Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product.

Circulating Tumor Cells (CTCs), interrogated by sampling blood from patients with cancer, contain multiple analytes, including intact RNA, high molecular weight DNA, proteins, and metabolic markers. However, the clinical utility of tumor cell-based liquid biopsy has been limited since CTCs are very rare, and current technologies cannot process the blood volumes required to isolate a sufficient number of tumor cells for in-depth assays. We previously described a high-throughput microfluidic prototype utilizing high-flow channels and amplification of cell sorting forces through magnetic lenses. Here, we apply this technology to analyze patient-derived leukapheresis products, interrogating a mean blood volume of 5.83 liters from patients with metastatic cancer, with a median of 2,799 CTCs purified per patient. Isolation of many CTCs from individual patients enables characterization of their morphological and molecular heterogeneity, including cell and nuclear size and RNA expression. It also allows robust detection of gene copy number variation, a definitive cancer marker with potential diagnostic applications. High-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies.

A microfluidic transistor for automatic control of liquids.

Microfluidics have enabled notable advances in molecular biology1,2, synthetic chemistry3,4, diagnostics5,6 and tissue engineering7. However, there has long been a critical need in the field to manipulate fluids and suspended matter with the precision, modularity and scalability of electronic circuits8-10. Just as the electronic transistor enabled unprecedented advances in the automatic control of electricity on an electronic chip, a microfluidic analogue to the transistor could enable improvements in the automatic control of reagents, droplets and single cells on a microfluidic chip. Previous works on creating a microfluidic analogue to the electronic transistor11-13 did not replicate the transistor's saturation behaviour, and could not achieve proportional amplification14, which is fundamental to modern circuit design15. Here we exploit the fluidic phenomenon of flow limitation16 to develop a microfluidic element capable of proportional amplification with flow-pressure characteristics completely analogous to the current-voltage characteristics of the electronic transistor. We then use this microfluidic transistor to directly translate fundamental electronic circuits into the fluidic domain, including the amplifier, regulator, level shifter, logic gate and latch. We also combine these building blocks to create more complex fluidic controllers, such as timers and clocks. Finally, we demonstrate a particle dispenser circuit that senses single suspended particles, performs signal processing and accordingly controls the movement of each particle in a deterministic fashion without electronics. By leveraging the vast repertoire of electronic circuit design, microfluidic-transistor-based circuits enable fluidic automatic controllers to manipulate liquids and single suspended particles for lab-on-a-chip platforms.

A Microfluidic Transistor for Liquid Signal Processing.

Microfluidics have enabled significant advances in molecular biology 1-3 , synthetic chemistry 4,5 , diagnostics 6,7 , and tissue engineering 8 . However, there has long been a critical need in the field to manipulate fluids and suspended matter with the precision, modularity, and scalability of electronic circuits 9-11 . Just as the electronic transistor enabled unprecedented advances in the control of electricity on an electronic chip, a microfluidic analogue to the transistor could enable improvements in the complex, scalable control of reagents, droplets, and single cells on an autonomous microfluidic chip. Prior works on creating a microfluidic analogue to the electronic transistor 12-14 could not replicate the transistor's saturation behavior, which is crucial to perform analog amplification 15 and is fundamental to modern circuit design 16 . Here we exploit the fluidic phenomenon of flow-limitation 17 to develop a microfluidic element with flow-pressure characteristics completely analogous to the current-voltage characteristics of the electronic transistor. As this microfluidic transistor successfully replicates all of the key operating regimes of the electronic transistor (linear, cut-off and saturation), we are able to directly translate a variety of fundamental electronic circuit designs into the fluidic domain, including the amplifier, regulator, level shifter, logic gate, and latch. Finally, we demonstrate a "smart" particle dispenser that senses single suspended particles, performs liquid signal processing, and accordingly controls the movement of said particles in a purely fluidic system without electronics. By leveraging the vast repertoire of electronic circuit design, microfluidic transistor-based circuits are easy to integrate at scale, eliminate the need for external flow control, and enable uniquely complex liquid signal processing and single-particle manipulation for the next generation of chemical, biological, and clinical platforms.

Targeting breast and pancreatic cancer metastasis using a dual-cadherin antibody.

