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Exosomes are believed to be secreted from multivesicular endosomes and containing proteins and nucleic acids, including mRNA and microRNAs, which have been implicated to play a role in neurodegenerative diseases. Neuron-derived exosomes at the circulation provide a unique potential as biomarkers towards assessment of Alzheimer's disease (AD), even at the pre-clinical stage. This review briefly discusses their biogenesis and transport, exosomal protein verses soluble protein, evidence for their role in AD, isolation of exosomes, and challenges and future directions to realize reliable blood-based biomarkers to meet phenomenal unmet clinical and pre-clinical need of AD.

When breast cancer patients start to exhibit resistance to hormonal therapy or chemotherapy, the mTOR inhibitor everolimus can be considered as an alternative therapeutic agent. Everolimus can deregulate the PI3K/AKT/mTOR pathway and affect a range of cellular functions. In some patients, the agent does not exhibit the desired efficacy and, even worse, not without the associated side effects. This study assessed the use of immunofluorescence (IF) as a modality to fill this unmet need of predicting the efficacy of everolimus prior to administration. Cell viability and MTT assays based on IF intensities of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 on breast cancer cells (Hs578T, MCF7, BT474, MDA-MB-231) and patient-derived cell culture from metastatic sites (ABC-82T and ABC-16TX1) were interrogated. Results show that independent pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 IF expressions can classify data into different groups: everolimus sensitive and resistant. The combined IF baseline intensity of these proteins is predictive of the efficacy of everolimus, and their intensities change dynamically when cells are resistant to everolimus. Furthermore, mTOR resistance is not only consequence of the AKT/mTOR pathway but also through the LKB1 or MAPK/ERK pathway. The LKB1 and pho-GSK3β may also be potential predictive markers for everolimus.

Therapeutic drug synergism intervened in cancer treatments has been demonstrated to be more effective than using a single effector. However, it remains inherently challenging, with a limited cell count from tumor samples, to achieve potent personalized drug cocktails. To address the issue above, we herein present a nanodroplet cell processing platform. The platform incorporates an automatic nanodroplet dispenser with cell array ParaStamp chips, which were fabricated by a new wax stamping approach derived from laser direct writing. Such approach enables not only the on-demand de-wetting with hydrophobic wax films on substrates but also the mask-less fabrication of non-planar microstructures (i.e. no photolithography process). The ParaStamp chip was pre-occupied with anti-cancer drugs and their associate mixtures, enabling for the spatially addressable screening of optimal drug combinations simultaneously. Each droplet with a critical volume of 200 nl containing with 100 cells was utilized. Results revealed that the optimal combination reduces approximate 28-folds of conducted doses compared with single drugs. Tumor inhibition with the optimally selected drug combination was further confirmed by using PC-3 tumor-bearing mouse models. Together, the nanodroplet cell processing platform could therefore offer new opportunities to power the personalized cancer medicine at early-stage drug screening and discovery.

When breast cancer patients start to exhibit resistance to hormonal therapy or chemotherapy, the mTOR inhibitor everolimus can be considered as an alternative therapeutic agent. Everolimus can deregulate the PI3K/AKT/mTOR pathway and affect a range of cellular functions. In some patients, the agent does not exhibit the desired efficacy and, even worse, not without the associated side effects. This study assessed the use of immunofluorescence (IF) as a modality to fill this unmet need of predicting the efficacy of everolimus prior to administration. Cell viability and MTT assays based on IF intensities of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 on breast cancer cells (Hs578T, MCF7, BT474, MDA-MB-231) and patient-derived cell culture from metastatic sites (ABC-82T and ABC-16TX1) were interrogated. Results show that independent pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 IF expressions can classify data into different groups: everolimus sensitive and resistant. The combined IF baseline intensity of these proteins is predictive of the efficacy of everolimus, and their intensities change dynamically when cells are resistant to everolimus. Furthermore, mTOR resistance is not only consequence of the AKT/mTOR pathway but also through the LKB1 or MAPK/ERK pathway. The LKB1 and pho-GSK3β may also be potential predictive markers for everolimus.

With respect to male fertility, sperm play a crucial role in the whole process of fertilization. Many technologies for manipulating sperm have been developed over the past few decades and these have focused on counting and sorting sperm. Microfludic technologies were introduced into this field at the beginning of the 21 st century. This paper reviews the history of microfluidic technologies and their application to forensic medicine and sperm counting and sorting. An attempt is made to establish an automatic platform for in vitro fertilization using microfluidic technologies.

Non-planar microstructure-based tissue culture devices have emerged as powerful tools to mimic in vivo physiological microenvironments in a wide range of medical applications. Here we report a spontaneous aqueous molding approach - inspired by Stenocara gracilipes beetles - to rapidly fabricate non-planar microstructure devices for facilitating tissue-based bioassays. The device fabrication is determined from the self-assembled liquid morphology, which is induced by condensation or guided by surface tension. Through experiments and modeling, we reveal that the molding mainly comprises two typical circular and striped domains, highlighting versatile applications for bioengineering. In addition, the molding characteristic is dependent on the geometry of the patterned wetting surfaces, the working volume of the liquid, and the interaction between the liquid and the substrate. The theoretical model, based on the geometry of the patterned liquid, is highly consistent with experimental data. We also demonstrate that our approach can facilitate the culturing of tumor spheroids incorporated with biomimic nano-cilia, rapid high-throughput drug screening, tumor spheroid migration assay, and in vitro modeling of blood vessels. Remarkably, the delivery of multiple concentrations of drugs and their associate mixtures (a total of 25 test spots in one device) can be carried out simultaneously within seconds. Taken together, these insights may offer new opportunities to tailor non-planar microstructures, and our proposed methodology can be applicable for the emerging needs in tumor cell biology and tissue engineering.

