Electrokinetic Analysis of Cell Translocation in Low-Cost Microfluidic Cytometry for Tumor Cell Detection and Enumeration.

PubMed

Guo, Jinhong; Pui, Tze Sian; Ban, Yong-Ling; Rahman, Abdur Rub Abdur; Kang, Yuejun

2013-12-01

Conventional Coulter counters have been introduced as an important tool in biological cell assays since several decades ago. Recently, the emerging portable Coulter counter has demonstrated its merits in point of care diagnostics, such as on chip detection and enumeration of circulating tumor cells (CTC). The working principle is based on the cell translocation time and amplitude of electrical current change that the cell induces. In this paper, we provide an analysis of a Coulter counter that evaluates the hydrodynamic and electrokinetic properties of polystyrene microparticles in a microfluidic channel. The hydrodynamic force and electrokinetic force are concurrently analyzed to determine the translocation time and the electrical current pulses induced by the particles. Finally, we characterize the chip performance for CTC detection. The experimental results validate the numerical analysis of the microfluidic chip. The presented model can provide critical insight and guidance for developing micro-Coulter counter for point of care prognosis.

Making the invisible visible: a microfluidic chip using a low refractive index polymer.

PubMed

Hanada, Yasutaka; Ogawa, Tatsuya; Koike, Kazuhiko; Sugioka, Koji

2016-07-07

Microfluidic frameworks known as micro-total-analysis-systems or lab-on-a-chip have become versatile tools in cell biology research, since functional biochips are able to streamline dynamic observations of various cells. Glass or polymers are generally used as the substrate due to their high transparency, chemical stability and cost-effectiveness. However, these materials are not well suited for the microscopic observation of cell migration at the fluid boundary due to the refractive index mismatch between the medium and the biochip material. For this reason, we have developed a new method of fabricating three-dimensional (3D) microfluidic chips made of the low refractive index fluoric polymer CYTOP. This novel fabrication procedure involves the use of a femtosecond laser for direct writing, followed by wet etching with a dilute fluorinated solvent and annealing, to create high-quality 3D microfluidic chips inside a polymer substrate. A microfluidic chip made in this manner enabled us to more clearly observe the flagellum motion of a Dinoflagellate moving in circles near the fluid surface compared to the observations possible using conventional microfluidic chips. We believe that CYTOP microfluidic chips made using this new method may allow more detailed analysis of various cell migrations near solid boundaries.

A nano-patterned self assembled monolayer (SAM) rutile titania cancer chip for rapid, low cost, highly sensitive, direct cancer analysis in MALDI-MS.

PubMed

Manikandan, M; Gopal, Judy; Hasan, Nazim; Wu, Hui-Fen

2014-12-01

We developed a cancer chip by nano-patterning a highly sensitive SAM titanium surface capable of capturing and sensing concentrations as low as 10 cancer cells/mL from the environment by Matrix Assisted Laser Desorption and Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). The current approach evades any form of pretreatment and sample preparation processes; it is time saving and does not require the (expensive) conventional MALDI target plate. The home made aluminium (Al) target holder cost, on which we loaded the cancer chips for MALDI-TOF MS analysis, is about 60 USD. While the conventional stainless steel MALDI target plate is more than 700 USD. The SAM surface was an effective platform leading to on-chip direct MALDI-MS detection of cancer cells. We compared the functionality of this chip with the unmodified titanium surfaces and thermally oxidized (TO) titanium surfaces. The lowest detectable concentration of the TO chip was 10(3) cells/mL, while the lowest detectable concentration of the control or unmodified titanium chips was 10(6) cells/mL. Compared to the control surface, the SAM cancer chip showed 100,000 times of enhanced sensitivity and compared with the TO chip, 1000 times of increased sensitivity. The high sensitivity of the SAM surfaces is attributed to the presence of the rutile SAM, surface roughness and surface wettability as confirmed by AFM, XRD, contact angle microscope and FE-SEM. This study opens a new avenue for the potent application of the SAM cancer chip for direct cancer diagnosis by MALDI-TOF MS in the near future. Copyright © 2014. Published by Elsevier B.V.

A Facile Droplet-Chip-Time-Resolved Inductively Coupled Plasma Mass Spectrometry Online System for Determination of Zinc in Single Cell.

PubMed

Wang, Han; Chen, Beibei; He, Man; Hu, Bin

2017-05-02

Single cell analysis is a significant research field in recent years reflecting the heterogeneity of cells in a biological system. In this work, a facile droplet chip was fabricated and online combined with time-resolved inductively coupled plasma mass spectrometry (ICPMS) via a microflow nebulizer for the determination of zinc in single HepG2 cells. On the focusing geometric designed PDMS microfluidic chip, the aqueous cell suspension was ejected and divided by hexanol to generate droplets. The droplets encapsulated single cells remain intact during the transportation into ICP for subsequent detection. Under the optimized conditions, the frequency of droplet generation is 3-6 × 10 6 min -1 , and the injected cell number is 2500 min -1 , which can ensure the single cell encapsulation. ZnO nanoparticles (NPs) were used for the quantification of zinc in single cells, and the accuracy was validated by conventional acid digestion-ICPMS method. The ZnO NPs incubated HepG2 cells were analyzed as model samples, and the results exhibit the heterogeneity of HepG2 cells in the uptake/adsorption of ZnO NPs. The developed online droplet-chip-ICPMS analysis system achieves stable single cell encapsulation and has high throughput for single cell analysis. It has the potential in monitoring the content as well as distribution of trace elements/NPs at the single cell level.

Finite element analysis of a micromechanical deformable mirror device

NASA Technical Reports Server (NTRS)

Sheerer, T. J.; Nelson, W. E.; Hornbeck, L. J.

1989-01-01

A monolithic spatial light modulator chip was developed consisting of a large number of micrometer-scale mirror cells which can be rotated through an angle by application of an electrostatic field. The field is generated by electronics integral to the chip. The chip has application in photoreceptor based non-impact printing technologies. Chips containing over 16000 cells were fabricated, and were tested to several billions of cycles. Finite Element Analysis (FEA) of the device was used to model both the electrical and mechanical characteristics.

Chip-based generation of carbon nanodots via electrochemical oxidation of screen printed carbon electrodes and the applications for efficient cell imaging and electrochemiluminescence enhancement

NASA Astrophysics Data System (ADS)

Xu, Yuanhong; Liu, Jingquan; Zhang, Jizhen; Zong, Xidan; Jia, Xiaofang; Li, Dan; Wang, Erkang

2015-05-01

A portable lab-on-a-chip methodology to generate ionic liquid-functionalized carbon nanodots (CNDs) was developed via electrochemical oxidation of screen printed carbon electrodes. The CNDs can be successfully applied for efficient cell imaging and solid-state electrochemiluminescence sensor fabrication on the paper-based chips.A portable lab-on-a-chip methodology to generate ionic liquid-functionalized carbon nanodots (CNDs) was developed via electrochemical oxidation of screen printed carbon electrodes. The CNDs can be successfully applied for efficient cell imaging and solid-state electrochemiluminescence sensor fabrication on the paper-based chips. Electronic supplementary information (ESI) available: Experimental section; Fig. S1. XPS spectra of the as-prepared CNDs after being dialyzed for 72 hours; Fig. S2. LSCM images showing time-dependent fluorescence signals of HeLa cells treated by the as-prepared CNDs; Tripropylamine analysis using the Nafion/CNDs modified ECL sensor. See DOI: 10.1039/c5nr01765c

Microfluidic Platform for the Long-Term On-Chip Cultivation of Mammalian Cells for Lab-On-A-Chip Applications.

PubMed

Bunge, Frank; Driesche, Sander van den; Vellekoop, Michael J

2017-07-10

Lab-on-a-Chip (LoC) applications for the long-term analysis of mammalian cells are still very rare due to the lack of convenient cell cultivation devices. The difficulties are the integration of suitable supply structures, the need of expensive equipment like an incubator and sophisticated pumps as well as the choice of material. The presented device is made out of hard, but non-cytotoxic materials (silicon and glass) and contains two vertical arranged membranes out of hydrogel. The porous membranes are used to separate the culture chamber from two supply channels for gases and nutrients. The cells are fed continuously by diffusion through the membranes without the need of an incubator and low requirements on the supply of medium to the assembly. The diffusion of oxygen is modelled in order to find the optimal dimensions of the chamber. The chip is connected via 3D-printed holders to the macroscopic world. The holders are coated with Parlyene C to ensure that only biocompatible materials are in contact with the culture medium. The experiments with MDCK-cells show the successful seeding inside the chip, culturing and passaging. Consequently, the presented platform is a step towards Lab-on-a-Chip applications that require long-term cultivation of mammalian cells.

Rapid analysis of protein interactions: On-chip micropurification of recombinant protein expressed in Esherichia coli.

PubMed

Natsume, Tohru; Taoka, Masato; Manki, Hiroshi; Kume, Shouen; Isobe, Toshiaki; Mikoshiba, Katsuhiko

2002-09-01

We describe a rapid analysis of interactions between antibodies and a recombinant protein present in total cell lysates. Using a surface plasmon resonance biosensor, a low concentration of glutathione-S-transferase (GST) fused protein expressed in small scale Esherichia coli culture was purified on an anti-GST antibody immobilized sensor chip. The 'on-chip purification' was verified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry by measuring the molecular masses of recombinant proteins purified on the sensor chip. The specific binding of monoclonal antibodies for the on-chip micropurified recombinant proteins can then be monitored, thus enabling kinetic analysis and epitope mapping of the bound antibodies. This approach reduced time, resources and sample consumption by avoiding conventional steps related to concentration and purification.

FISH-in-CHIPS: A Microfluidic Platform for Molecular Typing of Cancer Cells.

PubMed

Perez-Toralla, Karla; Mottet, Guillaume; Tulukcuoglu-Guneri, Ezgi; Champ, Jérôme; Bidard, François-Clément; Pierga, Jean-Yves; Klijanienko, Jerzy; Draskovic, Irena; Malaquin, Laurent; Viovy, Jean-Louis; Descroix, Stéphanie

2017-01-01

Microfluidics offer powerful tools for the control, manipulation, and analysis of cells, in particular for the assessment of cell malignancy or the study of cell subpopulations. However, implementing complex biological protocols on chip remains a challenge. Sample preparation is often performed off chip using multiple manually performed steps, and protocols usually include different dehydration and drying steps that are not always compatible with a microfluidic format.Here, we report the implementation of a Fluorescence in situ Hybridization (FISH) protocol for the molecular typing of cancer cells in a simple and low-cost device. The geometry of the chip allows integrating the sample preparation steps to efficiently assess the genomic content of individual cells using a minute amount of sample. The FISH protocol can be fully automated, thus enabling its use in routine clinical practice.

Microchip-based cell lysis and DNA extraction from sperm cells for application to forensic analysis.

PubMed

Bienvenue, Joan M; Duncalf, Natalie; Marchiarullo, Daniel; Ferrance, Jerome P; Landers, James P

2006-03-01

The current backlog of casework is among the most significant challenges facing crime laboratories at this time. While the development of next-generation microchip-based technology for expedited forensic casework analysis offers one solution to this problem, this will require the adaptation of manual, large-volume, benchtop chemistry to small volume microfluidic devices. Analysis of evidentiary materials from rape kits where semen or sperm cells are commonly found represents a unique set of challenges for on-chip cell lysis and DNA extraction that must be addressed for successful application. The work presented here details the development of a microdevice capable of DNA extraction directly from sperm cells for application to the analysis of sexual assault evidence. A variety of chemical lysing agents are assessed for inclusion in the extraction protocol and a method for DNA purification from sperm cells is described. Suitability of the extracted DNA for short tandem repeat (STR) analysis is assessed and genetic profiles shown. Finally, on-chip cell lysis methods are evaluated, with results from fluorescence visualization of cell rupture and DNA extraction from an integrated cell lysis and purification with subsequent STR amplification presented. A method for on-chip cell lysis and DNA purification is described, with considerations toward inclusion in an integrated microdevice capable of both differential cell sorting and DNA extraction. The results of this work demonstrate the feasibility of incorporating microchip-based cell lysis and DNA extraction into forensic casework analysis.

On-chip Magnetic Separation and Cell Encapsulation in Droplets†

PubMed Central

Chen, Aaron; Byvank, Tom; Chang, Woo-Jin; Bharde, Atul; Vieira, Greg; Miller, Brandon; Chalmers, Jeffrey J.; Bashir, Rashid; Sooryakumar, Ratnasingham

2014-01-01

The demand for high-throughput single cell assays is gaining importance because of the heterogeneity of many cell suspensions, even after significant initial sorting. These suspensions may display cell-to-cell variability at the gene expression level that could impact single cell functional genomics, cancer, stem-cell research and drug screening. The on-chip monitoring of individual cells in an isolated environment would prevent cross-contamination, provide high recovery yield, and enable study of biological traits at a single cell level. These advantages of on-chip biological experiments is a significant improvement for myriad of cell analyses over conventional methods, which require bulk samples providing only averaged information on cell metabolism. We report on a device that integrates mobile magnetic trap array with microfluidic technology to provide, combined functionality of separation of immunomagnetically labeled cells or magnetic beads and their encapsulation with reagents into pico-liter droplets. This scheme of simultaneous reagent delivery and compartmentalization of the cells immediately after sorting, all performed seamlessly within the same chip, offers unique advantages such as the ability to capture cell traits as originated from its native environment, reduced chance of contamination, minimal use and freshness of the reagent solution that reacts only with separated objects, and tunable encapsulation characteristics independent of the input flow. In addition to the demonstrated preliminary cell viability assay, the device can potentially be integrated with other up- or downstream on-chip modules to become a powerful single-cell analysis tool. PMID:23370785

Micromagnet arrays for on-chip focusing, switching, and separation of superparamagnetic beads and single cells.

PubMed

Rampini, S; Kilinc, D; Li, P; Monteil, C; Gandhi, D; Lee, G U

2015-08-21

Nonlinear magnetophoresis (NLM) is a novel approach for on-chip transport and separation of superparamagnetic (SPM) beads, based on a travelling magnetic field wave generated by the combination of a micromagnet array (MMA) and an applied rotating magnetic field. Here, we present two novel MMA designs that allow SPM beads to be focused, sorted, and separated on-chip. Converging MMAs were used to rapidly collect the SPM beads from a large region of the chip and focus them into synchronised lines. We characterise the collection efficiency of the devices and demonstrate that they can facilitate on-chip analysis of populations of SPM beads using a single-point optical detector. The diverging MMAs were used to control the transport of the beads and to separate them based on their size. The separation efficiency of these devices was determined by the orientation of the magnetisation of the micromagnets relative to the external magnetic field and the size of the beads and relative to that of micromagnets. By controlling these parameters and the rotation of the external magnetic field we demonstrated the controlled transport of SPM bead-labelled single MDA-MB-231 cells. The use of these novel MMAs promises to allow magnetically-labelled cells to be efficiently isolated and then manipulated on-chip for analysis with high-resolution chemical and physical techniques.

Anti-cancer activity of ZnO chips by sustained zinc ion release.

PubMed

Moon, Seong-Hee; Choi, Won Jin; Choi, Sik-Won; Kim, Eun Hye; Kim, Jiyeon; Lee, Jeong-O; Kim, Seong Hwan

2016-01-01

We report anti-cancer activity of ZnO thin-film-coated chips by sustained release of zinc ions. ZnO chips were fabricated by precisely tuning ZnO thickness using atomic layer deposition, and their potential to release zinc ions relative to the number of deposition cycles was evaluated. ZnO chips exhibited selective cytotoxicity in human B lymphocyte Raji cells while having no effect on human peripheral blood mononuclear cells. Of importance, the half-maximal inhibitory concentration of the ZnO chip on the viability of Raji cells was 121.5 cycles, which was comparable to 65.7 nM of daunorubicin, an anti-cancer drug for leukemia. Molecular analysis of cells treated with ZnO chips revealed that zinc ions released from the chips increased cellular levels of reactive oxygen species, including hydrogen peroxide, which led to the down-regulation of anti-apoptotic molecules (such as HIF-1α, survivin, cIAP-2, claspin, p-53, and XIAP) and caspase-dependent apoptosis. Because the anti-cancer activity of ZnO chips and the mode of action were comparable to those of daunorubicin, the development and optimization of ZnO chips that gradually release zinc ions might have clinical anti-cancer potential. A further understanding of the biological action of ZnO-related products is crucial for designing safe biomaterials with applications in disease treatment.

