High-throughput Microfluidic Device Research Articles

Cascaded nanozyme-based high-throughput microfluidic device integrating with glucometer and smartphone for point-of-care pheochromocytoma diagnosis

The development of point-of-care (POC) diagnostics devices for circulating tumor cells (CTCs) detection plays an important role in the early diagnosis of pheochromocytoma (PCC), especially in a low-resource setting. To further realize the rapid, portable, and high-throughput detection of CTCs, an Au@CuMOF cascade enzyme-based microfluidic device for instant point-of-care detection of CTCs was constructed by combining a smartphone application and a commercial portable glucose meter (PGM). In this microfluidic system, DOTA and norepinephrine (NE) modified Au@CuMOF signal probes and Fe3O4@SiO2 capture probes were used for the dual recognition and capture of rare PCC-CTCs. Then, the targeted binding of the Au@CuMOF cascade nanozymes to the CTCs endowed the cellular complexes with multienzyme mimetic activities (i.e., glucose oxidase-like and peroxidase-like activity) to catalyze glucose reduction as signal output for colorimetric and personal glucose meter (PGM) dual-mode detection of CTCs. The developed method has a linear range of 4 to 105 cells mL−1 and a detection limit of 3 cells mL−1. This method allows the simultaneous detection of six samples and demonstrates good applicability for CTCs detection in whole blood samples. More importantly, the combination of PGM, smartphone app and array microfluidic chips enables the rapid, portable, and high-throughput diagnoses of PCC, and providing provide a convenient and reliable alternative to traditional liquid biopsy diagnosis of various cancers.

High-resolution temporal profiling of E. coli transcriptional response

Understanding how cells dynamically adapt to their environment is a primary focus of biology research. Temporal information about cellular behavior is often limited by both small numbers of data time-points and the methods used to analyze this data. Here, we apply unsupervised machine learning to a data set containing the activity of 1805 native promoters in E. coli measured every 10 minutes in a high-throughput microfluidic device via fluorescence time-lapse microscopy. Specifically, this data set reveals E. coli transcriptome dynamics when exposed to different heavy metal ions. We use a bioinformatics pipeline based on Independent Component Analysis (ICA) to generate insights and hypotheses from this data. We discovered three primary, time-dependent stages of promoter activation to heavy metal stress (fast, intermediate, and steady). Furthermore, we uncovered a global strategy E. coli uses to reallocate resources from stress-related promoters to growth-related promoters following exposure to heavy metal stress.

A high-throughput microfluidic device based on controlled incremental filtration to enable centrifugation-free, low extracorporeal volume leukapheresis

Leukapheresis, the extracorporeal separation of white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), is a life-saving procedure used for treating patients with cancer and other conditions, and as the initial step in the manufacturing of cellular and gene-based therapies. Well-tolerated by adults, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of standard centrifugation-based machines represents a particularly large fraction of these patients’ total blood volume. Here we describe a novel high-throughput microfluidic device (with a void volume of 0.4 mL) based on controlled incremental filtration (CIF) technology that could replace centrifugation for performing leukapheresis. The CIF device was tested extensively using whole blood from healthy volunteers at multiple hematocrits (5–30%) and flow rates (10–30 mL/min). In the flow-through regime, the CIF device separated WBCs with > 85% efficiency and 10–15% loss of RBCs and PLTs while processing whole blood diluted with saline to 10% hematocrit at a flow rate of 10 mL/min. In the recirculation regime, the CIF device demonstrated a similar level of separation performance, virtually depleting WBCs in the recirculating blood (~ 98% reduction) by the end of a 3.5-hour simulated leukapheresis procedure. Importantly, the device operated without clogging or decline in separation performance, with minimal activation of WBCs and PLTs and no measurable damage to RBCs. Compared to the typical parameters of centrifugation-based leukapheresis, the CIF device had a void volume at least 100-fold smaller, removed WBCs about twice as fast, and lost ~ 2–3-fold fewer PLTs, while operating at a flow rate compatible with the current practice. The hematocrit and flow rate at which the CIF device operated were significantly higher than previously published for other microfluidic cell separation methods. Finally, this study is the first to demonstrate a highly efficient separation of cells from recirculating blood using a microfluidic device. Overall, these findings suggest the feasibility of using high-throughput microfluidic cell separation technology to ultimately enable centrifugation-free, low-ECV leukapheresis. Such a capability would be particularly useful in young children, a vulnerable group of patients who are currently underserved.