We present a microarray chip integrated with novel round bottom μ-well arrays and biomimetic nano-cilia, which can, for the first time, not only recapitulate in vivo-like tumor microenvironment but also be achieved in situ for hallmarks of tumor-based bioassays.

Cancer metastasis and drug resistance are important malignant tumor phenotypes that cause roughly 90% mortality in human cancers. Current therapeutic strategies, however, face substantial challenges partially due to a lack of applicable pre-clinical models and drug-screening platforms. Notably, microscale and three-dimensional (3D) tissue culture platforms capable of mimicking in vivo microenvironments to replicate physiological conditions have become vital tools in a wide range of cellular and clinical studies. Here, we present a microfluidic device capable of mimicking a configurable tumor microenvironment to study in vivo-like cancer cell migration as well as screening of inhibitors on both parental tumors and migratory cells. In addition, a novel evaporation-based paper pump was demonstrated to achieve adaptable and sustainable concentration gradients for up to 6 days in this model. This straightforward modeling approach allows for fast patterning of a wide variety of cell types in 3D and may be further integrated into biological assays. We also demonstrated cell migration from tumor spheroids induced by an epidermal growth factor (EGF) gradient and exhibited lowered expression of an epithelial marker (EpCAM) compared with parental cells, indicative of partial epithelial-mesenchymal transition (EMT) in this process. Importantly, pseudopodia protrusions from the migratory cells - critical during cancer metastasis - were demonstrated. Insights gained from this work offer new opportunities to achieve active control of in vitro tumor microenvironments on-demand, and may be amenable towards tailored clinical applications.

The planar patch-clamp technique has revolutionized ion channel study by increasing throughput and minimizing sophisticated operation. Nevertheless, the system design is often focused toward a particular objective, which sometimes limits the range of information output. Here, we present an integrated electrophysiological and optical approach for ion channel study in a microfluidic system allowing multi-content detection. Multiple fluidic injections enable both intra- and extracellular solution exchange to facilitate the study of the ion channel in a microfluidic chip with high-yield seal formation. Whole-cell and single cell recordings were validated for various cell lines, i.e . endogenous channels of HIT-T15, CHO-K1, HEK-293T, and RIN-m5f cells. Long-term recording of temporal fluorescent changes on a trapped RIN-m5F cell shows the ability of extracellular solution exchange and simultaneous optical observation. The integrated microdevice system serves as a practical tool for high-quality and multi-content ion channel electrophysiological study.

Recent studies have shown that specific rare cells in the blood can serve as an indicator of cancer prognosis, among other purposes. This article demonstrates the concept of separating and detecting rare cells from peripheral blood mononuclear cells via an economical microfluidic disk with a model system. MCF7, labeled with magnetic beads, was used to simulate circulating tumor cells as a target. Jurkat clone E6-1 was used to simulate leukocytes or other cells abundant in human blood. A tailored multistage magnet maximized the magnetic field to ensure optimal trapping efficiency. Results indicate that the yield of detected MCF7 was consistent at approximately 80% when fewer than hundreds of MCF7 cells were mixed in greater than 1 million Jurkat cells. The 80% yield also held for 10 MCF7 in 100 million Jurkat (rarity of 10 7 ). Compared with the results from autoMACS, the performance was at least 20% higher and was more independent of the number of Jurkat. The viability of the enriched cells was approximately 90 ± 20%, showing that this method caused little damage to trapped cells. The microfluidic disk should be applicable for separation and detection of various rare cells, such as circulating tumor cells and circulating endothelial cells in human blood.

BACKGROUND Circulating endothelial cells (CECs) in the blood are rare but have been shown to be associated with various diseases. With the ratio of CECs to peripheral blood mononuclear cells (PBMCs) less than 1 part per thousand, their separation from PBMCs and detection are challenging. We present a means of detecting CECs from PBMCs via an economical microfluidic disk with a model cell system [human umbilical vein endothelial cells (HUVECs) in PBMCs], along with demonstration of its efficacy clinically. METHODS To enrich these rare cells, we used immunomagnetic beads and a tailor-made magnet on the disk. CEC-simulating HUVECs, as target cells, were stained with primary anti–CD146-phycoerythrin antibody and bound with secondary antibody on antiphycoerythrin magnetic beads. PBMCs served as nontarget cells and were labeled with anti–CD45-FITC antibody. RESULTS When hundreds of HUVECs were mixed in 106 PBMCs, 95% of spiked HUVECs were detected. This yield also held for 60 HUVEC in CONCLUSIONS The count of CECs is a suitable marker for symptoms of systemic lupus erythematosus. The microfluidic disk system should be a viable platform for detection of CECs.