Continuous cell introduction and rapid dynamic lysis for high-throughput single-cell analysis on microfludic chips with hydrodynamic focusing.

PubMed

Xu, Chun-Xiu; Yin, Xue-Feng

2011-02-04

A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance. Copyright © 2010 Elsevier B.V. All rights reserved.

Epigenome analysis of pluripotent stem cells

PubMed Central

Ricupero, Christopher L.; Swerdel, Mavis R.; Hart, Ronald P.

2015-01-01

Summary Mis-regulation of gene expression due to epigenetic abnormalities has been linked with complex genetic disorders, psychiatric illness and cancer. In addition, the dynamic epigenetic changes that occur in pluripotent stem cells are believed to impact regulatory networks essential for proper lineage development. Chromatin immunoprecipitation (ChIP) is a technique used to isolate and enrich chromatin fragments using antibodies against specific chromatin modifications, such as DNA binding proteins or covalent histone modifications. Until recently, many ChIP protocols required millions of cells for each immunoprecipitation. This severely limited analysis of rare cell populations or post-mitotic, differentiated cell lines. Here, we describe a low cell number ChIP protocol with next generation sequencing and analysis, that has the potential to uncover novel epigenetic regulatory pathways that were previously difficult or impossible to obtain. PMID:23546758

Single-Cell Isolation of Circulating Tumor Cells from Whole Blood by Lateral Magnetophoretic Microseparation and Microfluidic Dispensing.

PubMed

Kim, Jinho; Cho, Hyungseok; Han, Song-I; Han, Ki-Ho

2016-05-03

This paper introduces a single-cell isolation technology for circulating tumor cells (CTCs) using a microfluidic device (the "SIM-Chip"). The SIM-Chip comprises a lateral magnetophoretic microseparator and a microdispenser as a two-step cascade platform. First, CTCs were enriched from whole blood by the lateral magnetophoretic microseparator based on immunomagnetic nanobeads. Next, the enriched CTCs were electrically identified by single-cell impedance cytometer and isolated as single cells using the microshooter. Using 200 μL of whole blood spiked with 50 MCF7 breast cancer cells, the analysis demonstrated that the single-cell isolation efficiency of the SIM-Chip was 82.4%, and the purity of the isolated MCF7 cells with respect to WBCs was 92.45%. The data also showed that the WBC depletion rate of the SIM-Chip was 2.5 × 10(5) (5.4-log). The recovery rates were around 99.78% for spiked MCF7 cells ranging in number from 10 to 90. The isolated single MCF7 cells were intact and could be used for subsequent downstream genetic assays, such as RT-PCR. Single-cell culture evaluation of the proliferation of MCF7 cells isolated by the SIM-Chip showed that 84.1% of cells at least doubled in 5 days. Consequently, the SIM-Chip could be used for single-cell isolation of rare target cells from whole blood with high purity and recovery without cell damage.

Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; Faley, Shannon; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell death is a stochastic process, often initiated and/or executed in a multi-pathway/multi-organelle fashion. Therefore, high-throughput single-cell analysis platforms are required to provide detailed characterization of kinetics and mechanisms of cell death in heterogeneous cell populations. However, there is still a largely unmet need for inert fluorescent probes, suitable for prolonged kinetic studies. Here, we compare the use of innovative adaptation of unsymmetrical SYTO dyes for dynamic real-time analysis of apoptosis in conventional as well as microfluidic chip-based systems. We show that cyanine SYTO probes allow non-invasive tracking of intracellular events over extended time. Easy handling and “stain–no wash” protocols open up new opportunities for high-throughput analysis and live-cell sorting. Furthermore, SYTO probes are easily adaptable for detection of cell death using automated microfluidic chip-based cytometry. Overall, the combined use of SYTO probes and state-of-the-art Lab-on-a-Chip platform emerges as a cost effective solution for automated drug screening compared to conventional Annexin V or TUNEL assays. In particular, it should allow for dynamic analysis of samples where low cell number has so far been an obstacle, e.g. primary cancer stems cells or circulating minimal residual tumors. PMID:19298813

Rapid, portable and cost-effective yeast cell viability and concentration analysis using lensfree on-chip microscopy and machine learning.

PubMed

Feizi, Alborz; Zhang, Yibo; Greenbaum, Alon; Guziak, Alex; Luong, Michelle; Chan, Raymond Yan Lok; Berg, Brandon; Ozkan, Haydar; Luo, Wei; Wu, Michael; Wu, Yichen; Ozcan, Aydogan

2016-11-01

Monitoring yeast cell viability and concentration is important in brewing, baking and biofuel production. However, existing methods of measuring viability and concentration are relatively bulky, tedious and expensive. Here we demonstrate a compact and cost-effective automatic yeast analysis platform (AYAP), which can rapidly measure cell concentration and viability. AYAP is based on digital in-line holography and on-chip microscopy and rapidly images a large field-of-view of 22.5 mm 2 . This lens-free microscope weighs 70 g and utilizes a partially-coherent illumination source and an opto-electronic image sensor chip. A touch-screen user interface based on a tablet-PC is developed to reconstruct the holographic shadows captured by the image sensor chip and use a support vector machine (SVM) model to automatically classify live and dead cells in a yeast sample stained with methylene blue. In order to quantify its accuracy, we varied the viability and concentration of the cells and compared AYAP's performance with a fluorescence exclusion staining based gold-standard using regression analysis. The results agree very well with this gold-standard method and no significant difference was observed between the two methods within a concentration range of 1.4 × 10 5 to 1.4 × 10 6 cells per mL, providing a dynamic range suitable for various applications. This lensfree computational imaging technology that is coupled with machine learning algorithms would be useful for cost-effective and rapid quantification of cell viability and density even in field and resource-poor settings.

Droplet-based microfluidic analysis and screening of single plant cells.

PubMed

Yu, Ziyi; Boehm, Christian R; Hibberd, Julian M; Abell, Chris; Haseloff, Jim; Burgess, Steven J; Reyna-Llorens, Ivan

2018-01-01

Droplet-based microfluidics has been used to facilitate high-throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems where phenotyping analyses typically are time-consuming and low-throughput. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 cells per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.

Gold-coated polydimethylsiloxane microwells for high-throughput electrochemiluminescence analysis of intracellular glucose at single cells.

PubMed

Xia, Juan; Zhou, Junyu; Zhang, Ronggui; Jiang, Dechen; Jiang, Depeng

2018-06-04

In this communication, a gold-coated polydimethylsiloxane (PDMS) chip with cell-sized microwells was prepared through a stamping and spraying process that was applied directly for high-throughput electrochemiluminescence (ECL) analysis of intracellular glucose at single cells. As compared with the previous multiple-step fabrication of photoresist-based microwells on the electrode, the preparation process is simple and offers fresh electrode surface for higher luminescence intensity. More luminescence intensity was recorded from cell-retained microwells than that at the planar region among the microwells that was correlated with the content of intracellular glucose. The successful monitoring of intracellular glucose at single cells using this PDMS chip will provide an alternative strategy for high-throughput single-cell analysis. Graphical abstract ᅟ.

Programmable lab-on-a-chip system for single cell analysis

NASA Astrophysics Data System (ADS)

Thalhammer, S.

2009-05-01

The collection, selection, amplification and detection of minimum genetic samples became a part of everyday life in medical and biological laboratories, to analyze DNA-fragments of pathogens, patient samples and traces on crime scenes. About a decade ago, a handful of researchers began discussing an intriguing idea. Could the equipment needed for everyday chemistry and biology procedures be shrunk to fit on a chip in the size of a fingernail? Miniature devices for, say, analysing DNA and proteins should be faster and cheaper than conventional versions. Lab-on-a-chip is an advanced technology that integrates a microfluidic system on a microscale chip device. The "laboratory" is created by means of channels, mixers, reservoirs, diffusion chambers, integrated electrodes, pumps, valves and more. With lab-ona- chip technology, complete laboratories on a square centimetre can be created. Here, a multifunctional programmable Lab-on-a-Chip driven by nanofluidics and controlled by surface acoustic waves (SAW) is presented. This system combines serial DNA-isolation-, amplification- and array-detection-process on a modified glass-platform. The fluid actuation is controlled via SAW by interdigital transducers implemented in the chemical modified chip surface. The chemical surface modification allows fluid handling in the sub-microliter range. Minute amount of sample material is extracted by laser-based microdissection out of e.g. histological sections at the single cell level. A few picogram of genetic material are isolated and transferred via a low-pressure transfer system (SPATS) onto the chip. Subsequently the genetic material inside single droplets, which behave like "virtual" beaker, is transported to the reaction and analysis centers on the chip surface via surface acoustic waves, mainly known as noise dumping filters in mobile phones. At these "biological reactors" the genetic material is processed, e.g. amplified via polymerase chain reaction methods, and genetically characterized.

High-throughput analysis of yeast replicative aging using a microfluidic system

PubMed Central

Jo, Myeong Chan; Liu, Wei; Gu, Liang; Dang, Weiwei; Qin, Lidong

2015-01-01

Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction. PMID:26170317

Microfluidic device for acoustic cell lysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Branch, Darren W.; Cooley, Erika Jane; Smith, Gennifer Tanabe

2015-08-04

A microfluidic acoustic-based cell lysing device that can be integrated with on-chip nucleic acid extraction. Using a bulk acoustic wave (BAW) transducer array, acoustic waves can be coupled into microfluidic cartridges resulting in the lysis of cells contained therein by localized acoustic pressure. Cellular materials can then be extracted from the lysed cells. For example, nucleic acids can be extracted from the lysate using silica-based sol-gel filled microchannels, nucleic acid binding magnetic beads, or Nafion-coated electrodes. Integration of cell lysis and nucleic acid extraction on-chip enables a small, portable system that allows for rapid analysis in the field.

Engineering cell-compatible paper chips for cell culturing, drug screening, and mass spectrometric sensing.

PubMed

Chen, Qiushui; He, Ziyi; Liu, Wu; Lin, Xuexia; Wu, Jing; Li, Haifang; Lin, Jin-Ming

2015-10-28

Paper-supported cell culture is an unprecedented development for advanced bioassays. This study reports a strategy for in vitro engineering of cell-compatible paper chips that allow for adherent cell culture, quantitative assessment of drug efficiency, and label-free sensing of intracellular molecules via paper spray mass spectrometry. The polycarbonate paper is employed as an excellent alternative bioscaffold for cell distribution, adhesion, and growth, as well as allowing for fluorescence imaging without light scattering. The cell-cultured paper chips are thus amenable to fabricate 3D tissue construction and cocultures by flexible deformation, stacks and assembly by layers of cells. As a result, the successful development of cell-compatible paper chips subsequently offers a uniquely flexible approach for in situ sensing of live cell components by paper spray mass spectrometry, allowing profiling the cellular lipids and quantitative measurement of drug metabolism with minimum sample pretreatment. Consequently, the developed paper chips for adherent cell culture are inexpensive for one-time use, compatible with high throughputs, and amenable to label-free and rapid analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recent lab-on-chip developments for novel drug discovery.

PubMed

Khalid, Nauman; Kobayashi, Isao; Nakajima, Mitsutoshi

2017-07-01

Microelectromechanical systems (MEMS) and micro total analysis systems (μTAS) revolutionized the biochemical and electronic industries, and this miniaturization process became a key driver for many markets. Now, it is a driving force for innovations in life sciences, diagnostics, analytical sciences, and chemistry, which are called 'lab-on-a-chip, (LOC)' devices. The use of these devices allows the development of fast, portable, and easy-to-use systems with a high level of functional integration for applications such as point-of-care diagnostics, forensics, the analysis of biomolecules, environmental or food analysis, and drug development. In this review, we report on the latest developments in fabrication methods and production methodologies to tailor LOC devices. A brief overview of scale-up strategies is also presented together with their potential applications in drug delivery and discovery. The impact of LOC devices on drug development and discovery has been extensively reviewed in the past. The current research focuses on fast and accurate detection of genomics, cell mutations and analysis, drug delivery, and discovery. The current research also differentiates the LOC devices into new terminology of microengineering, like organ-on-a-chip, stem cells-on-a-chip, human-on-a-chip, and body-on-a-chip. Key challenges will be the transfer of fabricated LOC devices from lab-scale to industrial large-scale production. Moreover, extensive toxicological studies are needed to justify the use of microfabricated drug delivery vehicles in biological systems. It will also be challenging to transfer the in vitro findings to suitable and promising in vivo models. WIREs Syst Biol Med 2017, 9:e1381. doi: 10.1002/wsbm.1381 For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

Validation and perspectives of a femtosecond laser fabricated monolithic optical stretcher

PubMed Central

Bellini, Nicola; Bragheri, Francesca; Cristiani, Ilaria; Guck, Jochen; Osellame, Roberto; Whyte, Graeme

2012-01-01

The combination of high power laser beams with microfluidic delivery of cells is at the heart of high-throughput, single-cell analysis and disease diagnosis with an optical stretcher. So far, the challenges arising from this combination have been addressed by externally aligning optical fibres with microfluidic glass capillaries, which has a limited potential for integration into lab-on-a-chip environments. Here we demonstrate the successful production and use of a monolithic glass chip for optical stretching of white blood cells, featuring microfluidic channels and optical waveguides directly written into bulk glass by femtosecond laser pulses. The performance of this novel chip is compared to the standard capillary configuration. The robustness, durability and potential for intricate flow patterns provided by this monolithic optical stretcher chip suggest its use for future diagnostic and biotechnological applications. PMID:23082304

Microfluidic Devices for Forensic DNA Analysis: A Review.

PubMed

Bruijns, Brigitte; van Asten, Arian; Tiggelaar, Roald; Gardeniers, Han

2016-08-05

Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10-20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook.

Monitoring dynamic interactions of tumor cells with tissue and immune cells in a lab-on-a-chip.

PubMed

Charwat, Verena; Rothbauer, Mario; Tedde, Sandro F; Hayden, Oliver; Bosch, Jacobus J; Muellner, Paul; Hainberger, Rainer; Ertl, Peter

2013-12-03

A complementary cell analysis method has been developed to assess the dynamic interactions of tumor cells with resident tissue and immune cells using optical light scattering and impedance sensing to shed light on tumor cell behavior. The combination of electroanalytical and optical biosensing technologies integrated in a lab-on-a-chip allows for continuous, label-free, and noninvasive probing of dynamic cell-to-cell interactions between adherent and nonadherent cocultures, thus providing real-time insights into tumor cell responses under physiologically relevant conditions. While the study of adherent cocultures is important for the understanding and suppression of metastatic invasion, the analysis of tumor cell interactions with nonadherent immune cells plays a vital role in cancer immunotherapy research. For the first time, the direct cell-to-cell interactions of tumor cells with bead-activated primary T cells were continuously assessed using an effector cell to target a cell ratio of 10:1.

Trapping and Collection of Lymphocytes Using Microspot Array Chip and Magnetic Beads

NASA Astrophysics Data System (ADS)

Hashioka, Shingi; Obata, Tsutomu; Tokimitsu, Yoshiharu; Fujiki, Satoshi; Nakazato, Hiroyoshi; Muraguchi, Atsushi; Kishi, Hiroyuki; Tanino, Katsumi

2006-04-01

A microspot array chip, which has microspots of a magnetic thin film patterned on a glass substrate, was fabricated for trapping individual cells and for measuring their cellular response. The chip was easily fabricated by conventional semiconductor fabrication techniques on a mass production level as a disposable medical device. When a solution of lymphocyte-bound-magnetic beads was poured into the magnetized chip, each lymphocyte was trapped on each microspot of the magnetic thin film. The trapped cells were easily recovered from the chip using a micromanipulator. The micro-spot array chip can be utilized for arraying live cells and for measuring the response of each cell. The chip will be useful for preparing on array of different kinds of cells and for analyzing cellular response at the single cell level. The chip will be particularly useful for detecting antigen-specific B-lymphocytes and antigen-specific antibody complementary deoxyribonucleic acid (cDNA).

