcription of certain genes in leukocytes (Novaes et al. 1997).
CTC enumeration without labeling has been reported using an on-chip integrated con-
Zhao Cong et al. : Isolation of circulating tumor cells 483
ductivity sensor following release from the capture surface (Adams et al. 2008). The isolated
CTCs were readily released from the antibody-functionalized surface using trypsin. The re-
leased CTCs were then enumerated on-chip using a label-free solution conductivity route
capable of detecting single tumor cells traveling through the detection electrodes. The con-
ductivity readout provided near 100% detection efficiency and high specificity for CTCs due
to favorable scaling factors and the non-optimal electrical properties of potential interfer-
ences (erythrocytes or leukocytes).
6.5 In vivo CTCs imaging with optical MEMS technology
Noninvasive imaging of CTCs in real time as they flow through the peripheral vascu-
lature could improve detection sensitivity by enabling analysis of significantly larger blood
volumes (potentially the entire blood volume of a patient); however, to date, such analyses
have proven successful only when cancer cells are labeled ex vivo prior to their injection
(Georgakoudi et al. 2004, Novak et al. 2004). Although mitochondria-containing cells and
apoptotic cells have been successfully labeled in vivo for detection in the vasculature (Wei
et al. 2005, Zhong et al. 2005), no method has yet been developed for in vivo labeling and
quantitation of CTCs. An intravital flow cytometry method has been reported whereby
CTCs are counted noninvasively in vivo as they flow through the peripheral vasculature (He
et al. 2007). The method involves intravenous injection of a tumor-specific fluorescently-
labeled ligand followed by fluorescence imaging of superficial blood vessels to quantitate the
flowing CTCs. Studies in mice with metastatic tumors suggest that CTCs can be counted
weeks before metastatic disease is detected by other means. Analysis of whole blood samples
from cancer patients further established that human CTCs can be selectively labeled and
counted when present at 2 CTCs per mL, opening opportunities for earlier detection of
metastatic disease.
Integration of optical MEMS technology is desirable for in vivo imaging of CTCs by
miniaturized conventional microscopy. Current optical MEMS imaging technology is focused
on the development of ultra-portable or needle-like MEMS-based optical micro-endoscopes
for cancer or cell diagnosis in vivo. Two main modalities that have already been success-
fully realized are confocal and two-photon microscopy. Confocal microscopy is an optical
imaging technique utilizing point by point illumination and a spatial pinhole to increase
resolution and contrast of specimens. It offers several advantages over conventional optical
microscopy, including controllable depth of field, elimination of out-of-focus image, and abil-
ity to collect serial optical sections from thick specimens (up to 150 microns) to re-construct
484 力 学 进 展 44 : 201412
three-dimensional images. Two-photon microscopy allows imaging of living tissue up to one
millimeter with sub-cellular to cellular resolution. Typically, a femtosecond laser is used to
enable the high photon density and flux required for two photons absorption. These imaging
modalities have allowed microscopes to obtain high-resolution images of living, intact tis-
sues and have become useful for in vivo imaging. Miniaturization of the two modalities rely
on both micro-fabricated two-dimensional MEMS scanners and miniature optics to allow
intravital and clinical imaging in vivo. Using optical MEMS based technology, both optical
imaging modalities have been miniaturized with very high acquisition frame rate (15 Hz),
which could potentially be used for in vivo CTCs imaging (Khemthongcharoen et al. 2014).
The capability of microendoscopes has been demonstrated for in vivo real-time vasculature
imaging in mice and cancer imaging in human patients (Piyawattanametha et al. 2009,
Piyawattanametha et al. 2012).
7 Summary and future perspectives
Despite the extensive advances that have been made, there are still drawbacks in
microfluidic-based CTC analytical technology. First, there are limitations in isolation effi-
ciency as each of the current capture strategies is liable to miss a portion of the CTCs. For
example, EpCAM-based isolation cannot adapt for EpCAM-negative CTCs, and filtration-
based methods cannot trap CTCs with small sizes. To overcome these limitations, it is
tempting to combine different targeting ligands, or even different capture strategies to de-
velop a more robust CTC isolation method.
Second, CTC characterization assays with high specificity are still highly desired. It has
been pointed out that the most widely used criteria for CTC characterization, namely cells
with CK(+), CD45() and a nucleus, cannot distinguish between circulating tumor cells and
circulating epithelial non-tumor cells. Since the most malignant tumor cells most likely lose
their “epithelial-specific antigens”, during EMT, defining CTCs as cells expressing epithelial
antigens may lead to a critical interpretation bias. Therefore, it is crucial to discover unique
biomarkers for CTC characterization, which are highly specific to cancer cells.
Third, improved throughput is necessary to meet the needs for clinical applications.
In pratical healthcare environment, tens or even hundreds of samples are to be processed
every day; hence, an ideal CTC detection assay should feature short analysis time and
parallel sample processing capacity. Lastly, the cost for CTC analysis should be largely
reduced such that CTC detection can become a routine process in cancer treatment. To
Zhao Cong et al. : Isolation of circulating tumor cells 485
date, Veridex CellSearchTM is still the only FDA-approved technology for CTC detection
and analysis. The CTC working group of the Cancer Steering Committee in the Biomarkers
Consortium (www.biomarkersconsortium.org) has been working on development of standard
language for describing CTCs and of Standard Operating Procedures (SOPs) for clinical
evaluation of CTC assays prior to any testing on patient specimens (Parkinson et al. 2012).