Mixing in micro-environment has been explored in a number of studies. This study presents a unique approach of efficient mixing of two heterogeneous streams via two counter-rotating recirculatory streams induced by in-plane resonance of a rectangular microplate actuated via Lorentz force. The generated time-mean flow structure was interrogated for mixing efficacy over a range of excitation voltage, Reynolds number, and Peclet number, along with numerical analysis to probe the time-mean flow physics. Results show that the recirculatory flow is generated at the plate’s edges due to local flow non-linearity, characteristic of acoustic streaming. The percentage of mixing, at one device length-scale downstream, attains 93% at a low Reynolds number of 0.0037 (based on mean velocity of 0.078 mm/s and channel height of 50 μm) at 8 V excitation. Further characterization via enhanced diffusivity shows a maximum of 80.7-fold increase. Comparison with other active mixers shows the current device achieves mixing in one of the shortest distances. The proposed approach is robust, tunable to attain desired mixing characteristics and essentially independent of the properties of the fluid medium, which should be useful in a number of microfluidic applications requiring fast mixing.

Planar patch clamp has revolutionized characterization of ion channel behavior in drug discovery primarily via advancement in high throughput. Lab use of planar technology, however, addresses different requirements and suffers from inflexibility to enable wide range of interrogation via a single cell. This work presents integration of planar patch clamp with microfluidics, achieving multiple solution exchanges for tailor-specific measurement and allowing rapid replacement of the cell-contacting aperture. Studies via endogenously expressed ion channels in HEK 293T cells were commenced to characterize the device. Results reveal the microfluidic concentration generator produces distinct solution/drug combination/concentrations on-demand. Volume-regulated chloride channel and voltage-gated potassium channels in HEK 293T cells immersed in generated solutions under various osmolarities or drug concentrations show unique channel signature under specific condition. Excitation and blockage of ion channels in a single cell was demonstrated via serial solution exchange. Robustness of the reversible bonding and ease of glass substrate replacement were proven via repeated usage of the integrated device. The present approach reveals the capability and flexibility of integrated microfluidic planar patch-clamp system for ion channel assays.

Cyto-analysis of rare cells often requires separation and detection with each procedure posing substantial challenges. This paper presents a disk-based microfluidic platform for both procedures via an immunomagnetic negative selection process. The microfluidic platform's unique features include a multistage magnetic gradient to trap labeled cells in double trapping areas, drainage of fluid to substantially shorten detection time, and a bin-like regions to capture target cells to facilitate a seamless enumeration process. Proof-of-concept was conducted using MCF7 as target rare cells (stained with anti-cytokeratin-FITC antibodies) spiked into Jurkat Clone E6-1 non-target cells (labeled with anti-CD45-PE and anti-PE BD magnetic beads). Then, mononuclear cells (MNC) from healthy blood donors were mixed with MCF7s, modeling rare cells, and tested in the disk. Results show a non-linear magnetic coupling effect of the multistage magnet substantially increased the trapping efficacy over that of a single magnet, contributing to the depletion rate of Jurkats, which reaches 99.96%. Detection time is extensively shortened by depletion of 95% of non-cell-containing fluid in the collection area. The average yield of detected MCF7 cells is near-constant 60 ± 10% over a wide range of rarity from 10(-3) to 10(-6) and this yield also holds for the MCF7/MNC complex mixture. Comparison with autoMACS and BD IMagnet separators revealed the average yield from the disk (60%) is superior to that of autoMACS (37.3%) and BD IMagnet (48.3%). The advantages of near-constant yield, roughly 30 min of operation, an acceptable level of cell loss, and potentially low cost system should aid in cyto-analysis of rare cells.

A home-use device that allows rapid and quantitative sperm quality analysis is desirable but not yet fully realized. To aid this effort, this article presents a microfluidic device capable of quantifying sperm quality in terms of two critical fertility-related parameters—motile sperm concentration and motility. The microdevice produces flow field for sperms to swim against, and sperms that overcame the flow within a specified time are propelled along in a separate channel and counted via resistive pulse technique. Data are compared to two control methods clinically utilized for sperm quality exam—hemocytometer and the sperm quality analyzer. Results reveal the numbers of pulses generated by passage of sperms correlates strongly with the two control methods: pulse number from 0 to 335 corresponds to progressively motile sperm concentrations from 0 to 19 × 106/ml (hemocytometer) and Sperm Motility Index from 0 to 204 (sperm quality analyzer). The microdevice should be applicable to facilitate self-assessment of sperm quality at home.

This paper presents hydrodynamic trapping of bioparticles in a microfluidic device. An in-plane oscillatory microplate, driven via Lorentz law, generates two counter-rotating microvortices, trapping the bioparticles within the confines of the microvortices. The force required to trap bioparticles is quantified by tuning the background flow and the microplate's excitation voltage. Trapping and releasing of 10-microm polystyrene beads, human embryonic kidney (HEK) cells, red blood cells (RBCs), and IgG antibodies were demonstrated. Results show the microvortices rotates at 0-6 Hz corresponding to 2-9 Vpp (peak-to-peak) excitation. At a particular rate of rotation (2-7 Vpp tested), a bioparticle is trapped until the background flow exceeds a limit. This flow limit increases with the rate of rotation, which defines the trap/release force boundary over the range of operation. This boundary is 12 +/- 2.0 pN for cell-size bioparticles and 160 +/- 50 fN for antibodies. Trapping of RBCs demonstrated microvortices' ability for nonspherical cells. Cell viability was studied via HEK cells that were trapped for 30 min and shown to be viable. This hydrodynamically controlled approach to trap a wide range of bioparticles should be useful as a microfluidic device for cellular and subcellular bioassay applications.