Surface transport and stable trapping of particles and cells by an optical waveguide loop.

PubMed

Hellesø, Olav Gaute; Løvhaugen, Pål; Subramanian, Ananth Z; Wilkinson, James S; Ahluwalia, Balpreet Singh

2012-09-21

Waveguide trapping has emerged as a useful technique for parallel and planar transport of particles and biological cells and can be integrated with lab-on-a-chip applications. However, particles trapped on waveguides are continuously propelled forward along the surface of the waveguide. This limits the practical usability of the waveguide trapping technique with other functions (e.g. analysis, imaging) that require particles to be stationary during diagnosis. In this paper, an optical waveguide loop with an intentional gap at the centre is proposed to hold propelled particles and cells. The waveguide acts as a conveyor belt to transport and deliver the particles/cells towards the gap. At the gap, the diverging light fields hold the particles at a fixed position. The proposed waveguide design is numerically studied and experimentally implemented. The optical forces on the particle at the gap are calculated using the finite element method. Experimentally, the method is used to transport and trap micro-particles and red blood cells at the gap with varying separations. The waveguides are only 180 nm thick and thus could be integrated with other functions on the chip, e.g. microfluidics or optical detection, to make an on-chip system for single cell analysis and to study the interaction between cells.

Dynamics of cells function on laser cell-chip system

NASA Astrophysics Data System (ADS)

Kushibiki, Toshihiro; Sano, Tomoko; Ishii, Katsunori; Yoshihashi-Suzuki, Sachiko; Awazu, Kunio

2006-02-01

A new type of cell-cultivation system based on laser processing has been developed for the on-chip cultivation of living cells. We introduce a "laser cell-chip", on which migration of cells, such as stem cells, tumor cells or immunocompetent cells, can be observed. A sheet prepared from epoxy resin was processed by KrF excimer laser (248 nm, 1.6 J/cm2) for preparation of microgrooved surfaces with various groove width, spacing, and depth. A laser cell-chip can make kinetic studies of cell migration depending on the concentration gradient of a chemoattractant. In this study, megakaryocytes were used for the migration on a groove of laser cell-chip by the concentration gradient of the stromal cell derived factor 1 (SDF-1/CXCL12). SDF-1/CXCL12 plays an important and unique role in the regulation of stem/progenitor cell trafficking. A megakaryocyte was migrated on a groove of laser cell-chip depending on the optical concentration gradient of SDF-1/CXCL12. Since SDF-1/CXCL12-induced migration of mature megakaryocyte was known to increase the platelet production in the bone marrow extravascular space, the diagnosis of cell migration on laser cell-chip could provide a new strategy to potentially reconstitute hematopoiesis and avoid life-threatening hemorrhage after myelosuppression or bone marrow failure.

Capture and On-chip analysis of Melanoma Cells Using Tunable Surface Shear forces

NASA Astrophysics Data System (ADS)

Tsao, Simon Chang-Hao; Vaidyanathan, Ramanathan; Dey, Shuvashis; Carrascosa, Laura G.; Christophi, Christopher; Cebon, Jonathan; Shiddiky, Muhammad J. A.; Behren, Andreas; Trau, Matt

2016-01-01

With new systemic therapies becoming available for metastatic melanoma such as BRAF and PD-1 inhibitors, there is an increasing demand for methods to assist with treatment selection and response monitoring. Quantification and characterisation of circulating melanoma cells (CMCs) has been regarded as an excellent non-invasive candidate but a sensitive and efficient tool to do these is lacking. Herein we demonstrate a microfluidic approach for melanoma cell capture and subsequent on-chip evaluation of BRAF mutation status. Our approach utilizes a recently discovered alternating current electrohydrodynamic (AC-EHD)-induced surface shear forces, referred to as nanoshearing. A key feature of nanoshearing is the ability to agitate fluid to encourage contact with surface-bound antibody for the cell capture whilst removing nonspecific cells from the surface. By adjusting the AC-EHD force to match the binding affinity of antibodies against the melanoma-associated chondroitin sulphate proteoglycan (MCSP), a commonly expressed melanoma antigen, this platform achieved an average recovery of 84.7% from biological samples. Subsequent staining with anti-BRAFV600E specific antibody enabled on-chip evaluation of BRAFV600E mutation status in melanoma cells. We believe that the ability of nanoshearing-based capture to enumerate melanoma cells and subsequent on-chip characterisation has the potential as a rapid screening tool while making treatment decisions.

Microfluidic-Based Enrichment and Retrieval of Circulating Tumor Cells for RT-PCR Analysis.

PubMed

Gogoi, Priya; Sepehri, Saedeh; Chow, Will; Handique, Kalyan; Wang, Yixin

2017-01-01

Molecular analysis of circulating tumor cells (CTCs) is hindered by low sensitivity and high level of background leukocytes of currently available CTC enrichment technologies. We have developed a novel device to enrich and retrieve CTCs from blood samples by using a microfluidic chip. The Celsee PREP100 device captures CTCs with high sensitivity and allows the captured CTCs to be retrieved for molecular analysis. It uses the microfluidic chip which has approximately 56,320 capture chambers. Based on differences in cell size and deformability, each chamber ensures that small blood escape while larger CTCs of varying sizes are trapped and isolated in the chambers. In this report, we used the Celsee PREP100 to capture cancer cells spiked into normal donor blood samples. We were able to show that the device can capture as low as 10 cells with high reproducibility. The captured CTCs were retrieved from the microfluidic chip. The cell recovery rate of this back-flow procedure is 100% and the level of remaining background leukocytes is very low (about 300-400 cells). RNA from the retrieved cells are extracted and converted to cDNA, and gene expression analysis of selected cancer markers can be carried out by using RT-PCR assays. The sensitive and easy-to-use Celsee PREP100 system represents a promising technology for capturing and molecular characterization of CTCs.

NANOG priming before full reprogramming may generate germ cell tumours.

PubMed

Grad, I; Hibaoui, Y; Jaconi, M; Chicha, L; Bergström-Tengzelius, R; Sailani, M R; Pelte, M F; Dahoun, S; Mitsiadis, T A; Töhönen, V; Bouillaguet, S; Antonarakis, S E; Kere, J; Zucchelli, M; Hovatta, O; Feki, A

2011-11-09

Reprogramming somatic cells into a pluripotent state brings patient-tailored, ethical controversy-free cellular therapy closer to reality. However, stem cells and cancer cells share many common characteristics; therefore, it is crucial to be able to discriminate between them. We generated two induced pluripotent stem cell (iPSC) lines, with NANOG pre-transduction followed by OCT3/4, SOX2, and LIN28 overexpression. One of the cell lines, CHiPS W, showed normal pluripotent stem cell characteristics, while the other, CHiPS A, though expressing pluripotency markers, failed to differentiate and gave rise to germ cell-like tumours in vivo. Comparative genomic hybridisation analysis of the generated iPS lines revealed that they were genetically more stable than human embryonic stem cell counterparts. This analysis proved to be predictive for the differentiation potential of analysed cells. Moreover, the CHiPS A line expressed a lower ratio of p53/p21 when compared to CHiPS W. NANOG pre-induction followed by OCT3/4, SOX2, MYC, and KLF4 induction resulted in the same tumour-inducing phenotype. These results underline the importance of a re-examination of the role of NANOG during reprogramming. Moreover, this reprogramming method may provide insights into primordial cell tumour formation and cancer stem cell transformation.

Isolation of circulating tumor cells using a microvortex-generating herringbone-chip.

PubMed

Stott, Shannon L; Hsu, Chia-Hsien; Tsukrov, Dina I; Yu, Min; Miyamoto, David T; Waltman, Belinda A; Rothenberg, S Michael; Shah, Ajay M; Smas, Malgorzata E; Korir, George K; Floyd, Frederick P; Gilman, Anna J; Lord, Jenna B; Winokur, Daniel; Springer, Simeon; Irimia, Daniel; Nagrath, Sunitha; Sequist, Lecia V; Lee, Richard J; Isselbacher, Kurt J; Maheswaran, Shyamala; Haber, Daniel A; Toner, Mehmet

2010-10-26

Rare circulating tumor cells (CTCs) present in the bloodstream of patients with cancer provide a potentially accessible source for detection, characterization, and monitoring of nonhematological cancers. We previously demonstrated the effectiveness of a microfluidic device, the CTC-Chip, in capturing these epithelial cell adhesion molecule (EpCAM)-expressing cells using antibody-coated microposts. Here, we describe a high-throughput microfluidic mixing device, the herringbone-chip, or "HB-Chip," which provides an enhanced platform for CTC isolation. The HB-Chip design applies passive mixing of blood cells through the generation of microvortices to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface. Efficient cell capture was validated using defined numbers of cancer cells spiked into control blood, and clinical utility was demonstrated in specimens from patients with prostate cancer. CTCs were detected in 14 of 15 (93%) patients with metastatic disease (median = 63 CTCs/mL, mean = 386 ± 238 CTCs/mL), and the tumor-specific TMPRSS2-ERG translocation was readily identified following RNA isolation and RT-PCR analysis. The use of transparent materials allowed for imaging of the captured CTCs using standard clinical histopathological stains, in addition to immunofluorescence-conjugated antibodies. In a subset of patient samples, the low shear design of the HB-Chip revealed microclusters of CTCs, previously unappreciated tumor cell aggregates that may contribute to the hematogenous dissemination of cancer.

Hazardous effects of fried potato chips on the development of retina in albino rats.

PubMed

El-Sayyad, Hassan I; Sakr, Saber A; Badawy, Gamal M; Afify, Hanaa S

2011-08-01

To evaluate the hazardous effects of fried potato chips upon the retina of two developmental stages of the albino rats aged 7 and 14 days from parturition. PREGNANT RATS WERE ARRANGED INTO TWO GROUPS: control pregnant rats and consequently their delivered newborns until reaching 7 and 14 days old from parturition and fried potato chips group in which pregnant rats at the 6th day of gestation maintained on diet formed of fried potato chips supplied from the market mixed with standard diet at a concentration of 50% per each till 7 and 14 post-partum. Three fold integrated approaches were adopted, namely, histological, ultrastructural and proteomic analysis. Histological examination of the retina of the experimental offsprings revealed many histopathological changes, including massive degeneration, vacuolization and cell loss in the ganglion cell layer, as well as general reduction in retinal size. At the ultrastructural level, the retina of experimental offsprings exhibited number of deformities, including ill differentiated and degenerated nuclear layer, malformed and vacuolated pigment epithelium with vesiculated and fragmented rough endoplasmic reticulum, degenerated outer segment of photoreceptors, as well as swollen choriocapillaris and loss of neuronal cells. Proteomic analysis of retina of the two experimental developmental stages showed variations in the expressed proteins as a result of intoxication which illustrated the adverse toxic effects of fried potato chips upon the retina. It can be concluded that the effect of fried potato chips on the development of retina in rats may be due to the presence of acrylamide or its metabolite.

Electrochemical cell-based chip for the detection of toxic effects of bisphenol-A on neuroblastoma cells.

PubMed

Kafi, Md Abdul; Kim, Tae-Hyung; An, Jeung Hee; Choi, Jeong-Woo

2011-03-15

A cell-based chip was fabricated for the electrochemical detection of the dose-dependent effects of bisphenol-A (BPA) on neuroblastoma cells (SH-SY5Y), which showed dual-mode correlation as a standard curve. Toxicity assessment of BPA became very important in environmental toxicants detection since BPA can be reached out easily from various common plastic-based product and give negative cellular effects on living organism. Cell chip was fabricated by immobilizing cells on C(RGD)(4) peptide coated electrode to detect the cytotoxicity of BPA electrochemically. Redox properties in living cells were determined by cyclic voltammetry using a home-made three-electrode system, and the cathodic peak current (I(pc)) was used as a parameter for measurement of the effect of BPA on cell viability. The peak current, I(pc) value increased with the concentration of BPA up to 300 nM and then decreased because of the stimulation of cancer cell activity at the concentration of BPA below 300nM and cytotoxicity at the concentration of BPA above 300 nM, respectively. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and optical microscopy-based morphological analysis confirmed the results of electrochemical study. This dual-mode correlation between the concentration of BPA and voltammetric signal intensity should be firstly considered to analyze its dose-dependent stimulus and cytotoxic effects on neuroblastoma cells by cell chip. Copyright © 2010 Elsevier B.V. All rights reserved.

Fishing on chips: up-and-coming technological advances in analysis of zebrafish and Xenopus embryos.

PubMed

Zhu, Feng; Skommer, Joanna; Huang, Yushi; Akagi, Jin; Adams, Dany; Levin, Michael; Hall, Chris J; Crosier, Philip S; Wlodkowic, Donald

2014-11-01

Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole-organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high-throughput cell-based in vitro assays and low-throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor-intensive. Recently, a new generation of miniaturized chip-based devices have been developed for zebrafish and Xenopus embryo on-chip culture and experimentation. In this work, we review the critical developments in the field of Lab-on-a-Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

Specific capture, recovery and culture of cancer cells using oriented antibody-modified polystyrene chips coated with agarose film.

PubMed

Jeong, Jiyun; Lee, Yeolin; Yoo, Yeongeun; Lee, Myung Kyu

2018-02-01

Agarose gel can be used for three dimensional (3D) cell culture because it prevents cell attachment. The dried agarose film coated on a culture plate also protected cell attachment and allowed 3D growth of cancer cells. We developed an efficient method for agarose film coating on an oxygen-plasma treated micropost polystyrene chip prepared by an injection molding process. The agarose film was modified to maleimide or Ni-NTA groups for covalent or cleavable attachment of photoactivatable Fc-specific antibody binding proteins (PFcBPs) via their N-terminal cysteine residues or 6xHis tag, respectively. The antibodies photocrosslinked onto the PFcBP-modified chips specifically captured the target cells without nonspecific binding, and the captured cells grew 3D modes on the chips. The captured cells on the cleavable antibody-modified chips were easily recovered by treatment of commercial trypsin-EDTA solution. Under fluidic conditions using an antibody-modified micropost chip, the cells were mainly captured on the micropost walls of the chip rather than on the bottom of it. The presented method will also be applicable for immobilization of oriented antibodies on various microfluidic chips with different structures. Copyright © 2017 Elsevier B.V. All rights reserved.

The Antitumor Effect of C-terminus of Hsp70-Interacting Protein via Degradation of c-Met in Small Cell Lung Cancer.

PubMed

Cho, Sung Ho; Kim, Jong In; Kim, Hyun Su; Park, Sung Dal; Jang, Kang Won

2017-06-01

The mesenchymal-epithelial transition factor (MET) receptor can be overexpressed in solid tumors, including small cell lung cancer (SCLC). However, the molecular mechanism regulating MET stability and turnover in SCLC remains undefined. One potential mechanism of MET regulation involves the C-terminus of Hsp70-interacting protein (CHIP), which targets heat shock protein 90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, we investigated the functional effects of CHIP expression on MET regulation and the control of SCLC cell apoptosis and invasion. To evaluate the expression of CHIP and c-Met, which is a protein that in humans is encoded by the MET gene (the MET proto-oncogene), we examined the expression pattern of c-Met and CHIP in SCLC cell lines by western blotting. To investigate whether CHIP overexpression reduced cell proliferation and invasive activity in SCLC cell lines, we transfected cells with CHIP and performed a cell viability assay and cellular apoptosis assays. We found an inverse relationship between the expression of CHIP and MET in SCLC cell lines (n=5). CHIP destabilized the endogenous MET receptor in SCLC cell lines, indicating an essential role for CHIP in the regulation of MET degradation. In addition, CHIP inhibited MET-dependent pathways, and invasion, cell growth, and apoptosis were reduced by CHIP overexpression in SCLC cell lines. CHIP is capable of regulating SCLC cell apoptosis and invasion by inhibiting MET-mediated cytoskeletal and cell survival pathways in NCI-H69 cells. CHIP suppresses MET-dependent signaling, and regulates MET-mediated SCLC motility.