Selected microfluidic CTC systems, such as silicon CTC chip proposed by Massachusetts
General Hospital/Harvard Medical School (On-Q-ity Inc., MA, USA), ClearCellTM System
(Clearbridge BioMedics, Singapore) have been in clinical trial. It will be expected that
some low-cost microfluidic CTC systems will replace the expensive CellSearchTM assay in
the future.
Acknowledgement This research was partially supported by Hong Kong RGC GRF grant
(16205314) and partially by the research grants from NSFC, China (81171418, 81371649).
WP is partially supported by grants from the Fraunhofer-Bessel Research Award from the
Alexander von Humboldt Foundation, Germany; the Newton Fund, British Council, UK;
the King Mongkut’s Institute of Technology Ladkrabang, Thailand; the Thailand Research
Fund, Thailand; and the National Research Council, Thailand.
References
Adams A A, Okagbare P I, Feng J, Hupert M L, Patterson D, G¨ottert J, McCarley R L, Nikitopoulos D,
Murphy M C, Soper S A. 2008. Highly efficient circulating tumor cell isolation from whole blood and
label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor. Journal
of the American Chemical Society,130: 8633-8641.
Adams C L, Nelson W J. 1998. Cytomechanics of cadherin-mediated cell-cell adhesion. Current Opinion in
Cell Biology,10: 572-577.
Allard, W J, Matera J, Miller M C, Repollet M, Connelly M C, Rao C, Tibbe A G, Uhr J W, Terstappen L
W. 2004. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects
or patients with nonmalignant diseases. Clin Cancer Res,10: 6897-6904.
Ashworth T R. 1869. A case of cancer in which cells similar to those in the tumours were seen in the blood
after death. Aust. Med. J.,14: 145-149.
Baeuerle P A, Gires O. 2007. Epcam (Cd326) finding its role in cancer. Br J Cancer,96: 417-423.
Bauer J. 1999. Advances in cell separation: Recent developments in counterflow centrifugal elutriation and
continuous flow cell separation. Journal of Chromatography B: Biomedical Sciences and Applications,
722: 55-69.
Becker F F, Wang X B, Huang Y, Pethig R, Vykoukal J, Gascoyne P R. 1995. Separation of human breast
cancer cells from blood by differential dielectric affinity. Proceedings of the National Academy of Sciences
of the United States of America,92: 860-864.
Birchmeier W, Behrens J. 1994. Cadherin expression in carcinomas: Role in the formation of cell junctions
and the prevention of invasiveness. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer,1198:
486 力 学 进 展 44 : 201412
11-26.
Blattert C, Jurischka R, Schoth A, Kerth P, Menz W. 2004. Separation of blood cells and plasma in
microchannel bend structures. In: Proceedings of 8th International Conference on Miniaturized Systems
for Chemistry and Life Sciences, Malmo, Sweden, 143-151.
Braun S, Kentenich C, Janni W, Hepp F, de Waal J, Willgeroth F, Sommer H Pantel K. 2000. Lack of effect
of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk
breast cancer patients. Journal of Clinical Oncology 18: 80.
Braun S, Pantel K, Muller P, Janni W, Hepp F, Kentenich C R, Gastroph S, Wischnik A, Dimpfl T,
Kindermann G, Riethm¨uller G, Schlimok G. 2000. Cytokeratin-positive cells in the bone marrow and
survival of patients with stage I, II, or III breast cancer. New England Journal of Medicine,342: 525-
533.
Brenner T, Haeberle S, Zengerle R, Ducree J. 2004. Continuous centrifugal separation of whole blood on a
disk. In: Proceedings of 8th International Conference on Miniaturized Systems for Chemistry and Life
Sciences, Malmo, Sweden, 566-568.
Butler T P, Gullino P M. 1975. Quantitation of cell shedding into efferent blood of mammary adenocarci-
noma. Cancer Res. 35: 512-516.
Carlson R H, Gabel C V, Chan S S, Austin R H, Brody J P, Winkelman J W. 1997. Self-sorting of white
blood cells in a lattice. Physical Review Letters,79: 2149-2152.
Chang W C, Lee L P, Liepmann D. 2005. Biomimetic technique for adhesion-based collection and separation
of cells in a microfluidic channel. Lab on a Chip,5: 64-73.
Chang Y S, di Tomaso E, McDonald D M, Jones R, Jain R K, Munn L L. 2000. Mosaic blood vessels in
tumors: Frequency of cancer cells in contact with flowing blood. PNAS,97: 14608-14613.
Chelly J, Concordet J P, Kaplan J C, Kahn A. 1989. Illegitimate transcription: Transcription of any gene
in any cell type. PNAS,86: 2617-2621.
Chen J, Li J, Sun Y. 2012. Microfluidic approaches for cancer cell detection, characterization, and separation.