Abstract 6031: Microfluidics: Simulating the gut microbiome for early cancer detection

Abstract Background: Cancer is the second leading cause of death globally and ~39.5% of people will be diagnosed with cancer at some point during their lifetimes. Thus, there is an unmet need to identify novel strategies for early cancer detection and prevention. The emerging evidence suggests that the gut microbiome has a role in promoting cancer. This microbiome including bacteria plays a vital role in maintaining homeostasis in the body. An imbalance in bacterial composition may cause diseases including cancer. Here we developed a microfluidic chip that can accurately simulate the gut microbiome to test the effects of bacteria and therapies on cancer cells. Methods and Results: To test the causal effect of bacteria on cancer, we developed a new high-throughput microfluidic device for simulating the environment of the gut. Initially, we used the photolithography technique where we designed the chip in AutoCAD and fabricated using photoresist resins and Polydimethylsiloxane (PDMS). Next, we tested the effect of bacteria on the growth of colorectal cancer cells. For this, we cultured colorectal cancer cells (HCT-116) with lipopolysaccharide (LPS), which is found in the outer membrane of bacteria, as well as the Bacillus bacteria in our microfluidics. Our data show that both LPS and Bacillus significantly accelerate the growth of cancer cells 2.02 times (p value = 0.012) and 1.58 times (p value = 0.011), respectively, over a 4 day culture period. These results show that the increased presence of certain bacteria can promote cancer cell growth and that our chip can be used to test the specific correlation between bacteria and cancer cell growth. The previously described method was inefficient and time-consuming. To overcome this limitation, we designed a new chip that allows running 16 samples at once with improved efficiency and accuracy. The template of the device that had 16 microfluidic channels was printed by a 3D printer and used for PDMS replica molding. The PDMS device was attached to the modified multiwell plate to feed media to and collect waste from each channel in a high-throughput manner. In the initial design, the bacteria grew faster than cancer cells taking over the chips. Our new design has dual layered chambers to keep bacteria and cancer cells separated by a membrane, allowing only bacterial secretions to pass through the membrane to cancer cells, mimicking the human gut. The new design also allowed the chip to maintain continuous microfluidic flow and a hypoxic environment. Conclusion: Our research demonstrates that the new microfluidic device has broader implications including simulating other body organs such as the lung and liver, and testing the impact of viruses such as influenza and COVID-19 on human cells. This device can be used to test both the effect of bacteria and new treatment on clinical samples for the identification of personalized therapy, thus reducing the need for mouse model testing, which is a lengthy and expensive process. Citation Format: Ekansh Mittal, Young Bock Kang. Microfluidics: Simulating the gut microbiome for early cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6031.

A high-throughput microfluidic device inspired by the Wheatstone bridge principle for characterizing the mechanical properties of single cells.

The mechanical properties of single cells have been recognized as biomarkers for identifying individual cells and diagnosing human diseases. Microfluidic devices based on the flow cytometry principle, which are not limited by the vision field of a microscope and can achieve a very high throughput, have been extensively adopted to measure the mechanical properties of single cells. However, these kinds of microfluidic devices usually required pressure-driven pumps with a very low flow rate and high precision. In this study, we developed a high-throughput microfluidic device inspired by the Wheatstone bridge principle for characterizing the mechanical properties of single cells. The microfluidic analogue of the Wheatstone bridge not only took advantage of flow cytometry, but also allowed precise control of a very low flow rate through the constricted channel with a higher input flow rate generated by a commercially available pressure-driven pump. Under different input flow rates of the pump, the apparent elastic moduli and the fluidity of osteosarcoma (U-2OS) cells and cervical carcinoma (HeLa) cells were measured by monitoring their dynamic deformations passing through the bridge-channel with different sizes of rectangular constrictions. The results showed that the input flow rate had little effect on measuring the mechanical properties of the cells, while the ratio of cell radius to effective constriction radius was different, i.e., for U-2OS cells it was 1.20 and for HeLa cells it was 1.09. Under this condition compared with predecessors, our statistic results of cell mechanical properties exhibited minimal errors. Furthermore, the cell viability after measurements was kept above 90% that demonstrated the non-destructive property of our proposed method.