Single-Cell Electric Lysis on an Electroosmotic-Driven Microfluidic Chip with Arrays of Microwells

PubMed Central

Jen, Chun-Ping; Amstislavskaya, Tamara G.; Liu, Ya-Hui; Hsiao, Ju-Hsiu; Chen, Yu-Hung

2012-01-01

Accurate analysis at the single-cell level has become a highly attractive tool for investigating cellular content. An electroosmotic-driven microfluidic chip with arrays of 30-μm-diameter microwells was developed for single-cell electric lysis in the present study. The cellular occupancy in the microwells when the applied voltage was 5 V (82.4%) was slightly higher than that at an applied voltage of 10 V (81.8%). When the applied voltage was increased to 15 V, the cellular occupancy in the microwells dropped to 64.3%. More than 50% of the occupied microwells contain individual cells. The results of electric lysis experiments at the single-cell level indicate that the cells were gradually lysed as the DC voltage of 30 V was applied; the cell was fully lysed after 25 s. Single-cell electric lysis was demonstrated in the proposed microfluidic chip, which is suitable for high-throughput cell lysis. PMID:22969331

Stem cell culture and differentiation in microfluidic devices toward organ-on-a-chip.

PubMed

Zhang, Jie; Wei, Xiaofeng; Zeng, Rui; Xu, Feng; Li, XiuJun

2017-06-01

Microfluidic lab-on-a-chip provides a new platform with unique advantages to mimic complex physiological microenvironments in vivo and has been increasingly exploited to stem cell research. In this review, we highlight recent advances of microfluidic devices for stem cell culture and differentiation toward the development of organ-on-a-chip, especially with an emphasis on vital innovations within the last 2 years. Various aspects for improving on-chip stem-cell culture and differentiation, particularly toward organ-on-a-chip, are discussed, along with microenvironment control, surface modification, extracellular scaffolds, high throughput and stimuli. The combination of microfluidic technologies and stem cells hold great potential toward versatile systems of 'organ-on-a-chip' as desired. Adapted with permission from [1-8].

Guided self-assembly of magnetic beads for biomedical applications

NASA Astrophysics Data System (ADS)

Gusenbauer, Markus; Nguyen, Ha; Reichel, Franz; Exl, Lukas; Bance, Simon; Fischbacher, Johann; Özelt, Harald; Kovacs, Alexander; Brandl, Martin; Schrefl, Thomas

2014-02-01

Micromagnetic beads are widely used in biomedical applications for cell separation, drug delivery, and hyperthermia cancer treatment. Here we propose to use self-organized magnetic bead structures which accumulate on fixed magnetic seeding points to isolate circulating tumor cells. The analysis of circulating tumor cells is an emerging tool for cancer biology research and clinical cancer management including the detection, diagnosis and monitoring of cancer. Microfluidic chips for isolating circulating tumor cells use either affinity, size or density capturing methods. We combine multiphysics simulation techniques to understand the microscopic behavior of magnetic beads interacting with soft magnetic accumulation points used in lab-on-chip technologies. Our proposed chip technology offers the possibility to combine affinity and size capturing with special antibody-coated bead arrangements using a magnetic gradient field created by Neodymium Iron Boron permanent magnets. The multiscale simulation environment combines magnetic field computation, fluid dynamics and discrete particle dynamics.

Microfluidic Devices for Forensic DNA Analysis: A Review

PubMed Central

Bruijns, Brigitte; van Asten, Arian; Tiggelaar, Roald; Gardeniers, Han

2016-01-01

Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10–20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook. PMID:27527231

Development of a cell microarray chip for detection of circulating tumor cells

NASA Astrophysics Data System (ADS)

Yamamura, S.; Yatsushiro, S.; Abe, K.; Baba, Y.; Kataoka, M.

2012-03-01

Detection of circulating tumor cells (CTCs) in the peripheral blood of metastatic cancer patients has clinical significance in earlier diagnosis of metastases. In this study, a novel cell microarray chip for accurate and rapid detection of tumor cells from human leukocytes was developed. The chip with 20,944 microchambers (105 μm diameter and 50 μm depth) was made from polystyrene, and the surface was rendered to hydrophilic by means of reactive-ion etching, which led to the formation of mono-layers of leukocytes on the microchambers. As the model of CTCs detection, we spiked human bronchioalveolar carcinoma (H1650) cells into human T lymphoblastoid leukemia (CEM) cells suspension and detected H1650 cells using the chip. A CEM suspension contained with H1650 cells was dispersed on the chip surface, followed by 10 min standing to allow the cells to settle down into the microchambers. About 30 CEM cells were accommodated in each microchamber, over 600,000 CEM cells in total being on a chip. We could detect 1 H1650 cell per 106 CEM cells on the microarray by staining with fluorescence-conjugated antibody (Anti-Cytokeratin) and cell membrane marker (DiD). Thus, this cell microarray chip has highly potential to be a novel tool of accurate and rapid detection of CTCs.

Microfluidic cell disruption system employing a magnetically actuated diaphragm.

PubMed

Huh, Yun Suk; Choi, Jong Hyun; Huh, Kyoung Ae Kim; Kim, Kyoung Ae; Park, Tae Jung; Hong, Yeon Ki; Kim, Do Hyun; Hong, Won Hi; Lee, Sang Yup

2007-12-01

A microfluidic cell lysis chip equipped with a micromixer and SPE unit was developed and used for quantitative analysis of intracellular proteins. This miniaturized sample preparation system can be employed for any purpose where cell disruption is needed to obtain intracellular constituents for the subsequent analysis. This system comprises a magnetically actuated micromixer to disrupt cells, a hydrophobic valve to manipulate the cell lysate, and a packed porous polymerized monolith chamber for SPE and filtering debris from the cell lysate. Using recombinant Escherichia coli expressing intracellular enhanced green fluorescent protein (EGFP) and lipase as model bacteria, we optimized the cell disruption condition with respect to the lysis buffer composition, mixing time, and the frequency of the diaphragm in the micromixer, which was magnetically actuated by an external magnetic stirrer in the micromixer chamber. The lysed sample prepared under the optimal condition was purified by the packed SPE in the microfluidic chip. At a frequency of 1.96 Hz, the final cell lysis efficiency and relative fluorescence intensity of EGFP after the cell disruption process were greater than 90 and 94%, respectively. Thus, this microfluidic cell disruption chip can be used for the efficient lysis of cells for further analysis of intracellular contents in many applications.

On-chip dynamic stress control for cancer cell evolution study

NASA Astrophysics Data System (ADS)

Liu, Liyu; Austin, Robert

2010-03-01

The growth and spreading of cancer in host organisms is an evolutionary process. Cells accumulate mutations that help them adapt to changing environments and to obtain survival fitness. However, all cancer--promoting mutations do not occur at once. Cancer cells face selective environmental pressures that drive their evolution in stages. In traditional cancer studies, environmental stress is usually homogenous in space and difficult to change in time. Here, we propose a microfluidic chip employing embedded dynamic traps to generate dynamic heterogeneous microenvironments for cancer cells in evolution studies. Based on polydimethylsiloxane (PDMS) flexible diaphragms, these traps are able to enclose and shield cancer cells or expose them to external environmental stress. Digital controls for each trap determine the nutrition, antibiotics, CO2/O2 conditions, and temperatures to which trapped cells are subjected. Thus, the stress applied to cells can be varied in intensity and duration in each trap independently. The chip can also output cells from specific traps for sequencing and other biological analysis. Hence our design simultaneously monitors and analyzes cell evolution behaviors under dynamic stresses.

Metabolic enzyme microarray coupled with miniaturized cell-culture array technology for high-throughput toxicity screening.

PubMed

Lee, Moo-Yeal; Dordick, Jonathan S; Clark, Douglas S

2010-01-01

Due to poor drug candidate safety profiles that are often identified late in the drug development process, the clinical progression of new chemical entities to pharmaceuticals remains hindered, thus resulting in the high cost of drug discovery. To accelerate the identification of safer drug candidates and improve the clinical progression of drug candidates to pharmaceuticals, it is important to develop high-throughput tools that can provide early-stage predictive toxicology data. In particular, in vitro cell-based systems that can accurately mimic the human in vivo response and predict the impact of drug candidates on human toxicology are needed to accelerate the assessment of drug candidate toxicity and human metabolism earlier in the drug development process. The in vitro techniques that provide a high degree of human toxicity prediction will be perhaps more important in cosmetic and chemical industries in Europe, as animal toxicity testing is being phased out entirely in the immediate future.We have developed a metabolic enzyme microarray (the Metabolizing Enzyme Toxicology Assay Chip, or MetaChip) and a miniaturized three-dimensional (3D) cell-culture array (the Data Analysis Toxicology Assay Chip, or DataChip) for high-throughput toxicity screening of target compounds and their metabolic enzyme-generated products. The human or rat MetaChip contains an array of encapsulated metabolic enzymes that is designed to emulate the metabolic reactions in the human or rat liver. The human or rat DataChip contains an array of 3D human or rat cells encapsulated in alginate gels for cell-based toxicity screening. By combining the DataChip with the complementary MetaChip, in vitro toxicity results are obtained that correlate well with in vivo rat data.

Nanoporous Glass Integrated in Volumetric Bar-Chart Chip for Point-of-Care Diagnostics of Non-Small Cell Lung Cancer.

PubMed

Li, Ying; Xuan, Jie; Song, Yujun; Qi, Wenjin; He, Bangshun; Wang, Ping; Qin, Lidong

2016-01-26

Point-of-care (POC) testing has the potential to enable rapid, low-cost, and large-scale screening. POC detection of a multiplexed biomarker panel can facilitate the early diagnosis of non-small cell lung cancer (NSCLC) and, thus, may allow for more timely surgical intervention for life-saving treatment. Herein, we report the nanoporous glass (NPG) integrated volumetric bar-chart chip (V-Chip) for POC detection of the three NSCLC biomarkers CEA, CYFRA 21-1, and SCCA, by the naked eye. The 3D nanostructures in the NPG membrane efficiently increase the number of binding sites for antibodies and decrease the diffusion distance between antibody and antigen, enabling the low detection limit and rapid analysis time of the NPG-V-Chip. We utilized the NPG-V-Chip to test the NSCLC biomarker panel and found that the limit of detection can reach 50 pg/mL (10-fold improvement over the original V-Chip), and the total assay time can be decreased from 4 to 0.5 h. We then detected CEA in 21 serum samples from patients with common cancers, and the on-chip results showed good correlation with the clinical results. We further assayed 10 lung cancer samples using the device and confirmed the results obtained using conventional ELISA methods. In summary, the NPG-V-Chip platform has the ability of multiplex, low detection limit, low cost, lack of need for accessory equipment, and rapid analysis time, which may render the V-Chip a useful platform for quantitative POC detection in resource-limited settings and personalized diagnostics.

A polymeric micro total analysis system for single-cell analysis

NASA Astrophysics Data System (ADS)

Lai, Hsuan-Hong

The advancement of microengineering has enabled the manipulation and analysis of single cells, which is critical in understanding the molecular mechanisms underlying the basic physiological functions from the point of view of modern biologists. Unfortunately, analysis of single cells remains challenging from a technical perspective, mainly because of the miniature nature of the cell and the high throughput requirements of the analysis. Lab-on-a-chip (LOC) emerges as a research field that shows great promise in this perspective. We have demonstrated a micro total analysis system (mu-TAS) combining chip-based electrophoretic separation, fluorescence detection, and a pulsed Nd:YAG laser cell lysis system, in a Poly(dimethylsiloxane) (PDMS) microfluidic analytical platform for the implementation of single-cell analysis. To accomplish the task, a polymeric microfluidic device was fabricated and UV graft polymerization surface modification techniques were used. To optimize the conditions for the surface treatment techniques, the modified surfaces of PDMS were characterized using AIR-IR spectrum and sessile water drop contact angle measurements, and in-channel surfaces were characterized by their electroosmotic flow mobility. Accurate single-cell analysis relies on rapid cell lysis and therefore an optical measure of fast cell lysis was implemented and optimized in a microscopic station. The influences of pulse energy and the location of the laser beam with respect to the cell in the microchannel were explored. The observation from the cell disruption experiments suggested that the cell lysis was enabled mainly via a thermo-mechanical instead of a plasma-mediated mechanism. Finally, after chip-based electrophoresis and a laser-induced fluorescence (LIF) detection system were incorporated with the laser lysis system in a microfluidic analytical station, a feasibility demonstration of single-cell analysis was implemented. The analytical platform exhibited the capability of fluidic transportation, optical lysis of single cells, separation, and analysis of the lysates by electrophoresis and LIF detection. In comparison with the control experiment, the migration times of the fluorescent signals for the cytosolic fluorophores were in good agreement with those for the standard fluorophores, which confirmed the feasibility of the analytical processes.

Nanostructured surfaces for analysis of anticancer drug and cell diagnosis based on electrochemical and SERS tools.

PubMed

El-Said, Waleed A; Yoon, Jinho; Choi, Jeong-Woo

2018-01-01

Discovering new anticancer drugs and screening their efficacy requires a huge amount of resources and time-consuming processes. The development of fast, sensitive, and nondestructive methods for the in vitro and in vivo detection of anticancer drugs' effects and action mechanisms have been done to reduce the time and resources required to discover new anticancer drugs. For the in vitro and in vivo detection of the efficiency, distribution, and action mechanism of anticancer drugs, the applications of electrochemical techniques such as electrochemical cell chips and optical techniques such as surface-enhanced Raman spectroscopy (SERS) have been developed based on the nanostructured surface. Research focused on electrochemical cell chips and the SERS technique have been reviewed here; electrochemical cell chips based on nanostructured surfaces have been developed for the in vitro detection of cell viability and the evaluation of the effects of anticancer drugs, which showed the high capability to evaluate the cytotoxic effects of several chemicals at low concentrations. SERS technique based on the nanostructured surface have been used as label-free, simple, and nondestructive techniques for the in vitro and in vivo monitoring of the distribution, mechanism, and metabolism of different anticancer drugs at the cellular level. The use of electrochemical cell chips and the SERS technique based on the nanostructured surface should be good tools to detect the effects and action mechanisms of anticancer drugs.

Nanostructured surfaces for analysis of anticancer drug and cell diagnosis based on electrochemical and SERS tools

NASA Astrophysics Data System (ADS)

El-Said, Waleed A.; Yoon, Jinho; Choi, Jeong-Woo

2018-04-01

Discovering new anticancer drugs and screening their efficacy requires a huge amount of resources and time-consuming processes. The development of fast, sensitive, and nondestructive methods for the in vitro and in vivo detection of anticancer drugs' effects and action mechanisms have been done to reduce the time and resources required to discover new anticancer drugs. For the in vitro and in vivo detection of the efficiency, distribution, and action mechanism of anticancer drugs, the applications of electrochemical techniques such as electrochemical cell chips and optical techniques such as surface-enhanced Raman spectroscopy (SERS) have been developed based on the nanostructured surface. Research focused on electrochemical cell chips and the SERS technique have been reviewed here; electrochemical cell chips based on nanostructured surfaces have been developed for the in vitro detection of cell viability and the evaluation of the effects of anticancer drugs, which showed the high capability to evaluate the cytotoxic effects of several chemicals at low concentrations. SERS technique based on the nanostructured surface have been used as label-free, simple, and nondestructive techniques for the in vitro and in vivo monitoring of the distribution, mechanism, and metabolism of different anticancer drugs at the cellular level. The use of electrochemical cell chips and the SERS technique based on the nanostructured surface should be good tools to detect the effects and action mechanisms of anticancer drugs.

Analysis of single mammalian cells on-chip.