Lab on a Chip,12: 1753-1767.
Cristofanilli M, Budd G T, Ellis M J, Stopeck A, Matera J, Miller M C, Reuben J M, Doyle G V, Allard
W J, Terstappen L W, Hayes D F. 2004. Circulating tumor cells, disease progression, and survival in
metastatic breast cancer. New England Journal of Medicine,351: 781-791.
Das T, Chakraborty S. 2013. Perspective: Flicking with flow: Can microfluidics revolutionize the cancer
research? Biomicrofluidics,7: 011811.
de Boer M, van Deurzen C H M, van Dijck J A A M, Borm G F, van Diest P J, Adang E M, Nortier J W,
Rutgers E J, Seynaeve C, Menke-Pluymers M B, Bult P, Tjan-Heijnen V C. 2009. Micrometastases or
isolated tumor cells and the outcome of breast cancer. New England Journal of Medicine,361: 653-663.
Dharmasiri U, Balamurugan S, Adams A A, Okagbare P I, Obubuafo A, Soper S A. 2009. Highly efficient
capture and enumeration of low abundance prostate cancer cells using prostate-specific membrane antigen
aptamers immobilized to a polymeric microfluidic device. Electrophoresis,30: 3289-3300.
Dharmasiri U, Njoroge S K, Witek M A, Adebiyi M G, Kamande J W, Hupert M L, Barany F, Soper S
A. 2011. High-throughput selection, enumeration, electrokinetic manipulation, and molecular profiling of
low-abundance circulating tumor cells using a microfluidic system. Analytical Chemistry,83: 2301-2309.
Dong Y, Skelley A M, Merdek K D, Sprott K M, Jiang C, Pierceall W E, Lin J. 2013. Microfluidics and
circulating tumor cells. The Journal of Molecular Diagnostics,15: 149-157.
Zhao Cong et al. : Isolation of circulating tumor cells 487
Du Z, Cheng K H, Vaughn M W, Collie N L, Gollahon L S. 2007. Recognition and capture of breast
cancer cells using an antibody-based platform in a microelectromechanical systems device. Biomedical
Microdevices,9: 35-42.
Fan Z H, Xu Y, Phillips J A, Yan J L, Li Q G, Tan W H. 2009. Aptamer-based microfluidic device for
enrichment, sorting, and detection of multiple cancer cells. Analytical Chemistry,81: 7436-7442.
Fehm T, Sagalowsky A, Clifford E, Beitsch P, Saboorian H, Euhus D, Meng S, Morrison L, Tucker T, Lane N,
Ghadimi BM, Heselmeyer-Haddad K, Ried T, Rao C, Uhr J. 2002. Cytogenetic evidence that circulating
epithelial cells in patients with carcinoma are malignant. Clin Cancer Res,8: 2073-2084.
Fiedler S, Shirley S G, Schnelle T, Fuhr G. 1998. Dielectrophoretic sorting of particles and cells in a
microsystem. Analytical Chemistry,70: 1909-1915.
Frixen U H, Behrens J, Sachs M, Eberle G, Voss B, Warda A, L¨ochner D, Birchmeier W. 1991. E-cadherin-
mediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J Cell Biol,113: 173-185.
Fukuda S, Schmid-Schonbein G W. 2002. Centrifugation attenuates the fluid shear response of circulating
leukocytes. Journal of Leukocyte Biology,72: 133-139.
Garrod D, Chidgey M, North A. 1996. Desmosomes: Differentiation, development, dynamics and disease.
Current Opinion in Cell Biology,8: 670-678.
Gascoyne P, Mahidol C, Ruchirawat M, Satayavivad J, Watcharasit P, Becker F. 2002. Microsample prepa-
ration by dielectrophoresis: Isolation of malaria. Lab on a Chip,2: 70-75.
Gascoyne P R C, Vykoukal J. 2002. Particle separation by dielectrophoresis. Electrophoresis,23: 1973-1983.
Gascoyne P R C, Vykoukal J V. 2004. Dielectrophoresis-based sample handling in general-purpose pro-
grammable diagnostic instruments. Proceedings of the IEEE,92: 22-42.
Georgakoudi I, Solban N, Novak J, Rice W L, Wei X, Hasan T, Lin C P. 2004. In vivo flow cytometry: A
new method for enumerating circulating cancer cells. Cancer Res,64: 5044-5047.
Goldsby R A, Kindt T J, Osborne B A, Kuby J. 2003. Immunology. New York: W.H. Freeman. A-1-A-12.
Greenburg G, Hay E D. 1982. Epithelia suspended in collagen gels can lose polarity and express character-
istics of migrating mesenchymal cells. J Cel l Biol,95: 333-339.
Griffith A W, Cooper J M. 1998. Single-cell measurements of human neutrophil activation using electroro-
tation. Analytical Chemistry,70: 2607-2612.
Hallden G, Nopp A, Ihre E, Peterson C, Lundahl J. 1999. Conditions in blood sampling procedures that
extend the ex vivo stability of eosinophil activity markers in peripheral blood from allergic patients and
healthy controls. Annals of Allergy, Asthma &Immunology,83: 413-421.
