Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting.

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 µm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument.

High-throughput single-microparticle imaging flow analyzer.

Optical microscopy is one of the most widely used diagnostic methods in scientific, industrial, and biomedical applications. However, while useful for detailed examination of a small number (< 10,000) of microscopic entities, conventional optical microscopy is incapable of statistically relevant screening of large populations (> 100,000,000) with high precision due to its low throughput and limited digital memory size. We present an automated flow-through single-particle optical microscope that overcomes this limitation by performing sensitive blur-free image acquisition and nonstop real-time image-recording and classification of microparticles during high-speed flow. This is made possible by integrating ultrafast optical imaging technology, self-focusing microfluidic technology, optoelectronic communication technology, and information technology. To show the system's utility, we demonstrate high-throughput image-based screening of budding yeast and rare breast cancer cells in blood with an unprecedented throughput of 100,000 particles/s and a record false positive rate of one in a million.

Magnetic microparticle concentration and collection using a mechatronic magnetic ratcheting system.

Magnetic ratcheting cytometry is a promising approach to separate magnetically-labeled cells and magnetic particles based on the quantity of magnetic material. We have previously reported on the ability of this technique to separate magnetically-labeled cells. Here, with a new chip design, containing high aspect ratio permalloy micropillar arrays, we demonstrate the ability of this technique to rapidly concentrate and collect superparamagnetic iron oxide particles. The platform consists of a mechatronic wheel used to generate and control a cycling external magnetic field that impinges on a "ratcheting chip." The ratcheting chip is created by electroplating a 2D array of high aspect ratio permalloy micropillars onto a glass slide, which is embedded in a thin polymer layer to create a planar surface above the micropillars. By varying magnetic field frequency and direction through wheel rotation rate and angle, we direct particle movement on chip. We explore the operating conditions for this system, identifying the effects of varying ratcheting frequency, along with time, on the dynamics and resulting concentration of these magnetic particles. We also demonstrate the ability of the system to rapidly direct the movement of superparamagnetic iron oxide particles of varying sizes. Using this technique, 2.8 µm, 500 nm, and 100 nm diameter superparamagnetic iron oxide particles, suspended within an aqueous fluid, were concentrated. We further define the ability of the system to concentrate 2.8 µm superparamagnetic iron oxide particles, present in a liquid suspension, into a small chip surface area footprint, achieving a 100-fold surface area concentration, and achieving a concentration factor greater than 200%. The achieved concentration factor of greater than 200% could be greatly increased by reducing the amount of liquid extracted at the chip outlet, which would increase the ability of achieving highly sensitive downstream analytical techniques. Magnetic ratcheting-based enrichment may be useful in isolating and concentrating subsets of magnetically-labeled cells for diagnostic automation.

Continuous and Quantitative Purification of T-Cell Subsets for Cell Therapy Manufacturing Using Magnetic Ratcheting Cytometry.

T-cell-based immunotherapies represent a growing medical paradigm that has the potential to revolutionize contemporary cancer treatments. However, manufacturing bottlenecks related to the enrichment of therapeutically optimal T-cell subpopulations from leukopak samples impede scale-up and scale-out efforts. This is mainly attributed to the challenges that current cell purification platforms face in balancing the quantitative sorting capacity needed to isolate specific T-cell subsets with the scalability to meet manufacturing throughputs. In this work, we report a continuous-flow, quantitative cell enrichment platform based on a technique known as ratcheting cytometry that can perform complex, multicomponent purification targeting various subpopulations of magnetically labeled T cells directly from apheresis or peripheral blood mononuclear cell (PBMC) samples. The integrated ratcheting cytometry instrument and cartridge demonstrated enrichment of T cells directly from concentrated apheresis samples with a 97% purity and an 85% recovery of magnetically tagged cells. Magnetic sorting of different T-cell subpopulations was also accomplished on chip by multiplexing cell surface targets onto particles with differing magnetic strengths. We believe that ratcheting cytometry's quantitative capacity and throughput scalability represents an excellent technology candidate to alleviate cell therapy manufacturing bottlenecks.

Correction: Unsupervised capture and profiling of rare immune cells using multi-directional magnetic ratcheting.

Correction for 'Unsupervised capture and profiling of rare immune cells using multi-directional magnetic ratcheting' by Coleman Murray et al., Lab Chip, 2018, 18, 2396-2409.

Unsupervised capture and profiling of rare immune cells using multi-directional magnetic ratcheting.