Microenvironment mimicking biological situation is a vital issue in tissue regeneration. With much progress being made, one of the major challenges remains to develop a convenient method to fabricate the scaffold microenvironment suitable for cell attachment and proliferation. This article demonstrates the efficacy of microcontact printed laminin, an extracellular matrix protein, on three different oxygen plasma treatment substrates-tissue culture polystyrene, poly(methyl methacrylate) films, and chitosan films-for alignment and growth of the Schwann cells in in vitro culturing. Replica molding of polydimethylsiloxane elastomeric stamps, fabricated from patterned SU-8 structure on silicon master, was used to print laminin on the three substrates. Pattern and growth of Schwann cells for low (10(3) cells/cm(2)) and increased cell density (2 x 10(4) cells/cm(2)) on the varied substrates with and without microcontact printed laminin were characterized. Results of in vitro cell culture of Schwann cells showed a high degree of cell orientation on the laminin-micropatterned substrates for both cell densities. However, different cell seeding densities will strongly impact the morphology and orientation of Schwann cells. Microcontact printing proves to be a convenient means to pattern cell-recognition molecules on scaffold for cell-guilded growth in tissue regeneration.

Drug-based treatments are the most widely used interventions for cancer management. Personalized drug response profiling remains inherently challenging with low cell count harvested from tumour sample. We present a 96well-formatted microfluidic plate with built-in micro-gap that preserves up to 99.2% of cells during multiple assay/wash operation and only 9,000 cells needed for a single reagent test (i.e. 1,000 cells per test spot x 3 selected concentration x triplication), enabling drug screening and compatibility with conventional automated workstations. Results with MCF7 and MDA-MB-231 cell lines showed that no statistical significance was found in dose-response between the device and conventional 96-well plate control. Primary tumour samples from breast cancer patients tested in the device also showed good IC50 prediction. With drug screening of primary cancer cells must consider a wide range of scenarios, e.g. suspended/attached cell types and rare/abundant cell availability, the device enables high throughput screening even for suspended cells with low cell count since the signature microfluidic cell-trapping feature ensures cell preservation in a multiple solution exchange protocol.

Microfabricated devices for cell lysis have demonstrated many advantages over conventional approaches. Among various design of microdevices that employ electroporation for cytolysis, most utilize Ag/AgCl wires or 2D planar electrodes. Although, simple in fabrication the electric field generated by 2D electrodes decays exponentially, resulting in rather non-uniform forcing on the cell membrane. This paper investigates the effect of electric field generated by 3D cylindrical electrodes to perform cell lysis via electroporation in a microfluidic platform, and compared with that by 2D design. Computational results of the electric field for both 2D and 3D electrode geometries showed that the 3D configuration demonstrated a significantly higher effective volume ratio-volume which electric field is sufficient for cell lysis to that of net throughflow volume. Hence, the efficacy of performing cell lysis is substantially greater for cells passing through 3D than 2D electrodes. Experimentally, simultaneous multi-pores were observed on leukocytes lysed with 3D electrodes, which is indicative of enhanced uniformity of the electric field generated by 3D design. Additionally, a single row of 3D electrode demonstrated a substantially higher lysing percentage (30%) than that of 2D (8%) under that same flow condition. This work should aid in the design of electrodes in performing cell lysis via electroporation.

A heat-driven thermoacoustic refrigerator has been designed and tested. A detailed thermal model of the device is presented. Energy balances within the system are discussed using external, heat exchanger, and stack control volumes in order to clarify the relationships of work and heat fluxes below and above onset. Thermal modeling is discussed as a tool for performance analysis as well as for determining system heat losses and finding input heat flows required by a thermoacoustic code. A method of using the control volume balance equations to find stack work and device efficiencies is presented. Experimental measurements are compared to DELTAE thermoacoustic modeling predictions. Modeling results show that viscous losses within the system have a significant impact on the device performance as well as on the ability of DELTAE to accurately predict performance. Modeling has led to an understanding of system performance and highlighted loss sources that are areas for improvement in a redesign.

This paper addresses the vibratory mechanics associated with frequency discrimination of basilar membrane within the cochlear of the inner ear. Periodic excitation is provided to the oval window, which results in generation of waves within the fluid-filled cochlear traveling towards the apex. These waves interact with the compliant basilar membrane structure causing its vibratory motion. Solution procedure of the fluid/structure model consists of a two-step process. First, a finite element calculation (ANSYS) solves for the membrane vibration with an initial harmonic pressure distribution. Second, a control volume analysis links the resultant vibratory motion with the fluid pressure acting on the basilar membrane, thus a pressure feedback loop is accomplished. Results show that dominant factors affecting vibratory characteristics of the basilar membrane are its structural geometry and attenuation of pressure wave as it travels away from the oval window. Calculations clearly capture the designed function of the basilar membrane, principally its frequency discrimination behavior.

