Multiscale immunomagnetic enrichment of circulating tumor cells: from tubes to microchips.

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Multiscale immunomagnetic enrichment of circulating tumor cells: from tubes to microchips Cite this: DOI: 10.1039/c3lc51107c

Peng Chen, Yu-Yen Huang,† Kazunori Hoshino and Xiaojing Zhang* We review the rare cancer cell sorting technologies, with a focus on multiscale immunomagnetic approaches. Starting from the conventional magnetic activated cell sorting system, we derive the scaling Received 30th September 2013, Accepted 12th November 2013 DOI: 10.1039/c3lc51107c www.rsc.org/loc

laws of immunomagnetic assay and justify the recent trend of using downscaled systems for CTC studies. Furthermore, we introduce recent work on combining the immunomagnetic assay with microfluidic technology for enhanced separation. We summarize different types of in-channel micro-magnetic structures that can further increase the local magnetic field without lowering the system throughput. Related design concepts, principles, and microfabrication techniques are presented and evaluated.

Introduction Metastasis, which is the major cause of carcinoma-related death in cancer patients, occurs often through blood circulation and the lymphatic system. During this process, cancer cells spread from a primary tumor site to distant organs, which leads to the development of another solid tumor. In 1889, the English surgeon Stephen Paget introduced the “seed and soil” theory to describe metastasis, in which cancer

Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas, USA. E-mail: [email protected] † The authors contributed equally to this work.

Peng Chen received his B.S. degree in Biomedical Engineering from Zhejiang University, Hangzhou, China, in 2010. As an undergraduate student, he worked on the development of LAPS based biosensors for taste cell characterization. He is currently a Ph.D. student studying in the Department of Biomedical Engineering at the University of Texas at Austin. His current research includes theoretical modeling Peng Chen and multi-physical optimization of the microchip based immunomagnetic assay for rare circulating tumor cell detection.

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cells were considered to be the seed, while the organ microenvironments were considered to be the soil.1–4 Circulating Tumor Cells (CTCs) are cells that have escaped from the primary tumor site and now circulate in the peripheral bloodstream. Viable CTCs cause the carcinoma metastasis through a series of steps, namely local invasion, intravasation, circulation, arrest, extravasation, proliferation, and angiogenesis.5 The presence of CTCs is believed to be an important indicator of carcinoma progression and metastasis. Accurate counting of CTCs contributes to cancer diagnosis, prognosis, and therapeutic response monitoring.6,7 Due to the natural rareness of CTCs in the peripheral blood sample, only 1–100 CTCs can be found from 1 mL of whole blood, which usually contains one billion normal blood cells.8,9 Enriching, characterizing,

Yu-Yen Huang received his B.S. degree in Mechanical Engineering from the National Chung Cheng University, Chia-yi, Taiwan, in 2003, and his M.S. degree in Engineering Science from the National Cheng Kung University, Tainan, Taiwan, in 2005. He is currently a Ph.D. student in the Department of Biomedical Engineering, The University of Texas at Austin, TX, USA. His main research is focused Yu-Yen Huang on the development of the immunomagnetic microfluidic chip based detection system for rare cancer cell study. He has more than 10 years' experience in the design and fabrication of micro-electro-mechanical system (MEMS) technology for optical and biological applications.

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and analyzing CTCs in such low concentrations is challenging. Therefore, an effective CTC detection system with high sensitivity, high throughput, high purity, and low cost is needed for advancing biological and clinical cancer studies. In the past decade, emerging CTC separation technologies have attracted significant attention from the scientific community and biotechnology industry. Various mechanisms have been proposed and successfully demonstrated, as summarized in previous reviews.10–12 Based on the separation principles, these methods can be categorized as (1) separation based on physical parameters (sizes and density gradients,13–18 dielectrophoretic separation,19,20 and hydrodynamic separation)21–24 and (2) affinity mediated separation (immunoassay25,26 and immunomagnetic separation).7,27–30 Separation based on physical parameters distinguishes CTCs from normal blood cells by relying on one or more intrinsic properties of the CTCs (such as size, density, deformability, and electrical charges) without external labels. The size filtration method works under the assumption that carcinoma cells are generally larger than normal blood cells.31–33 Density gradient centrifugation for the enrichment of cells is based on the differences in cellular buoyant density.34,35 Other techniques based on cell deformability can provide additional information for cell separation.15,16 Although filter based approaches are not limited by cell types, these techniques cannot differentiate between cancer cells and normal blood cells that are physically similar. When passing through filtration systems, CTCs may potentially become damaged or lost due to the increased shear stress. Additionally, low pore density and pore fusion of filter structures are other disadvantages that fundamentally limit the system throughput.8,36,37 A promising alternative to the filtration systems is to separate cells in a streamline by manipulating the hydrodynamic forces acting on cells. Cells with different sizes or shapes

