基礎講座 電気泳動(Electrophoresis)

Liquid biopsy has been introduced as a new diagnostic concept predicated on the analysis of circulating tumour cells (CTCs) or circulating tumour-derived factors, in particular, cell-free tumour DNA (ctDNA). Highly sensitive liquid biopsy assays have been developed that can now be applied to detect and characterize minimal residual disease (MRD), which reflects the presence of tumour cells disseminated from the primary lesion to distant organs in patients who lack any clinical or radiological signs of metastasis or residual tumour cells left behind after local therapy that eventually lead to local recurrence. This application is the new frontier of liquid biopsy analyses, which are challenged by the very low concentrations of CTCs and ctDNA in blood samples. In this Review, we discuss the key technologies that can be used to detect and characterize CTCs in surveillance of MRD and provide a brief overview of similar roles of ctDNA analyses. We then focus on the current clinical data on the use of CTCs and ctDNA in the detection and monitoring of MRD and in obtaining information on therapeutic targets and resistance mechanisms relevant to the management of individual patients with cancer. In this Review, Pantel and Alix-Panabieres provide an overview of approaches for the detection and characterization of minimal residual disease (MRD) using circulating tumour cells and circulating tumour DNA. They also discuss the clinical implications of such liquid biopsy approaches to MRD monitoring for the management of patients with cancer.

Liquid biopsy and minimal residual disease - latest advances and implications for cure.

This article reviews the recent developments in microfluidic technologies for in vitro cancer diagnosis. We summarize the working principles and experimental results of key microfluidic platforms for cancer cell detection, characterization, and separation based on cell-affinity micro-chromatography, magnetic activated micro-sorting, and cellular biophysics (e.g., cell size and mechanical and electrical properties). We examine the advantages and limitations of each technique and discuss future research opportunities for improving device throughput and purity, and for enabling on-chip analysis of captured cancer cells.

Microfluidic approaches for cancer cell detection, characterization, and separation

Rare cells are low-abundance cells in a much larger population of background cells. Conventional benchtop techniques have limited capabilities to isolate and analyze rare cells because of their generally low selectivity and significant sample loss. Recent rapid advances in microfluidics have been providing robust solutions to the challenges in the isolation and analysis of rare cells. In addition to the apparent performance enhancements resulting in higher efficiencies and sensitivity levels, microfluidics provides other advanced features such as simpler handling of small sample volumes and multiplexing capabilities for high-throughput processing. All of these advantages make microfluidics an excellent platform to deal with the transport, isolation, and analysis of rare cells. Various cellular biomarkers, including physical properties, dielectric properties, as well as immunoaffinities, have been explored for isolating rare cells. In this Focus article, we discuss the design considerations of representative microfluidic devices for rare cell isolation and analysis. Examples from recently published works are discussed to highlight the advantages and limitations of the different techniques. Various applications of these techniques are then introduced. Finally, a perspective on the development trends and promising research directions in this field are proposed.

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Rare cell isolation and analysis in microfluidics

Magnetic sorting using magnetic beads has become a routine methodology for the separation of key cell populations from biological suspensions. Due to the inherent ability of magnets to provide forces at a distance, magnetic cell manipulation is now a standardized process step in numerous processes in tissue engineering, medicine, and in fundamental biological research. Herein we review the current status of magnetic particles to enable isolation and separation of cells, with a strong focus on the fundamental governing physical phenomena, properties and syntheses of magnetic particles and on current applications of magnet-based cell separation in laboratory and clinical settings. We highlight the contribution of cell separation to biomedical research and medicine and detail modern cell-separation methods (both magnetic and non-magnetic). In addition to a review of the current state-of-the-art in magnet-based cell sorting, we discuss current challenges and available opportunities for further research, development and commercialization of magnetic particle-based cell-separation systems.

http://iopscience.iop.org/article/10.1088/0034-4885/78/1/016601/pdf

Fundamentals and application of magnetic particles in cell isolation and enrichment: a review

Rare Cell Capture in Microfluidic Devices

Prevailing commercialized cardiac platforms for in vitro drug development utilize planar microelectrode arrays to map action potentials, or impedance sensing to record contraction in real time, but cannot record both functions on the same chip with high spatial resolution. Here we report a novel cardiac platform that can record cardiac tissue adhesion, electrophysiology, and contractility on the same chip. The platform integrates two independent yet interpenetrating sensor arrays: a microelectrode array for field potential readouts and an interdigitated electrode array for impedance readouts. Together, these arrays provide real-time, non-invasive data acquisition of both cardiac electrophysiology and contractility under physiological conditions and under drug stimuli. Human induced pluripotent stem cell-derived cardiomyocytes were cultured as a model system, and used to validate the platform with an excitation–contraction decoupling chemical. Preliminary data using the platform to investigate the effect of the drug norepinephrine are combined with computational efforts. This platform provides a quantitative and predictive assay system that can potentially be used for comprehensive assessment of cardiac toxicity earlier in the drug discovery process.