Towards a Closed System for Extracting Lymphocytes from Whole Blood

Introduction: Cellular therapies promise new treatment options and potentially curative efficacy for a wide array of acute and chronic diseases. The ability to obtain viable, uncontaminated lymphocyte concentrates from peripheral blood is in high demand. A major barrier for translating lymphocyte-based therapies into clinical practice is the poor scalability of manual, labor- and time-consuming ‘open system’ methods (e.g. Ficoll density gradient centrifugation) currently being used for isolating lymphocytes. To address the well-known limitations of existing separation methods, we developed a two-stage cell isolation approach that enables highly-efficient extraction of lymphocytes from whole blood (WB) without the use of centrifugation or any open-air manipulations by (1) depleting ~99% of red blood cells (RBCs) from the sample via rapid sedimentation induced by HES (6% hydroxyethyl starch in 0.9% sodium chloride), and then (2) passing the leukocyte-rich supernatant through a high-throughput microfluidic device (based on our newly-developed approach of ‘controlled incremental filtration,‘ CIF) to separate lymphocytes from platelets and residual RBCs.Materials and Methods: WB was collected from healthy consenting volunteers under an IRB approved protocol. An optimization study was performed to identify parameters of the sedimentation protocol with the highest lymphocyte yield and minimal contamination. This optimal protocol was then used to compare performance of (i) the standard Ficoll density gradient separation method, and (ii) our new two-stage separation approach. Each WB sample was divided into two equal parts - one half was processed via the conventional density gradient method, and the other half was first mixed with HES, allowed to sediment, and then the supernatant was passed through a CIF-based microfluidic device (designed for high-precision separation of lymphocytes from smaller cells, and ~15-fold reduction of the sample volume) at a flow rate of 0.5 mL/min. For both separation methods, product yield and purity were quantified by performing CBC with a 5-part differential on all samples. Flow cytometry was used to measure viability of isolated lymphocytes, and activation level of residual platelets via P-selectin and phosphatidylserine (PS) exposure. Separated lymphocytes were cultured for 4 days using standard proliferation techniques. Total number of viable cells was determined using Trypan Blue exclusion dye and manual counting with a hemocytometer.Results and Discussion: The optimized sedimentation protocol, which involved mixing WB with 10% HES (v/v) and waiting 30 min to complete, yielded the best combination of lymphocyte recovery (72.1±2.2%) and RBC depletion (99.48±0.07%) for the first stage of our separation approach. In the split-sample study, our two-stage method overall recovered 70.4±0.7% of lymphocytes from the initial WB sample, with 98.2±0.6% viability, while depleting 92.4±0.4% of platelets and 99.62±0.1% of RBCs. In comparison, the Ficoll density gradient centrifugation method removed 99.95±0.2% of RBCs and 94.2±0.1% of platelets, while recovering 74.5±2.40% of lymphocytes, with 98.3±0.7% viability. Lymphocytes produced by our method showed a significnatly higher prolifiration capacity than cells separated via density gradient centrifugation (Fig. 1). Markers of activation for residual platelets in the lymphocyte concentrate produced by our two-stage method were several fold lower than for Ficoll density gradient separation: 1.9±0.1% (our method) vs. 8.4±7.0% (Ficoll) for P-selectin, and 2.0±1.5% (our method) vs. 4.4±1.5% (Ficoll) for PS exposure. Importantly, lymphocyte isolation using our two-stage method was 3-fold faster than the Ficoll density graduate separation for the same amount of WB sample: <60 min (our method) vs. >180 min (Ficoll).Conclusion: We developed an easy-to-use approach for extracting lymphocytes from peripheral blood with purity, viability and yield meeting the current standard for cellular therapies. Since our new approach is scalable, compatible with ‘closed system’ operation, produces lymphocytes with higher proliferation capacity, and is much faster than conventional density gradient separation, it could have a potentially transformative impact by accelerating research into new cellular therapies, and simplifying translation of these treatments into clinical practice. [Display omitted] DisclosuresGifford:Halcyon Biomedical Incorporated: Employment, Equity Ownership. Shevkoplyas:Halcyon Biomedical Incorporated: Employment, Equity Ownership, Patents & Royalties: U.S. Patent Appl. 61/929,357, Research Funding; New Health Sciences, Inc.: Consultancy, Research Funding.

A Novel High-Throughput Microfluidic Technology for Isolating Lymphocytes from Buffy Coats for Allogeneic Cellular Therapies