The successful application of antibody-based therapeutics in either primary or metastatic cancer depends upon the selection of rare cell surface epitopes that distinguish cancer cells from surrounding normal epithelial cells. By contrast, as circulating tumor cells (CTCs) transit through the bloodstream, they are surrounded by hematopoietic cells with dramatically distinct cell surface proteins, greatly expanding the number of targetable epitopes. Here, we show that an antibody (23C6) against cadherin proteins effectively suppresses blood-borne metastasis in mouse isogenic and xenograft models of triple negative breast and pancreatic cancers. The 23C6 antibody is remarkable in that it recognizes both the epithelial E-cadherin (CDH1) and mesenchymal OB-cadherin (CDH11), thus overcoming considerable heterogeneity across tumor cells. Despite its efficacy against single cells in circulation, the antibody does not suppress primary tumor formation, nor does it elicit detectable toxicity in normal epithelial organs, where cadherins may be engaged within intercellular junctions and hence inaccessible for antibody binding. Antibody-mediated suppression of metastasis is comparable in matched immunocompetent and immunodeficient mouse models. Together, these studies raise the possibility of antibody targeting CTCs within the vasculature, thereby suppressing blood-borne metastasis.

Isolation of circulating tumor cells.

Circulating tumor cells (CTCs) enter the vasculature from solid tumors and disseminate widely to initiate metastases. Mining the metastatic-enriched molecular signatures of CTCs before, during, and after treatment holds unique potential in personalized oncology. Their extreme rarity, however, requires isolation from large blood volumes at high yield and purity, yet they overlap leukocytes in size and other biophysical properties. Additionally, many CTCs lack EpCAM that underlies much of affinity-based capture, complicating their separation from blood. Here, we provide a comprehensive introduction of CTC isolation technology, by analyzing key separation modes and integrated isolation strategies. Attention is focused on recent progress in microfluidics, where an accelerating evolution is occurring in high-throughput sorting of cells along multiple dimensions.

Negative-Selection Enrichment of Circulating Tumor Cells from Peripheral Blood Using the Microfluidic CTC-iChip.

The ability to isolate and analyze rare circulating tumor cells (CTCs) holds the potential to increase our understanding of cancer evolution and allows monitoring of disease and therapeutic responses through a relatively non-invasive blood-based biopsy. While many methods have been described to isolate CTCs from the blood, the vast majority rely on size-based sorting or positive selection of CTCs based on surface markers, which introduces bias into the downstream product by making assumptions about these heterogenous cells. Here we describe a negative-selection protocol for enrichment of CTCs through removal of blood components including red blood cells, platelets, and white blood cells. This procedure results in a product that is amenable to downstream single-cell analytics including RNA-Seq, ATAC-Seq and DNA methylation, droplet digital PCR (ddPCR) for tumor specific transcripts, staining and extensive image analysis, and ex vivo culture of patient-derived CTCs.

Microfluidic capture of chromatin fibres measures neutrophil extracellular traps (NETs) released in a drop of human blood.

Neutrophils are the largest population of white blood cells in the circulation, and their primary function is to protect the body from microbes. They can release the chromatin in their nucleus, forming characteristic web structures and trap microbes, contributing to antimicrobial defenses. The chromatin webs are known as neutrophil extracellular traps (NETs). Importantly, neutrophils can also release NETs in pathological conditions related to rheumatic diseases, atherosclerosis, cancer, and sepsis. Thus, determining the concentration of NETs in the blood is increasingly important for monitoring patients, evaluating treatment efficacy, and understanding the pathology of various diseases. However, traditional methods for measuring NETs require separating cells and plasma from blood, are prone to sample preparation artifacts, and cannot distinguish between intact and degraded NETs. Here, we design a microfluidic analytical tool that captures NETs mechanically from a drop of blood and measures the amount of intact NETs unbiased by the presence of degraded NETs in the sample.

Ultrahigh-throughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells.

Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.

In-flow measurement of cell-cell adhesion using oscillatory inertial microfluidics.