Simultaneous electrical recording of cardiac electrophysiology and contraction on chip

We report the positive dielectrophoretic (pDEP) characterization of wild-type and ethambutol-treated Mycobacterium smegmatis populations via automated pDEP cell trapping experiments. The automated technique was validated by measurements of carboxylate-modified polystyrene microspheres and Escherichia coli. The characterization of M. smegmatis identifies a key frequency regime where the membrane-specific action of ethambutol leads to a change in the cellular dielectrophoretic response. This work represents the first such characterization of Mycobacteria and highlights the potential for DEP measurements to measure changes in mycobacterial membrane properties associated with chemical treatments or genetic mutation.

Automated dielectrophoretic characterization of Mycobacterium smegmatis.

The isolation of circulating tumor cells (CTCs) from cancer patient blood is a technical challenge that is often addressed by microfluidic approaches. Two of the most prominent techniques for rare cancer cell separation, immunocapture and dielectrophoresis (DEP), are currently limited by a performance tradeoff between high efficiency and high purity. The development of a platform capable of these two performance criteria can potentially be facilitated by incorporating both DEP and immunocapture. We present a hybrid DEP-immunocapture system to characterize how DEP controls the shear-dependent capture of a prostate cancer cell line, LNCaP, and the nonspecific adhesion of peripheral blood mononuclear cells (PBMCs). Characterization of cell adhesion with and without DEP effects was performed in a Hele-Shaw flow cell that was functionalized with the prostate-specific monoclonal antibody, J591. In this model system designed to make nonspecific PBMC adhesion readily apparent, we demonstrate LNCaP enrichment from PBMCs by precisely tuning the applied AC electric field frequency to enhance immunocapture of LNCaPs and reduce nonspecific adhesion of PBMCs with positive and negative DEP, respectively. Our work shows that DEP and immunocapture techniques can work synergistically to improve cancer cell capture performance, and it informs the design of future hybrid DEP-immunocapture systems with improved CTC capture performance to facilitate research on cancer metastasis and drug therapies.

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Enrichment of prostate cancer cells from blood cells with a hybrid dielectrophoresis and immunocapture microfluidic system

We used automated electrorotation to measure the cytoplasmic permittivity, cytoplasmic conductivity, and specific membrane capacitance of pancreatic cancer cells under environmental perturbation to evaluate the effects of serum starvation, epithelial-to-mesenchymal transition, and evolution of chemotherapy resistance which may be associated with the development and dissemination of cancer. First, we compared gemcitabine-resistant BxPC3 subclones with gemcitabine-naive parental cells. Second, we serum-starved BxPC3 and PANC-1 cells and compared them to untreated counterparts. Third, we induced the epithelial-to-mesenchymal transition in PANC-1 cells and compared them to untreated PANC-1 cells. We also measured the electrorotation spectra of white blood cells isolated from a healthy donor. The properties from fit electrorotation spectra were used to compute dielectrophoresis (DEP) spectra and crossover frequencies. For all three experiments, the median crossover frequency for both treated and untreated pancreatic cancer cells remained significantly lower than the median crossover frequency for white blood cells. The robustness of the crossover frequency to these treatments indicates that DEP is a promising technique for enhancing capture of circulating cancer cells.

Automated electrorotation shows electrokinetic separation of pancreatic cancer cells is robust to acquired chemotherapy resistance, serum starvation, and EMT

Chao Huang

Papers

Rare Cell Capture in Microfluidic Devices

Simultaneous electrical recording of cardiac electrophysiology and contraction on chip

Automated dielectrophoretic characterization of Mycobacterium smegmatis.

Enrichment of prostate cancer cells from blood cells with a hybrid dielectrophoresis and immunocapture microfluidic system

Automated electrorotation shows electrokinetic separation of pancreatic cancer cells is robust to acquired chemotherapy resistance, serum starvation, and EMT