微流控芯片系统在循环肿瘤细胞分离检测中的应用进展

循环肿瘤细胞(CTCs)的分离分析一直是肿瘤相关研究中的热点方向,作为液体活检的重要标志物之一,其在外周血中的含量与癌症病发状况密切相关.然而人体血液中CTCs的含量非常低,通常来说仅有0～10个/mL,因此在开展临床血液样本中CTCs的检测前,往往需要对样本进行前处理,以实现CTCs的分离和富集.微流控芯片技术凭借样...     

Integration of Lateral Filter Arrays with Immunoaffinity for Circulating‐Tumor‐Cell Isolation

Circulating tumor cells (CTCs) are an important biomarker for cancer prognosis and treatment monitoring. However, the heterogeneity of the physical and biological properties of CTCs limits the efficiency of various approaches used to isolate small numbers of CTCs from billions of normal blood cells. To address this challenge, we developed a lateral filter array microfluidic (LFAM) device to integrate size‐based separation with immunoaffinity‐based CTC isolation. The LFAM device consists of a serpentine main channel, through which most of a sample passes, and an array of lateral filters for CTC isolation. The unique device design produces a two‐dimensional flow, which reduces nonspecific, geometric capture of normal cells as typically observed in vertical filters. The LFAM device was further functionalized by immobilizing antibodies that are specific to the target cells. The resulting devices captured pancreatic cancer cells spiked in blood samples with (98.7±1.2) % efficiency and were used to isolate CTCs from patients with metastatic colorectal cancer.     

Label-free enrichment of MCF7 breast cancer cells from leukocytes using continuous flow dielectrophoresis

Circulating tumor cells (CTCs) present in the bloodstream are strongly linked to the invasive behavior of cancer; therefore, their detection holds great significance for monitoring disease progression. Currently available CTC isolation tools are often based on tumor-specific antigen or cell size approaches. However, these techniques are limited due to the lack of a unique and universal marker for CTCs, and the overlapping size between CTCs and regular blood cells. Dielectrophoresis (DEP), governed by the intrinsic dielectric properties of the particles, is a promising marker-free, accurate, fast, and low-cost technique that enables the isolation of CTCs from blood cells. This study presents a continuous flow, antibody-free DEP-based microfluidic device to concentrate MCF7 breast cancer cells, a well-established CTC model, in the presence of leukocytes extracted from human blood samples. The enrichment strategy was determined according to the DEP responses of the corresponding cells, obtained in our previously reported DEP spectrum study. It was based on the positive-DEP integrated with hydrodynamic focusing under continuous flow. In the proposed device, the parylene microchannel with two inlets and outlets was built on top of rectangular and equally spaced isolated planar electrodes rotated certain degree relative to the main flow (13°). The recovery of MCF7 cells mixed with leukocytes was 74%–98% at a frequency of 1 MHz and a magnitude of 10–12 V_pp. Overall, the results revealed that the presented system successfully concentrates MCF7 cancer cells from leukocytes, ultimately verifying our DEP spectrum study, in which the enrichment frequency and separation strategy of the microfluidic system were determined.     

Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation

Blood is a highly complex bio-fluid with cellular components making up >40% of the total volume, thus making its analysis challenging and time-consuming. In this work, we introduce a high-throughput size-based separation method for processing diluted blood using inertial microfluidics. The technique takes advantage of the preferential cell focusing in high aspect-ratio microchannels coupled with pinched flow dynamics for isolating low abundance cells from blood. As an application of the developed technique, we demonstrate the isolation of cancer cells (circulating tumor cells (CTCs)) spiked in blood by exploiting the difference in size between CTCs and hematologic cells. The microchannel dimensions and processing parameters were optimized to enable high throughput and high resolution separation, comparable to existing CTC isolation technologies. Results from experiments conducted with MCF-7 cells spiked into whole blood indicate >80% cell recovery with an impressive 3.25 × 10(5) fold enrichment over red blood cells (RBCs) and 1.2 × 10(4) fold enrichment over peripheral blood leukocytes (PBL). In spite of a 20× sample dilution, the fast operating flow rate allows the processing of ～10(8) cells min(-1) through a single microfluidic device. The device design can be easily customized for isolating other rare cells from blood including peripheral blood leukocytes and fetal nucleated red blood cells by simply varying the 'pinching' width. The advantage of simple label-free separation, combined with the ability to retrieve viable cells post enrichment and minimal sample pre-processing presents numerous applications for use in clinical diagnosis and conducting fundamental studies.     