Haukanes B I, Kvam C. 1993. Application of magnetic beads in bioassays. Nat Biotech,11: 60-63.
He W, Wang H, Hartmann L C, Cheng J X, Low P S. 2007. In vivo quantitation of rare circulating tumor
cells by multiphoton intravital flow cytometry. PNAS,104: 11760-11765.
Helo P, Cronin A M, Danila D C, Wenske S, Gonzalez-Espinoza R, Anand A, Koscuiszka M, V¨an¨anen R M,
Pettersson K, Chun F K, Steuber T, Huland H, Guillonneau B D, Eastham J A, Scardino P T, Fleisher
M, Scher H I, Lilja H. 2009. Circulating prostate tumor cells detected by reverse transcription-pcr in men
with localized or castration-refractory prostate cancer: Concordance with cellsearch assay and association
with bone metastases and with survival. Clin Chem,55: 765-773.
Hirohashi S. 1998. Inactivation of the E-Cadherin-Mediated Cell Adhesion System in Human Cancers. The
American Journal of Pathology,153: 333-339.
Holcomb K, Delatorre R, Pedemonte B, McLeod C, Anderson L, Chambers J. 2002. E-cadherin expression
488 力 学 进 展 44 : 201412
in endometrioid, papillary serous, and clear cell carcinoma of the endometrium. Obstetrics &Gynecology,
100: 1290-1295.
Holmes D, Green N G, Morgan F. 2003. Microdevices for dielectrophoretic flow–through cell separation.
Engineering in Medicine and Biology Magazine, IEEE,22: 85-90.
Holmes D, Morgan H. 2003. Cell sorting and separation using dielectrophoresis. In: Electrostatics 2003.
Bristol, UK: Inst. Phys. Publ. 107-112.
Hoshino K, Huang Y Y, Lane N, Huebschman M, Uhr J W, Frenkel E P, Zhang X. 2011. Microchip-based
immunomagnetic detection of circulating tumor cells. Lab on a Chip,11: 3449-3457.
Hosokawa M, Hayata T, Fukuda Y, Arakaki A, Yoshino T, Tanaka T, Matsunaga T. 2010. Size-selective
microcavity array for rapid and efficient detection of circulating tumor cells. Analytical Chemistry,82:
6629-6635.
Huang L R, Cox E C, Austin R H, Sturm J C. 2004. Continuous particle separation through deterministic
lateral displacement. Science,304: 987-990.
Huang Y, Joo S, Duhon M, Heller M, Wallace B, Xu X. 2002. Dielectrophoretic cell separation and gene
expression profiling on microelectronic chip arrays. Analytical Chemistry,74: 3362-3371.
Hughes M P. 2002. Strategies for dielectrophoretic separation in laboratory-on-a-chip systems. Electrophore-
sis,23: 2569-2582.
Imhof B A, Vollmers H P, Goodman S L, Birchmeier W. 1983. Cell-cell interaction and polarity of epithelial
cells: Specific perturbation using a monoclonal antibody. Cell,35: 667-675.
Isselbacher K J, Stott S L, Hsu C H, Tsukrov D I, Yu M, Miyamoto D T, Waltman B A, Rothenberg S M,
Shah A M, Smas M E, Korir G K, Floyd F P Jr, Gilman A J, Lord J B, Winokur D, Springer S, Irimia D,
Nagrath S, Sequist L V, Lee R J, Isselbacher K J, Maheswaran S, Haber D A, Toner M. 2010. Isolation
of circulating tumor cells using a microvortex-generating herringbone-chip. PNAS,107: 18392-18397.
Kahn H, Presta A, Yang L Y, Blondal J, Trudeau M, Lickley L, Holloway C, McCready D R, Maclean D,
Marks A. 2004. Enumeration of circulating tumor cells in the blood of breast cancer patients after filtration
enrichment: Correlation with disease stage. Breast Cancer Research and Treatment,86: 237-247.
Kalluri R, Weinberg R A. 2009. The basics of epithelial-mesenchymal transition. The Journal of Clinical
Investigation,119: 1420-1428.
Kang J Y, Cho H, Kwak S M, Yoon D S, Kim T S. 2004. Novel particle separation using spiral channel
and centrifugal force for plasma preparation from whole blood. In: Proceedings of 8th International
Conference on Miniaturized Systems for Chemistry and Life Sciences, Malmo, Sweden, 614-616.
Khemthongcharoen N, Jolivot R, Rattanavarin S, Piyawattanametha W. 2014. Advances in imaging probes
and optical microendoscopic imaging techniques for early in vivo cancer assessment. Advanced Drug
Delivery Reviews,74: 53-74.
Kost G J, Ehrmeyer S S, Chernow B, Winkelman J W, Zaloga G P, Dellinger R P, Shirey T. 1999. The
laboratory-clinical interface: Point-of-care testing. Chest,115: 1140-1154.
Kowalczyk A P, Bornslaeger E A, Norvell S M, Palka H L, Green K J. 1998. Desmosomes: Intercellular
adhesive junctions specialized for attachment of intermediate filaments. International Review of Cytology,
185: 237-302.