PubMed

Sims, Christopher E; Allbritton, Nancy L

2007-04-01

A goal of modern biology is to understand the molecular mechanisms underlying cellular function. The ability to manipulate and analyze single cells is crucial for this task. The advent of microengineering is providing biologists with unprecedented opportunities for cell handling and investigation on a cell-by-cell basis. For this reason, lab-on-a-chip (LOC) technologies are emerging as the next revolution in tools for biological discovery. In the current discussion, we seek to summarize the state of the art for conventional technologies in use by biologists for the analysis of single, mammalian cells, and then compare LOC devices engineered for these same single-cell studies. While a review of the technical progress is included, a major goal is to present the view point of the practicing biologist and the advances that might increase adoption by these individuals. The LOC field is expanding rapidly, and we have focused on areas of broad interest to the biology community where the technology is sufficiently far advanced to contemplate near-term application in biological experimentation. Focus areas to be covered include flow cytometry, electrophoretic analysis of cell contents, fluorescent-indicator-based analyses, cells as small volume reactors, control of the cellular microenvironment, and single-cell PCR.

Parallel multipoint recording of aligned and cultured neurons on corresponding Micro Channel Array toward on-chip cell analysis.

PubMed

Tonomura, W; Moriguchi, H; Jimbo, Y; Konishi, S

2008-01-01

This paper describes an advanced Micro Channel Array (MCA) so as to record neuronal network at multiple points simultaneously. Developed MCA is designed for neuronal network analysis which has been studied by co-authors using MEA (Micro Electrode Arrays) system. The MCA employs the principle of the extracellular recording. Presented MCA has the following advantages. First of all, the electrodes integrated around individual micro channels are electrically isolated for parallel multipoint recording. Sucking and clamping of cells through micro channels is expected to improve the cellular selectivity and S/N ratio. In this study, hippocampal neurons were cultured on the developed MCA. As a result, the spontaneous and evoked spike potential could be recorded by sucking and clamping the cells at multiple points. Herein, we describe the successful experimental results together with the design and fabrication of the advanced MCA toward on-chip analysis of neuronal network.

Optical cell monitoring system for underwater targets

NASA Astrophysics Data System (ADS)

Moon, SangJun; Manzur, Fahim; Manzur, Tariq; Demirci, Utkan

2008-10-01

We demonstrate a cell based detection system that could be used for monitoring an underwater target volume and environment using a microfluidic chip and charge-coupled-device (CCD). This technique allows us to capture specific cells and enumerate these cells on a large area on a microchip. The microfluidic chip and a lens-less imaging platform were then merged to monitor cell populations and morphologies as a system that may find use in distributed sensor networks. The chip, featuring surface chemistry and automatic cell imaging, was fabricated from a cover glass slide, double sided adhesive film and a transparent Polymethlymetacrylate (PMMA) slab. The optically clear chip allows detecting cells with a CCD sensor. These chips were fabricated with a laser cutter without the use of photolithography. We utilized CD4+ cells that are captured on the floor of a microfluidic chip due to the ability to address specific target cells using antibody-antigen binding. Captured CD4+ cells were imaged with a fluorescence microscope to verify the chip specificity and efficiency. We achieved 70.2 +/- 6.5% capturing efficiency and 88.8 +/- 5.4% specificity for CD4+ T lymphocytes (n = 9 devices). Bright field images of the captured cells in the 24 mm × 4 mm × 50 μm microfluidic chip were obtained with the CCD sensor in one second. We achieved an inexpensive system that rapidly captures cells and images them using a lens-less CCD system. This microfluidic device can be modified for use in single cell detection utilizing a cheap light-emitting diode (LED) chip instead of a wide range CCD system.

Enhancement of local surface plasmon resonance (LSPR) effect by biocompatible metal clustering based on ZnO nanorods in Raman measurements.

PubMed

Lee, Sanghwa; Lee, Seung Ho; Paulson, Bjorn; Lee, Jae-Chul; Kim, Jun Ki

2018-06-20

The development of size-selective and non-destructive detection techniques for nanosized biomarkers has many reasons, including the study of living cells and diagnostic applications. We present an approach for Raman signal enhancement on biocompatible sensing chips based on surface enhancement Raman spectroscopy (SERS). A sensing chip was fabricated by forming a ZnO-based nanorod structure so that the Raman enhancement occurred at a gap of several tens to several hundred nanometers. The effect of coffee-ring formation was eliminated by introducing the porous ZnO nanorods for the bio-liquid sample. A peculiarity of this approach is that the gold sputtered on the ZnO nanorods initially grows at their heads forming clusters, as confirmed by secondary electron microscopy. This clustering was verified by finite element analysis to be the main factor for enhancement of local surface plasmon resonance (LSPR). This clustering property and the ability to adjust the size of the nanorods enabled the signal acquisition points to be refined using confocal based Raman spectroscopy, which could be applied directly to the sensor chip based on the optimization process in this experiment. It was demonstrated by using common cancer cell lines that cell growth was high on these gold-clad ZnO nanorod-based surface-enhanced Raman substrates. The porosity of the sensing chip, the improved structure for signal enhancement, and the cell assay make these gold-coated ZnO nanorods substrates promising biosensing chips with excellent potential for detecting nanometric biomarkers secreted by cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Single cell array impedance analysis in a microfluidic device

NASA Astrophysics Data System (ADS)

Altinagac, Emre; Taskin, Selen; Kizil, Huseyin

2016-10-01

Impedance analysis of single cells is presented in this paper. Following the separation of a target cell type by dielectrophoresis in our previous work, this paper focuses on capturing the cells as a single array and performing impedance analysis to point out the signature difference between each cell type. Lab-on-a-chip devices having a titanium interdigitated electrode layer on a glass substrate and a PDMS microchannel are fabricated to capture each cell in a single form and perform impedance analysis. HCT116 (homosapiens colon colorectal carcin) and HEK293 (human embryonic kidney) cells are used in our experiments.

Development of sugar chain-binding single-chain variable fragment antibody to adult T-cell leukemia cells using glyco-nanotechnology and phage display method.

PubMed

Muchima, Kaname; Todaka, Taro; Shinchi, Hiroyuki; Sato, Ayaka; Tazoe, Arisa; Aramaki, Rikiya; Kakitsubata, Yuhei; Yokoyama, Risa; Arima, Naomichi; Baba, Masanori; Wakao, Masahiro; Ito, Yuji; Suda, Yasuo

2018-04-01

Adult T-cell leukemia (ATL) is an intractable blood cancer caused by the infection of human T-cell leukemia virus type-1, and effective medical treatment is required. It is known that the structure and expression levels of cell surface sugar chains vary depending on cell states such as inflammation and cancer. Thus, it is expected that the antibody specific for ATL cell surface sugar chain would be an effective diagnostic tool and a strong candidate for the development of an anti-ATL drug. Here, we developed a stable sugar chain-binding single-chain variable fragment antibody (scFv) that can bind to ATL cells using a fibre-type Sugar Chip and phage display method. The fiber-type Sugar Chips were prepared using O-glycans released from ATL cell lines. The scFv-displaying phages derived from human B cells (diversity: 1.04 × 108) were then screened using the fiber-type Sugar Chips, and an O-glycan-binding scFv was obtained. The flow cytometry analysis revealed that the scFv predominantly bound to ATL cell lines. The sugar chain-binding properties of the scFv was evaluated by array-type Sugar Chip immobilized with a library of synthetic glycosaminoglycan disaccharide structures. Highly sulphated disaccharide structures were found to have high affinity to scFv.

Development of an Integrated Chip for Automatic Tracking and Positioning Manipulation for Single Cell Lysis

PubMed Central

Young, Chao-Wang; Hsieh, Jia-Ling; Ay, Chyung

2012-01-01

This study adopted a microelectromechanical fabrication process to design a chip integrated with electroosmotic flow and dielectrophoresis force for single cell lysis. Human histiocytic lymphoma U937 cells were driven rapidly by electroosmotic flow and precisely moved to a specific area for cell lysis. By varying the frequency of AC power, 15 V AC at 1 MHz of frequency configuration achieved 100% cell lysing at the specific area. The integrated chip could successfully manipulate single cells to a specific position and lysis. The overall successful rate of cell tracking, positioning, and cell lysis is 80%. The average speed of cell driving was 17.74 μm/s. This technique will be developed for DNA extraction in biomolecular detection. It can simplify pre-treatment procedures for biotechnological analysis of samples. PMID:22736957

Development of an integrated chip for automatic tracking and positioning manipulation for single cell lysis.

PubMed

Young, Chao-Wang; Hsieh, Jia-Ling; Ay, Chyung

2012-01-01

This study adopted a microelectromechanical fabrication process to design a chip integrated with electroosmotic flow and dielectrophoresis force for single cell lysis. Human histiocytic lymphoma U937 cells were driven rapidly by electroosmotic flow and precisely moved to a specific area for cell lysis. By varying the frequency of AC power, 15 V AC at 1 MHz of frequency configuration achieved 100% cell lysing at the specific area. The integrated chip could successfully manipulate single cells to a specific position and lysis. The overall successful rate of cell tracking, positioning, and cell lysis is 80%. The average speed of cell driving was 17.74 μm/s. This technique will be developed for DNA extraction in biomolecular detection. It can simplify pre-treatment procedures for biotechnological analysis of samples.

Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms.

PubMed

Zhang, Qiang; Wang, Tingting; Zhou, Qian; Zhang, Peng; Gong, Yanhai; Gou, Honglei; Xu, Jian; Ma, Bo

2017-01-23

Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and single-cell cultivation, especially for small-size microbes. Herein, a facile droplet microfluidic platform was developed to dispense individual microbial cells into conventional standard containers for downstream analysis. Functional parts for cell encapsulation, droplet inspection and sorting, as well as a chip-to-tube capillary interface were integrated on one single chip with simple architecture, and control of the droplet sorting was achieved by a low-cost solenoid microvalve. Using microalgal and yeast cells as models, single-cell isolation success rate of over 90% and single-cell cultivation success rate of 80% were demonstrated. We further showed that the individual cells isolated can be used in high-quality DNA and RNA analyses at both gene-specific and whole-genome levels (i.e. real-time quantitative PCR and genome sequencing). The simplicity and reliability of the method should improve accessibility of single-cell analysis and facilitate its wider application in microbiology researches.

Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms

PubMed Central

Zhang, Qiang; Wang, Tingting; Zhou, Qian; Zhang, Peng; Gong, Yanhai; Gou, Honglei; Xu, Jian; Ma, Bo

2017-01-01

Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and single-cell cultivation, especially for small-size microbes. Herein, a facile droplet microfluidic platform was developed to dispense individual microbial cells into conventional standard containers for downstream analysis. Functional parts for cell encapsulation, droplet inspection and sorting, as well as a chip-to-tube capillary interface were integrated on one single chip with simple architecture, and control of the droplet sorting was achieved by a low-cost solenoid microvalve. Using microalgal and yeast cells as models, single-cell isolation success rate of over 90% and single-cell cultivation success rate of 80% were demonstrated. We further showed that the individual cells isolated can be used in high-quality DNA and RNA analyses at both gene-specific and whole-genome levels (i.e. real-time quantitative PCR and genome sequencing). The simplicity and reliability of the method should improve accessibility of single-cell analysis and facilitate its wider application in microbiology researches. PMID:28112223

Cytometer on a Chip

NASA Technical Reports Server (NTRS)

Fernandez, Salvador M.

2011-01-01

A cytometer now under development exploits spatial sorting of sampled cells on a microarray chip followed by use of grating-coupled surface-plasmon-resonance imaging (GCSPRI) to detect the sorted cells. This cytometer on a chip is a prototype of contemplated future miniature cytometers that would be suitable for rapidly identifying pathogens and other cells of interest in both field and laboratory applications and that would be attractive as alternatives to conventional flow cytometers. The basic principle of operation of a conventional flow cytometer requires fluorescent labeling of sampled cells, stringent optical alignment of a laser beam with a narrow orifice, and flow of the cells through the orifice, which is subject to clogging. In contrast, the principle of operation of the present cytometer on a chip does not require fluorescent labeling of cells, stringent optical alignment, or flow through a narrow orifice. The basic principle of operation of the cytometer on a chip also reduces the complexity, mass, and power of the associated laser and detection systems, relative to those needed in conventional flow cytometry. Instead of making cells flow in single file through a narrow flow orifice for sequential interrogation as in conventional flow cytometry, a liquid containing suspended sampled cells is made to flow over the front surface of a microarray chip on which there are many capture spots. Each capture spot is coated with a thin (approximately 50-nm) layer of gold that is, in turn, coated with antibodies that bind to cell-surface molecules characteristic of one the cell species of interest. The multiplicity of capture spots makes it possible to perform rapid, massively parallel analysis of a large cell population. The binding of cells to each capture spot gives rise to a minute change in the index of refraction at the surface of the chip. This change in the index of refraction is what is sensed in GCSPRI, as described briefly below. The identities of the various species in a sample of cells is spatially encoded in the chip by the pattern of capture spots. The number of cells of a particular species is determined from the magnitude of the GCSPRI signal from that spot. GCSPRI as used here can be summarized as follows: The cytometer chip is fabricated with a diffraction grating on its front surface. The chip is illuminated with a light emitting diode (LED) from the front. By proper choice of grating parameters and of the wavelength and the angle of incidence of a laser beam, laser light can be made to be coupled into an electromagnetic mode that resonates with surface plasmons and thus couples light into surface plasmons. Coupling of light into a surface plasmon at a given location reduces the amount of incident light reflected from that location. A change in the index of refraction at the surface of a capture spot gives rise to a change in the resonance condition. Depending on the specific design, the change in the index of refraction could manifest itself as a brightening or darkening, a change in the wavelength needed to excite the plasmon at a given angle of incidence, or a change in the angle of incidence needed to excite the plasmon at a given wavelength. Whereas a multiwavelength laser system with multichannel detection would be needed to detect multiple species in conventional flow cytometry, it suffices to use an LED and a single detector channel in the GCSPRI approach: this contributes significantly to reductions in cost, complexity, size, mass, and power. GCSPRI cytometer chips could be made of plastic and could be mass-produced cheaply by use of molding and other methods adopted from the manufacture of digital video disks. These methods are amenable to a high degree of miniaturization: such additional features as fluidic channels, reaction chambers, and fluid-coupling ports could readily be incorporated into the chips, without incurring substantial additional costs.

Cytometer on a Chip

NASA Technical Reports Server (NTRS)

Fernandez, Salvador M.

2011-01-01

A cytometer now under development exploits spatial sorting of sampled cells on a microarray chip followed by use of grating-coupled surface-plasmon-resonance imaging (GCSPRI) to detect the sorted cells. This cytometer on a chip is a prototype of contemplated future miniature cytometers that would be suitable for rapidly identifying pathogens and other cells of interest in both field and laboratory applications and that would be attractive as alternatives to conventional flow cytometers. The basic principle of operation of a conventional flow cytometer requires fluorescent labeling of sampled cells, stringent optical alignment of a laser beam with a narrow orifice, and flow of the cells through the orifice, which is subject to clogging. In contrast, the principle of operation of the present cytometer on a chip does not require fluorescent labeling of cells, stringent optical alignment, or flow through a narrow orifice. The basic principle of operation of the cytometer on a chip also reduces the complexity, mass, and power of the associated laser and detection systems, relative to those needed in conventional flow cytometry. Instead of making cells flow in single file through a narrow flow orifice for sequential interrogation as in conventional flow cytometry, a liquid containing suspended sampled cells is made to flow over the front surface of a microarray chip on which there are many capture spots. Each capture spot is coated with a thin (.50-nm) layer of gold that is, in turn, coated with antibodies that bind to cell-surface molecules characteristic of the cell species of interest. The multiplicity of capture spots makes it possible to perform rapid, massively parallel analysis of a large cell population. The binding of cells to each capture spot gives rise to a minute change in the index of refraction at the surface of the chip. This change in the index of refraction is what is sensed in GCSPRI, as described briefly below. The identities of the various species in a sample of cells is spatially encoded in the chip by the pattern of capture spots. The number of cells of a particular species is determined from the magnitude of the GCSPRI signal from that spot. GCSPRI as used here can be summarized as follows: The cytometer chip is fabricated with a diffraction grating on its front surface. The chip is illuminated with a light emitting diode (LED) from the front. By proper choice of grating parameters and of the wavelength and the angle of incidence of a laser beam, laser light can be made to be coupled into an electromagnetic mode that resonates with surface plasmons and thus couples light into surface plasmons. Coupling of light into a surface plasmon at a given location reduces the amount of incident light reflected from that location. A change in the index of refraction at the surface of a capture spot gives rise to a change in the resonance condition. Depending on the specific design, the change in the index of refraction could manifest itself as a brightening or darkening, a change in the wavelength needed to excite the plasmon at a given angle of incidence, or a change in the angle of incidence needed to excite the plasmon at a given wavelength. Whereas a multiwavelength laser system with multichannel detection would be needed to detect multiple species in conventional flow cytometry, it suffices to use an LED and a single detector channel in the GCSPRI approach: this contributes significantly to reductions in cost, complexity, size, mass, and power. GCSPRI cytometer chips could be made of plastic and could be mass-produced cheaply by use of molding and other methods adopted from the manufacture of digital video disks. These methods are amenable to a high degree of miniaturization: such additional features as fluidic channels, reaction chambers, and fluid-coupling ports could readily be incorporated into the chips, without incurring substantial additional costs.