Introduction: Adoptive immunotherapies involving infusion of third-party donor-derived T lymphocytes (T-cells) to treat life-threatening viral infections (including BK virus, adenovirus, cytomegalovirus, and Epstein-Barr virus) in immunosuppressed recipients have shown potentially curative efficacy in recent clinical trials. Currently, allogeneic T-cells are typically obtained by extracting mononuclear cells via Ficoll density gradient centrifugation of peripheral blood, or via leukapheresis collected from an HLA-matched, virus-seropositive donor. A library of cryopreserved HLA-typed allogeneic T-cell concentrates would provide a much more sustainable source of T-cells, and enable producing T-cell based therapies on-demand quickly. An abundant source of T-cells for generating such a library could be the ‘buffy coat’ (BC); the layer of white blood cells (WBCs) between the red blood cells (RBCs) and the platelet-rich plasma, which is produced when donated whole blood is processed into components via the 'buffy coat method.' However, the U.S. standard favors the alternative 'PRP method,' therefore WBCs (including T-cells) are typically trapped in leukoreduction filters and subsequently discarded. Here we present the initial proof-of-concept for applying our recently-developed microfluidic technique (controlled incremental filtration, CIF) to purification and concentration of lymphocytes from BC units, at practical flowrates.Materials and Methods: BC units were obtained from MD Anderson Blood Bank (Houston, TX). A preliminary study was performed to determine the optimal dilution factor and device design parameters to achieve the highest lymphocyte recovery with minimal RBC contamination from our CIF approach. Several individual CIF-based microfluidic modules were multiplexed in parallel to achieve high throughput. The microfluidic devices were fabricated via soft-lithography techniques. Diluted BC samples were passed through the CIF-based microfluidic cell separator (Run 1) - the lymphocyte-rich product was collected, re-diluted to its original volume, and passed through the same device again (Run 2), using a flow rate of 6 mL/min for both runs (Fig. 1). A CBC with 5-part differential was performed on all blood samples using a hematology analyzer to quantify the recovery of lymphocytes and removal of RBCs and platelets.Results and Discussion: Our preliminary study revealed that a starting WBC count of ~10-15×103/µL gives the best balance between lymphocyte recovery and RBC removal at a flow rate of 6 mL/min. BC units were typically 35-50 mL, with initial WBC count between 20-75×103/µL, therefore units required 2-5-fold dilution. In a subsequent separation study, Run 1 through the CIF-based microfluidic device recovered 92.6±0.6% of lymphocytes while removing 92.0±0.5% of RBCs from the diluted BC suspensions. During Run 2, the CIF device recovered 93.0±0.4% of remaining lymphocytes, and removed 83.3±0.3% of residual RBCs. Overall, processing a BC unit through the CIF-based microfluidic device took approximately 1-1.5 hours. Combined, the two runs achieved a total lymphocyte recovery of 86.1±0.9% (final concentration of 51.7±5.2×103/µL) and a total RBC removal of 98.6±0.1% (reducing RBC count from 2.6±0.1×106/µL to 0.13±0.01×106/µL), making the final product cryopreservation-ready without the need for further purification or concentration. These results are on par with those typically achieved via Ficoll density gradient separation, while avoiding the associated drawbacks of laborious workflow, reagent cost, and reliance on large-footprint machinery (e.g. a centrifuge).Conclusion: In this study we demonstrated the feasibility of using a simple, disposable, high-throughput microfluidic device for rapidly purifying and concentrating lymphocytes from buffy coat units. This capability could simplify the creation of cryopreserved HLA-typed lymphocyte lines from donated blood, thereby substantially improving treatment options for patients who need allogeneic T-cell therapy by providing clinicians with HLA-compatible cells, on-demand. [Display omitted] DisclosuresGifford:Halcyon Biomedical Incorporated: Employment, Equity Ownership. Shevkoplyas:Halcyon Biomedical Incorporated: Employment, Equity Ownership, Patents & Royalties: U.S. Patent Appl. 61/929,357, Research Funding; New Health Sciences, Inc.: Consultancy, Research Funding.

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

Age-dependent decline in stress response capacity revealed by proteins dynamics analysis

The aging process is regarded as the progressive loss of physiological integrity, leading to impaired biological functions and the increased vulnerability to death. Among various biological functions, stress response capacity enables cells to alter gene expression patterns and survive when facing internal and external stresses. Here, we explored changes in stress response capacity during the replicative aging of Saccharomyces cerevisiae. To this end, we used a high-throughput microfluidic device to deliver intermittent pulses of osmotic stress and tracked the dynamic changes in the production of downstream stress-responsive proteins, in a large number of individual aging cells. Cells showed a gradual decline in stress response capacity of these osmotic-related downstream proteins during the aging process after the first 5 generations. Among the downstream stress-responsive genes and unrelated genes tested, the residual level of response capacity of Trehalose-6-Phosphate Synthase (TPS2) showed the best correlation with the cell remaining lifespan. By monitor dynamics of the upstream transcription factors and mRNA of Tps2, it was suggested that the decline in downstream stress response capacity was caused by the decline of translational rate of these proteins during aging.

A High-Throughput Microfluidic Magnetic Separation (µFMS) Platform for Water Quality Monitoring.