Multicellular clusters in circulation can exhibit a substantially different function and biomarker significance compared to individual cells. Notably, clusters of circulating tumor cells (CTCs) are much more effective initiators of metastasis than single CTCs, and correlate with worse patient prognoses. Measuring the cell-cell adhesion strength of CTC clusters is a critical step towards understanding their subsistence in the circulation and mechanism of elevated tumorigenicity. However, measuring cell-cell adhesion forces in flow is elusive using existing methods. Here, we report an oscillatory inertial microfluidics system which exerts a repeating fluidic force profile on suspended cell doublets to determine their cell-cell adhesion strength (Fs), without any biophysical modifications to the cell surface and physiological morphology. Using our system, we analyzed a large number (N > 500) of doublets from a patient-derived breast cancer CTC line. We discovered that the cell-cell adhesion strength of CTC doublets varied almost 20-fold between the weakly adhered (Fs < 28 nN) and strongly bound subpopulations (Fs > 542 nN). Our system can be used with other cancer or noncancer cells without restrictions, and may be used for rapid screening of drugs aiming to disrupt the highly-metastatic CTC clusters in circulation.

Microfluidic concentration and separation of circulating tumor cell clusters from large blood volumes.

Circulating tumor cells (CTCs) are extremely rare in the blood, yet they account for metastasis. Notably, it was reported that CTC clusters (CTCCs) can be 50-100 times more metastatic than single CTCs, making them particularly salient as a liquid biopsy target. Yet they can split apart and are even rarer, complicating their recovery. Isolation by filtration risks loss when clusters squeeze through filter pores over time, and release of captured clusters can be difficult. Deterministic lateral displacement is continuous but requires channels not much larger than clusters, leading to clogging. Spiral inertial focusing requires large blood dilution factors (or lysis). Here, we report a microfluidic chip that continuously isolates untouched CTC clusters from large volumes of minimally (or undiluted) whole blood. An array of 100 µm-wide channels first concentrates clusters in the blood, and then a similar array transfers them into a small volume of buffer. The microscope-slide-sized PDMS device isolates individually-spiked CTC clusters from >30 mL per hour of whole blood with 80% efficiency into enumeration (fluorescence imaging), and on-chip yield approaches 100% (high speed video). Median blood cell removal (in base-10 logs) is 4.2 for leukocytes, 5.5 for red blood cells, and 4.9 for platelets, leaving less than 0.01% of leukocytes alongside CTC clusters in the product. We also demonstrate that cluster configurations are preserved. Gentle, high throughput concentration and separation of circulating tumor cell clusters from large blood volumes will enable cluster-specific diagnostics and speed the generation of patient-specific CTC cluster lines.

Oscillatory inertial focusing in infinite microchannels.

Inertial microfluidics (i.e., migration and focusing of particles in finite Reynolds number microchannel flows) is a passive, precise, and high-throughput method for microparticle manipulation and sorting. Therefore, it has been utilized in numerous biomedical applications including phenotypic cell screening, blood fractionation, and rare-cell isolation. Nonetheless, the applications of this technology have been limited to larger bioparticles such as blood cells, circulating tumor cells, and stem cells, because smaller particles require drastically longer channels for inertial focusing, which increases the pressure requirement and the footprint of the device to the extent that the system becomes unfeasible. Inertial manipulation of smaller bioparticles such as fungi, bacteria, viruses, and other pathogens or blood components such as platelets and exosomes is of significant interest. Here, we show that using oscillatory microfluidics, inertial focusing in practically "infinite channels" can be achieved, allowing for focusing of micron-scale (i.e. hundreds of nanometers) particles. This method enables manipulation of particles at extremely low particle Reynolds number (Rep < 0.005) flows that are otherwise unattainable by steady-flow inertial microfluidics (which has been limited to Rep > ∼10-1). Using this technique, we demonstrated that synthetic particles as small as 500 nm and a submicron bacterium, Staphylococcus aureus, can be inertially focused. Furthermore, we characterized the physics of inertial microfluidics in this newly enabled particle size and Rep range using a Peclet-like dimensionless number (α). We experimentally observed that α >> 1 is required to overcome diffusion and be able to inertially manipulate particles.

Anti-thrombotic strategies for microfluidic blood processing.