A combined micromagnetic-microfluidic device for rapid capture and culture of rare circulating tumor cells

Here we describe a combined microfluidic-micromagnetic cell separation device that has been developed to isolate, detect and culture circulating tumor cells (CTCs) from whole blood, and demonstrate its utility using blood from mammary cancer-bearing mice. The device was fabricated from polydimethylsiloxane and contains a microfluidic architecture with a main channel and redundant 'double collection' channel lined by two rows of dead-end side chambers for tumor cell collection. The microdevice design was optimized using computational simulation to determine dimensions, magnetic forces and flow rates for cell isolation using epithelial cell adhesion molecule (EpCAM) antibody-coated magnetic microbeads (2.8 μm diameter). Using this device, isolation efficiencies increased in a linear manner and reached efficiencies close to 90% when only 2 to 80 breast cancer cells were spiked into a small volume (1.0 mL) of blood taken from wild type mice. The high sensitivity visualization capabilities of the device also allowed detection of a single cell within one of its dead-end side chambers. When blood was removed from FVB C3(1)-SV40 T-antigen mammary tumor-bearing transgenic mice at different stages of tumor progression, cells isolated in the device using anti-EpCAM-beads and magnetically collected within the dead-end side chambers, also stained positive for pan-cytokeratin-FITC and DAPI, negative for CD45-PerCP, and expressed SV40 large T antigen, thus confirming their identity as CTCs. Using this isolation approach, we detected a time-dependent rise in the number of CTCs in blood of female transgenic mice, with a dramatic increase in the numbers of metastatic tumor cells appearing in the blood after 20 weeks when tumors transition to invasive carcinoma and exhibit increased growth of metastases in this model. Importantly, in contrast to previously described CTC isolation methods, breast tumor cells collected from a small volume of blood removed from a breast tumor-bearing animal remain viable and they can be easily removed from these devices and expanded in culture for additional analytical studies or potential drug sensitivity testing.     

Microfluidic applications on circulating tumor cell isolation and biomimicking of cancer metastasis

The prognosis of malignant tumors is challenged by insufficient means to effectively detect tumors at early stage. Liquid biopsy using circulating tumor cells (CTCs) as biomarkers demonstrates a promising solution to tackle the challenge, because CTCs play a critical role in cancer metastatic process via intravasation, circulation, extravasation, and formation of secondary tumor. However, the effectiveness of the solution is compromised by rarity, heterogeneity, and vulnerability associated with CTCs. Among a plethora of novel approaches for CTC isolation and enrichment, microfluidics leads to isolation and detection of CTCs in a cost-effective and operation-friendly way. Development of microfluidics also makes it feasible to model the cancer metastasis in vitro using a microfluidic system to mimick the in vivo microenvironment, thereby enabling analysis and monitor of tumor metastasis. This paper aims to review the latest advances for exploring the dual-roles microfluidics has played in early cancer diagnosis via CTC isolation and investigating the role of CTCs in cancer metastasis; the merits and drawbacks for dominating microfluidics-based CTC isolation methods are discussed; biomimicking cancer metastasis using microfluidics are presented with example applications on modelling of tumor microenvironment, tumor cell dissemination, tumor migration, and tumor angiogenesis. The future perspectives and challenges are discussed.     

Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP)

Circulating tumor cells (CTCs) are highly correlated with the invasive behavior of cancer, so their isolations and quantifications are important for biomedical applications such as cancer prognosis and measuring the responses to drug treatments. In this paper, we present the development of a microfluidic device for the separation of CTCs from blood cells based on the physical properties of cells. For use as a CTC model, we successfully separated human breast cancer cells (MCF-7) from a spiked blood cell sample by combining multi-orifice flow fractionation (MOFF) and dielectrophoretic (DEP) cell separation technique. Hydrodynamic separation takes advantage of the massive and high-throughput filtration of blood cells as it can accommodate a very high flow rate. DEP separation plays a role in precise post-processing to enhance the efficiency of the separation. The serial combination of these two different sorting techniques enabled high-speed continuous flow-through separation without labeling. We observed up to a 162-fold increase in MCF-7 cells at a 126 μL min(-1) flow rate. Red and white blood cells were efficiently removed with separation efficiencies of 99.24% and 94.23% respectively. Therefore, we suggest that our system could be used for separation and detection of CTCs from blood cells for biomedical applications.     

Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells

Microfluidic technology has been successfully applied to isolate very rare tumor-derived epithelial cells (circulating tumor cells, CTCs) from blood with relatively high yield and purity, opening up exciting prospects for early detection of cancer. However, a major limitation of state-of-the-art CTC-chips is their inability to characterize the behavior and function of captured CTCs, for example to obtain information on proliferative and invasive properties or, ultimately, tumor re-initiating potential. Although CTCs can be efficiently immunostained with markers reporting phenotype or fate (e.g. apoptosis, proliferation), it has not yet been possible to reliably grow captured CTCs over long periods of time and at single cell level. It is challenging to remove CTCs from a microchip after capture, therefore such analyses should ideally be performed directly on-chip. To address this challenge, we merged CTC capture with three-dimensional (3D) tumor cell culture on the same microfluidic platform. PC3 prostate cancer cells were isolated from spiked blood on a transparent PDMS CTC-chip, encapsulated on-chip in a biomimetic hydrogel matrix (QGel?) that was formed in situ, and their clonal 3D spheroid growth potential was assessed by microscopy over one week in culture. The possibility to clonally expand a subset of captured CTCs in a near-physiological in vitro model adds an important element to the expanding CTC-chip toolbox that ultimately should improve prediction of treatment responses and disease progression.     

Characterization of a hybrid dielectrophoresis and immunocapture microfluidic system for cancer cell capture

The capture of circulating tumor cells (CTCs) from cancer patient blood enables early clinical assessment as well as genetic and pharmacological evaluation of cancer and metastasis. Although there have been many microfluidic immunocapture and electrokinetic techniques developed for isolating rare cancer cells, these techniques are often limited by a capture performance tradeoff between high efficiency and high purity. We present the characterization of shear‐dependent cancer cell capture in a novel hybrid DEP–immunocapture system consisting of interdigitated electrodes fabricated in a Hele‐Shaw flow cell that was functionalized with a monoclonal antibody, J591, which is highly specific to prostate‐specific membrane antigen expressing prostate cancer cells. We measured the positive and negative DEP response of a prostate cancer cell line, LNCaP, as a function of applied electric field frequency, and showed that DEP can control capture performance by promoting or preventing cell interactions with immunocapture surfaces, depending on the sign and magnitude of the applied DEP force, as well as on the local shear stress experienced by cells flowing in the device. This work demonstrates that DEP and immunocapture techniques can work synergistically to improve cell capture performance, and it will aid in the design of future hybrid DEP–immunocapture systems for high‐efficiency CTC capture with enhanced purity.     