The long-term aim of this work is to develop a biosensing system that rapidly detects bacterial targets of interest, such as Escherichia coli, in drinking and recreational water quality monitoring. For these applications, a standard sample size is 100 mL, which is quite large for magnetic separation microfluidic analysis platforms that typically function with <20 µL/s throughput. Here, we report the use of 1.5-µm-diameter magnetic microdisc to selectively tag target bacteria, and a high-throughput microfluidic device that can potentially isolate the magnetically tagged bacteria from 100 mL water samples in less than 15 min. Simulations and experiments show ~90% capture efficiencies of magnetic particles at flow rates up to 120 µL/s. Also, the platform enables the magnetic microdiscs/bacteria conjugates to be directly imaged, providing a path for quantitative assay.

Intracellular Delivery of Exogenous Macromolecules into Human Mesenchymal Stem Cells by Double Deformation of the Plasma Membrane

AbstractPhysical techniques for intracellular delivery of exogeneous materials offer an attractive strategy to enhance the therapeutic efficiency of stem cells. However, these methods are currently limited by poor delivery efficiency as well as cytotoxic effects. Here, a high throughput microfluidic device is designed for efficient (≈85%) cytosolic delivery of exogenous macromolecules with minimal cell death (less than 10%). The designed microfluidic device enables the generation of transient pores as the cells pass through the micron‐sized constrictions (6–10 µm) leading to the passive diffusion of extracellular cargos into the cell cytosol. Specifically, the microfluidic system is designed to induce a double deformation on the cell membrane at the squeezing zones to maximize intracellular delivery. Additionally, the flow rate, ionic concentration, and the molecular weight of the cargo are optimized for maximum efficiency. The optimized device enables cytosolic diffusion of small (3 kDa) and large molecules (70 kDa) without inducing any apoptotic effect. Overall, this double cell deformation platform offers new opportunities to rapidly and efficiently deliver extracellular cargo into stem cells without affecting their viability and functionality.

A high-throughput microfluidic device for probing calcium dynamics of single cells squeezing through narrow channels

To probe intracellular calcium response while single cells squeeze through narrow channels, we built a high-throughput microfluidic device where single cells can be trapped efficiently and stimulated mechanically. With this device, dozens of single cells’ dynamic morphologies and intracellular [Ca2+] responses under dynamic mechanical stimuli can be monitored simultaneously. We observed a two-peak [Ca2+] response, which was closely coupled together with the dynamic cellular squeezing process. This type of [Ca2+] response, to our knowledge, was observed for the first time. We also investigated the role of the cytoskeleton in the [Ca2+] response and found that the cytoskeleton was an important regulator of [Ca2+] signaling during the cellular squeezing process. In addition, we investigated the difference between the two-peak [Ca2+] responses of Hela cells and HUVECs and found that one characteristic parameter could distinguish Hela cells from HUVECs.

Abstract 444: Continuous, high throughput microfluidic device to monitor circulating tumor cells in cancer patients

Abstract Analysis of circulating tumor biomarkers, designated “liquid biopsies” are less invasive and lower risk to the patient and might replace or complement standard tissue biopsies to determine prognosis, predict benefit from specific therapies, or monitor patients with cancer. Circulating tumor cells (CTCs) are extremely rare, with only 1-10 CTCs per mL of blood. Current technologies typically process 1-10 mL of blood ex vivo. We report an in vivo, indwelling CTC-capture device that is analogous to a Holter monitor used to interrogate cardiac rhythm abnormalities over long periods of time. The system incorporates a dual lumen intravenous catheter that directs blood from the vein into a small (~ the size of a wristwatch) capture carriage by virtue of a peristaltic pump. The carriage contains a CTC capture chip which separates CTCs from whole blood based on flow differences and solid phase anti-EpCAM capture, while the remaining whole blood is returned to the venous system via the dual lumen catheter. The chip is then removed from the carriage for further enumeration and characterization of the CTCs. In a proof of principle set of experiments, the system was placed into investigational canines into which fluorescently labeled human breast cancer cells were injected intravenously. Although the vast majority of these xeno-cancer cells were rapidly removed from circulation, the system captured “CTCs” over a 2 hour time period more successfully than was seen in simultaneous blood draws for ex vivo enumeration over the same time. No adverse effects were observed in the subjects at the time of the experiments or over the succeeding 48 hours after removal of the IV system.Our system permits interrogation of a much larger volume of blood than can be processed with standard blood draws. The larger processed volume will drastically increase the number of cells isolated which in turn increases the number of downstream assays that can be performed. This study is focused on developing a high throughput microfluidic device that can detect CTCs and implementing it in a canine model. Citation Format: Kaylee Smith, Tae Hyun Kim, Costanza Paoletti, Douglas H. Thamm, Daniel F. Hayes, Sunitha Nagrath. Continuous, high throughput microfluidic device to monitor circulating tumor cells in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 444.