behave differently in the streamline and can be sorted accordingly.21 The continuous flow deterministic array is one of the earliest methods used to separate blood components by hydrodynamic size.23 In asymmetric pinched flow fractionation, cells with different sizes and shapes move along different streamlines and enter different collection outlets.38 When micro-scale vortices are applied to separate CTCs using size as the biomarker, target cells with larger diameters migrate to and are enriched in the expansion–contraction trapping reservoirs.22,39 Spiral microchannels focus larger CTCs against the inner wall of a curvilinear chamber and use the inherent Dean vortex flow to collect the CTCs in the inner outlet.40,41 A stream based separation operates with high flow rates and does not suffer from an increased shear stress because no physical filtration is present. However, to keep the carrier liquid consistent in properties such as viscosity, the sample usually needs to be diluted appreciably before being screened, which limits the separation efficiency. Electrical properties of cells can also be used to perform separation. When an external alternating current electric field is applied, cells are polarized by the field and affected by the applied dielectrophoretic (DEP) force. The magnitude of the applied DEP force is dependent on the electric field, the fluid medium, dielectric, and density of the cells.42 CTCs and normal blood are increased to different heights and are transported with different velocities in a parabolic flow profile. Blood cells move faster near the center, whereas CTCs move slower near the edge, and become separated thereby.19,43 DEP systems are appealing because a large amount of blood sample can be treated in a flow-through device without labeling, and target cells can be collected for further analysis. However, to increase the separation resolution, a large chamber size is preferred, and the DEP system requires an external power supply, both of which lower the portability of the system.

Dr. Kazunori Hoshino obtained his Ph.D. from the University of Tokyo in 2000. He worked for the University of Tokyo from 2003 to 2006 as a lecturer in the Department of MechanoInformatics. In 2006, he joined the University of Texas at Austin, where he currently works as a Senior Research Associate in the Department of Biomedical Engineering. His research interests include Dr. Kazunori Hoshino (1) nano/micro-electro-mechanical systems based detection and analysis of cancer cells, and (2) nano/microscale mechanical sensing and optical imaging. He has more than 100 peer reviewed publications and is the inventor of 6 US patents and 12 Japanese patents.

Dr. Xiaojing (John) Zhang is an Associate Professor at the University of Texas of Austin in the Department of Biomedical Engineering. He received his Ph.D. from Stanford University and was a Research Scientist at Massachusetts Institute of Technology (MIT). Zhang's research focuses on exploring bio-inspired nanomaterials, scale-dependent biophysics, and nanofabrication technology, towards developing Dr. John X. J. Zhang new diagnostic devices and methods on quantitative biological measurements critical to understand development and diseases. Dr. Zhang is a recipient of the Wallace H. Coulter Foundation Early Career Award in Biomedical Engineering, the British Council Early Career RXP Award, NSF CAREER Award, and DARPA Young Faculty Award.

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Other than the physical parameters, CTCs can also be differentiated from normal blood cells based on the surface markers. In affinity mediated immunoassays, affinity ligands provide direct capture force and usually display better selectivity than physical separation due to the nature of antibody– antigen interactions. Affinity immunoassay is first demonstrated by coating the three-dimensional micro-post structures with anti-EpCAM antibodies.25 Afterwards, a number of devices with carefully engineered three-dimensional structures are introduced to help increase the separation efficiency by taking advantage of the enhanced surface to volume ratio.26,44 However, the affinity mediated immunoassay is limited by the relatively slow transport of target cells to the capture surface. Besides, selecting the appropriate shear stress that facilitates reactions between CTCs and surface molecules, dissociates the non-specific bonds and removes non-target cells is difficult. To address these challenges, a fluid-permeable nanoporous membrane has been recently integrated to the immunoassay to promote surface reactions and reduce nontarget fouling.45,46 Meanwhile, affinity ligands can be used solely as labels in mechanisms such as fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS). FACS has been widely used in past decades, and the method sorts a heterogeneous mixture of cells into multiple containers based on fluorescent signals.47 Recent developments in nanotechnology have enabled miniaturized FACS systems based on microfluidics and pulsed lasers.48,49 However, FACS is usually designed to sort cells with large sub-populations and lacks the sensitivity to separate cells as rare as CTCs. In a MACS system, magnetic force is applied to separate CTCs labeled with magnetic tags.50 Magnetic tags, such as magnetic microbeads or nanoparticles, are usually conjugated specifically to cancer cells through antibody–antigen binding without affecting normal blood cells. Depending on the desired field intensity, either permanent magnets29 or electromagnets51 are used to provide the magnetic field. Compared with other CTC enrichment methodologies, an immunomagnetic assay has several advantages that make it especially suitable for rare CTC separation. (a) Selectivity: similar to affinity mediated immunoassays, immunomagnetic assays have good sensitivity that arises from antibody–antigen binding. Currently, many immunoassays are based on the over-expression of an epithelial cell adhesion molecule (EpCAM), a common biomarker found on epithelium-derived cancer cells. However, EpCAM may be down-regulated during metastasis. Increased separation sensitivity can be achieved by expanding the type of antigens that can distinguish CTCs and by using magnetic tags labeled with multiple antibodies. As a recent example, we have successfully demonstrated the capture of A-431 (skin cancer cell) using EpCAM/EGFR, SK-BR-3 (breast cancer cell) using EpCAM/HER2, and BT-20 (breast cancer cell) using EpCAM/MUC1.52 (b) Specificity: using magnetic force as the retaining force, an immunomagnetic assay increases the contrast between


