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.

High-Throughput and Dosage-Controlled Intracellular Delivery of Large Cargos by an Acoustic-Electric Micro-Vortices Platform.

A high-throughput non-viral intracellular delivery platform is introduced for the transfection of large cargos with dosage-control. This platform, termed Acoustic-Electric Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with 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. Intracellular delivery of a wide range of molecule sizes (<1 kDa to 2 MDa) with high efficiency (>90%), cell viability (>80%), and uniform dosages (<60% coefficient of variation (CV)) simultaneously into 1 million cells min-1 per single chip is demonstrated. AESOP is successfully applied to two gene editing applications that require the delivery of large plasmids: i) enhanced green fluorescent protein (eGFP) plasmid (6.1 kbp) transfection, and ii) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and single guide RNA (sgRNA). Compared to alternative platforms, this platform offers dosage-controlled intracellular delivery of large plasmids simultaneously to large populations of cells while maintaining cell viability at comparable delivery efficiencies.

Rapid isolation of circulating cancer associated fibroblasts by acoustic microstreaming for assessing metastatic propensity of breast cancer patients.

We demonstrate a label free and high-throughput microbubble-based acoustic microstreaming technique to isolate rare circulating cells such as circulating cancer associated fibroblasts (cCAFs) in addition to circulating tumor cells (CTCs) and immune cells (i.e. leukocytes) from clinically diagnosed patients with a capture efficiency of 94% while preserving cell functional integrity within 8 minutes. The microfluidic device is self-pumping and was optimized to increase flow rate and achieve near perfect capturing of rare cells enabled by having a trapping capacity above the acoustic vortex saturation concentration threshold. Our approach enables rapid isolation of CTCs, cCAFs and their associated clusters from blood samples of cancer patients at different stages. By examining the combined role of cCAFs and CTCs in early cancer onset and metastasis progression, the device accurately diagnoses both cancer and the metastatic propensity of breast cancer patients. This was confirmed by flow cytometry where we observed that metastatic breast cancer blood samples had significantly higher percentage of exhausted CD8+ T cells expressing programmed cell death protein 1 (PD1), higher number of CD4+ T regulatory cells and T helper cells. We show for the first time that our lateral cavity acoustic transducers (LCATs)-based approach can thus be developed into a metastatic propensity assay for clinical usage by elucidating cancer immunological responses and the complex relationships between CTCs and its companion tumor microenvironment.

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.

On the transport of particles/cells in high-throughput deterministic lateral displacement devices: Implications for circulating tumor cell separation.

Deterministic lateral displacement (DLD), which takes advantage of the asymmetric bifurcation of laminar flow around the embedded microposts, has shown promising capabilities in separating cells and particles of different sizes. Growing interest in utilizing high-throughput DLD devices for practical applications, such as circulating tumor cell separation, necessitates employing higher flow rates in these devices, leading to operating in moderate to high Reynolds number (Re) regimes. Despite extensive research on DLD devices in the creeping regime, limited research has focused on the physics of flow, critical size of the device, and deformable cell behavior in DLD devices at moderate to high Re. In this study, the transport behavior of particles/cells is investigated in realistic high-throughput DLD devices with hundreds of microposts by utilizing multiphysics modeling. A practical formula is proposed for the prediction of the device critical size, which could serve as a design guideline for high-throughput DLD devices. Then, the complex hydrodynamic interactions between a deformable cell and DLD post arrays are investigated. A dimensionless index is utilized for comparing different post designs to quantify the cell-post interaction. It is shown that the separation performances in high-throughput devices are highly affected by Re as well as the micropost shapes. These findings can be utilized for the design and optimization of high-throughput DLD microfluidic devices.

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.

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.

Lipoplex-Mediated Single-Cell Transfection via Droplet Microfluidics.