Easy-to-use microfluidic chip for long-term 3D-cell cultures

NASA Astrophysics Data System (ADS)

Bunge, Frank; van den Driesche, Sander; Vellekoop, Michael J.

2017-05-01

We present a microfluidic chip for an easy setup of a 3D-culture of mammalian cells. The chip contains feeding structures and gas supply for long-term cultivation of mammalian cells. The device is fabricated out of hard materials like silicon and glass that are all highly biocompatible. The chip uses the concept of surficial phaseguides that allows the partial filling of a microfluidic chip with liquids based on hydrophobic and hydrophilic surfaces. Here, a suspension of mammalian cells and melted agarose is filled into the chip and is pulled by the capillary pressure on the hydrophilic areas but not on the hydrophobic phaseguides. Consequently, only a part of the chip is filled with the agarose which gels by cooling a form the 3D-cell culture. The unfilled areas are used as supply structures for nutrition and gases. So the supply is based on diffusion and the supply of nutrition and gases is controlled independently. We cultured HaCaT-cells over 24 hours in our device and achieve a good viability.

Electrical lysis: dynamics revisited and advances in On-chip operation.

PubMed

Morshed, Bashir; Shams, Maitham; Mussivand, Tofy

2013-01-01

Electrical lysis (EL) is the process of breaking the cell membrane to expose the internal contents under an applied high electric field. Lysis is an important phenomenon for cellular analysis, medical treatment, and biofouling control. This paper aims to review, summarize, and analyze recent advancements on EL. Major databases including PubMed, Ei Engineering Village, IEEE Xplore, and Scholars Portal were searched using relevant keywords. More than 50 articles published in English since 1997 are cited in this article. EL has several key advantages compared to other lysis techniques such as chemical, mechanical, sonication, or laser, including rapid speed of operation, ability to control, miniaturization, low cost, and low power requirement. A variety of cell types have been investigated for including protoplasts, E. coli, yeasts, blood cells, and cancer cells. EL has been developed and applied for decontamination, cytology, genetics, single-cell analysis, cancer treatment, and other applications. On-chip EL is a promising technology for multiplexed automated implementation of cell-sample preparation and processing with micro- or nanoliter reagents.

CHIP, a carboxy terminus HSP-70 interacting protein, prevents cell death induced by endoplasmic reticulum stress in the central nervous system.

PubMed

Cabral Miranda, Felipe; Adão-Novaes, Juliana; Hauswirth, William W; Linden, Rafael; Petrs-Silva, Hilda; Chiarini, Luciana B

2014-01-01

Endoplasmic reticulum (ER) stress and protein misfolding are associated with various neurodegenerative diseases. ER stress activates unfolded protein response (UPR), an adaptative response. However, severe ER stress can induce cell death. Here we show that the E3 ubiquitin ligase and co-chaperone Carboxyl Terminus HSP70/90 Interacting Protein (CHIP) prevents neuron death in the hippocampus induced by severe ER stress. Organotypic hippocampal slice cultures (OHSCs) were exposed to Tunicamycin, a pharmacological ER stress inducer, to trigger cell death. Overexpression of CHIP was achieved with a recombinant adeno-associated viral vector (rAAV) and significantly diminished ER stress-induced cell death, as shown by analysis of propidium iodide (PI) uptake, condensed chromatin, TUNEL and cleaved caspase 3 in the CA1 region of OHSCs. In addition, overexpression of CHIP prevented upregulation of both CHOP and p53 both pro-apoptotic pathways induced by ER stress. We also detected an attenuation of eIF2a phosphorylation promoted by ER stress. However, CHIP did not prevent upregulation of BiP/GRP78 induced by UPR. These data indicate that overexpression of CHIP attenuates ER-stress death response while maintain ER stress adaptative response in the central nervous system. These results indicate a neuroprotective role for CHIP upon UPR signaling. CHIP emerge as a candidate for clinical intervention in neurodegenerative diseases associated with ER stress.

A novel mast cell co-culture microfluidic chip for the electrochemical evaluation of food allergen.

PubMed

Jiang, Hui; Jiang, Donglei; Zhu, Pei; Pi, Fuwei; Ji, Jian; Sun, Chao; Sun, Jiadi; Sun, Xiulan

2016-09-15

In this study a novel cell-to-cell electrochemical microfluidic chip was developed for qualitative and quantitative analysis of food allergen. Microfluidic cell culture, food allergen-induced cell morphological changes, and cell metabolism measurements were performed simultaneously using the aforementioned device. RBL-2H3 mast cells and ANA-1 macrophages have been used within a cell co-culture model to observe their allergic response when they are introduced to the antigen stimulus. Two cell cultivation microfluidic channels are located in the microfluidic chip, which is fabricated with four groups of gold electrodes, with an additional "capillary". In order to detect the allergic response, the cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) without anti-DNP IgE incubation. When exocytosis occurs, the cell-secreted inflammatory cytokines were measured by enzyme-linked immuno sorbent assay (ELISA) and cell impedance changes were detected using cell-based electrochemical assay. Results indicate that the real-time cell allergic response are accurately monitored by this electrochemical microfluidic chip, which provides a general example of rapidly prototyped low-cost biosensor technology for applications in both food allergen detection and investigation. Copyright © 2016 Elsevier B.V. All rights reserved.

Lensfree On-Chip Microscopy and Tomography for Bio-Medical Applications

PubMed Central

Isikman, Serhan O.; Bishara, Waheb; Mudanyali, Onur; Sencan, Ikbal; Su, Ting-Wei; Tseng, Derek; Yaglidere, Oguzhan; Sikora, Uzair; Ozcan, Aydogan

2012-01-01

Lensfree on-chip holographic microscopy is an emerging technique that offers imaging of biological specimens over a large field-of-view without using any lenses or bulky optical components. Lending itself to a compact, cost-effective and mechanically robust architecture, lensfree on-chip holographic microscopy can offer an alternative toolset addressing some of the emerging needs of microscopic analysis and diagnostics in low-resource settings, especially for telemedicine applications. In this review, we summarize the latest achievements in lensfree optical microscopy based on partially coherent on-chip holography, including portable telemedicine microscopy, cell-phone based microscopy and field-portable optical tomographic microscopy. We also discuss some of the future directions for telemedicine microscopy and its prospects to help combat various global health challenges. PMID:24478572

Real-time Image Processing for Microscopy-based Label-free Imaging Flow Cytometry in a Microfluidic Chip.

PubMed

Heo, Young Jin; Lee, Donghyeon; Kang, Junsu; Lee, Keondo; Chung, Wan Kyun

2017-09-14

Imaging flow cytometry (IFC) is an emerging technology that acquires single-cell images at high-throughput for analysis of a cell population. Rich information that comes from high sensitivity and spatial resolution of a single-cell microscopic image is beneficial for single-cell analysis in various biological applications. In this paper, we present a fast image-processing pipeline (R-MOD: Real-time Moving Object Detector) based on deep learning for high-throughput microscopy-based label-free IFC in a microfluidic chip. The R-MOD pipeline acquires all single-cell images of cells in flow, and identifies the acquired images as a real-time process with minimum hardware that consists of a microscope and a high-speed camera. Experiments show that R-MOD has the fast and reliable accuracy (500 fps and 93.3% mAP), and is expected to be used as a powerful tool for biomedical and clinical applications.

Functional differentiation of human pluripotent stem cells on a chip.

PubMed

Giobbe, Giovanni G; Michielin, Federica; Luni, Camilla; Giulitti, Stefano; Martewicz, Sebastian; Dupont, Sirio; Floreani, Annarosa; Elvassore, Nicola

2015-07-01

Microengineering human "organs-on-chips" remains an open challenge. Here, we describe a robust microfluidics-based approach for the differentiation of human pluripotent stem cells directly on a chip. Extrinsic signal modulation, achieved through optimal frequency of medium delivery, can be used as a parameter for improved germ layer specification and cell differentiation. Human cardiomyocytes and hepatocytes derived on chips showed functional phenotypes and responses to temporally defined drug treatments.

Chip-Based Dynamic Real-Time Quantification of Drug-Induced Cytotoxicity in Human Tumor Cells

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; McGuinness, Dagmara; Faley, Shannon; Kolch, Walter; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell cytotoxicity tests are among the most common bioassays using flow cytometry and fluorescence imaging analysis. The permeability of plasma membranes to charged fluorescent probes serves, in these assays, as a marker distinguishing live from dead cells. Since it is generally assumed that probes, such as propidium iodide (PI) or 7-amino-actinomycin D (7-AAD), are themselves cytotoxic, they are currently generally used only as the end-point markers of assays for live versus dead cells. In the current study, we provide novel insights into potential applications of these classical plasma membrane integrity markers in the dynamic tracking of drug-induced cytotoxicity. We show that treatment of a number of different human tumor cell lines in cultures for up to 72 h with the PI, 7-AAD, SYTOX Green (SY-G), SYTOX Red (SYR), TO-PRO, and YO-PRO had no effect on cell viability assessed by the integrity of plasma membrane, cell cycle progression, and rate of proliferation. We subsequently explore the potential of dynamic labeling with these markers in real-time analysis, by comparing results from both conventional cytometry and microfluidic chips. Considering the simplicity of the staining protocols and their low cost combined with the potential for real-time data collection, we show how that real-time fluorescent imaging and Lab-on-a-Chip platforms have the potential to be used for automated drug screening routines. PMID:19572560

Single-Cell mRNA-Seq Using the Fluidigm C1 System and Integrated Fluidics Circuits.

PubMed

Gong, Haibiao; Do, Devin; Ramakrishnan, Ramesh

2018-01-01

Single-cell mRNA-seq is a valuable tool to dissect expression profiles and to understand the regulatory network of genes. Microfluidics is well suited for single-cell analysis owing both to the small volume of the reaction chambers and easiness of automation. Here we describe the workflow of single-cell mRNA-seq using C1 IFC, which can isolate and process up to 96 cells. Both on-chip procedure (lysis, reverse transcription, and preamplification PCR) and off-chip sequencing library preparation protocols are described. The workflow generates full-length mRNA information, which is more valuable compared to 3' end counting method for many applications.

Microfluidic Gut-liver chip for reproducing the first pass metabolism.

PubMed

Choe, Aerim; Ha, Sang Keun; Choi, Inwook; Choi, Nakwon; Sung, Jong Hwan

2017-03-01

After oral intake of drugs, drugs go through the first pass metabolism in the gut and the liver, which greatly affects the final outcome of the drugs' efficacy and side effects. The first pass metabolism is a complex process involving the gut and the liver tissue, with transport and reaction occurring simultaneously at various locations, which makes it difficult to be reproduced in vitro with conventional cell culture systems. In an effort to tackle this challenge, here we have developed a microfluidic gut-liver chip that can reproduce the dynamics of the first pass metabolism. The microfluidic chip consists of two separate layers for gut epithelial cells (Caco-2) and the liver cells (HepG2), and is designed so that drugs go through a sequential absorption in the gut chamber and metabolic reaction in the liver chamber. We fabricated the chip and showed that the two different cell lines can be successfully co-cultured on chip. When the two cells are cultured on chip, changes in the physiological function of Caco-2 and HepG2 cells were noted. The cytochrome P450 metabolic activity of both cells were significantly enhanced, and the absorptive property of Caco-2 cells on chip also changed in response to the presence of flow. Finally, first pass metabolism of a flavonoid, apigenin, was evaluated as a model compound, and co-culture of gut and liver cells on chip resulted in a metabolic profile that is closer to the reported profile than a monoculture of gut cells. This microfluidic gut-liver chip can potentially be a useful platform to study the complex first pass metabolism of drugs in vitro.

Single-cell printer: automated, on demand, and label free.

PubMed

Gross, Andre; Schöndube, Jonas; Niekrawitz, Sonja; Streule, Wolfgang; Riegger, Lutz; Zengerle, Roland; Koltay, Peter

2013-12-01

Within the past years, single-cell analysis has developed into a key topic in cell biology to study cellular functions that are not accessible by investigation of larger cell populations. Engineering approaches aiming to access single cells to extract information about their physiology, phenotype, and genotype at the single-cell level are going manifold ways, meanwhile allowing separation, sorting, culturing, and analysis of individual cells. Based on our earlier research toward inkjet-like printing of single cells, this article presents further characterization results obtained with a fully automated prototype instrument for printing of single living cells in a noncontact inkjet-like manner. The presented technology is based on a transparent microfluidic drop-on-demand dispenser chip coupled with a camera-assisted automatic detection system. Cells inside the chip are detected and classified with this detection system before they are expelled from the nozzle confined in microdroplets, thus enabling a "one cell per droplet" printing mode. To demonstrate the prototype instrument's suitability for biological and biomedical applications, basic experiments such as printing of single-bead and cell arrays as well as deposition and culture of single cells in microwell plates are presented. Printing efficiencies greater than 80% and viability rates about 90% were achieved.

Three levels of neuroelectronic interfacing: silicon chips with ion channels, nerve cells, and brain tissue.

PubMed

Fromherz, Peter

2006-12-01

We consider the direct electrical interfacing of semiconductor chips with individual nerve cells and brain tissue. At first, the structure of the cell-chip contact is studied. Then we characterize the electrical coupling of ion channels--the electrical elements of nerve cells--with transistors and capacitors in silicon chips. On that basis it is possible to implement signal transmission between microelectronics and the microionics of nerve cells in both directions. Simple hybrid neuroelectronic systems are assembled with neuron pairs and with small neuronal networks. Finally, the interfacing with capacitors and transistors is extended to brain tissue cultured on silicon chips. The application of highly integrated silicon chips allows an imaging of neuronal activity with high spatiotemporal resolution. The goal of the work is an integration of neuronal network dynamics with digital electronics on a microscopic level with respect to experiments in brain research, medical prosthetics, and information technology.

Pulsatile plasma filtration and cell-free DNA amplification using a water-head-driven point-of-care testing chip.

PubMed

Lee, Yonghun; Kim, Dong-Min; Li, Zhenglin; Kim, Dong-Eun; Kim, Sung-Jin

2018-03-13

We demonstrate a microfiltration chip that separates blood plasma by using water-head-driven pulsatile pressures rather than any external equipment and use it for on-chip amplification of nucleic acids. The chip generates pulsatile pressures to significantly reduce filter clogging without hemolysis, and consists of an oscillator, a plasma-extraction pump, and filter units. The oscillator autonomously converts constant water-head pressure to pulsatile pressure, and the pump uses the pulsatile pressure to extract plasma through the filter. Because the pulsatile pressure can periodically clear blood cells from the filter surface, filter clogging can be effectively reduced. In this way, we achieve plasma extraction with 100% purity and 90% plasma recovery at 15% hematocrit. During a 10 min period, the volume of plasma extracted was 43 μL out of a 243 μL extraction volume at 15% hematocrit. We also studied the influence of the pore size and diameter of the filter, blood loading volume, oscillation period, and hematocrit level on the filtration performance. To demonstrate the utility of our chip for point-of-care testing (POCT) applications, we successfully implemented on-chip amplification of a nucleic acid (miDNA21) in plasma filtered from blood. We expect our chip to be useful not only for POCT applications but also for other bench-top analysis tools using blood plasma.