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target and non-target cells in terms of the surface attachment. High shear stress can be applied in the flushing steps to remove the non-target cells. (c) Throughput: unlike an affinity mediated immunoassay where direct contact between CTCs and surface molecules is essential for successful capture, an immunomagnetic assay can attract cells over a broader spatial domain. Larger separation chamber space and higher flow rates (up to tens of ml h−1)29 can be adopted without sacrificing the separation performance. (d) Tunability: compared to the fixed filtration structure or the surface molecule immobilization, the magnetic field can be easily and accurately modulated, especially when an electromagnet is used as the magnetic field source. The field intensity and distribution can be adjusted based on the cell types and the magnetic tag properties. (e) Integration: a magnetic field can be introduced without direct contact with cells within the immunomagnetic assay, and the platform can be integrated seamlessly with other separation methods. It has been demonstrated that integrating the immunomagnetic assay with deterministic flow and inertial focusing in series can notably increase the separation efficiency by removing red blood cells (RBCs) and platelets in advance.53

Macroscopic magnetic activated separation system Various types of immunomagnetic assays for cell separation have been invented over the past decades. In conventional magnetic-activated cell isolation systems, samples are stored in conical tubes and the screening/flushing steps are usually done manually. Fig. 1(a) shows the principle of a MACS system. Cells labeled with supermagnetic microbeads are attracted to the tube wall and unlabeled cells are eluted. Once the external magnetic field is removed, the labeled cells can be released.27 Bulky permanent magnets provide the magnetic field and are arranged either as dipole or quadrupole separators. Dipole separators drive cells across the streamlines with a constant magnetostatic energy gradient, while quadrupole separators deflect and capture cells in the radical direction.54 Commercial magnetic-activated cell sorting (MACS) systems have been developed (such as Manual MACS by Miltenyi Biotec), in which a high magnetic field gradient is generated in a column tube. Separating rare cell samples with a conventional MACS system is usually a time-consuming process with low throughput because the system is manually operated and the magnetic field intensity is limited. Recently, a new magnetic cell sorting system design called the MagSweeper technology was reported. In the system, magnetic rods covered with plastic sheaths are swept through the well to magnetically attract microbead-labeled target cells.28,55 Fig. 1(b) shows the operation principles of the MagSweeper system. The diluted blood samples are pre-labeled with magnetic particles, and the samples are loaded into the capture wells. Sheath-covered magnetic rods are swept a few millimeters above the bottom of the wells.

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Fig. 1 Major macroscopic cell separation technologies using magnetic force. (a) MACS system. Cells labeled with superparamagnetic beads (black dots) are attracted to the tube wall by the external magnetic field, while unlabeled cells (white dots) are eluted. The attracted cells can be released once the magnets are removed. (b) Operational schematic of the MagSweeper system used to isolate rare CTCs. Magnetic rods covered with plastic sheaths are swept through the well to magnetically attract microbead-labeled target cells. (c) Work flow of the FDA-proved CTC detection system – CellSearch. Reproduced from ref. 27, 55 and 8 with permissions from Wiley and Elsevier.

Loosely bound contaminating cells are removed, while the sheathed rods are washed. An external magnetic field is applied to facilitate the release of labeled cells and excessive magnetic particles. Using multiple rods, the system can process different samples at the same time. However, during the screening process, sheaths considerably reduce the magnetic force on the magnetic rods and may lower the capture efficiency. CellSearch™ is a commercial system used to detect CTCs and is based on the MACS method. CellSearch™ is among the few systems that have been approved by the U.S. Food and Drug Administration (FDA) for clinical diagnostics of breast, colorectal, and lung cancers. The system consists of a CellSave™ preservative tube (Immunicon, Huntingdon Valley, PA) for sample collection, preservation, and transportation. A CellSearch™ profile kit (Veridex) containing ferrofluid nanoparticles and a capture enhancement reagent is used for the screening test. Fig. 1(c) illustrates the working process of the CellSearch™ system.8 CTCs labeled with ferrofluids conjugated with EpCAM are isolated from unlabeled blood cells. A semi-automated microscope is utilized for sample scanning