微流控芯片分选富集循环肿瘤细胞的研究进展

近年来,循环肿瘤细胞(CTCs)研究得到了越来越多的关注,许多研究报告已经证实其在肿瘤转移的早期诊断、治疗方案选择、个体化治疗及探索肿瘤转移机制等方面具有潜在的价值,然而CTCs在循环系统中的含量极低,这成为限制其临床相关应用的主要难点。微流控芯片技术具有低成本、快速、高通量及操作简单等优势,利用微流控芯片可实现CTCs的高速、高回收率、高纯度的分选富集,近年来得到广泛的关注。本文综述了近年来在微流控芯片内进行CTCs分选富集的研究并探讨了各种方法的优缺点,并在本研究团队的研究基础上进行了展望。     

Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies

Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper, we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells, we develop an image processing methodology with a recognition capability of multiple features, e.g., cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern, in which single as well as multiple laser traps are employed for cell transportation, and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach.     

Nanoparticle‐Mediated Binning and Profiling of Heterogeneous Circulating Tumor Cell Subpopulations

The analysis of circulating tumor cells (CTCs) is an important capability that may lead to new approaches for cancer management. CTC capture devices developed to date isolate a bulk population of CTCs and do not differentiate subpopulations that may have varying phenotypes with different levels of clinical relevance. Here, we present a new device for CTC spatial sorting and profiling that sequesters blood‐borne tumor cells with different phenotypes into discrete spatial bins. Validation data are presented showing that cancer cell lines with varying surface expression generate different binning profiles within the device. Working with patient blood samples, we obtain profiles that elucidate the heterogeneity of CTC populations present in cancer patients and also report on the status of CTCs within the epithelial‐to‐mesenchymal transition (EMT).     

循环肿瘤细胞体内检测技术及其应用研究

从实体瘤脱落进入血液循环系统的肿瘤细胞即循环肿瘤细胞(CTCs)与肿瘤转移密切相关,因此CTCs检测对癌症患者的诊断、治疗监测、病情评估以及肿瘤转移机制研究具有重要意义.由于CTCs在体内含量极少、异质性、分布不均一,通过体外采血发展的CTCs检测技术虽然已取得很大进展,但仍然面临肿瘤细胞损失、失活、失真以及灵敏度低等...     

On the design of deterministic dielectrophoresis for continuous separation of circulating tumor cells from peripheral blood cells

Detection and analysis of circulating tumor cells (CTCs) have emerged as a promising way to diagnose cancer, study its cellular mechanism, and test or develop potential treatments. However, the rarity of CTCs among peripheral blood cells is a big challenge toward CTC detection. In addition, in cases where there is similar size range between certain types of CTCs (e.g. breast cancer cells) and white blood cells (WBCs), high‐resolution techniques are needed. In the present work, we propose a deterministic dielectrophoresis (DEP) method that combines the concept of deterministic lateral displacement (DLD) and insulator‐based dielectrophoresis (iDEP) techniques that rely on physical markers such as size and dielectric properties to differentiate different type of cells. The proposed deterministic DEP technology takes advantage of frequency‐controlled AC electric field for continuous separation of CTCs from peripheral blood cells. Utilizing numerical modeling, different aspects of coupled DLD‐DEP design such as the required applied voltages, velocities, and geometrical parameters of DLD arrays of microposts are investigated. Regarding the inevitable difference and uncertainty ranges for the reported crossover frequencies of cells, a comprehensive analysis is conducted on applied electric field frequency as design's determinant factor. Deterministic DEP design provides continuous sorting of CTCs from WBCs even with similar size and has the future potential for high throughput and efficiency.     

Examination of the dielectrophoretic spectra of MCF7 breast cancer cells and leukocytes

The detection of circulating tumor cells (CTCs) in blood is crucial to assess metastatic progression and to guide therapy. Dielectrophoresis (DEP) is a powerful cell surface marker-free method that allows intrinsic dielectric properties of suspended cells to be exploited for CTC enrichment/isolation from blood. Design of a successful DEP-based CTC enrichment/isolation system requires that the DEP response of the targeted particles should accurately be known. This paper presents a DEP spectrum method to investigate the DEP spectra of cells without directly analyzing their membrane and cytoplasmic properties in contrast to the methods in literature, which employ theoretical assumptions and complex modeling. Integrating electric field simulations based on DEP theory with the experimental data enables determination of the DEP spectra of leukocyte subpopulations, polymorphonuclear and mononuclear leukocytes, and MCF7 breast cancer cells as a model of CTC due to their metastatic origin over the frequency range 100 kHz–50 MHz at 10 V_pp. In agreement with earlier findings, differential DEP responses were detected for mononuclear and polymorphonuclear leukocytes due to the richness of the cell surface features and morphologies of the different leukocyte types. The data reveal that the strength of the DEP force exerted on MCF7 cells was particularly high between 850 kHz and 20 MHz. These results illustrate that the proposed technique has the potential to provide a generic platform to identify DEP responses of different biological particles.     