While lipoplex (cationic lipid-nucleic acid complex)-mediated intracellular delivery is widely adopted in mammalian cell transfection, its transfection efficiency for suspension cells, e.g., lymphatic and hematopoietic cells, is reported at only ≈5% or even lower. Here, efficient and consistent lipoplex-mediated transfection is demonstrated for hard-to-transfect suspension cells via a single-cell, droplet-microfluidics approach. In these microdroplets, monodisperse lipoplexes for effective gene delivery are generated via chaotic mixing induced by the serpentine microchannel and co-confined with single cells. Moreover, the cell membrane permeability increases due to the shear stress exerted on the single cells when they pass through the droplet pinch-off junction. The transfection efficiency, examined by the delivery of the pcDNA3-EGFP plasmid, improves from ≈5% to ≈50% for all three tested suspension cell lines, i.e., K562, THP-1, Jurkat, and with significantly reduced cell-to-cell variation, compared to the bulk method. Efficient targeted knockout of the TP53BP1 gene for K562 cells via the CRISPR (clustered regularly interspaced short palindromic repeats)-CAS9 (CRISPR-associated nuclease 9) mechanism is also achieved using this platform. Lipoplex-mediated single-cell transfection via droplet microfluidics is expected to have broad applications in gene therapy and regenerative medicine by providing high transfection efficiency and low cell-to-cell variation for hard-to-transfect suspension cells.

It's Electric: When Technology Gives a Boost to Stem Cell Science.

Purpose of Review: Advanced technologies can aid discoveries in stem cell science in surprising ways. The application of electrokinetic techniques, which use electric fields to interrogate or separate cells, to the study of stem cells has yielded important insights into stem cell function. These techniques probe inherent cell properties, obviating the need for cell-type specific labels. Recent Findings: Analysis of a variety of stem cell types including hematopoietic, mesenchymal and adipose-derived, neural, and pluripotent stem cells by electrokinetic techniques has revealed fate-specific signatures of cells. Distinct inherent cell properties are sufficient for their label-free enrichment without causing cell damage or toxicity. Summary: The successful application of label-free techniques to the analysis and sorting of stem cells open new avenues for exploring the basic biology of stem cells and optimizing their use in regenerative medicine applications.

Effects of electrothermal vortices on insulator-based dielectrophoresis for circulating tumor cell separation.

Insulator-based dielectrophoresis (iDEP) is a powerful technique for separation and manipulation of bioparticles. In recent years, iDEP designs using arrays of insulating posts have shown promising results toward reaching high-efficiency bioparticle manipulation. Joule heating (JH) and electrothermal (ET) flows have been observed in iDEP microdevices and significantly affecting their performances. In this research, we utilize mathematical modeling to study, iDEP technique and the effects of JH and ET flow on device performance and propose a separation scenario for selective trapping of circulating tumor cells (CTCs). A robust numerical model is developed to calculate the distribution of electric and fluid flow fields in the presence of JH and ET flow, and predict the cells' trajectory inside the system. Our results indicate that JH not only induces temperature rise in the system, but also may alter the design iDEP separation scenario by inducing ET vortices that affect the cell's trajectory. To investigate the impact of JH-induced ET flow characteristics and vortex generation on separation efficiency, we introduce a dimensionless force ratio encompassing the effects of electrical field, drag forces, JH, and ET flow. Interestingly, it was found that ET flows can be used to significantly enhance the separation efficiency, even in higher inlet flow rates. Lastly, the effect of post geometry has been discussed.

Deformability-based circulating tumor cell separation with conical-shaped microfilters: Concept, optimization, and design criteria.

Circulating tumor cells (CTCs) separation technology has made positive impacts on cancer science in many aspects. The ability of detecting and separating CTCs can play a key role in early cancer detection and treatment. In recent years, there has been growing interest in using deformability-based CTC separation microfilters due to their simplicity and low cost. Most of the 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 the cell separation process in the filters. While experimental efforts face challenges, especially microfabrication difficulties, we adopt numerical simulation here to study conical-shaped microfilters using deformability difference between CTCs and blood cells for the separation process. We use the liquid drop model for modeling a CTC passing through such microfilters. The accuracy of the model in predicting the pressure signature of the system is validated by comparing it with previous experiments. Pressure-deformability analysis of the cell going through the channel is then carried out in detail in order to better understand how a CTC behaves throughout the filtration process. Different system design criteria such as system throughput and unclogging of the system are discussed. Specifically, pressure behavior under different system throughput is analyzed. Regarding the unclogging issue, we define pressure ratio as a key parameter representing the ability to overcome clogging in such CTC separation devices and investigate the effect of conical angle on the optimum pressure ratio. Finally, the effect of unclogging applied pressure on the system performance is examined. Our study provides detailed understandings of the cell separation process and its characteristics, which can be used for developing more efficient CTC separation devices.