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and data acquisition (CellSpotter Analyzer™, Veridex).56,57 There have been a number of studies carried out using CellSearch™ to study the correlation between the CTC count level and the survival rates of cancer patients.58–64 The limitations of the CellSearch™ system include the fixed target assay designed only for EpCAM surface marker expression which may lead to low capture efficiency in many clinical cases. Besides, instead of collecting captured cells, the system re-suspends the captured cells in the original screening solution after fluorescence imaging. It is difficult for the suspended cells to be retrieved on slides for further analyses. Conventional magnetic activated cell sorting systems play significant roles in cancer biology and clinical studies, as revealed by the high level of interest and research activities in CTCs. The field has been expanding remarkably fast in the past few years, and the enrichment techniques are improving.65,66 At present, the conventional MACS system cannot provide the performance needed for sophisticated molecular studies. In addition, the bulky size and low portability restrict their applications for point-of-care medical systems. New technology is needed to provide more control of


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the magnetic and hydrodynamic forces acting on the target cells to improve separation.

Scaling law of immunomagnetic separation The physical principles of immunomagnetic assays evolve over the length-scale. To elucidate key design parameters, we discuss the scaling laws of the immunomagnetic assay. Considering a virtual immunomagnetic capture region as illustrated in Fig. 2, the motion of target cells in a flow environment, under an external magnetic field, is affected by the magnetic force (Fmag), hydrodynamic drag force (Fdrag), gravitational force (G), and buoyancy force (Fbuo). According to our previous estimation based on the properties of the nanoparticles and the labeling efficiency, the magnetic force (on the order of 10−10 N) is nearly 100 times larger than the gravitational force (on the order of 10−12 N) on the CTCs.67 Therefore, to simplify the calculation, we only consider Fmag and Fdrag. More specifically, Fdrag is closely related to the flow field inside the capture region, which is determined by the flow rate (FR) and the dimensions of the capture region, including length (L), width (W) and height (H), whereas Fmag is dependent on the magnetic field generated by the magnetic flux source (B0), the thickness of the substrate (S), the magnetic susceptibility of the magnetic tags (Δχp), and the labeling efficiency which is represented as the number of tags on each cell (N). Biological properties of the screening samples and other parameters may also influence the system, but they are beyond the scope of the discussions here. If the dimensions of the permanent magnets are much greater than the capture region, the magnetic field within the

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capture region falls off inversely with the square of the distance to the magnet (y) and can be represented as: B y 

B0

 S  y 2

0  y  H 

(1)

The average time (τ) a cell travels inside the capture region can be estimated using the flow rate and the dimensions of the region: W H L FR



(2)

The magnetic force acting on a nanoparticle (Fp) is dependent on the magnetic dipole m of the particles and the magnetic field B, given by the equation:68 Fp = (m · ∇)B

(3)

The total magnetic moment of a nanoparticle can be expressed using: m

Vp  p

0

B

(4)

Here, Vp is the volume of a single nanoparticle, μ0 = 4π × 10−7 T m A−1 is the magnetic permeability of vacuum. We assume ∇ × B = 0, and the magnetic force on a magnetic nanoparticle can be simplified to: Fp 

Vp  p 2 0

B 2

(5)

Eventually, the magnetic force applied to a nanoparticlelabeled cell (Fc) is simply the summation of the forces from all the nanoparticles. Fc = N × Fp

(6)

When the magnetic force drives cells along the laminar flow inside the capture region, we assume a quasi-static motion, equating Stokes' drag force (Fdrag = 6πηRΔν) to the magnetic force. Therefore, the instant relative velocity of the cells can be calculated as v 

Rc 2  c B 2 9 0

(7)

where Rc is the radius of the cell, η is the viscosity of the medium, and Δχc is the effective magnetic susceptibility of the cell, represented as Fig. 2 Physical model and scaling laws of the immunomagnetic cell separation system. Cell motion is affected by the magnetic force, gravitational force, buoyancy force and hydrodynamic force. However, the magnetic force is approximately 2 orders of magnitude larger than the gravitational force, hence dominating the movements of the cells. Reducing the height (H) or increasing the cross-section area (W × L) of the capture region can help improve the immunomagnetic separation efficiency, as shown in eqn (10).


 c  N

Rp3 Rc3

 p

(8)

Here, Rp is the radius of the nanoparticle. Therefore, the average velocity of cells inside the channel can be calculated by averaging across the height of the channel

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vave 

1 Rc 2  c H 9 0



H

0

 B  x  dx 2

(9)

To determine the field strength required for a successful capture, we equate the vertical distance moved by the cells to the height of the channel (τ · νave = H), yielding B0 

9 0 H  FR 1   Rc2  c W  L  S  H 4  S 4

(10)

Enhancing the magnetic force by increasing the magnetic field intensity can improve the cell capture rate. For other parts of the immunomagnetic system, we make the assessment based on the required minimum magnetic field. The lower this value, the easier the cells are captured. There are several ways to reduce this value, such as decreasing the screening flow rate (FR) or using stronger magnetic tags and increasing the labeling efficiency. However, from the perspective of scale, the immunomagnetic separation system can be optimized by decreasing the height (H) of the capture region and increasing the cross-section area (W × L) of the device. The result indicates that using miniaturized immunomagnetic devices may be critical to increase CTC detection efficiency.