An experimental study is presented of flow control of the rotor wake in a rotor/stator axial compressor. The objective is to quantify the reduction in the stator forced response due to flow blowing from the rotor trailingedge. The experiment was conducted in a low-speed, large-scale axial compressor at both near-design and high time-mean blade loading. The blowing cases considered are 35, 72, and 105% of the nonblowing wake momentum defect, which ranges from 1.3 to 3.7% of the compressor mass flow at near-design loading. Instrumentation include slanted hot wire to acquire three-dimensional ensemble-averaged rotor wake profile and fast-response pressure gauges embedded within the stator blades at midspan. Results show that the development of the near wake (15% chord downstream of trailing edge) is very much loading dependent; the same amount of blowing momentum has an effect on the velocity defect at near-design loading but not at high loading. Farther downstream, trailing-edge blowing is effective in energizing the wake. The decrease in magnitude of transverse gust is essentially linear with the wake momentum increase due to the blowing flow. Data show reduction in the stator unsteady force amplitude for all blowing cases tested. With the wake momentum at 72% of that for the wakeless flow, force amplitude reduction of 25 and 35% are realized for the near-design and high loading cases, respectively.

Circulating tumor cells (CTC) are believed to be the culprit of metastasis and studies have shown that their enumeration has prognostic value in wide range of solid tumors. Although much progress has been made in CTC technology, their rarity in blood and their inherent heterogeneity still provide much challenge towards maturity of the technology. This paper presents semi-automated enrichment of CTC via density-based approach in a novel microfluidic disk, followed by multi-step on-disk immunofluorescence staining (pan-CK, EpCAM, Hoechst and CD45), fluorescence microscopy for image capture, and software image analysis. To characterize the performance of the system, we spiked 1 to 300 cells (mean 105, median 125) from six cell lines (DLD-1, Huh-7, MCF7, PC3, MDA-MB-231 and PC-9) from five cancers into whole blood from healthy donors. The recovery rate of the system is 87.4%±3.7% (R2=0.958) regardless of EpCAM expression levels of the cell lines. To interrogate the limitation of the technology, this data set included ultra-low cell counts spiked of 1 to 13 cells (mean 5.7, median 5) which might be indicative of CTC from metastatic breast cancer (MBC) patients. The recovery rate for this low cell count is 90%±10%. Notably, one single cell was spiked into healthy whole blood, processed via the technology, and one target cell was detected. This test was repeated in triplicate with all three tests recovered one cell each. Furthermore, the microfluidic disk technology enables operation over a range of blood volume (from 2 to 7.5ml) with no statistically significant difference in recovery rate. Recovered MCF-7 cells spiked in blood were subsequently cultured for 6 days and showed good viability with cell proliferation. We tested the technology in MBC patients along with CA15-3 and CT imaging. A total 34 of blood samples were collected from 21 patients over the course of their systemic treatment. Results showed CTC were detected in 28 samples (82%). The target CTC detected ranges from 0 to 138 (mean 16, median 4 CTCs per 7.5mL). 15 out of 34 samples (44%) had CTC number ≥ 5/7.5mL). In one patient with triple negative diagnosis, CTC count, CA15-3 and CT imaging were monitored during the course of chemotherapy. The CTC count remained zero at the end of the first and second treatment course, elevated to 24 CTCs at the end of the third course, and continued its elevation to 31 by the end of the fourth treatment course. During this entire treatment course, CA15-3 did not vary significantly. However, CT image confirmed the metastasized liver tumor grew from 6.15cm at the beginning of the second chemo course to 8.09cm at the beginning of the fourth course. Taken together, our label-free CTC enrichment technology has high analytical sensitivity (87%) over a wide range of cancers, able to handle a flexible blood volume (2mL to 7.5mL), and amenable to detect a high percentage (82%) of CTC in MBC patients. Citation Format: Yu-Jen Chang, Chen-Lin Chen, Wei-Fan Hsu, Meng-Ze Li, Wei-Yuan Ma, Ken-Chao Chen, Guan-Syuan Huang, Wai-Sang Wong, Jhan-Yu Syu, Thomas, Yo-Yan Huang, Ching-Hung Lin, Andrew M. Wo, Chiun-Sheng Huang. Label-free enrichment and detection of circulating tumor cells in metastatic breast cancer patients show 82% of cohorts have detectable targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 798. doi:10.1158/1538-7445.AM2017-798

Engineered materials have been employed as versatile tools to explore fundamental processes in cell biology/drug development as well as an approach towards intelligent devices, thereby becoming key components in modern technology. Herein, a ParaStamp technique is revealed to possess applications for cell patterning, drug screening, and rewritable functional patterning. The ParaStamp includes a micropatterned polydimethylsiloxane master and a liquid-phased paraffin oil generated at high temperature, which can transfer the patterned paramembrane onto varied material surfaces such as glass, polystyrene, and flexible foil. This technique is simple and cost-effective to meet the high-throughput requirement for industries. Taken together, the findings herein should provide general insights in cell biology, biodetection, and the development of smart hydrophobic surfaces.

Three-dimensional near-wake structure behind a rotor was measured using slanted hot-wire technique in a large-scale, low-speed, rotor/stator axial compressor. Unsteady flow interaction between blade rows was varied by setting the axial gap between rows at 10% and 30% of rotor chord. Results show that stronger flow interactions between blade rows, or closer axial gap, produce more pronounced time variation within the rotor wake. All parameters measured – three component velocities, yaw and pitch angles – varied strongly within the wake, and are quantified.