The redundant mechanisms involved in blood coagulation are crucial for rapid hemostasis. Yet they also create challenges in blood processing in medical devices and lab-on-a-chip systems. In this work, we investigate the effects of both shear stress and hypothermic blood storage on thrombus formation in microfluidic processing. For fresh blood, thrombosis occurs only at high shear, and the glycoprotein IIb/IIIa inhibitor tirofiban is highly effective in preventing thrombus formation. Blood storage generally activates platelets and primes them towards thrombosis via multiple mechanisms. Thrombus formation of stored blood at low shear can be adequately inhibited by glycoprotein IIb/IIIa inhibitors. At high shear, von Willebrand factor-mediated thrombosis contributes significantly and requires additional treatments with thiol-containing antioxidants-such as N acetylcysteine and reduced glutathione-that interfere with von Willebrand factor polymerization. We further demonstrate the effectiveness of these anti-thrombotic strategies in microfluidic devices made of cyclic olefin copolymer, a popular material used in the healthcare industry. This work identifies effective anti-thrombotic strategies that are applicable in a wide range of blood- and organ-on-a-chip applications.

Non-equilibrium Inertial Separation Array for High-throughput, Large-volume Blood Fractionation.

Microfluidic blood processing is used in a range of applications from cancer therapeutics to infectious disease diagnostics. As these applications are being translated to clinical use, processing larger volumes of blood in shorter timescales with high-reliability and robustness is becoming a pressing need. In this work, we report a scaled, label-free cell separation mechanism called non-equilibrium inertial separation array (NISA). The NISA mechanism consists of an array of islands that exert a passive inertial lift force on proximate cells, thus enabling gentler manipulation of the cells without the need of physical contact. As the cells follow their size-based, deterministic path to their equilibrium positions, a preset fraction of the flow is siphoned to separate the smaller cells from the main flow. The NISA device was used to fractionate 400 mL of whole blood in less than 3 hours, and produce an ultrapure buffy coat (96.6% white blood cell yield, 0.0059% red blood cell carryover) by processing whole blood at 3 mL/min, or ∼300 million cells/second. This device presents a feasible alternative for fractionating blood for transfusion, cellular therapy and blood-based diagnostics, and could significantly improve the sensitivity of rare cell isolation devices by increasing the processed whole blood volume.

Monolithic Chip for High-throughput Blood Cell Depletion to Sort Rare Circulating Tumor Cells.

Circulating tumor cells (CTCs) are a treasure trove of information regarding the location, type and stage of cancer and are being pursued as both a diagnostic target and a means of guiding personalized treatment. Most isolation technologies utilize properties of the CTCs themselves such as surface antigens (e.g., epithelial cell adhesion molecule or EpCAM) or size to separate them from blood cell populations. We present an automated monolithic chip with 128 multiplexed deterministic lateral displacement devices containing ~1.5 million microfabricated features (12 µm-50 µm) used to first deplete red blood cells and platelets. The outputs from these devices are serially integrated with an inertial focusing system to line up all nucleated cells for multi-stage magnetophoresis to remove magnetically-labeled white blood cells. The monolithic CTC-iChip enables debulking of blood samples at 15-20 million cells per second while yielding an output of highly purified CTCs. We quantified the size and EpCAM expression of over 2,500 CTCs from 38 patient samples obtained from breast, prostate, lung cancers, and melanoma. The results show significant heterogeneity between and within single patients. Unbiased, rapid, and automated isolation of CTCs using monolithic CTC-iChip will enable the detailed measurement of their physicochemical and biological properties and their role in metastasis.

Bacterial Ice Nucleation in Monodisperse D2O and H2O-in-Oil Emulsions.

Ice nucleation is of fundamental significance in many areas, including atmospheric science, food technology, and cryobiology. In this study, we investigated the ice-nucleation characteristics of picoliter-sized drops consisting of different D2O and H2O mixtures with and without the ice-nucleating bacteria Pseudomonas syringae. We also studied the effects of commonly used cryoprotectants such as ethylene glycol, propylene glycol, and trehalose on the nucleation characteristics of D2O and H2O mixtures. The results show that the median freezing temperature of the suspension containing 1 mg/mL of a lyophilized preparation of P. syringae is as high as -4.6 °C for 100% D2O, compared to -8.9 °C for 100% H2O. As the D2O concentration increases every 25% (v/v), the profile of the ice-nucleation kinetics of D2O + H2O mixtures containing 1 mg/mL Snomax shifts by about 1 °C, suggesting an ideal mixing behavior of D2O and H2O. Furthermore, all of the cryoprotectants investigated in this study are found to depress the freezing phenomenon. Both the homogeneous and heterogeneous freezing temperatures of these aqueous solutions depend on the water activity and are independent of the nature of the solute. These findings enrich our fundamental knowledge of D2O-related ice nucleation and suggest that the combination of D2O and ice-nucleating agents could be a potential self-ice-nucleating formulation. The implications of self-nucleation include a higher, precisely controlled ice seeding temperature for slow freezing that would significantly improve the viability of many ice-assisted cryopreservation protocols.