Microchip based immunomagnetic assay With the recent advancement of nanotechnologies and microfabrication techniques, researchers are able to make miniaturized tools to observe, measure and manipulate extremely small objects. For rare cell detections using microfluidics, such microsystems provide precise control of the flow behavior, transportation, and biological interactions in the microchannel environment. Integration of microchip technology with immunomagnetic assay has been well pursued for separation of rare cells. Similar to the conventional MACS system, a microchip based immunomagnetic assay uses magnetic beads/particles that are conjugated with cancer specific antibodies to label target cells and uses an external magnetic field for capturing. Depending on the direction of the magnetic field and the final status of the target cells, the microchip based immunomagnetic assays work either in a retaining mode, where CTCs are captured and fixed on the substrate,29 or in a deflection mode, where CTCs are magnetically driven to different streamlines to be collected at different outlets as cell suspension.51,69 There are also hybrid microchannels being reported that consist of multiple functionalities or separating mechanisms integrated on the same chip.53,70 Early work on combining microfluidic and immunomagnetic assays to study cells in the retaining mode began with analytical magnetapheresis, which was proposed to compare magnetic properties of iron-rich protein (ferritin)-labeled human lymphocyte and magnetite-doped dynabeads.68 Fig. 3a shows the

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setup of the analytical magnetapheresis. The samples are stored in a syringe before they are pumped, at a controlled flow rate, into a carrier medium-filled microchannel, which is placed over permanent magnets. Magnetized cells and dynabeads are magnetically attracted to the interpolar gap of the magnets. Cell samples fixed and stained on the slides can be used for microscopic analysis. Magnetic susceptibility of the ferritin-labeled lymphocyte and the necessary number of ferritin molecules per lymphocyte for the magnetic separation are measured and calculated using the developed analytical magnetapheresis. Recent work on a microchip-based immunomagnetic separation system in retaining mode has been successfully demonstrated with CTCs (Fig. 3b).30 The automated system yields high capture efficiency and high throughput with samples from breast, prostate, and lung cancer patients. CTCs are labeled with ferrofluids, which are conjugated with antiEpCAM. During the screening process, nanoparticle-labeled CTCs suspended in whole blood sample are pumped into the inverted microchip and are captured on the channel substrate. Other cells such as red blood cells (RBCs) and white blood cells (WBCs) escaping the magnetic entrapment are collected in the waste syringe. The CTCs are permanently fixed on the channel substrate for immunofluorescence staining and microscopic observation/identification. Clusters of CTCs, which might be clinically important, are found in patient samples using this system. The system takes advantage of the cell sedimentation and a motion-control program to keep the system working in the optimum inverted orientation. The deflection channel is also a popular design to collect rare CTCs. A microchip system is developed based on continuous-flow ferrofluid hydrodynamics to sort a mixture of particles and live cells simultaneously.71 Fig. 3c shows the sorting device with a stack of permanent magnets placed close to a microfluidic channel on the side face to sort different sizes of cells based on ferrohydrodynamics. The deflection system manipulates cells within the ferrofluid suspension under the external magnetic fields in the latter part of the channel. Escherichia coli (strain MG1655), Saccharomyces cerevisiae (Baker's yeast), and two different sizes of fluorescent polystyrene microparticles are tested for the sorting experiments. The device shows high throughput (107 cells h−1) and high capture efficiency (~100%). However, the device needs to be further optimized in terms of separation resolution for target cells with smaller differences for clinical applications. Viable CTCs can be retrieved using an immunomagnetic assay for cell culture. Integration of microstructures with a microfluidic magnetic separation device is developed to isolate CTCs from mammary cancer-bearing mice suspended in whole blood sample, followed by the culturing of isolated CTCs.70 Fig. 3d shows the device composed of a tilt inlet channel at a certain angle in a main microfluidic channel with two rows of dead-end side chambers to store attracted CTCs and protect them from being damaged by shear stress. A permanent magnet is placed directly beneath the lower