Microfluidic Analysis for Separating and Measuring the Deformability of Cancer Cell Subpopulations.

Increased deformability and softness endow tumor cells with highly invasive and metastatic capabilities. We exploited these characteristics to fabricate a high-throughput microfluidic device to measure cell deformability and separate cancer cells. Driven by hydrodynamic forces, the cells with better deformability passed through the chip faster, whereas stiffer cells passed through the device over a longer time period. The MDA-MB-231 and MCF-7 cell lines were used to evaluate the device because their metastatic potentials were known. We found that MDA-MB-231 cells, which were softer and exhibited stronger deformability, passed through the device more quickly. HeLa cells were also successfully separated into softer and stiffer subpopulations, whose distinct mechanical properties were confirmed by atomic force microscopy. We also measured the expression of metastasis-associated proteins (epidermal growth factor receptor and integrin β 1) and found that subpopulations with varied deformabilities had different expression levels. Our results suggested that this high-throughput microfluidic device could be used to screen and evaluate the curative effects of drug and cancer progression by simultaneously testing cell deformability and expression levels of metastasis-associated proteins in separated cell subpopulations.

Cell-free gene-regulatory network engineering with synthetic transcription factors

Gene-regulatory networks are ubiquitous in nature and critical for bottom-up engineering of synthetic networks. Transcriptional repression is a fundamental function that can be tuned at the level of DNA, protein, and cooperative protein-protein interactions, necessitating high-throughput experimental approaches for in-depth characterization. Here, we used a cell-free system in combination with a high-throughput microfluidic device to comprehensively study the different tuning mechanisms of a synthetic zinc-finger repressor library, whose affinity and cooperativity can be rationally engineered. The device is integrated into a comprehensive workflow that includes determination of transcription-factor binding-energy landscapes and mechanistic modeling, enabling us to generate a library of well-characterized synthetic transcription factors and corresponding promoters, which we then used to build gene-regulatory networks de novo. The well-characterized synthetic parts and insights gained should be useful for rationally engineering gene-regulatory networks and for studying the biophysics of transcriptional regulation.

Erythrocyte Deformability As a Potential Biomarker for Chronic Fatigue Syndrome

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is arguably the last major disease we know almost nothing about. It is a multi-systemic illness of unknown etiology affecting millions of individuals worldwide, with the capacity to persist for several years. ME/CFS is characterized by disabling fatigue of at least 6 months, accompanied serious fatigue and musculoskeletal pain, in addition to impaired short-term memory or concentration, and unrefreshing sleep or extended post-exertional. While the etiology of the disease is still debated, evidence suggest oxidative damage to immune and hematological systems as one of the pathophysiological mechanisms of the disease. Erythrocytes are potent scavengers of oxidative stress, and their shape changes appreciably in response to oxidative stress and certain inflammatory conditions including obesity and diabetes. The shape of erythrocytes change from biconcave discoid to an ellipsoid due shear flow in microcapillaries that provides a larger specific surface area-to-volume ratio for optimal microvascular perfusion and tissue oxygenation establishing the importance not only of total hematocrit but also of the capacity for large deformations in physiology. Clinically, ME/CFS patients show normal arterial oxygen saturation but nothing much is known about microvascular perfusion. In this work, we tested the hypothesis that the erythrocyte deformability in ME/CFS is adversely affected, using a combination of biophysical and biochemical techniques.We tested the deformability of RBCs using a high-throughput microfluidic device which mimics blood flow through microcapillaries. We perfused RBCs (suspension in plasma) from ME/CFS patients and from age and sex matched healthy controls (n=9 pairs of donors) through a high-throughput microfluidic platform of 5µm width and 3-5 µm height. We recorded the movement of the cells at high speed (4000 fps), followed by image analysis to assess the following parameters: entry time (time required by the cells to completely enter the test channels), average transit velocity (velocity of the cells inside the test channels) and elongation index (ratio of the major diameter before and after deformation in the test channel). We observed that RBCs from ME/CFS patients had higher entry time (~12%, p<0.0001), lower average transit velocity (~17%, p<0.0001) and lower elongation index (~14%, p<0.0001) as compared to RBCs from healthy controls. Taken together, this data shows that RBCs from ME/CFS patients have reduced deformability. To corroborate our findings, we also measured the erythrocyte sedimentation rate (ESR) for these donors which show that the RBCs from ME/CFS patients had lower (~40%, p<0.01) sedimentation rates.To understand the basis for differences in deformability, we investigated the changes in the fluidity of the membrane using a lateral diffusion assay using pyrenedecanoic acid (PDA), and observed that RBCs from ME/CFS patients have lower membrane fluidity (~30%, p<0.01). Apart from the fluidity, Zeta potential measurements showed that ME/CFS patients had lower net negative surface charge on the RBC plasma membrane (~18%, p<0.0001). Higher levels of reactive oxygen species (ROS) in RBCs from ME/CFS patients (~30%, p<0.008) were also observed, as compared to healthy controls. Using scanning electron microscopy (SEM), we also observed changes in RBC morphology between ME/CFS patients and healthy controls (presence of different morphological subclasses like biconcave disc, leptocyte, acanthocyte and burr cells; area and aspect ratio; levels of RBC aggregation). Despite these changes in RBC physiology, the hemoglobin levels remained comparable between healthy donors and ME/CFS patients. Finally, preliminary studies show that RBCs from recovering ME/CFS patients do not show such differences in cellular physiology, suggesting a connection between RBC deformability and disease severity.Taken together, our data demonstrates that the significant decrease in deformability of RBCs from ME/CFS patients may have origins in oxidative stress, and suggests that altered microvascular perfusion can be a possible cause for ME/CFS symptoms. Our data also suggests that RBC deformability may serve as a potential biomarker for ME/CFS, albeit further studies are necessary for non-specific classification of the disease. DisclosuresNo relevant conflicts of interest to declare.