Kuure S, Vuolteenaho R, Vainio S. 2000. Kidney morphogenesis: Cellular and molecular regulation. Mech-
anisms of Development,92: 31-45.
Lankiewicz S, Rivero B, Bocher O. 2006. Quantitative real-time rt-pcr of disseminated tumor cells in
combination with immunomagnetic cell enrichment. Molecular Biotechnology,34: 15-27.
Zhao Cong et al. : Isolation of circulating tumor cells 489
Lapizco-Encinas B H, Simmons B A, Cummings E B, Fintschenko Y. 2004. Dielectrophoretic concentration
and separation of live and dead bacteria in an array of insulators. Analytical Chemistry,76: 1571-1579.
Lee R J, Stott S L, Nagrath S, Ulkus L E, Dahl D M. 2009. Analyses of circulating tumor cell (ctc)
dynamics and treatment response in prostate cancer using the ctc-chip microfluidic device. Journal of
Clinical Oncology,27: 5149.
Lianidou E S, Markou A. 2011. Circulating tumor cells in breast cancer: detection systems, molecular
characterization, and future challenges. Clin Chem,57: 1242-1255.
Lundahl J, Hallden G, Hallgren M, Skold C M, Hed J. 1995. Altered expression of Cd11b/Cd18 and Cd62l
on human monocytes after cell preparation procedures. Journal of Immunological Methods,180: 93-100.
Ma W, Liu D, Shagoshtasbi H, Shukla A, Nugroho E S, Zohar Y, Lee Y K. 2013. Experimental and the-
oretical study of hydrodynamic cell lysing of cancer cells in a high-throughput circular multi-channel
microfiltration device. In: Proceedings of the 8th IEEE International Conference on Nano/Micro Engi-
neered and Molecular Systems (IEEE NEMS ’13), (Suzhou, China: IEEE.) 412-415.
Ma W, Zeng R, Liu D, Zohar Y, Lee Y K. 2014. Soi Technology-Based Microfiltration System for Circulating
Tumor Cells Isolation and Enumeration. In: Proceedings of the 9th IEEE International Conference on
Nano/Micro Engineered and Molecular Systems (IEEE NEMS ’14), (Hawaii, USA: IEEE.) 333-336.
Maheswaran S, Sequist L V, Nagrath S, Ulkus L, Brannigan B, Collura C V, Inserra E, Diederichs S, Iafrate
A J, Bell D W, Digumarthy S, Muzikansky A, Irimia D, Settleman J, Tompkins R G, Lynch T J, Toner M,
Haber D A. 2008. Detection of mutations in egfr in circulating lung-cancer cells. New England Journal
of Medicine,359: 366-377.
Mauk M G, Ziober B L, Chen Z, Thompson J A, Bau H H. 2007. Lab-on-a-chip technologies for oral-based
cancer screening and diagnostics. Annals of the New York Academy of Sciences,1098: 467-475.
Mishra A, Verma M. 2010. Cancer biomarkers: Are we ready for the prime time? Cancers,2: 190-208.
Mohamed H, McCurdy L D, Szarowski D H, Duva S, Turner J N, Caggana M. 2004. Development of a rare
cell fractionation device: Application for cancer detection. IEEE Transactions on NanoBioscience,3:
251-256.
Mohamed H, Murray M, Turner J N, Caggana M. 2009. Isolation of tumor cells using size and deformation.
Journal of Chromatography A,1216: 8289-8295.
Muller T, Schnelle T, Gradl G, Shirley S G, Fuhr G. 2000. Microdevice for cell and particle separation using
dielectrophoretic field-flow fractionation. Journal of Liquid Chromatography &Related Technologies,23:
47-59.
Nagrath S, Sequist L V, Maheswaran S, Bell D W, Irimia D, Ulkus L, Smith M R, Kwak E L, Digumarthy S,
Muzikansky A, Ryan P, Balis U J, Tompkins R G, Haber D A, Toner M. 2007. Isolation of rare circulating
tumour cells in cancer patients by microchip technology. Nature,450: 1235-1239.
Naume B, Borgen E, Kvalheim G, Karesen R, Qvist H, Sauer T, Kumar T, Nesland J M. 2001. Detection
of isolated tumor cells in bone marrow in early-stage breast carcinoma patients: Comparison with preop-
erative clinical parameters and primary tumor characteristics. Clinical Cancer Research,7: 4122-4129.
Nelson N J. 2010. Circulating tumor cells: Will they be clinically useful? Journal of the National Cancer
Institute,102: 146-148.
Neurauter A A, Bonyhadi M, Lien E, Nøleby L, Ruud E, Camacho S, Aarvak T. 2007. Cell isolation and
expansion using dynabeads. In: Cell Separation (eds). Berlin Heidelberg, Springer, 41-73.
Novaes M, Bendit I, Garicochea B, del Giglio A. 1997. Reverse transcriptase-polymerase chain reaction
analysis of cytokeratin 19 expression in the peripheral blood mononuclear cells of normal female blood
490 力 学 进 展 44 : 201412
donors. Molecular Pathology,50: 209.
