13 research outputs found
Label-Free Metabolic Classification of Single Cells in Droplets Using the Phasor Approach to Fluorescence Lifetime Imaging Microscopy.
Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of fluorescent tags and cell lysis followed by genomic profiling to identify the cellular heterogeneity. Identifying individual cells in a noninvasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet microfluidic technology together with the phasor approach to fluorescence lifetime imaging microscopy to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells-a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based noninvasive and label-free method to distinguish individual cells based on their metabolic states, which could be used as an upstream phenotypic platform to correlate with genomic statistics. © 2018 International Society for Advancement of Cytometry
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High-throughput continuous dielectrophoretic separation of neural stem cells.
We created an integrated microfluidic cell separation system that incorporates hydrophoresis and dielectrophoresis modules to facilitate high-throughput continuous cell separation. The hydrophoresis module consists of a serpentine channel with ridges and trenches to generate a diverging fluid flow that focuses cells into two streams along the channel edges. The dielectrophoresis module is composed of a chevron-shaped electrode array. Separation in the dielectrophoresis module is driven by inherent cell electrophysiological properties and does not require cell-type-specific labels. The chevron shape of the electrode array couples with fluid flow in the channel to enable continuous sorting of cells to increase throughput. We tested the new system with mouse neural stem cells since their electrophysiological properties reflect their differentiation capacity (e.g., whether they will differentiate into astrocytes or neurons). The goal of our experiments was to enrich astrocyte-biased cells. Sorting parameters were optimized for each batch of neural stem cells to ensure effective and consistent separations. The continuous sorting design of the device significantly improved sorting throughput and reproducibility. Sorting yielded two cell fractions, and we found that astrocyte-biased cells were enriched in one fraction and depleted from the other. This is an advantage of the new continuous sorting device over traditional dielectrophoresis-based sorting platforms that target a subset of cells for enrichment but do not provide a corresponding depleted population. The new microfluidic dielectrophoresis cell separation system improves label-free cell sorting by increasing throughput and delivering enriched and depleted cell subpopulations in a single sort
Pacemakers and Defibrillators Implantation
Since the introduction of pacemakers and defibrillators in the 1960s, many lives have been saved. The technologies used in the development and implantation of such devices are constantly improving, making the procedures increasingly effective and safe. However, the complexity of such implantations makes it one of the most important procedures that need high levels of expertise, knowledge, and experience on the part of the entire surgery team. There is a wide range of devices used for different purposes with various features and characteristics to suit different patients. They range from single-chamber and dual-chamber pacemakers to pulse generators and biventricular pacemakers. The present review chapter seeks to elaborate on the steps of pacemakers and defibrillators implantation, starting from patient selection to post-surgery care and patient education. It outlines all necessary measures in the preoperative, intraoperative, and postoperative stages to ensure the utmost safety, prevent infection, and avoid and treat further complications. The procedures used by our team have demonstrated satisfactory results for patients with a wide variety of conditions
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Microfluidic Technology for Cell Engineering and Analysis
In recent years, cellular and gene therapies have been transforming medicine. With the 2018 Nobel Prize awarded to pioneers in the field of cancer immunotherapy, more and more advances in cell engineering are being developed to produce genetic-modified and reprogrammed cells for cellular and gene therapies. One promising category is ex-vivo cell/gene therapy in which the target cells are isolated from patients, the therapy is administrated to the cells outside of the body in vitro, and the cells are then transferred back into the body. However, challenges remain in terms of (i) isolating the target cells to be engineered, (ii) developments of efficient, safe, and controllable methods for intracellular delivery of gene-editing cargos, and (iii) development of efficient quality control (QC) approaches based on single-cell analysis of engineered cells. This dissertation is set out to develop microfluidic technologies to address the challenges in cell engineering and analysis.First, a high-throughput non-viral intracellular delivery platform is introduced for the transfection of large cargos with dosage-control. This platform, termed Acoustic-Electrical Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with uniform poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. With this platform, we demonstrated large-plasmid (>9kbp) transfection for CRISPR-Cas9 at 1 million cells/min per single chip.
Second, toward development of more efficient 1–1 droplet encapsulation methods for single-cell analysis, the mechanism of particle trapping and release at the flow-focusing microfluidic droplet generation junction, utilizing the hydrodynamic micro-vortices generated in the dispersed phase, is described. This technique is based solely on our unique flow-focusing geometry and the flow control of the two immiscible phases and, thus, does not require any on-chip active components. The effectiveness of this technique to be used for particle trapping and the subsequent size selective release into the droplets depends on the fundamental understanding of the nature of the vortex streamlines. Therefore, theoretical, computational, and experimental fluid dynamics were utilized to study in detail these micro-vortices and parameters affecting their formation, trajectory, and magnitude.