Trapping DNA with a high throughput microfluidic device

Long strands of DNA can be trapped and concentrated near the inlet of a microfluidic channel by applying a pressure gradient and an opposing electric field. The mechanism for trapping involves a migration of DNA perpendicular to both the fluid flow and the electric field. Migration leads to a highly nonuniform distribution of DNA within a cross section of the channel, with the bulk of the DNA concentrated in a thin (10 μm) layer next to the walls of the channel. This highly concentrated layer generates an electrophoretic flux toward the inlet to the device, despite the much larger fluid flow in the opposite direction. In this paper, the extent to which DNA can be trapped and concentrated by this means has been characterized by fluorescence measurements. At short times (<2 hours) nearly all the incoming DNA remains trapped within the device until the electric field is turned off. The DNA largely accumulates near the inlet, but after 30-60 minutes additional DNA starts to accumulate deeper into the channel. Eventually DNA leaks from the device itself, but ≈80% of the incoming DNA can be retained for up to 5 hours. Optimizing the electric field strength can increase the amount of DNA that can be trapped, but the efficiency is not affected by the channel cross-section.

Abstract A21: High-throughput isolation of circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC) patients for personalized treatments

Abstract It has been recognized that circulating tumor cells (CTCs) shed by a primary tumor can be a valuable surrogate to tissue biopsies. Our project goal is to develop a CTC-based liquid biopsy platform as a noninvasive method for identifying primary and secondary mutations in non-small lung cancer to direct personalized therapies. The high-throughput, label-free and size-based inertial microfluidic separation device “Labyrinth” developed in the Nagrath lab has been used for simultaneous isolation of epithelial (EpCs) and mesenchymal (EMTCs) CTCs from lung cancer patients. The total channel length of Labyrinth is 637 mm, with 500 mm in width and 100 mm in height. The device consists of complex of small and high ratio of curvature structures (11 long loops and sharp right-angle 56 corners) to enhance differential focusing of both CTCs and white blood cells into separate fluid streams in the outlet area. The high recoveries and advantage of size-based biomarker independent separation are crucial for the downstream applications including molecular characterization of CTCs (mutation and mRNA profiling) and expansion of CTCs in vitro. Labyrinth was optimized and tested for inertial separation of cancer cells using the human lung cancer cell line PC-9. The percentage of recovery and purity was &amp;gt;93% with 2400 µL/min flow rate. We have enrolled 15 metastatic patients for this study and isolated CTCs from baseline (n=15) and post-therapy follow-up visits (range 1-4). To be able to detect different subpopulations of CTCs including EpCs and EMTCs from patients, we have employed immunofluorescence to label EpCAM (for EpCs) and Vim (for EMTCs), in addition to PanCK (tumor marker) and CD45 (leukocyte marker). CTCs were defined as EpCAM+/-/ Vim+/-/ PanCK+/ CD45-. All 15 patients, with different mutations/fusion, including EGFR, RET, ROS1, and ALK, had detectable CTCs at all visits, including 156±175 (at baseline, n=15), 141±133 (at visit 1, n=14), 255±218 (at visit 2, n=8), 84±81 (at visit 3, n=4), and 157±177 per mL (at visit 4, n=2). Patients with EGFR mutants had an average of 112±97 total CTCs per mL at their baseline and 66±57 after they were treated with either erlotinib or osimertanib. Patients with ROS1 fusion had slightly higher total CTCs per mL at their baseline (185±250) and during treatment with crizotinib (120±113) compared to EGFR mutant patients. To expand patient-derived CTCs for ex vivo drug testing, three different CTC culture conditions were tested: a plain, a “Fibronectin coated” (2D), and a “Matrigel-collagen-irradiated mice fibroblast coated” (3D) 48-well plate. After 6 weeks in culture, the growth rate was significantly higher in the 3D model. For CTC expansion, the enrichment methods require further optimization prior to adopting the 3D culture model for future samples. The cell surface marker independent microfluidic device, Labyrinth, isolated heterogeneous CTC subpopulations that were characterized and expanded in vitro. This could allow ex vivo drug testing to direct patient-specific therapies. Citation Format: Mina Zeinali, Arthi Nadhan, Anvya Mathur, Maggie Lee, Wei Huang, Mathias Hafner, Nithya Ramnath, Sunitha Nagrath. High-throughput isolation of circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC) patients for personalized treatments [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A21.