Liquid biopsy on chip: a paradigm shift towards the understanding of cancer metastasis.

PubMed

Tadimety, Amogha; Syed, Abeer; Nie, Yuan; Long, Christina R; Kready, Kasia M; Zhang, John X J

2017-01-23

This comprehensive review serves as a guide for developing scalable and robust liquid biopsies on chip for capture, detection, and analysis of circulating tumor cells (CTCs). Liquid biopsy, the detection of biomarkers from body fluids, has proven challenging because of CTC rarity and the heterogeneity of CTCs shed from tumors. The review starts with the underlying biological mechanisms that make liquid biopsy a challenge before moving into an evaluation of current technological progress. Then, a framework for evaluation of the technologies is presented with special attention to throughput, capture rate, and cell viability for analysis. Technologies for CTC capture, detection, and analysis will be evaluated based on these criteria, with a focus on current approaches, limitations and future directions. The paper provides a critical review for microchip developers as well as clinical investigators to build upon the existing progress towards the goal of designing CTC capture, detection, and analysis platforms.

Spectroscopic imaging system for high-throughput viability assessment of ovarian spheroids or microdissected tumor tissues (MDTs) in a microfluidic chip

NASA Astrophysics Data System (ADS)

St-Georges-Robillard, A.; Masse, M.; Kendall-Dupont, J.; Strupler, M.; Patra, B.; Jermyn, M.; Mes-Masson, A.-M.; Leblond, F.; Gervais, T.

2016-02-01

There is a growing effort in the biomicrosystems community to develop a personalized treatment response assay for cancer patients using primary cells, patient-derived spheroids, or live tissues on-chip. Recently, our group has developed a technique to cut tumors in 350 μm diameter microtissues and keep them alive on-chip, enabling multiplexed in vitro drug assays on primary tumor tissue. Two-photon microscopy, confocal microscopy and flow cytometry are the current standard to assay tissue chemosensitivity on-chip. While these techniques provide microscopic and molecular information, they are not adapted for high-throughput analysis of microtissues. We present a spectroscopic imaging system that allows rapid quantitative measurements of multiple fluorescent viability markers simultaneously by using a liquid crystal tunable filter to record fluorescence and transmittance spectra. As a proof of concept, 24 spheroids composed of ovarian cancer cell line OV90 were formed in a microfluidic chip, stained with two live cell markers (CellTrackerTM Green and Orange), and imaged. Fluorescence images acquired were normalized to the acquisition time and gain of the camera, dark noise was removed, spectral calibration was applied, and spatial uniformity was corrected. Spectral un-mixing was applied to separate each fluorophore's contribution. We have demonstrated that rapid and simultaneous viability measurements on multiple spheroids can be achieved, which will have a significant impact on the prediction of a tumor's response to multiple treatment options. This technique may be applied as well in drug discovery to assess the potential of a drug candidate directly on human primary tissue.

Enhanced tenogenic differentiation and tendon-like tissue formation by CHIP overexpression in tendon-derived stem cells.

PubMed

Han, Weifeng; Chen, Lei; Liu, Junpeng; Guo, Ai

2017-04-01

The carboxyl terminus of Hsc70-interacting protein (CHIP, also known as STUB1) plays critical roles in the proliferation and differentiation of many types of cells. The potential function of CHIP in tendon-derived stem cells (TDSCs) remains largely unknown at present. Here, we investigated the effects of CHIP on tenogenic differentiation of TDSCs via lentivirus-mediated overexpression. Forced expression of CHIP induced morphological changes and significantly enhanced cell proliferation, as well as tendon differentiation in vitro. Upon stimulation with differentiation induction medium, CHIP-overexpressing TDSCs displayed significant inhibition of differentiation into osteogenic and adipogenic lineages. Subsequent implantation of TDSCs overexpressing CHIP with collagen sponges into nude mice induced a marked increase in ectopic tendon formation in vivo, compared with the control group. Our findings collectively suggest that CHIP is an important contributory factor to tenogenic tissue formation. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Multicolor fluorescence microscopic imaging of cancer cells on the plasmonic chip (Presentation Recording)

NASA Astrophysics Data System (ADS)

Tawa, Keiko; Sasakawa, Chisato; Yamamura, Shohei; Shibata, Izumi; Kataoka, Masatoshi

2015-09-01

A plasmonic chip which is a metal coated substrate with grating structure can provide the enhanced fluorescence by the grating-coupled surface plasmon field. In our previous studies, bright epi-fluorescence microscopic imaging of neuron cells and sensitive immunosesnsing have been reported. In this study, two kinds of breast cancer cells, MCF-7 and MDA-MB231, were observed with epi-fluorescence microscope on the plasmonic chip with 2D hole-arrays . They were multicolor stained with 4', 6-diamidino-2-phenylindole (DAPI) and allophycocyanin (APC)-labeled anti-epithelial cell adhesion molecule (EpCAM) antibody. Our plasmonic chip provided the brighter fluorescence images of these cells compared with the glass slide. Even in the cells including few EpCAM, the distribution of EpCAM was clearly observed in the cell membrane. It was found that the plasmonic chip can be one of the powerful tools to detect the marker protein existing around the chip surface even at low concentration.

The Ubiquitin Ligase CHIP Prevents SirT6 Degradation through Noncanonical Ubiquitination

PubMed Central

Ronnebaum, Sarah M.; Wu, Yaxu; McDonough, Holly

2013-01-01

The ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) regulates protein quality control, and CHIP deletion accelerates aging and reduces the life span in mice. Here, we reveal a mechanism for CHIP's influence on longevity by demonstrating that CHIP stabilizes the sirtuin family member SirT6, a lysine deacetylase/ADP ribosylase involved in DNA repair, metabolism, and longevity. In CHIP-deficient cells, SirT6 protein half-life is substantially reduced due to increased proteasome-mediated degradation, but CHIP overexpression in these cells increases SirT6 protein expression without affecting SirT6 transcription. CHIP noncanonically ubiquitinates SirT6 at K170, which stabilizes SirT6 and prevents SirT6 canonical ubiquitination by other ubiquitin ligases. In CHIP-depleted cells, SirT6 K170 mutation increases SirT6 half-life and prevents proteasome-mediated degradation. The global decrease in SirT6 expression in the absence of CHIP is associated with decreased SirT6 promoter occupancy, which increases histone acetylation and promotes downstream gene transcription in CHIP-depleted cells. Cells lacking CHIP are hypersensitive to DNA-damaging agents, but DNA repair and cell viability are rescued by enforced expression of SirT6. The discovery of this CHIP-SirT6 interaction represents a novel protein-stabilizing mechanism and defines an intersection between protein quality control and epigenetic regulation to influence pathways that regulate the biology of aging. PMID:24043303

Imaging Neuronal Seal Resistance on Silicon Chip using Fluorescent Voltage-Sensitive Dye

PubMed Central

Braun, Dieter; Fromherz, Peter

2004-01-01

The electrical sheet resistance between living cells grown on planar electronic contacts of semiconductors or metals is a crucial parameter for bioelectronic devices. It determines the strength of electrical signal transduction from cells to chips and from chips to cells. We measured the sheet resistance by applying AC voltage to oxidized silicon chips and by imaging the voltage change across the attached cell membrane with a fluorescent voltage-sensitive dye. The phase map of voltage change was fitted with a planar core-coat conductor model using the sheet resistance as a free parameter. For nerve cells from rat brain on polylysine as well as for HEK293 cells and MDCK cells on fibronectin we find a similar sheet resistance of 10 MΩ. Taking into account the independently measured distance of 50 nm between chip and membrane for these cells, we obtain a specific resistance of 50 Ωcm that is indistinguishable from bulk electrolyte. On the other hand, the sheet resistance for erythrocytes on polylysine is far higher, at ∼1.5 GΩ. Considering the distance of 10 nm, the specific resistance in the narrow cleft is enhanced to 1500 Ωcm. We find this novel optical method to be a convenient tool to optimize the interface between cells and chips for bioelectronic devices. PMID:15298937

Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye.

PubMed

Braun, Dieter; Fromherz, Peter

2004-08-01

The electrical sheet resistance between living cells grown on planar electronic contacts of semiconductors or metals is a crucial parameter for bioelectronic devices. It determines the strength of electrical signal transduction from cells to chips and from chips to cells. We measured the sheet resistance by applying AC voltage to oxidized silicon chips and by imaging the voltage change across the attached cell membrane with a fluorescent voltage-sensitive dye. The phase map of voltage change was fitted with a planar core-coat conductor model using the sheet resistance as a free parameter. For nerve cells from rat brain on polylysine as well as for HEK293 cells and MDCK cells on fibronectin we find a similar sheet resistance of 10 MOmega. Taking into account the independently measured distance of 50 nm between chip and membrane for these cells, we obtain a specific resistance of 50 Omegacm that is indistinguishable from bulk electrolyte. On the other hand, the sheet resistance for erythrocytes on polylysine is far higher, at approximately 1.5 GOmega. Considering the distance of 10 nm, the specific resistance in the narrow cleft is enhanced to 1500 Omegacm. We find this novel optical method to be a convenient tool to optimize the interface between cells and chips for bioelectronic devices.

Development of plasma-on-chip: Plasma treatment for individual cells cultured in media

NASA Astrophysics Data System (ADS)

Kumagai, Shinya; Chang, Chun-Yao; Jeong, Jonghyeon; Kobayashi, Mime; Shimizu, Tetsuji; Sasaki, Minoru

2016-01-01

A device consisting of Si microwells and microplasma sources has been fabricated for plasma treatment of individual cells cultured in media. We named the device plasma-on-chip. The microwells have through-holes at the bottom where gas-liquid interfaces form when they are filled with media containing biological samples. The microplasma sources, which supply reactive species, are located on the back of each microwell. Through the gas-liquid interface, the reactive species are supplied to the cells. Chlorella cells were used to demonstrate the feasibility of the device and after three minutes of plasma treatment, the fluorescence intensity of Chlorella cells appeared to be decreased. Optical emission spectroscopy identified O and OH radicals in the plasma, which can affect the cells. In the analysis of biological samples such as human cells or tissues, this device raises the possibility of revealing the mechanisms of plasma medicine in more detail.

Nanomaterial-based Microfluidic Chips for the Capture and Detection of Circulating Tumor Cells.

PubMed

Sun, Duanping; Chen, Zuanguang; Wu, Minhao; Zhang, Yuanqing

2017-01-01

Circulating tumor cells (CTCs), a type of cancer cells that spreads from primary or metastatic tumors into the bloodstream, can lead to a new fatal metastasis. As a new type of liquid biopsy, CTCs have become a hot pursuit and detection of CTCs offers the possibility for early diagnosis of cancers, earlier evaluation of chemotherapeutic efficacy and cancer recurrence, and choice of individual sensitive anti-cancer drugs. The fundamental challenges of capturing and characterizing CTCs are the extremely low number of CTCs in the blood and the intrinsic heterogeneity of CTCs. A series of microfluidic devices have been proposed for the analysis of CTCs with automation capability, precise flow behaviors, and significant advantages over the conventional larger scale systems. This review aims to provide in-depth insights into CTCs analysis, including various nanomaterial-based microfluidic chips for the capture and detection of CTCs based on the specific biochemical and physical properties of CTCs. The current developmental trends and promising research directions in the establishment of microfluidic chips for the capture and detection of CTCs are also discussed.

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

PubMed

Yamagishi, J; Isobe, R; Takebuchi, T; Bando, H

2003-03-01

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

Immunolocalization of aquaporin CHIP in the guinea pig inner ear.

PubMed

Stanković, K M; Adams, J C; Brown, D

1995-12-01

Aquaporin CHIP (AQP-CHIP) is a water channel protein previously identified in red blood cells and water transporting epithelia. The inner ear is an organ of hearing and balance whose normal function depends critically on maintenance of fluid homeostasis. In this study, AQP-CHIP, or a close homologue, was found in specific cells of the inner ear, as assessed by immunocytochemistry with the use of affinity-purified polyclonal antibodies against AQP-CHIP.AQP-CHIP was predominantly found in fibrocytes in close association with bone, including most of the cells lining the bony labyrinth and in fibrocytes lining the endolymphatic duct and sac. AQP-CHIP-positive cells not directly apposing bone include cells under the basilar membrane, some type III fibrocytes of the spiral ligament, fibrocytes of the spiral limbus, and the trabecular perilymphatic tissue extending from the membranous to the bony labyrinth. AQP-CHIP was also found in the periosteum of the middle ear and cranial bones, as well as in chondrocytes of the oval window and stapes. The distribution of AQP-CHIP in the inner ear suggests that AQP-CHIP may have special significance for maintenance of bone and the basilar membrane, and for function of the spiral ligament.

Skin integrated with perfusable vascular channels on a chip.

PubMed

Mori, Nobuhito; Morimoto, Yuya; Takeuchi, Shoji

2017-02-01

This paper describes a method for fabricating perfusable vascular channels coated with endothelial cells within a cultured skin-equivalent by fixing it to a culture device connected to an external pump and tubes. A histological analysis showed that vascular channels were constructed in the skin-equivalent, which showed a conventional dermal/epidermal morphology, and the endothelial cells formed tight junctions on the vascular channel wall. The barrier function of the skin-equivalent was also confirmed. Cell distribution analysis indicated that the vascular channels supplied nutrition to the skin-equivalent. Moreover, the feasibility of a skin-equivalent containing vascular channels as a model for studying vascular absorption was demonstrated by measuring test molecule permeation from the epidermal layer into the vascular channels. The results suggested that this skin-equivalent can be used for skin-on-a-chip applications including drug development, cosmetics testing, and studying skin biology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Recombinant drugs-on-a-chip: The usage of capillary electrophoresis and trends in miniaturized systems - A review.

PubMed

Morbioli, Giorgio Gianini; Mazzu-Nascimento, Thiago; Aquino, Adriano; Cervantes, Cesar; Carrilho, Emanuel

2016-09-07

We present here a critical review covering conventional analytical tools of recombinant drug analysis and discuss their evolution towards miniaturized systems foreseeing a possible unique recombinant drug-on-a-chip device. Recombinant protein drugs and/or pro-drug analysis require sensitive and reproducible analytical techniques for quality control to ensure safety and efficacy of drugs according to regulatory agencies. The versatility of miniaturized systems combined with their low-cost could become a major trend in recombinant drugs and bioprocess analysis. Miniaturized systems are capable of performing conventional analytical and proteomic tasks, allowing for interfaces with other powerful techniques, such as mass spectrometry. Microdevices can be applied during the different stages of recombinant drug processing, such as gene isolation, DNA amplification, cell culture, protein expression, protein separation, and analysis. In addition, organs-on-chips have appeared as a viable alternative to testing biodrug pharmacokinetics and pharmacodynamics, demonstrating the capabilities of the miniaturized systems. The integration of individual established microfluidic operations and analytical tools in a single device is a challenge to be overcome to achieve a unique recombinant drug-on-a-chip device. Copyright © 2016 Elsevier B.V. All rights reserved.

Two-stage microfluidic chip for selective isolation of circulating tumor cells (CTCs).