Immunotherapies (IT) require induction, expansion, and maintenance of specific changes to a patient's immune cell repertoire which yield a therapeutic benefit. Recently, mechanistic understanding of these changes at the cellular level has revealed that IT results in complex phenotypic transitions in target cells, and that therapeutic effectiveness may be predicted by monitoring these transitions during therapy. However, monitoring will require unique tools that enable capture, manipulation, and profiling of rare immune cell populations. In this study, we introduce a method of automated and unsupervised separation and processing of rare immune cells, using high-force and multidimensional magnetic ratcheting (MR). We demonstrate capture of target immune cells using samples with up to 1 : 10 000 target cell to background cell ratios from input volumes as small as 25 microliters (i.e. a low volume and low cell frequency sample sparing assay interface). Cell capture is shown to achieve up to 90% capture efficiency and purity, and captured cell analysis is shown using both on-chip culture/activity assays and off-chip ejection and nucleic acid analysis. These results demonstrate that multi-directional magnetic ratcheting offers a unique separation system for dealing with blood cell samples that contain either rare cells or significantly small volumes, and the "sample sparing" capability leads to an expanded spectrum of parameters that can be measured. These tools will be paramount to advancing techniques for immune monitoring under conditions in which both the sample volume and number of antigen-specific target cells are often exceedingly small, including during IT and treatment of allergy, asthma, autoimmunity, immunodeficiency, cell based therapy, transplantation, and infection.

Modulating motility of intracellular vesicles in cortical neurons with nanomagnetic forces on-chip.

Vesicle transport is a major underlying mechanism of cell communication. Inhibiting vesicle transport in brain cells results in blockage of neuronal signals, even in intact neuronal networks. Modulating intracellular vesicle transport can have a huge impact on the development of new neurotherapeutic concepts, but only if we can specifically interfere with intracellular transport patterns. Here, we propose to modulate motion of intracellular lipid vesicles in rat cortical neurons based on exogenously bioconjugated and cell internalized superparamagnetic iron oxide nanoparticles (SPIONs) within microengineered magnetic gradients on-chip. Upon application of 6-126 pN on intracellular vesicles in neuronal cells, we explored how the magnetic force stimulus impacts the motion pattern of vesicles at various intracellular locations without modulating the entire cell morphology. Altering vesicle dynamics was quantified using, mean square displacement, a caging diameter and the total traveled distance. We observed a de-acceleration of intercellular vesicle motility, while applying nanomagnetic forces to cultured neurons with SPIONs, which can be explained by a decrease in motility due to opposing magnetic force direction. Ultimately, using nanomagnetic forces inside neurons may permit us to stop the mis-sorting of intracellular organelles, proteins and cell signals, which have been associated with cellular dysfunction. Furthermore, nanomagnetic force applications will allow us to wirelessly guide axons and dendrites by exogenously using permanent magnetic field gradients.

Induction of Calcium Influx in Cortical Neural Networks by Nanomagnetic Forces.

Nanomagnetic force stimulation with ferromagnetic nanoparticles was found to trigger calcium influx in cortical neural networks without observable cytotoxicity. Stimulated neural networks showed an average of 20% increment in calcium fluorescence signals and a heightened frequency in calcium spiking. These effects were also confined spatially to areas with engineered high magnetic field gradients. Furthermore, blockage of N-type calcium channels inhibited the stimulatory effects of the nanomagnetic forces, suggesting the role of mechano-sensitive ion channels in mediating calcium influx.

Research highlights: microfluidic analysis of antimicrobial susceptibility.

Antimicrobials remain an integral part of the treatment of patients with an infection. New microfluidic technologies are poised to help clinicians prescribe the right antimicrobials, sooner, reducing long hospital stays and improving outcomes. Given that current microbiologic diagnostic testing methods require a significant turnaround time (days), clinicians, in general, initially empirically determine a suitable therapy. After review of laboratory data, including information regarding the susceptibility of the microbial pathogen to specific anti-infectives, a clinician will then make alterations in therapy as appropriate, to direct therapy toward the pathogen involved in the illness. Important steps needed to quickly ascertain this information include the timely isolation of the microorganism, followed by direct antibiotic susceptibility tests (ASTs) or determination of the presence within the microorganism of any resistance genes or proteins that will impair the activity of a potential therapy. Recent microfluidic technologies highlighted here that can intrinsically interface at the scale of the organisms are starting to address these challenges by improving the speed and accuracy of tests aimed at helping physicians to give the right antimicrobials sooner.

Flexible and stretchable micromagnet arrays for tunable biointerfacing.

A process to surface pattern polydimethylsiloxane (PDMS) with ferromagnetic structures of varying sizes (micrometer to millimeter) and thicknesses (>70 µm) is developed. Their flexibility and magnetic reach are utilized to confer dynamic, additive properties to a variety of substrates, such as coverslips and Eppendorf tubes. It is found that these substrates can generate additional modes of magnetic droplet manipulation, and can tunably steer magnetic-cell organization.