Novak J, Georgakoudi I, Wei X, Prossin A, Lin C P. 204. In vivo flow cytometer for real-time detection and
quantification of circulating cells. Optical Letters,29: 77-79.
Pantel K, Alix-Panabies C. 2010. Circulating tumour cells in cancer patients: challenges and perspectives.
Trends in Molecular Medicine,16: 398-406.
Pantel K, Cote R J, Fodstad O. 1999. Detection and clinical importance of micrometastatic disease. Journal
of the National Cancer Institute,91: 1113-1124.
Park T, Jensen T, Park D, Guy J, Datta P, Soper S A, Murphy M C. 2007. Capture of very rare circulating
tumor cells for human breast cancer diagnosis. In: Proc. ASME IMECE 2007, Seattle, USA IMECE2007-
42425.
Parkinson D, Dracopoli N, Petty B, Compton C, Cristofanilli M, Deisseroth A, Hayes D F, Kapke G, Kumar
P, Lee JSh, Liu M C, McCormack R, Mikulski S, Nagahara L, Pantel K, Pearson-White S, Punnoose
E A, Roadcap L T, Schade A E, Scher H I, Sigman C C, Kelloff G J. 2012. Considerations in the
development of circulating tumor cell technology for clinical use. Journal of Translational Medicine,10:
138 (http://dx.doi.org/10.1186/1479-5876-10-138).
Paterlini-Brechot P, Benali N L. 2007. Circulating tumor cells (ctc) detection: clinical impact and future
directions. Cancer Letters,253: 180-204.
Pethig R. 1996. Dielectrophoresis: Using inhomogeneous ac electrical fields to separate and manipulate cells.
Critical Reviews in Biotechnology,16: 331-348.
Piyawattanametha W, Cocker E D, Burns L D, Barretto R P J, Jung J C, Ra H, Solgaard O, Schnitzer M
J. 2009. In vivo brain imaging using a portable 2.9 g two-photon microscope based on a microelectrome-
chanical systems scanning mirror. Optics Letters,34: 2309-2311.
Piyawattanametha W, Ra H, Qiu Z, Friedland S, Liu J T C, Loewke K, Kino G S, Solgaard O, Wang T D,
Mandella M J, Contag C H. 2012. In vivo near-infrared dual-axis confocal microendoscopy in the human
lower gastrointestinal tract. Journal of Biomedical Optics,17: 0211021:1-4.
ˇ
SafaˇıK I, ˇ
SafaˇıKov´a M. 1999. Use of magnetic techniques for the isolation of cells. Journal of Chromatog-
raphy B: Biomedical Sciences and Applications,722: 33-53.
Saliba A E, Saias L, Psychari E, Minc N, Simon D, Bidard F C, Mathiot C, Pierga J Y, Fraisier V, Salamero
J, Saada V, Farace F, Vielh P, Malaquin L, Viovy J L. 2010. Microfluidic sorting and multimodal typing
of cancer cells in self-assembled magnetic arrays. PNAS,107: 14524-14529.
Santana S M, Liu H, Bander N H, Gleghorn J P, Kirby B J. 2011. Immunocapture of prostate cancer
cells with anti-psma antibodies in microdevices. In: Proceedings of 2011 IEEE 37th Annual Northeast
Bioengineering Conference (NEBEC), (Troy, New York: IEEE.) 1-2.
Sequist L V, Nagrath S, Toner M, Haber D A, Lynch T J. 2009. The Ctc-chip: An exciting new tool to
detect circulating tumor cells in lung cancer patients. Journal of Thoracic Oncology,4: 281-283.
Shelby J P, White J, Ganesan K, Rathod P K, Chiu D T. 2003. A microfluidic model for single-cell capillary
obstruction by plasmodium falciparum-infected erythrocytes. 14618-14622.
Smerage J B, Hayes D F. 2005. The measurement and therapeutic implications of circulating tumour cells
in breast cancer. Br J Cancer,94: 8-12.
Stakenborg T, Liu C, Henry O, O’Sullivan C K, Fermer C, Roeser T, Ritzi-Lehnert M, Hauch S, Borgen
E, Laddach N, Lagae L. 2010. Lab-on-a-chip for the isolation and characterization of circulating tumor
cells. In: Proc IEEE Eng Med Biol Soc, Buenos Aires: IEEE. 292-294.
Zhao Cong et al. : Isolation of circulating tumor cells 491
Stoker M, Perryman M. 1985. An epithelial scatter factor released by embryo fibroblasts. Journal of Cell
Science,77: 209-223.
Talasaz A A H, Powell A A, Stahl P, Ronaghi M, Jeffrey S S, Mindrinos M, Davis R W. 2006. Cell trapping
in activated micropores for functional analysis. In: Proceedings of 28th Annual International Conference
of the IEEE Engineering in Medicine and Biology Society (IEEE EMBS ’06), New York, NY: IEEE.
1838-1841.
Tan S, Yobas L, Lee G, Ong C, Lim C. 2009. Microdevice for the isolation and enumeration of cancer cells
from blood. Biomedical Microdevices,11: 883-892.