Three‐dimensional microfluidic chip with twin‐layer herringbone structure for high efficient tumor cell capture and release via antibody‐conjugated magnetic microbeads

Harvesting rare circulating tumor cells (CTCs) from human blood is distinctly substantial to monitor tumor stage and evaluate therapeutic efficacy. As a proof‐of‐concept study, a microfluidic chip with twin‐layer herringbone grooves was developed to isolate and recover tumor cells with high efficiency based on the immunoreaction between cells and antibody‐conjugated microbeads (MBs) under local magnetic field. Functional MBs were initially localized onto the internal channel wall through the magnetic guidance. Then, infused tumor cells were deviated into the herringbone groove via passive microvortex and were further trapped through an irreversible interaction with MBs. Upon the removal of magnet, the captured cells and residual MBs were released from the channel and collected for further analysis in cell adhesion and proliferation in vitro. Capture efficiency of tumor cells reached up to ∼90% and limit of detection was down to 50 cells per mL based on this approach. Furthermore, recovery rate of tumor cells was as high as ∼94%, and potencies of cell attachment and proliferation was well maintained in retrieved cells. Hence, the present technique has a great potential for the isolation, quantitation and recovery of CTCs for cancer theranostic guidance and biomolecular analysis.     

Spectrally Combined Encoding for Profiling Heterogeneous Circulating Tumor Cells Using a Multifunctional Nanosphere‐Mediated Microfluidic Platform

Comprehensive phenotypic profiling of heterogeneous circulating tumor cells (CTCs) at single‐cell resolution has great importance for cancer management. Herein, a novel spectrally combined encoding (SCE) strategy was proposed for multiplex biomarker profiling of single CTCs using a multifunctional nanosphere‐mediated microfluidic platform. Different cellular biomarkers uniquely labeled by multifunctional nanosphere barcodes, possessing identical magnetic tags and distinct optical signatures, enabled isolation of heterogeneous CTCs with over 91.6 % efficiency and in situ SCE of phenotypes. By further trapping individual CTCs in ordered microstructures on chip, composite single‐cell spectral signatures were conveniently and efficiently obtained, allowing reliable spectral‐readout for multiplex biomarker profiling. This SCE strategy exhibited great potential in multiplex profiling of heterogeneous CTC phenotypes, offering new avenues for cancer study and precise medicine.     

Isolation,Detection, and Antigen‐Based Profiling of Circulating Tumor Cells Using a Size‐Dictated Immunocapture Chip

Even though the diagnostic and prognostic value of circulating tumor cells (CTCs) has been demonstrated, their clinical utility and widespread adoption have been limited. Herein, we describe a new device, size‐dictated immunocapture chip (SDI‐Chip), for efficient, sensitive, and spatially resolved capture and detection of CTCs. SDI‐Chip enables selective, frequent, and extended interaction of CTCs with hydrodynamically optimized immunocoated micropillar surfaces. CTCs with different antigen expression levels can be efficiently captured and spatially resolved around the micropillars. Capture efficiency greater than 92 % with a purity of 82 % was achieved with blood samples. CTCs were detected in non‐metastasis colorectal (CRC) patients, while none was detected from healthy volunteers. We believe that SDI‐Chip will facilitate the transition of tumor diagnosis from anatomical pathology to molecular pathology in localized CRC patients.