Engineering cortical neuron polarity with nanomagnets on a chip.

Intra- and extracellular signaling play critical roles in cell polarity, ultimately leading to the development of functional cell-cell connections, tissues, and organs. In the brain, pathologically oriented neurons are often the cause for disordered circuits, severely impacting motor function, perception, and memory. Aside from control through gene expression and signaling pathways, it is known that nervous system development can be manipulated by mechanical stimuli (e.g., outgrowth of axons through externally applied forces). The inverse is true as well: intracellular molecular signals can be converted into forces to yield axonal outgrowth. The complete role played by mechanical signals in mediating single-cell polarity, however, remains currently unclear. Here we employ highly parallelized nanomagnets on a chip to exert local mechanical stimuli on cortical neurons, independently of the amount of superparamagnetic nanoparticles taken up by the cells. The chip-based approach was utilized to quantify the effect of nanoparticle-mediated forces on the intracellular cytoskeleton as visualized by the distribution of the microtubule-associated protein tau. While single cortical neurons prefer to assemble tau proteins following poly-L-lysine surface cues, an optimal force range of 4.5-70 pN by the nanomagnets initiated a tau distribution opposed to the pattern cue. In larger cell clusters (groups comprising six or more cells), nanoparticle-mediated forces induced tau repositioning in an observed range of 190-270 pN, and initiation of magnetic field-directed cell displacement was observed at forces above 300 pN. Our findings lay the groundwork for high-resolution mechanical encoding of neural networks in vitro, mechanically driven cell polarization in brain tissues, and neurotherapeutic approaches using functionalized superparamagnetic nanoparticles to potentially restore disordered neural circuits.

Research highlights: printing the future of microfabrication.

In this issue we highlight emerging microfabrication approaches suitable for microfluidic systems with a focus on "additive manufacturing" processes (i.e. printing). In parallel with the now-wider availability of low cost consumer-grade 3D printers (as evidenced by at least three brands of 3D printers for sale in a recent visit to an electronics store in Akihabara, Tokyo), commercial-grade 3D printers are ramping to higher and higher resolution with new capabilities, such as printing of multiple materials of different transparency, and with different mechanical and electrical properties. We highlight new work showing that 3D printing (stereolithography approaches in particular) has now risen as a viable technology to print whole microfluidic devices. Printing on 2D surfaces such as paper is an everyday experience, and has been used widely in analytical chemistry for printing conductive materials on paper strips for glucose and other electrochemical sensors. We highlight recent work using electrodes printed on paper for digital microfluidic droplet actuation. Finally, we highlight recent work in which printing of membrane-bound droplets that interconnect through bilayer membranes may open up an entirely new approach to microfluidic manufacturing of soft devices that mimic physiological systems.

Rapid prototyping polymers for microfluidic devices and high pressure injections.

Multiple methods of fabrication exist for microfluidic devices, with different advantages depending on the end goal of industrial mass production or rapid prototyping for the research laboratory. Polydimethylsiloxane (PDMS) has been the mainstay for rapid prototyping in the academic microfluidics community, because of its low cost, robustness and straightforward fabrication, which are particularly advantageous in the exploratory stages of research. However, despite its many advantages and its broad use in academic laboratories, its low elastic modulus becomes a significant issue for high pressure operation as it leads to a large alteration of channel geometry. Among other consequences, such deformation makes it difficult to accurately predict the flow rates in complex microfluidic networks, change flow speed quickly for applications in stop-flow lithography, or to have predictable inertial focusing positions for cytometry applications where an accurate alignment of the optical system is critical. Recently, other polymers have been identified as complementary to PDMS, with similar fabrication procedures being characteristic of rapid prototyping but with higher rigidity and better resistance to solvents; Thermoset Polyester (TPE), Polyurethane Methacrylate (PUMA) and Norland Adhesive 81 (NOA81). In this review, we assess these different polymer alternatives to PDMS for rapid prototyping, especially in view of high pressure injections with the specific example of inertial flow conditions. These materials are compared to PDMS, for which magnitudes of deformation and dynamic characteristics are also characterized. We provide a complete and systematic analysis of these materials with side-by-side experiments conducted in our lab that also evaluate other properties, such as biocompatibility, solvent compatibility, and ease of fabrication. We emphasize that these polymer alternatives, TPE, PUMA and NOA, have some considerable strengths for rapid prototyping when bond strength, predictable operation at high pressure, or transitioning to commercialization are considered important for the application.