Tan S J, Lakshmi R L, Chen P, Lim W T, Yobas L, Lim C T. 2010. Versatile label free biochip for the de-
tection of circulating tumor cells from peripheral blood in cancer patients. Biosensors and Bioelectronics,
26: 1701-1705.
Thiery J P. 2002b. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer,2: 442-454.
Toner M, Irimia D. 2005. Blood-on-a-chip. Annual Review of Biomedical Engineering,7: 77-103.
Vankrunkelsven S, Clicq D, Pappaert K, Ranson W, De Tandt C, Ottevaere H, Thienpont H, Baron G V,
Desmet G. 2004. A novel microstep device for the size separation of cells. Electrophoresis,25: 1714-1722.
Vilkner T, Janasek D, Manz A. 2004. Micro total analysis systems. recent developments. Analytical
Chemistry,76: 3373-3386.
Voldman J, Gray M L, Schmidt M A. 1999. Microfabrication in biology and medicine. Annual Review of
Biomedical Engineering,1: 401-425.
Voldman J, Gray M L, Toner M, Schmidt M A. 2002. A microfabrication-based dynamic array cytometer.
Analytical Chemistry,74: 3984-3990.
Vona G, Sabile A, Louha M, Sitruk V, Romana S, Scutze K, Capron F, Franco D, Pazzagli M, Vekemans M,
Lacour B, Br´echot C, Paterlini-Br´echot P. 2000. Isolation by size of epithelial tumor cells: A new method
for the immunomorphological and molecular characterization of circulating tumor cells. The American
Journal of Pathology,156: 57-63.
Wang J Y, Li L, Liu W M, Wang J C, Tu Q, Liu R, Wang J. 2010. Lectin-aided separation of circulat-
ing tumor cells and assay of their response to an anticancer drug in an integrated microfluidic device.
Electrophoresis,31: 3159-3166.
Wang N, Shi L, Li H, Hu Y, Du W, Liu W, Zheng J, Huang S, Qu X. 2012. Detection of circulating tumor
cells and tumor stem cells in patients with breast cancer by using flow cytometry. Tumor Biology,33:
561-569.
Wang S, Liu K, Liu J, Yu Z T F, Xu X, Zhao L, Lee T, Lee E K, Reiss J, Lee Y K, Chung L W, Huang
J, Rettig M, Seligson D, Duraiswamy K N, Shen C K, Tseng H R. 2011. Highly efficient capture of
circulating tumor cells by using nanostructured silicon substrates with integrated chaotic micromixers.
Angewandte Chemie International Edition,50: 3084-3088.
Wang S, Wang H, Jiao J, Chen K J, Owens G E, Kamei K, Sun J, Sherman D J, Behrenbruch C P, Wu
H, Tseng H R. 2009. Three-dimensional nanostructured substrates toward efficient capture of circulating
tumor cells. Angew Chem Int Ed Engl,48: 8970-8973.
Wei X, Sipkins D A, Lin C P. 2005. Real-time detection of circulating apoptotic cells by in vivo flow
cytometry. Mol Imaging,4: 415-416.
Went P T H, Lugli A, Meier S, Bundi M, Mirlacher M, Sauter G, Dirnhofer S. 2004. Frequent epcam protein
expression in human carcinomas. Human Pathology,35: 122-128.
492 力 学 进 展 44 : 201412
Wilding P, Kricka L J, Cheng J, Hvichia G, Shoffner M A, Fortina P. 1998. Integrated cell isolation and
polymerase chain reaction analysis using silicon microfilter chambers. Analytical Biochemistry,257:
95-100.
Willipinski-Stapelfeldt B, Riethdorf S, Assmann V, Woelfle U, Rau T, Sauter G, Heukeshoven J, Pantel K.
2005. Changes in cytoskeletal protein composition indicative of an epithelial-mesenchymal transition in
human micrometastatic and primary breast carcinoma cells. Clin Cancer Res,11: 8006-8014.
Wintrobe M M. 1980. Blood, Pure and Eloquent: A Story of Discovery, of People, and of Ideas. New York:
McGraw-Hill.
Xia J, Chen X, Zhou C Z, Li Y G, Peng Z H. 2011. Development of a low-cost magnetic microfluidic chip
for circulating tumour cell capture. IET Nanobiotechnology,5: 114-120.
Xu T, Lu B, Tai Y C, Goldkorn A. 2010. A cancer detection platform which measures telomerase activity
from live circulating tumor cells captured on a microfilter. Cancer Res,70: 6420-6.
Yamada M, Nakashima M, Seki M. 2004. Pinched flow fractionation: continuous size separation of particles
utilizing a laminar flow profile in a pinched microchannel. Analytical Chemistry,76: 5465-5471.
Yang J, Huang Y, Wang X B, Becker F F, Gascoyne P R C. 1999. Cell separation on microfabricated
electrodes using dielectrophoretic/gravitational field-flow fractionation. Analytical Chemistry,71: 911-
918.
