Jon F. Edd

Controlled encapsulation of single-cells into monodisperse picolitre drops

Encapsulation of cells within picolitre-size monodisperse drops provides new means to perform quantitative biological studies on a single-cell basis for large cell populations. Variability in the number of cells per drop due to stochastic cell loading is a major barrier to these techniques. We overcome this limitation by evenly spacing cells as they travel within a high aspect-ratio microchannel; cells enter the drop generator with the frequency of drop formation.

Equilibrium Separation and Filtration of Particles Using Differential Inertial Focusing

Rapid separation and filtration of particles in solution has a wide range of applications including blood cell separation, ultrasound contrast agent preparation, and purification of fermentation products. However, current techniques that provide quick processing rates are high in complexity. We present a rapid microfluidic filtration technology capable of separating particles based on size, with purities from 90 to 100% and high-volume throughputs of 1 mL/min. Data for separation of rigid particles, deformable emulsions, and platelets from whole blood are presented. The system is based upon differential inertial focusing of particles of varying sizes and allows continuous separation based only on intrinsic hydrodynamic forces developed in a flow through an asymmetrically curved channel. A theoretical description of the underlying forces is developed, and in combination with data determining a size cutoff for separation, a semiempirical relationship describing how channel geometry is related to this cutoff is shown. Cascading separations in series is shown to be useful for increasing purity and yield. This type of microfluidic system can filter deformable particles, is largely independent of particle density, and can provide throughputs typical of macroscale filtration in a compact format, enabling applications in blood filtration and particle concentration.

Mathematical modeling of irreversible electroporation for treatment planning.

Irreversible Electroporation (IRE) is a new drug-free method to ablate undesirable tissue of particular use in cancer therapy. IRE achieves cell death within the targeted tissue through a series of electric pulses that elevate the transmembrane potentials to an extent that permanently damages the lipid bilayers throughout the treated region. Although the IRE procedure is easy to perform, treatment planning is complicated by the fact that the electric field distribution within the tissue, the greatest single factor controlling the extents of IRE, depends non-trivially on the electrode configuration, pulse parameters and any tissue heterogeneities. To address this difficulty, we instruct on how to properly model IRE and discuss the benefit of modeling in designing treatment protocols. The necessary theoretical basis is introduced and discussed through the detailed analysis of two classic dual-electrode configurations from electrochemotherapy: coaxial disk electrodes and parallel needle electrodes. Dimensionless figures for these cases are also provided that allow cell constants, treated areas, and the details of heating to be determined for a wide range of conditions, for uniform tissues, simply by plugging in the appropriate physical property values and pulse parameters such as electrode spacing, size, and pulse amplitude. Complexities, such as heterogeneous tissues and changes in conductivity due to electroporation, are also discussed. The synthesis of these details can be used directly by surgeons in treatment planning. Irreversible electroporation is a promising new technique to treat cancer in a targeted manner without the use of drugs; however, it does require a detailed understanding of how electric currents flow within biological tissues. By providing the understanding and tools necessary to design an IRE protocol, this study seeks to facilitate the translation of this new and exciting cancer therapy into clinical practice.

Differential inertial focusing of particles in curved low-aspect-ratio microchannels

Microfluidic-based manipulation of particles is of great interest due to the insight it provides into the physics of hydrodynamic forces. Here, we study a particle-size-dependent phenomenon based on differential inertial focusing that utilizes the flow characteristics of curved, low aspect ratio (channel width ≫ height), microfluidic channels. We report the emergence of two focusing points along the height of the channel (z-plane), where different sized particles are focused and ordered in evenly spaced trains at correspondingly different lateral positions within the channel cross-section. We applied the system for continuous ordering and separation of suspension particles.

A review of the theory, methods and recent applications of high-throughput single-cell droplet microfluidics

Most cell biology experiments are performed in bulk cell suspensions where cell secretions become diluted and mixed in a contiguous sample. Confinement of single cells to small, picoliter-sized droplets within a continuous phase of oil provides chemical isolation of each cell, creating individual microreactors where rare cell qualities are highlighted and otherwise undetectable signals can be concentrated to measurable levels. Recent work in microfluidics has yielded methods for the encapsulation of cells in aqueous droplets and hydrogels at kilohertz rates, creating the potential for millions of parallel single-cell experiments. However, commercial applications of high-throughput microdroplet generation and downstream sensing and actuation methods are still emerging for cells. Using fluorescence-activated cell sorting (FACS) as a benchmark for commercially available high-throughput screening, this focused review discusses the fluid physics of droplet formation, methods for cell encapsulation in liquids and hydrogels, sensors and actuators and notable biological applications of high-throughput single-cell droplet microfluidics.

Differential effect of three-repeat and four-repeat tau on mitochondrial axonal transport

Tau protein is present in six different splice forms in the human brain and interacts with microtubules via either 3 or 4 microtubule binding repeats. An increased ratio of 3 repeat to 4 repeat isoforms is associated with neurodegeneration in inherited forms of frontotemporal dementia. Tau over‐expression diminishes axonal transport in several systems, but differential effects of 3 repeat and 4 repeat isoforms have not been studied. We examined the effects of tau on mitochondrial transport and found that both 3 repeat and 4 repeat tau change normal mitochondrial distribution within the cell body and reduce mitochondrial localization to axons; 4 repeat tau has a greater effect than 3 repeat tau. Further, we observed that the 3 repeat and 4 repeat tau cause different alterations in retrograde and anterograde transport dynamics with 3 repeat tau having a slightly stronger effect on axon transport dynamics. Our results indicate that tau‐induced changes in axonal transport may be an underlying theme in neurodegenerative diseases associated with isoform specific changes in tau’s interaction with microtubules.