The effect of blade row axial spacing on vortical and potential disturbances and gust response is studied for a compressor stator/rotor configuration near design and at high loadings using two-dimensional incompressible Navier–Stokes and potential codes, both written for multistage calculations. First, vortical and potential disturbances downstream of the isolated stator in the moving frame are defined; these disturbances exclude blade row interaction effects. Then, vortical and potential disturbances for the stator/rotor configuration are calculated for axial gaps of 10, 20, and 30 percent chord. Results show that the potential disturbance is uncoupled locally; the potential disturbance calculated from the isolated stator configuration is a good approximation for that from the stator/rotor configuration upstream of the rotor leading edge at the locations studied. The vortical disturbance depends strongly on blade row interactions. Low-order modes of vortical disturbance are of substantial magnitude and decay much more slowly downstream than do those of potential disturbance. Vortical disturbance decays linearly with increasing mode except very close to the stator trailing edge. For a small axial gap, e.g., 10 percent chord, both vortical and potential disturbances must be included to determine the rotor gust response.

The objective of this paper is to decompose the unsteady force on the rotor of a stator/rotor axial compressor into vortical and potential contributions, for axial gaps of 10, 20, and 30% chord between blade rows. Three methods of decomposition are proposed. The Ž nal method adopted requires two steps. First, the potential contributed gust within the gap region is found using a panel code for stator/rotor conŽ guration and the vortical contributed gust from the difference between that calculated by a Navier

Three-dimensional (3D) tissue culture platforms that are capable of mimicking in vivo microenvironments to replicate physiological conditions are vital tools in a wide range of cellular and clinical studies. Here, learning from the nature of cilia in lungs - clearing mucus and pathogens from the airway - we develop a 3D culture approach via flexible and kinetic copolymer-based chains (nano-cilia) for diminishing cell-to-substrate adhesion. Multicellular spheroids or colonies were tested for 3-7 days in a microenvironment consisting of generated cells with properties of putative cancer stem cells (CSCs). The dynamic and reversible regulation of epithelial-mesenchymal transition (EMT) was examined in spheroids passaged and cultured in copolymer-coated dishes. The expression of CSC markers, including CD44, CD133, and ABCG2, and hypoxia signature, HIF-1α, was significantly upregulated compared to that without the nano-cilia. In addition, these spheroids exhibited chemotherapeutic resistance in vitro and acquired enhanced metastatic propensity, as verified from microfluidic chemotaxis assay designed to replicate in vivo-like metastasis. The biomimetic nano-cilia approach and microfluidic device may offer new opportunities to establish a rapid and cost-effective platform for the study of anti-cancer therapeutics and CSCs.

BACKGROUND Semen analysis is essential for evaluating male infertility. Besides sperm concentration, other properties, such as motility and morphology, are critical indicators in assessing sperm quality. Nevertheless, rapid and complete assessment of these measures still presents considerable difficulty and involves a range of complex issues. Here we present a microfluidic device capable of quantifying a range of properties of human sperm via the resistive pulse technique (RPT). METHODS An aperture, designed as a long channel, was used to allow the quantification of various properties as sperm swam through. RESULTS The time trace of the voltage drop across the aperture during sperm passage contained a wealth of information: the sperm volume was presented by the amplitude of the induced pulse, the swim velocity was evaluated via the duration, and the beat frequency was calculated from the voltage undulation superposed on the pulse signal. The RPT measurement of swim velocity and beat frequency showed a correlation with the same observation in a microscope (R2 = 0.94 and 0.70, respectively). CONCLUSIONS The proposed proof of principle enables substantial quantification of the motion-dependent properties of sperm. Because this approach requires only a current/voltage source and data analysis, it is economically advantageous compared with optical methods for characterizing sperm motion. Furthermore, this approach may be used to characterize sperm morphology.

Communication between cells and extracellular environments is of interest because of its critical roles in cell development and differentiation. Particularly, this signal transduction is commonly believed to rely on the contact and binding of the participating molecules/proteins, suggesting that the binding distance needed is less than a few nanometers. However, it is difficult to precisely match the rapidly binding interaction which depends on the probability of molecular collision in living systems, raising a hypothesis that another mechanism exists, could promote this signal communication, and remains unknown. Here we report that a long-range signal delivery over 10-μm and 20-μm polydimethylsiloxane(PDMS) barriers can be observed in microfluidically tetracycline (Tet) inducible expression systems. Results show that a significant increment of the long-range induced green fluorescent protein in human embryonic kidney 293T (HEK 293T) cells by the stimulation of Tet is demonstrated, and that such a signal induction is not dominated by Tet diffusion and displays a specific bindingless property. In addition, our experimental results, combined with theoretical modeling, suggest that this communication exhibits a bump-shaped characteristic depending on barrier thickness, materially structural property, surface roughness, and agonist concentration. It strongly relies on the PDMS barrier to delivery signal; therefore, we call such a mechanism as “wired” cell communication instead of wireless. These results could ignite interests in the novel and “wired” cell communication, which we call it X-signal, and in the use of such systems for the study of cellular biology and development of new drug.

We present a microfluidic device to generate either statically spatial or dynamically temporal logarithmic concentrations. The temporal logarithmic concentration generator was also integrated with planar patch-clamp chips for dose-response assays on ion channels. Proposed serial dilution principle controls the flow pattern at each branching point via designing the flow resistance of microchannels. Simple and linear ratios of the flow resistance results in desired logarithmic concentration at outlets, where the concentrations can be dynamically altered by different combination of valve actuations, were demonstrated. Single-cell pharmacology on ion channels was implemented by sequentially applying logarithmic drug concentrations to patched cells. Inhibitory activity of potassium channels of human embryonic kidney cells was examined by tetraethylammonium solutions. Resulted IC(50) and Hill slope reveal excellent agreement with assays from manually prepared drug concentrations showing the practicability and preciseness of the present approach. Applications include cellular analysis under various drugs and/or logarithmic concentrations at the single-cell level.