Yang J, Huang Y, Wang X B, Becker F F, Gascoyne P R C. 2000. Differential analysis of human leukocytes
by dielectrophoretic field-flow-fractionation. Biophysical Journal,78: 2680-2689.
Yang X, Hibara A, Sato K, Tokeshi M, Morishima K, Kikutani Y, Kimura H, Kitamori T. 2004. Inter-
channel microstructure for separation and analyses of plasma from whole blood. In: Proceedings of
8th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Malmo, Sweden,
120-122.
Yoshida R, Kimura N, Harada Y, Ohuchi N. 2001. The loss of e-cadherin, A- and B-catenin expression
is associated with metastasis and poor prognosis in invasive breast cancer. International Journal of
Oncology 18: 513-520.
Zheng S, Lin H K, Lu B, Williams A, Datar R, Cote R J, Tai Y C. 2011. 3d microfilter device for viable
circulating tumor cell (Ctc) enrichment from blood. Biomedical Microdevices,13: 203-213.
Zhong C F, Thomas T, Ye J, Cao Z, Myc A, et al. 2005. Multiphoton in Vivo Flow Cytometry. In:
Proceedings of Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science and
Photonic Applications Systems Technologies, Baltimore, Maryland: Optical Society of America. 2145-
2147.
Zieglschmid V, Hollmann C, Bocher O. 2005. Detection of disseminated tumor cells in peripheral blood.
Critical Reviews in Clinical Laboratory Sciences,42: 155-196.
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Zhao Cong et al. : Isolation of circulating tumor cells 493
基于微流控技术在流体动力载荷下分离循环肿瘤细胞
1李贻昆 1,2, 1 3刘大渔 4
2PIYAWATTANAMETHA Wibool5,6ZOHAR Yitshak7
1香港科技大学生物医学工程学部,香港
2香港科技大学机械及航空航天工程系,香港
3香港科技大学生命科学部,香港
4广州市第一人民医院,广州 510180
5Department of Electronics, King Mongkut’s Institute of Technology, Bangkok, Thailand
6Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
7Department of Aerospace and Mechanical Engineering,
University of Arizona, Tucson, AZ, USA
摘 要 在世界范围内,癌症的死亡率仍在逐年上升.循环肿瘤细胞 (circulating tumor
cells, CTCs) 是指从原发肿瘤脱落并进入血液循环系统的细胞,可能引发肿瘤转移并
入侵其他正常组织和器官.因此, CTCs 的检测结果可以作为癌症病人疗效和预后的
评价指标.CTCs 的数量及其稀少,使得 CTCs 的检测尤为困难.在癌症转移的病
人中,每毫升血液约含有 10100 CTCs. 利用经生物活性材料表面修饰的微流控器
,可以从血中分离出 CTCs. 是一跨学科的挑战,要来 不同学科景的
家们共同参与,如细胞生物学、表面化学、流体力学及微纳加工技术等.该文首先介
绍了 CTCs 胞生学基,然后总结了当前分离 CTCs 主要流控,包括
基于 细胞 -- 配体作用、磁力作用和过滤等,最后综述了基于微流控技术的 CTC 检测
计数 CTC 成像等最新研究进展.
关键词 循环肿瘤细胞, CTC, 微流控,,微系统,微机电系统,上皮间质转化,
转移
收稿日期: 2014-05-20; 接收日期: 2014-10-08; 在线出版日期: 2014-10-20
E-mail: meyklee@ust.hk
引用方: Zhao C, Lee Y-K, Xu R, Liang C, Liu D Y, Ma W, Piyawattanametha W, Zohar Y. Isolation
of circulating tumor cells under hydrodynamic loading using microfluidic technology. 力学进,
2014, 44: 201412.
c
《力 学进 版权 所有
494 力 学 进 展 44 : 201412
李贻,, 1970 年生. 1992 1995 年先后获得台湾大学学士及应用力学
硕士 学位. 2001 年在加州大学洛杉矶分校 (UCLA) 械工 系取 得微
系统 (MEMS) 士学,师从美国工程院何志明院士. 2001 年任香港科技大
学助理教授, 2007 年升机械工程副教授至今, 2007 年任香港科大集成微系统
研究所副所长至今. 2008 任香港科大生物工程暨生物医疗仪器中心副主任,
2011 年美国加州理工学院 (Caltech) 客座副教授, 2011 年并聘为香港科大生
物医学工程学部副教授至今. 2014 3任香港力学学会会.主要研究
向为生物微纳米机电系统及微纳米流控芯片技术.
... Circulating tumor cells (CTCs) are tumor cells that are shed from in situ tumor lesions and enter the peripheral blood circulation, which is a necessary prerequisite for tumor metastasis. However, the number of CTCs in peripheral blood is extremely low: In 10 mL of whole blood, there are approximately 80 billion red blood cells (RBCs), approximately 500 million white blood cells (WBCs), and only 1-100 CTCs [5], which poses a great challenge to the capture and counting technologies. Because the size of the microfluidic channel of the microfluidic chip is of the micrometer scale, and fluid at the micrometer scale has a unique laminar flow effect, which is conducive to the sorting of cells in the microfluid, this technology has great development potential and wide application prospects in the detection of CTCs. ...
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