A perfused human blood\u2013brain barrier on-a-chip for high-throughput assessment of barrier function and antibody transport

BackgroundReceptor-mediated transcytosis is one of the major routes for drug delivery of large molecules into the brain. The aim of this study was to develop a novel model of the human blood–brain barrier (BBB) in a high-throughput microfluidic device. This model can be used to assess passage of large biopharmaceuticals, such as therapeutic antibodies, across the BBB.MethodsThe model comprises human cell lines of brain endothelial cells, astrocytes, and pericytes in a two-lane or three-lane microfluidic platform that harbors 96 or 40 chips, respectively, in a 384-well plate format. In each chip, a perfused vessel of brain endothelial cells was grown against an extracellular matrix gel, which was patterned by means of surface tension techniques. Astrocytes and pericytes were added on the other side of the gel to complete the BBB on-a-chip model. Barrier function of the model was studied using fluorescent barrier integrity assays. To test antibody transcytosis, the lumen of the model’s endothelial vessel was perfused with an anti-transferrin receptor antibody or with a control antibody. The levels of antibody that penetrated to the basal compartment were quantified using a mesoscale discovery assay.ResultsThe perfused BBB on-a-chip model shows presence of adherens and tight junctions and severely limits the passage of a 20 kDa FITC-dextran dye. Penetration of the antibody targeting the human transferrin receptor (MEM-189) was markedly higher than penetration of the control antibody (apparent permeability of 2.9 × 10−5 versus 1.6 × 10−5 cm/min, respectively).ConclusionsWe demonstrate successful integration of a human BBB microfluidic model in a high-throughput plate-based format that can be used for drug screening purposes. This in vitro model shows sufficient barrier function to study the passage of large molecules and is sensitive to differences in antibody penetration, which could support discovery and engineering of BBB-shuttle technologies.

Rheotaxis-based separation of sperm with progressive motility using a microfluidic corral system

The separation of motile sperm from semen samples is sought after for medical infertility treatments. In this work, we demonstrate a high-throughput microfluidic device that can passively isolate motile sperm within corrals inside a fluid channel, separating them from the rest of the diluted sample. Using finite element method simulations and proposing a model for sperm motion, we investigated how flow rate can provide a rheotaxis zone in front of the corral for sperm to move upstream/downstream depending on their motility. Using three different flow rates that provided shear rates above the minimum value within the rheotaxis zone, we experimentally tested the device with human and bovine semen. By taking advantage of the rheotactic behavior of sperm, this microfluidic device is able to corral motile sperm with progressive velocities in the range of 48-93 μm⋅s-1 and 51-82 μm⋅s-1 for bovine and human samples, respectively. More importantly, we demonstrate that the separated fractions of both human and bovine samples feature 100% normal progressive motility. Furthermore, by extracting the sperm swimming distribution within the rheotaxis zone and sperm velocity distribution inside the corral, we show that the minimum velocity of the corralled sperm can be adjusted by changing the flow rate; that is, we are able to control the motility of the separated sample. This microfluidic device is simple to use, is robust, and has a high throughput compared with traditional methods of motile sperm separation, fulfilling the needs for sperm sample preparation for medical treatments, clinical applications, and fundamental studies.