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Fig. 3 Microchip based immunomagnetic assay for cell isolation purposes. (a) Setup of the analytical magnetapheresis system, which is used to study human lymphocyte. Cells are trapped at the inter-polar gap of the magnets. (b) Microchip based immunomagnetic CTC screening device in retaining mode. Permanent magnets are placed beneath the microchannel to capture and retain the cells on the substrate. (c) Microchip based immunomagnetic CTC separation system in deflecting mode. Permanent magnets are placed to the side-wall of the microfluidic channel to sort different cell lines into different streamlines and are collected at the end of the channel. (d) Immunomagnetic microchip integrated with a cell storage chamber to retrieve and culture CTCs. (e) Hybrid immunomagnetic microchip for CTC detection, including hydrodynamic cell sorting, inertial focusing, and magnetophoresis separation. RBCs, platelets and other blood components are removed in advance. Reproduced from ref. 68, 30, 71, 70, and 53 with permissions from ACS, Springer, RSC and AAAS.

row of side chambers to attract and collect magnetic beadbinded CTCs. The device displays good isolation efficiency (87%) using spiked mouse metastatic M6C breast cancer cells. Collected CTCs along with RBCs are cultured for molecular analysis (RT-PCR) after the RBC lysis. The developed isolation platform can be scaled up to process large sample


volumes, multiple types of CTCs, and other molecules for clinical applications. Immunomagnetic assays can be assembled with other separating mechanisms to achieve better performance. A microfluidic chip (CTC-iChip) containing three separation stages, namely debulking, inertial focusing, and immunomagnetic

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separation, is fabricated for CTC detection.53 The system (Fig. 3e) incorporates three microfluidic functions to replace bulk RBC lysis and centrifugation, hydrodynamic sheath flow in flow cytometry, and magnetic-activated cell sorting. Hydrodynamic size-based filtration is performed in the first stage using an array of micropillar structures, in which RBCs, platelets, plasma proteins, free magnetic beads, and other blood components are discarded through the top outlet. The remaining CTCs and WBCs are then flowed to the second stage for inertial focusing before going to the third stage where immunomagnetic separation is performed. The developed CTC-iChip is capable of immunomagnetically sorting epithelial and non-epithelial cancer cells in both negative and positive modes. The captured cells are then used for RNA-based single-cell molecular analysis. The microfluidic device exhibits high capture efficiency for different human cancer cell lines expressing different levels of EpCAM. From conventional MACS to microchip based immunomagnetic assay, the downscaled systems provide better confinement of the flow field and magnetic field. Using a miniaturized microchip with a short vertical height and a large cross-sectional area helps the magnetic capture. However, the miniaturization is limited by our need to avoid cell clogging and a desire to maintain laminar flow inside the channel. In addition, the channel has to be made large enough to keep a substantial system throughput.

Approaches based on micromagnets To enhance the separation efficiency while maintaining the throughput, researchers have come up with different approaches, including the local magnetic field control by integrating small-scale magnets inside the microchannel. This is to precisely modulate the magnetic field gradient and facilitate cell distribution after capture. The micro- and nanoscale magnets (to simplify the notation, the term “micromagnets” is used later in the paper to represent all the micro/nanoscale magnetic structures) are engineered to generate a periodic, strong localized magnetic field. The array of magnetic elements inside the immunomagnetic channel enhances the interactions between the cells and magnetic field.29,30,67 Integration of these micro-flux sources opens up a new way to optimize CTC separation without sacrificing the system throughput. Micromagnetic field generation There are two ways to induce the localized micro-magnetic field: using micro-electromagnets72,73 or static micromagnets.74–76 For chip-scale implementations, the micro-electromagnets often involve the fabrication of 3-D micro-coils on the planar substrate inside the microfluidic channel77 and the usage of an additional current source to actuate the coil and generate magnetic field. Using electromagnets, the intensity and distribution of the magnetic field can be easily tailored by controlling the actuation current. However, the external power source