The off-design performance in a centrifugal pump is investigated experimentally. The objective is to identify flow features which lead to the onset of surge as the fundamental pumping system instability. Results show that there are primarily two reasons for the onset of surge as the flow is reduced in the pump studied: (a) adverse flow in the tongue region and (b) destabilizing effect of the pipe diffuser. The former is due to premature diffusion of the flow entering the tongue region, which is manifested by increased flow recirculation through the tongue/impeller gap and flow separation on the volute outer side-wall opposite the tongue. These effects in the tongue region flow coupled with the destabilizing behavior of the pipe diffuser lead to the eventual unstable pump operation.Copyright © 1993 by ASME

Cyto-analysis of rare cells often requires separation and detection with either procedure possesses substantial challenge. This paper presents a disk-based microfluidic platform for both procedures via immunomagnetic negative selection process. Proof-of-concept was conducted using wide range of MCF7 as target rare cells and spiked into Jurkat as non-target cells. Then, mononuclear cells (MNC) from healthy blood donors were mixed with MCF7s, modeling rare cells, and tested in the disk. Results show the average yield of detected MCF7 is near-constant 60±10% over a wide range of rarity from 10−3 to 10−6 and this yield also holds for MCF7/MNC complex mixture. Comparison with autoMACS and BD IMagnet separators revealed the average yield from the disk (60%) is superior to that of autoMACS (37.3%) and BD IMagnet (48.3%).

This paper presents a magnetically driven valve via a permanent magnet pressing a spacer against deformable polydimethylsiloxane (PDMS) to fully close a microchannel. Its ability for electrical isolation, time response, and resistance to backpressure are interrogated. Simulation of the valve closing process was commenced along with experimental verification. Effects of PDMS thickness, and dimension and aspect ratio of microchannels were characterized. Up to 10 GΩ electrical isolation was demonstrated, as well as 50–70 ms valve response and ∼200 kPa resistible pressure. On-demand actuation for arbitrary flow patterns further quantifies its utility. With advantages of simple fabrication, flexible valving location, and no external power requirement, the on/off valve could be leveraged for proof-of-concept microfluidic devices and other applications.

In the conventional bench-top approach, the DNA recombination process is time- and effort-consuming due to laborious procedures lasting from several hours to a day. A novel DNA selection and direct extraction process has been proposed, integrated and tested on chip. The integrative microfluidic chip can perform the whole procedure of DNA recombination, including DNA digestion, gel electrophoresis, DNA extraction and insert-vector ligation within 1 h. In this high-throughput design, the manual gel cutting was replaced by an automatic processing system that performed high-quality and high-recovery efficiency in DNA extraction process. With no need of gel-dissolving reagents and manipulation, the application of selection and direct extraction process could significantly eliminate the risks from UV and EtBr and also facilitate DNA recombination. Reliable output with high success rate of cloning has been achieved with a significant reduction in operational hazards, required materials, efforts and time.

This report presents a study of a novel mechanism (microvortices) to generate lift towards application in micro air vehicle (MAV). The mechanism of vortex generation considered is in-plane oscillation of a flat plate with suitable structure on the plate's top surface. Methodologically, experimental approach is first commenced followed by compution to study a wide range of parameter space. The microdevice leverages on Lorentz force to drive the suspended MEMs-based microplate to in-plane resonance.

This paper reports a hydrodynamic single-cell trapping platform for cellular assays. This approach utilizes an oscillatory microplate (100μm × 100μm) via Lorentz force to generate microvortices for cell trapping. The microdevice can trap a single cell in suspended state, which should aid analysis of a single cell. Results obtained with this platform show a single Jurkat cell can be trapped and labeled on CD45 antibodies under real-time monitoring.

The travelling wave dielectrophoretic pump studied here is essentially a rectangular straight micro-channel with an electrode array on part of its wall, and operated under an ac voltage with phase shift at neighbouring electrodes. The travelling wave dielectrophoretic force drives the cells, which drag the plasma, and after some sophisticated interaction between conventional dielectrophoresis, travelling wave dielectrophoresis and fluid mechanics, the whole blood is delivered. The pump was fabricated using MEMS techniques and studied in details for different parameters. It is found that the pumping velocity is maximized at an intermediate frequency around 20-30 MHz (varies with phase shift), and at an intermediate channel height at about 40 microm. The quasi-static average cell velocity can reach 15 microm s(-1) for a pump with 1 mm length and 16 electrodes (total array length 465 microm) operated at 5 V and 20 MHz with 90 degrees phase shift.