Nucleation and solidification in static arrays of monodisperse drops

The precise measurement of nucleation and non-equilibrium solidification are vital to fields as diverse as atmospheric science, food processing, cryopreservation and metallurgy. The emulsion technique, where the phase under study is partitioned into many droplets suspended within an immiscible continuous phase, is a powerful method for uncovering rates of nucleation and dynamics of phase changes as it isolates nucleation events to single droplets. However, averaging the behavior of many drops in a bulk emulsion leads to the loss of any drop-specific information, and drop polydispersity clouds the analysis. Here we adapt a microfluidic technique for trapping monodisperse drops in planar arrays to characterize solidification of highly supercooled aqueous solutions of glycerol. This system measured rates of nucleation between 10(-5) and 10(-2) pL(-1) s(-1), yielded an ice-water interfacial energy of 33.4 mJ m(-2) between -38 and -35 degrees C, and enabled the specific dynamics of solidification to be observed for over a hundred drops in parallel without any loss of specificity. In addition to the physical insights gained, the ability to observe the time and temperature of nucleation and subsequent growth of the solid phase in static arrays of uniform drops provides a powerful tool to discover thermodynamic protocols that generate desirable crystal structures.

Visualization of microscale particle focusing in diluted and whole blood using particle trajectory analysis

Inertial microfluidics has demonstrated the potential to provide a rich range of capabilities to manipulate biological fluids and particles to address various challenges in biomedical science and clinical medicine. Various microchannel geometries have been used to study the inertial focusing behavior of particles suspended in simple buffer solutions or in highly diluted blood. One aspect of inertial focusing that has not been studied is how particles suspended in whole or minimally diluted blood respond to inertial forces in microchannels. The utility of imaging techniques (i.e., high-speed bright-field imaging and long exposure fluorescence (streak) imaging) primarily used to observe particle focusing in microchannels is limited in complex fluids such as whole blood due to interference from the large numbers of red blood cells (RBCs). In this study, we used particle trajectory analysis (PTA) to observe the inertial focusing behavior of polystyrene beads, white blood cells, and PC-3 prostate cancer cells in physiological saline and blood. Identification of in-focus (fluorescently labeled) particles was achieved at mean particle velocities of up to 1.85 m s(-1). Quantitative measurements of in-focus particles were used to construct intensity maps of particle frequency in the channel cross-section and scatter plots of particle centroid coordinates vs. particle diameter. PC-3 cells spiked into whole blood (HCT = 45%) demonstrated a novel focusing mode not observed in physiological saline or diluted blood. PTA can be used as an experimental frame of reference for understanding the physical basis of inertial lift forces in whole blood and discover inertial focusing modes that can be used to enable particle separation in whole blood.

Microfluidic Isolation of Circulating Tumor Cell Clusters by Size and Asymmetry

Circulating tumor cell clusters (CTC clusters) are potent initiators of metastasis and potentially useful clinical markers for patients with cancer. Although there are numerous devices developed to isolate individual circulating tumor cells from blood, these devices are ineffective at capturing CTC clusters, incapable of separating clusters from single cells and/or cause cluster damage or dissociation during processing. The only device currently able to specifically isolate CTC clusters from single CTCs and blood cells relies on the batch immobilization of clusters onto micropillars which necessitates long residence times and causes damage to clusters during release. Here, we present a two-stage continuous microfluidic chip that isolates and recovers viable CTC clusters from blood. This approach uses deterministic lateral displacement to sort clusters by capitalizing on two geometric properties: size and asymmetry. Cultured breast cancer CTC clusters containing between 2–100 + cells were recovered from whole blood using this integrated two-stage device with minimal cluster dissociation, 99% recovery of large clusters, cell viabilities over 87% and greater than five-log depletion of red blood cells. This continuous-flow cluster chip will enable further studies examining CTC clusters in research and clinical applications.

High-throughput co-encapsulation of self-ordered cell trains: cell pair interactions in microdroplets

Droplet microfluidics is a booming sub-field of microfluidics that adds the benefits of confinement, including signal accumulation and isolation, to cell analysis. However, controlling the number of cells per droplet has been limited to using Poisson (random) encapsulation for the highest throughputs. The Poisson probability of a droplet containing one and only one cell is limited to 36.8%, and the probability of pairing two distinct cell types in a droplet is limited to 13.5%. Combining droplet microfluidics with inertial microfluidics, we present a device which efficiently co-encapsulates cell pairs in droplets at rates on the order of 6 kHz. We demonstrate particle co-encapsulation where 64% of droplets contained the correct one-to-one pairing, representing a nearly fivefold improvement to Poisson co-encapsulation. We also apply the device to encapsulate two separate strains of Chlamydomonas reinhardtii. C. reinhardtii is a single-cell microalgae with applications as a model organism, recombinant protein source, and potential source of multiple biofuels. After inducing gametogenesis by nitrogen starvation and thermally inducing flagella loss, we co-encapsulate separate mating-type plus (mt+) and mating-type minus (mt−) C. reinhardtii cells in droplets. Here, 29% of droplets contained one and only one cell of each mating type, over a twofold improvement to the Poisson co-encapsulation probability of 13%. Approximately one hour following deflagellation, gametes regained flagellar motility and mating ability within the droplets. The mated zygotes were stored in emulsion form without nutrient replenishment. After 17 days, both zygospores and, remarkably, some unmated gametes remained viable. When the emulsion was broken and plated on full-nutrient agar, zygospore germination, tetrad hatching, and then mitosis followed. In addition to algae, the device has the potential for confined interaction studies for a variety of cell types.