Retina-on-a-chip: a microfluidic platform for point access signaling studies.

We report on a microfluidic platform for culture of whole organs or tissue slices with the capability of point access reagent delivery to probe the transport of signaling events. Whole mice retina were maintained for multiple days with negative pressure applied to tightly but gently bind the bottom of the retina to a thin poly-(dimethylsiloxane) membrane, through which twelve 100 µm diameter through-holes served as fluidic access points. Staining with toluidine blue, transport of locally applied cholera toxin beta, and transient response to lipopolysaccharide in the retina demonstrated the capability of the microfluidic platform. The point access fluidic delivery capability could enable new assays in the study of various kinds of excised tissues, including retina.

Inertio-elastic focusing of bioparticles in microchannels at high throughput.

Controlled manipulation of particles from very large volumes of fluid at high throughput is critical for many biomedical, environmental and industrial applications. One promising approach is to use microfluidic technologies that rely on fluid inertia or elasticity to drive lateral migration of particles to stable equilibrium positions in a microchannel. Here, we report on a hydrodynamic approach that enables deterministic focusing of beads, mammalian cells and anisotropic hydrogel particles in a microchannel at extremely high flow rates. We show that on addition of micromolar concentrations of hyaluronic acid, the resulting fluid viscoelasticity can be used to control the focal position of particles at Reynolds numbers up to Re≈10,000 with corresponding flow rates and particle velocities up to 50 ml min(-1) and 130 m s(-1). This study explores a previously unattained regime of inertio-elastic fluid flow and demonstrates bioparticle focusing at flow rates that are the highest yet achieved.

A microfluidic cell co-culture platform with a liquid fluorocarbon separator.

A microfluidic cell co-culture platform that uses a liquid fluorocarbon oil barrier to separate cells into different culture chambers has been developed. Characterization indicates that the oil barrier could be effective for multiple days, and a maximum pressure difference between the oil barrier and aqueous media in the cell culture chamber could be as large as ~3.43 kPa before the oil barrier fails. Biological applications have been demonstrated with the separate transfection of two groups of primary hippocampal neurons with two different fluorescent proteins and subsequent observation of synaptic contacts between the neurons. In addition, the quality of the fluidic seal provided by the oil barrier is shown to be greater than that of an alternative solid-PDMS valve barrier design by testing the ability of each device to block low molecular weight CellTracker dyes used to stain cells in the culture chambers.

High throughput single-cell and multiple-cell micro-encapsulation.

UNLABELLED: Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 µm polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency P(k=1) of 83.7% and a double-particle encapsulation efficiency P(k=2) of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. INTRODUCTION: Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies. The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes. Encapsulation of cells in drops has been utilized to improve detection of protein expression, antibodies, enzymes, and metabolic activity for high throughput screening, and could be used to improve high throughput cytometry. Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry and targeted surface cell coatings. Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells. To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge. For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Q(oil) and Q(aq). As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle. When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops D(k) containing k cells is dictated by Poisson statistics, where D(k) = λ(k) exp(-λ)/(k!) and λ is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using P(k) = (k x D(k))/Σ(k' x D(k)'). The subtle difference between the two metrics is that D(k) relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and P(k) relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low λ) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric P(k) would be high, the majority of drops would be empty (low D(k)), thus requiring a sorting mechanism to remove empty drops, also reducing throughput. Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number). Here, the Reynolds number Re =uD(h)/ν and particle Reynolds number Rep =Re(a/D(h))², where u is a characteristic flow velocity, D(h) [=2wh/(w+h)] is the hydraulic diameter, ν is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Re(p) increase. Note that the high Re and Re(p) requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity. However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells. Because very few studies address inter-particle train spacing, determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz, a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz. In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average. In previous controlled encapsulation work, the average number of particles per drop λ was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when λ is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

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.