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hinders the portability of the entire system. Strength of the micro-electromagnet is limited due to the relatively weak magnetic field and heating issues. In practice, the static magnetic approach is more widely adopted due to its simplicity in implementation. In recent studies, soft magnetic materials are directly integrated onto the substrate of the microchannel using conventional semiconductor fabrication techniques such as photolithography and deposition.74–76 Upon application of an external magnetic field, these static micromagnets can be easily magnetized and can generate a localized magnetic field that is 100 times stronger than those magnetic fields produced by electromagnets. Using strong permanent magnets (like the NdFeB magnet) as the magnetization field source, the static magnetic approach can be made compact. Static micromagnets exhibit great potential for serving as a portable diagnostic system and will be the main scope of our discussions below. Micromagnet fabrications Recent micromagnet fabrication technologies presented in the literatures include 1) semiconductor fabrication techniques, which usually consist of photo-patterning using a photoresist to define the locations of the micromagnets. The micromagnet elements are integrated through techniques like sputtering, thermal deposition or electroplating, depending on the required size, thickness and resolution.72–76,78–80 2) Shrink induced micromagnets, in which Ni is deposited onto shape memory polymer films. Upon heating, the polymer film shrinks in lateral dimension, causing the Ni film to buckle and wrinkle and producing the micromagnets.81 3) Thermomagnetically patterned micromagnets, which use heat irradiation through a mask to selectively switch the magnetization direction of a thin film magnet and form an array of oppositely magnetized micromagnets.82,83 4) Ferromagnetic material encapsulation, in which PDMS is used to encapsulate ferroferric oxide powder into the master template made with SU-8. A micromagnet array is formed upon PDMS demoulding from the master template.84 Given the scales of these fabricated micromagnets, they can be easily integrated within microfluidic channels to fulfill target sorting, focusing, and isolating functions. The targets are usually magnetic microbeads, nanoparticles or cells labeled with magnetic tags.74,78,83,85 To choose the proper technique for immunomagnetic cell isolation purposes, we should consider various factors such as material costs, fabrication equipment availability, reproducibility, and large scale medical/clinical applications. Micromagnet-based designs and applications For chip-scale immunomagnetic cell separation, micromagnet structures are particularly important in the sense that they affect the magnitude and distribution of the local magnetic field, which directly determines the surface retaining force. For example, for small targets with weak magnetic response, large micromagnet elements are needed to provide sufficient


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attracting and retaining force. Therefore, it is essential to adapt the structures for specific applications, depending on the size, magnetic properties of the target, dimensions of the microchannels, and flow rates. As illustrated in Fig. 4, the demonstrated structures include (1) micro-strips: the Ni strip is sputtered onto the substrate to alter the directions of cells with different magnetic labeling. Leukocytes are successfully separated and collected at the end of the microchannel.75,78,85,86 (2) Micropillars: ferromagnetic post structures are integrated inside the microchannel and can be used to capture targets moving past them in the flow. The system is demonstrated by separating magnetic micro-beads from non-magnetic beads.84,87–90 (3) Micro-grooves: the groove structure is formed using the shrinking mechanism and aligned randomly on the substrate. The system can work either in positive extraction mode, where targets are directly captured inside the channel, or in negative collection mode, where non-targets are retained. DNA can be collected from a small amount of solution for profiling purposes.81 (4) Micro-chessboard: fabricated using heat irradiation, the resolution and alignment can be well controlled in an alternating pattern. Similarly, the system has only been demonstrated to be capable of separating mixed

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solutions of magnetic and non-magnetic microparticles.82,83,91 (5) Patterned thin-film: compared with previous structures, a patterned thin film has been specially designed for and successfully applied for CTC separation. The thin film micromagnet, with a thickness of 200 nm, can generate sufficient magnetic force to fix the CTCs in a certain range. The patterned array not only helped increase the capture rate, but also distributed the cells to make full usage of the space inside the channel.74,92 To combine the micromagnet with the immunomagnet CTC detection assay, the fundamental requirement is to enhance local magnetic force to facilitate surface cell retaining. Additionally, an appropriate micromagnet system should (1) minimize possible physical damage to the target cells inside the microchannel; (2) reduce the aggregation of cells and free nanoparticles; (3) provide retrievable cells after capture for subsequent cellular imaging and downstream molecular studies; and (4) have robust fabrication processes so we can scale up for mass production. We summarize the fabrication principles, materials, micromagnet dimensions, capture range and applications in Table 1. Here, the capture range is the maximal distance away from the magnetic element where the targets can still be attracted in the immunomagnetic assay.

Fig. 4 Micromagnet technologies used for cell separation. (a) Micromagnetic strips used to guide and sort cells. (b) Micro-pillar structure made of nickel inside a PDMS microchannel. (c) Groove micromagnet made using thermal shrinking techniques. (d) Chessboard micromagnets fabricated using thermal patterning techniques. (e) The patterned thin film made using thermal deposition. It has been used to successfully separate CTCs from blood. Reproduced from ref. 85, 97, 81, 82, and 92 with permission from AIP and IEEE.


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Table 1 Comparison of different micromagnet structures

Micromagnet structure


Strips85 87

Pillars

Chessboard83 81

Grooves

Micromagnet array92 a

∇B2 (T2 m−1)

Capture range (μm)

Demonstrated applications

10 μm wide, 2 μm thick, 35 μm period distance 7 μm height, 15 μm diameter

~50

10

~500

50

Separation of leukocyte and red blood cells Microbeads filtration

Feature size 50–100 μm, reversal depth 1.1 μm ± .12 μm 20 μm height

~5

40–70

Microparticles separation

~30

60–70

200 nm thickness, 20 μm × 20 μm lateral dimension

~45

20–30

Microbeads separation and DNA extraction Detection of CTCs

Fabrication technology

Materials

Dimensions

Etching and sputter coating Soft lithography, electrodeposition Deposition, pulse laser irradiation Deposition, heating and shrinking Photolithography and thermal deposition

Ni Ni NdFeB film Ni, polyolefin (PO) film Ni

a

Dimensions are found from the respective paper cited.