Uniform patterning of cells is highly desirable for most cellular studies involving cell–cell interactions but is often difficult in an in vitro environment. This paper presents the development of a collagen-coated planar interdigitated ring electrode (PIRE) array utilizing positive dielectrophoresis to pattern cells uniformly. Key features of the PIRE design include: (1) maximizing length along the edges where the localized maximum in the electric field exists; (2) making the inner gap slightly smaller than the outer gap in causing the electric field strength near the center of a PIRE being generally stronger than that near the outer edge of the same PIRE. Results of human hepatocellular carcinoma cells, HepG2, adhered on a 6 × 6 PIRE array show that cells patterned within minutes with good uniformity (48 ± 6 cells per PIRE). Cell viability test revealed healthy patterned cells after 24 h that were still confined to the collagen-coated PIREs. Furthermore, quantification of fluorescence intensity of living cells shows an acceptable reproducibility of cell viability among PIREs (mean normalized intensity per PIRE was 1 ± 0.138). The results suggest that the PIRE array would benefit applications that desire uniform cellular patterning, and improve both response and reproducibility of cell-based biosensors. © 2008 Elsevier B.V. All rights reserved.

We present a fast and easy fabrication method to create an hourglass-shaped aperture for cellular electrophysiology. A series of short pulses of CO2 laser were applied to drill on borosilicate cover glass. Special reflow phenomenon of melted glass occurred at the rear of the surface resulted in the unique tailored-contour aperture. Smooth and debris-free surface of the aperture reveals the capability of planar patch-clamp application. In seal resistance characterization, calculated seal resistance was 1.2MOmega before and 862MOmega after cell attached to a 4mum aperture. These values are promising and they are most likely due to freshly activated contact surface during seal formation. This efficient and economic approach could be extended to array configuration and integrate with microfluidic system.

This paper presents new results on manipulation of undiluted whole blood using traveling wave dielectrophoresis (twDEP). Although twDEP is not new, data on whole blood manipulation is rare. The twDEP in this work consists of four 2D electrodes of 20/spl mu/m width operating at 10MHz sinusoidal wave with 90 degree phase apart. Results show that whole blood bulk flow can be manipulated at-will in either direction. Moreover, simultaneous bi-direction motion is also observed at different height of the 25 /spl mu/m flow channel. This flow feature might be utilized for separation purpose.

The cellular signal transduction is commonly believed to rely on the direct “contact” or “binding” of the participating molecule reaction that depends positively on the corresponding molecule concentrations. In living systems, however, it is somewhat difficult to precisely match the corresponding rapid “binding,” depending on the probability of molecular collision, existing in the cellular receptor-ligand interactions. Thus, a question arises that if there is another mechanism (i.e., bindingless) that could promote this signal communication. According to this hypothesis, we report a cellular model based on the examination of intracellular calcium concentration to explore whether the unidentified signal delivery in cells exists, via a microfluidic device. This device was designed to isolate the cells from directly contacting with the corresponding ligands/molecules by the particular polydimethylsiloxane (PDMS) membranes with different thicknesses. Results show a significant increment of calcium mobilization in human prostate cancer PC-3 cells by the stimulation of endothelin-1, even up to a separated distance of 95 μm. In addition, these stimulated signals exhibited a bump-shaped characteristics depending on the membrane thickness. When the PDMS membrane is capped by SiO2, a particular trait that resembles the ballistic signal conduction was observed. A theoretical model was developed to describe the signal transport process across the PDMS membrane. Taken together, these results indicate that the unidentified signal (ligand structural information) delivery could occur in cells and be examined by the proposed approach, exhibiting a bindingless communication manner. Moreover, this approach and our finding may offer new opportunities to establish a robust and cost-effective platform for the study of cellular biology and new drug development.

This paper addresses the decay of rotor wake vorticity for a rotor/stator axial compressor, with the axial gap between blade rows being 10, 20 and 30 percent chord, and at both design and high loading levels. Experiments were conducted in a large-scale, low-speed axial compressor. Navier-Stokes calculations were also executed. Both data and Navier-Stokes results reveal that the decay of rotor wake vorticity increases substantially as the axial gap decreases; the decay for 10 percent gap is about twice that of 30 percent. Increased time-mean blade loading causes the vorticity decay to also increase, with this effect more pronounced for large axial gap than small. At the stator inlet mid-pitch location, the wake maximum vorticity for 10 and 30 percent chord gap cases being nearly the same (differ by 3.8%) at design loading. The corresponding stator unsteady force agrees within 5.2%. Variation of vorticity decay with axial gap is directly linked to the change in potential disturbance by the downstream stator on the rotor wake due to the change in gap spacing. This suggests that the stator potential disturbance causes the upstream rotor wake to decay at an increased rate which, in turns, results in a lowered level of stator response compared to that without this stator/wake interaction effect. Thus, in this context, blade row interaction is considered beneficial.

This paper demonstrates reduction of stator unsteady loading due to forced response in a large-scale, low-speed, rotor/stator/rotor axial compressor rig by clocking the downstream rotor. Data from the rotor/stator configuration showed that the stator response due to the upstream vortical disturbance reaches a maximum when the wake impinges against the suction surface immediately downstream of the leading edge. Results from the stator/rotor configuration revealed that the stator response due to the downstream potential disturbance reaches a minimum with a slight time delay after the rotor sweeps pass the stator trailing edge. For the rotor/stator/rotor configuration, with Gap1 = 10 percent chord and Gap2 = 30 percent chord, results showed a 60 percent reduction in the stator force amplitude by clocking the downstream rotor so that the time occurrence of the maximum force due to the upstream vortical disturbance coincides with that of the minimum force due to the downstream potential disturbance. This is the first time, the authors believe, that beneficial use of flow unsteadiness is definitively demonstrated to reduce the blade unsteady loading.