Third, an integrated microfluidic platform is presented that provides 3-part differential sorting of WBCs from whole blood. The proposed system accomplishes 3-part differential sorting of WBCs by: (1) On-chip lysis of RBCs from the blood sample, and (2) Downstream isolation of subpopulation of WBCs using dielectrophoresis (DEP) technology. The developed platform is capable of efficient isolation of viable monocytes, granulocytes, and lymphocytes from undiluted whole blood sample with volumes as low as 50 ul
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Design and optimization of circulating tumor cells separation devices
Circulating tumor cells (CTCs) separation technology has had positive impacts on the cancer science in many aspects. The ability of detecting and separating CTCs can play a key role in early cancer detection and treatment. Most of previous studies in this area are mainly based on experimental work. Although experimental research provides useful insights in designing CTC separation devices, there is still a lack of design guidelines based on fundamental understandings of cell separation process in such devices. While experimental efforts face challenges, especially microfabrication difficulties, we adopt numerical simulation here to study the fundamental concepts and provide design guidelines for two different CTC separation techniques. First, we investigate, in detail, the label-free deformability-based CTC separation technique using conicalshaped microfilters. To achieve this, we develop numerical models to predict the passing event of CTCs and normal blood cells through such microfilters. Using numerical simulations, we study fundamentally the concept of the deformability-based microfiltration technique and investigate comprehensively different parameters affecting system performance. Furthermore, the most important design criteria of the system such as system throughput and clogging issue are introduced and discussed in detail. Regarding the performance optimization, the effect of different geometrical and operational parameters, such as applied pressure profile and other possible microfilter geometries, on the system performance are studied. In the second part of the thesis, we propose a novel label-free CTC separation method by combining the concept of dielectrophoresis (DEP) and deterministic lateral displacement (DLD) CTC separation techniques. A numerical model is developed to study the concept and evaluate the performance of the proposed coupled DLD-DEP method. In addition, we provide design guidelines on choosing appropriate operation parameters such as flow rate and electric field characteristics. Finally, Joule heating and the corresponding electrothermal flows, as one of the important phenomena associated with the coupled DLD-DEP method, are modelled and discussed thoroughly
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A High-Throughput Microfluidic Cell Sorter Using a Three-Dimensional Coupled Hydrodynamic-Dielectrophoretic Pre-Focusing Module.
Dielectrophoresis (DEP) is a powerful tool for label-free sorting of cells, even those with subtle differences in morphological and dielectric properties. Nevertheless, a major limitation is that most existing DEP techniques can efficiently sort cells only at low throughputs (<1 mL h-1). Here, we demonstrate that the integration of a three-dimensional (3D) coupled hydrodynamic-DEP cell pre-focusing module upstream of the main DEP sorting region enables cell sorting with a 10-fold increase in throughput compared to conventional DEP approaches. To better understand the key principles and requirements for high-throughput cell separation, we present a comprehensive theoretical model to study the scaling of hydrodynamic and electrostatic forces on cells at high flow rate regimes. Based on the model, we show that the critical cell-to-electrode distance needs to be ≤10 µm for efficient cell sorting in our proposed microfluidic platform, especially at flow rates ≥ 1 mL h-1. Based on those findings, a computational fluid dynamics model and particle tracking analysis were developed to find optimum operation parameters (e.g., flow rate ratios and electric fields) of the coupled hydrodynamic-DEP 3D focusing module. Using these optimum parameters, we experimentally demonstrate live/dead K562 cell sorting at rates as high as 10 mL h-1 (>150,000 cells min-1) with 90% separation purity, 85% cell recovery, and no negative impact on cell viability
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Label-Free Metabolic Classification of Single Cells in Droplets Using the Phasor Approach to Fluorescence Lifetime Imaging Microscopy.
Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of fluorescent tags and cell lysis followed by genomic profiling to identify the cellular heterogeneity. Identifying individual cells in a noninvasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet microfluidic technology together with the phasor approach to fluorescence lifetime imaging microscopy to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells-a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based noninvasive and label-free method to distinguish individual cells based on their metabolic states, which could be used as an upstream phenotypic platform to correlate with genomic statistics. © 2018 International Society for Advancement of Cytometry