Different designs use different targets in their demonstration experiments. To eliminate the impact of these variations of the targets and only compare the capabilities of generating the retaining force of different micromagnet structures objectively, we estimate the magnetic gradient ∇B2, which can be considered as a direct reflection of the magnetic force in eqn (5). In a conventional MACS system, like CellSearch™, fluorescent signal is the only available outcome. However, by using a microchip-based immunomagnetic detection assay and by integrating in-channel magnetic structures, CTCs can be captured and retrieved, allowing more in-depth analysis. Meanwhile, with the development of single cell profiling techniques, CTCs have made significant contributions to oncology and clinical studies. Potential downstream molecular studies for CTC can be performed using a set of advanced tools including polymerase chain reaction (PCR), reverse transcription– polymerase chain reaction (RT-PCR), quantitative reverse transcription–polymerase chain reaction (qRT-PCR), digital PCR, and fluorescence in situ hybridization (FISH) on selected cells.25,26,53,93,94 The correlation between primary tumors and corresponding CTCs95 or heterogeneity of different cancer cell lines55,96 may have clinical implications for discoveries of new drugs and targeted therapeutic strategies in patients. Moreover, breakthroughs regarding the gene expression of CTCs97 and epithelial–mesenchymal transition (EMT)65 could help provide new insight into metastasis.

Conclusion Magnetic cell separation plays a significant role in biology and medicine, where cells can be sorted under the combined force of biological bindings and magnetic fields. It is particularly valuable for rare cell separation such as for Circulating Tumor Cells or CTCs, which poses a unique challenge in terms of the required high sensitivity and specificity for precise clinical interpretations, without tremendous consumption of samples and assay. In this paper, we review the rare cancer cell sorting technologies based on magneticactivated and immunomagnetic assays over multiple scales.

Lab Chip

We begin the discussions on the macroscopic cell detection systems such as magnetic-activated cell sorting systems, followed by the scaling laws on miniaturization towards microfluidic chip based immunomagnetic assay for cell separation. Furthermore, we introduce recent work that integrates micro-magnets in separation experiments. Related design concepts, principles, and microfabrication techniques are presented and evaluated. Effective enrichment of rare tumor cells across glass slides, combined with downstream molecular analyses, may provide a minimally invasive tool to monitor cancer diagnosis and prognosis.

Acknowledgements We thank our collaborators Professor Konstantin V. Sokolov at the University of Texas at Austin, Professor Eugene P. Frenkel, Professor Jonathan W. Uhr, Nancy Lane, and Dr. Michael Huebschman at the University of Texas Southwestern Medical Center for the discussions related to nanoparticles development and clinical applications. We are grateful for the financial support from the National Institutes of Health (NIH) National Cancer Institute (NCI) Cancer Diagnosis Program under grant 1R01CA139070.

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Lab Chip

Inkjet-Print Micromagnet Array on Glass Slides for Immunomagnetic Enrichment of Circulating Tumor Cells.

Microfluidic platform for negative enrichment of circulating tumor cells.

Nanotechnology for enrichment and detection of circulating tumor cells.

Circulating tumor cells in pancreatic cancer patients: enrichment and cultivation.

Ex vivo expansion of circulating lung tumor cells based on one-step microfluidics-based immunomagnetic isolation.

Cell lines from circulating tumor cells.

Enrichment and enumeration of circulating tumor cells by efficient depletion of leukocyte fractions.

EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer.

An ultra-high-throughput spiral microfluidic biochip for the enrichment of circulating tumor cells.

Correction: EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer.

Detection of circulating tumor cells using targeted surface-enhanced Raman scattering nanoparticles and magnetic enrichment.

Clinical test on circulating tumor cells in peripheral blood of lung cancer patients, based on novel immunomagnetic beads.

A chip assisted immunomagnetic separation system for the efficient capture and in situ identification of circulating tumor cells.

Circulating Tumor Cells: Back to the Future.

Aptamer-Mediated Transparent-Biocompatible Nanostructured Surfaces for Hepotocellular Circulating Tumor Cells Enrichment.

Enrichment of circulating tumor cells in tumor-bearing mouse blood by a deterministic lateral displacement microfluidic device.

Detection of circulating tumor cells.

Analysis of circulating tumor cells derived from advanced gastric cancer.

Enrichment of circulating melanoma cells (CMCs) using negative selection from patients with metastatic melanoma.

[Circulating tumor cells: liquid biopsy].

Metastasis and Circulating Tumor Cells.

Genetic engineering of platelets to neutralize circulating tumor cells.

Looking back, to the future of circulating tumor cells.

Clinical validation of an ultra high-throughput spiral microfluidics for the detection and enrichment of viable circulating tumor cells.