PubMed

Hyun, Kyung-A; Lee, Tae Yoon; Lee, Su Hyun; Jung, Hyo-Il

2015-05-15

Over the past few decades, circulating tumor cells (CTCs) have been studied as a means of overcoming cancer. However, the rarity and heterogeneity of CTCs have been the most significant hurdles in CTC research. Many techniques for CTC isolation have been developed and can be classified into positive enrichment (i.e., specifically isolating target cells using cell size, surface protein expression, and so on) and negative enrichment (i.e., specifically eluting non-target cells). Positive enrichment methods lead to high purity, but could be biased by their selection criteria, while the negative enrichment methods have relatively low purity, but can isolate heterogeneous CTCs. To compensate for the known disadvantages of the positive and negative enrichments, in this study we introduced a two-stage microfluidic chip. The first stage involves a microfluidic magnetic activated cell sorting (μ-MACS) chip to elute white blood cells (WBCs). The second stage involves a geometrically activated surface interaction (GASI) chip for the selective isolation of CTCs. We observed up to 763-fold enrichment in cancer cells spiked into 5 mL of blood sample using the μ-MACS chip at 400 μL/min flow rate. Cancer cells were successfully separated with separation efficiencies ranging from 10.19% to 22.91% based on their EpCAM or HER2 surface protein expression using the GASI chip at a 100 μL/min flow rate. Our two-stage microfluidic chips not only isolated CTCs from blood cells, but also classified heterogeneous CTCs based on their characteristics. Therefore, our chips can contribute to research on CTC heterogeneity of CTCs, and, by extension, personalized cancer treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

Carboxy terminus of heat shock protein (HSP) 70-interacting protein (CHIP) inhibits HSP70 in the heart.

PubMed

Zhao, Bijun; Sun, Guocheng; Feng, Guanli; Duan, Weixun; Zhu, Xiaoling; Chen, Shaoyang; Hou, Lichao; Jin, Zhenxiao; Yi, Dinghua

2012-12-01

Heat shock protein (HSP) 70 plays a critical role in protecting the heart from various stressor-induced cell injuries; the mechanism remains to be further understood. The present study aims to elucidate the effect of a probiotics-derived protein, LGG-derived protein p75 (LGP), in alleviating the ischemia/reperfusion (I/R)-induced heart injury. We treated rats with the I/R with or without preadministration with LGP. The levels of HSP70 and carboxy terminus of HSP70-interacting protein (CHIP) in the heart tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The effect of CHIP on suppression of HSP70 and the effect of LGP on suppression of CHIP were investigated with an I/R rat model and a cell culture model. The results showed that I/R-induced infarction in the heart could be alleviated by pretreatment with LGP. HSP70 was detected in naïve rat heart tissue extracts. I/R treatment significantly suppressed the level of HSP70 and increased the levels of CHIP in the heart. A complex of CHIP/HSP70 was detected in heart tissue extracts. The addition of recombinant CHIP to culture inhibited HSP70 in heart cells. LGP was bound CHIP in heart cells and prevented the CHIP from binding HSP70. In summary, I/R can suppress HSP70 and increase CHIP in heart cells. CHIP can suppress HSP70 that can be prevented by pretreatment with LGP. The results imply that CHIP may be a potential target in the prevention of I/R-induced heart cell injury.

Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

PubMed

Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

2016-10-07

Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

Microfluidic on-chip biomimicry for 3D cell culture: a fit-for-purpose investigation from the end user standpoint

PubMed Central

Liu, Ye; Gill, Elisabeth; Shery Huang, Yan Yan

2017-01-01

A plethora of 3D and microfluidics-based culture models have been demonstrated in the recent years with the ultimate aim to facilitate predictive in vitro models for pharmaceutical development. This article summarizes to date the progress in the microfluidics-based tissue culture models, including organ-on-a-chip and vasculature-on-a-chip. Specific focus is placed on addressing the question of what kinds of 3D culture and system complexities are deemed desirable by the biological and biomedical community. This question is addressed through analysis of a research survey to evaluate the potential use of microfluidic cell culture models among the end users. Our results showed a willingness to adopt 3D culture technology among biomedical researchers, although a significant gap still exists between the desired systems and existing 3D culture options. With these results, key challenges and future directions are highlighted. PMID:28670465

Microfluidic on-chip biomimicry for 3D cell culture: a fit-for-purpose investigation from the end user standpoint.

PubMed

Liu, Ye; Gill, Elisabeth; Shery Huang, Yan Yan

2017-06-01

A plethora of 3D and microfluidics-based culture models have been demonstrated in the recent years with the ultimate aim to facilitate predictive in vitro models for pharmaceutical development. This article summarizes to date the progress in the microfluidics-based tissue culture models, including organ-on-a-chip and vasculature-on-a-chip. Specific focus is placed on addressing the question of what kinds of 3D culture and system complexities are deemed desirable by the biological and biomedical community. This question is addressed through analysis of a research survey to evaluate the potential use of microfluidic cell culture models among the end users. Our results showed a willingness to adopt 3D culture technology among biomedical researchers, although a significant gap still exists between the desired systems and existing 3D culture options. With these results, key challenges and future directions are highlighted.

Microvalve controlled multi-functional microfluidic chip for divisional cell co-culture.

PubMed

Li, Rui; Zhang, Xingjian; Lv, Xuefei; Geng, Lina; Li, Yongrui; Qin, Kuiwei; Deng, Yulin

2017-12-15

Pneumatic micro-valve controlled microfluidic chip provides precise fluidic control for cell manipulation. In this paper, a multi-functional microfluidic chip was designed for three separate experiments: 1. Different cell lines were dispensed and cultured; 2. Three transfected SH-SY5Y cells were introduced and treated with methyl-phenyl-pyridinium (MPP + ) as drug delivery mode; 3. Specific protection and interaction were observed among cell co-culture after nerve damage. The outcomes revealed the potential and practicability of our entire multi-functional pneumatic chip system on different cell biology applications. Copyright © 2017. Published by Elsevier Inc.

CHIP promotes thyroid cancer proliferation via activation of the MAPK and AKT pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Li; Liu, Lianyong; Department of Endocrinology, Shanghai Punan Hospital, Shanghai 200125

The carboxyl terminus of Hsp70-interacting protein (CHIP) is a U box-type ubiquitin ligase that plays crucial roles in various biological processes, including tumor progression. To date, the functional mechanism of CHIP in thyroid cancer remains unknown. Here, we obtained evidence of upregulation of CHIP in thyroid cancer tissues and cell lines. CHIP overexpression markedly enhanced thyroid cancer cell viability and colony formation in vitro and accelerated tumor growth in vivo. Conversely, CHIP knockdown impaired cell proliferation and tumor growth. Notably, CHIP promoted cell growth through activation of MAPK and AKT pathways, subsequently decreasing p27 and increasing cyclin D1 and p-FOXO3a expression. Ourmore » findings collectively indicate that CHIP functions as an oncogene in thyroid cancer, and is therefore a potential therapeutic target for this disease. - Highlights: • CHIP is significantly upregulated in thyroid cancer cells. • Overexpression of CHIP facilitates proliferation and tumorigenesis of thyroid cancer cells. • Silencing of CHIP inhibits the proliferation and tumorigenesis of thyroid cancer cells. • CHIP promotes thyroid cancer cell proliferation via activating the MAPK and AKT pathways.« less

Cloning and characterization of carboxyl terminus of heat shock cognate 70-interacting protein gene from the silkworm, Bombyx mori.

PubMed

Ohsawa, Takeshi; Fujimoto, Shota; Tsunakawa, Akane; Shibano, Yuka; Kawasaki, Hideki; Iwanaga, Masashi

2016-11-01

Carboxyl terminus of heat shock cognate 70-interacting protein (CHIP) is an evolutionarily conserved E3 ubiquitin ligase across different eukaryotic species and is known to play a key role in protein quality control. CHIP has two distinct functional domains, an N-terminal tetratricopeptide repeat (TPR) and a C-terminal U-box domain, which are required for the ubiquitination of numerous labile client proteins that are chaperoned by heat shock proteins (HSPs) and heat shock cognate proteins (HSCs). During our screen for CHIP-like proteins in the Bombyx mori databases, we found a novel silkworm gene, Bombyx mori CHIP. Phylogenetic analysis showed that BmCHIP belongs to Lepidopteran lineages. Quantitative reverse transcription-PCR analysis indicated that BmCHIP was relatively highly expressed in the gonad and fat body. A pull-down experiment and auto-ubiquitination assay showed that BmCHIP interacted with BmHSC70 and had E3 ligase activity. Additionally, immunohistochemical analysis revealed that BmCHIP was partially co-localized with ubiquitin in BmN4 cells. These data support that BmCHIP plays an important role in the ubiquitin proteasome system as an E3 ubiquitin ligase in B. mori. Copyright © 2016 Elsevier Inc. All rights reserved.

On-chip wavelength multiplexed detection of cancer DNA biomarkers in blood

PubMed Central

Cai, H.; Stott, M. A.; Ozcelik, D.; Parks, J. W.; Hawkins, A. R.; Schmidt, H.

2016-01-01

We have developed an optofluidic analysis system that processes biomolecular samples starting from whole blood and then analyzes and identifies multiple targets on a silicon-based molecular detection platform. We demonstrate blood filtration, sample extraction, target enrichment, and fluorescent labeling using programmable microfluidic circuits. We detect and identify multiple targets using a spectral multiplexing technique based on wavelength-dependent multi-spot excitation on an antiresonant reflecting optical waveguide chip. Specifically, we extract two types of melanoma biomarkers, mutated cell-free nucleic acids —BRAFV600E and NRAS, from whole blood. We detect and identify these two targets simultaneously using the spectral multiplexing approach with up to a 96% success rate. These results point the way toward a full front-to-back chip-based optofluidic compact system for high-performance analysis of complex biological samples. PMID:28058082

A droplet-to-digital (D2D) microfluidic device for single cell assays.

PubMed

Shih, Steve C C; Gach, Philip C; Sustarich, Jess; Simmons, Blake A; Adams, Paul D; Singh, Seema; Singh, Anup K

2015-01-07

We have developed a new hybrid droplet-to-digital microfluidic platform (D2D) that integrates droplet-in-channel microfluidics with digital microfluidics (DMF) for performing multi-step assays. This D2D platform combines the strengths of the two formats-droplets-in-channel for facile generation of droplets containing single cells, and DMF for on-demand manipulation of droplets including control of different droplet volumes (pL-μL), creation of a dilution series of ionic liquid (IL), and parallel single cell culturing and analysis for IL toxicity screening. This D2D device also allows for automated analysis that includes a feedback-controlled system for merging and splitting of droplets to add reagents, an integrated Peltier element for parallel cell culture at optimum temperature, and an impedance sensing mechanism to control the flow rate for droplet generation and preventing droplet evaporation. Droplet-in-channel is well-suited for encapsulation of single cells as it allows the careful manipulation of flow rates of aqueous phase containing cells and oil to optimize encapsulation. Once single cell containing droplets are generated, they are transferred to a DMF chip via a capillary where they are merged with droplets containing IL and cultured at 30 °C. The DMF chip, in addition to permitting cell culture and reagent (ionic liquid/salt) addition, also allows recovery of individual droplets for off-chip analysis such as further culturing and measurement of ethanol production. The D2D chip was used to evaluate the effect of IL/salt type (four types: NaOAc, NaCl, [C2mim] [OAc], [C2mim] [Cl]) and concentration (four concentrations: 0, 37.5, 75, 150 mM) on the growth kinetics and ethanol production of yeast and as expected, increasing IL concentration led to lower biomass and ethanol production. Specifically, [C2mim] [OAc] had inhibitory effects on yeast growth at concentrations 75 and 150 mM and significantly reduced their ethanol production compared to cells grown in other ILs/salts. The growth curve trends obtained by D2D matched conventional yeast culturing in microtiter wells, validating the D2D platform. We believe that our approach represents a generic platform for multi-step biochemical assays such as drug screening, digital PCR, enzyme assays, immunoassays and cell-based assays.

Microfluidic immunomagnetic cell separation from whole blood.

PubMed

Bhuvanendran Nair Gourikutty, Sajay; Chang, Chia-Pin; Puiu, Poenar Daniel

2016-02-01

Immunomagnetic-based separation has become a viable technique for the separation of cells and biomolecules. Here we report on the design and analysis of a simple and efficient microfluidic device for high throughput and high efficiency capture of cells tagged with magnetic particles. This is made possible by using a microfluidic chip integrated with customized arrays of permanent magnets capable of creating large magnetic field gradients, which determine the effective capturing of the tagged cells. This method is based on manipulating the cells which are under the influence of a combination of magnetic and fluid dynamic forces in a fluid under laminar flow through a microfluidic chip. A finite element analysis (FEA) model is developed to analyze the cell separation process and predict its behavior, which is validated subsequently by the experimental results. The magnetic field gradients created by various arrangements of magnetic arrays have been simulated using FEA and the influence of these field gradients on cell separation has been studied with the design of our microfluidic chip. The proof-of-concept for the proposed technique is demonstrated by capturing white blood cells (WBCs) from whole human blood. CD45-conjugated magnetic particles were added into whole blood samples to label WBCs and the mixture was flown through our microfluidic device to separate the labeled cells. After the separation process, the remaining WBCs in the elute were counted to determine the capture efficiency, and it was found that more than 99.9% WBCs have been successfully separated from whole blood. The proposed design can be used for positive selection as well as for negative enrichment of rare cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Low temperature co-fired ceramic packaging of CMOS capacitive sensor chip towards cell viability monitoring.

PubMed

Halonen, Niina; Kilpijärvi, Joni; Sobocinski, Maciej; Datta-Chaudhuri, Timir; Hassinen, Antti; Prakash, Someshekar B; Möller, Peter; Abshire, Pamela; Kellokumpu, Sakari; Lloyd Spetz, Anita

2016-01-01

Cell viability monitoring is an important part of biosafety evaluation for the detection of toxic effects on cells caused by nanomaterials, preferably by label-free, noninvasive, fast, and cost effective methods. These requirements can be met by monitoring cell viability with a capacitance-sensing integrated circuit (IC) microchip. The capacitance provides a measurement of the surface attachment of adherent cells as an indication of their health status. However, the moist, warm, and corrosive biological environment requires reliable packaging of the sensor chip. In this work, a second generation of low temperature co-fired ceramic (LTCC) technology was combined with flip-chip bonding to provide a durable package compatible with cell culture. The LTCC-packaged sensor chip was integrated with a printed circuit board, data acquisition device, and measurement-controlling software. The packaged sensor chip functioned well in the presence of cell medium and cells, with output voltages depending on the medium above the capacitors. Moreover, the manufacturing of microfluidic channels in the LTCC package was demonstrated.

Product assurance technology for procuring reliable, radiation-hard, custom LSI/VLSI electronics

NASA Technical Reports Server (NTRS)

Buehler, M. G.; Allen, R. A.; Blaes, B. R.; Hicks, K. A.; Jennings, G. A.; Lin, Y.-S.; Pina, C. A.; Sayah, H. R.; Zamani, N.

1989-01-01

Advanced measurement methods using microelectronic test chips are described. These chips are intended to be used in acquiring the data needed to qualify Application Specific Integrated Circuits (ASIC's) for space use. Efforts were focused on developing the technology for obtaining custom IC's from CMOS/bulk silicon foundries. A series of test chips were developed: a parametric test strip, a fault chip, a set of reliability chips, and the CRRES (Combined Release and Radiation Effects Satellite) chip, a test circuit for monitoring space radiation effects. The technical accomplishments of the effort include: (1) development of a fault chip that contains a set of test structures used to evaluate the density of various process-induced defects; (2) development of new test structures and testing techniques for measuring gate-oxide capacitance, gate-overlap capacitance, and propagation delay; (3) development of a set of reliability chips that are used to evaluate failure mechanisms in CMOS/bulk: interconnect and contact electromigration and time-dependent dielectric breakdown; (4) development of MOSFET parameter extraction procedures for evaluating subthreshold characteristics; (5) evaluation of test chips and test strips on the second CRRES wafer run; (6) two dedicated fabrication runs for the CRRES chip flight parts; and (7) publication of two papers: one on the split-cross bridge resistor and another on asymmetrical SRAM (static random access memory) cells for single-event upset analysis.

Human bone perivascular niche-on-a-chip for studying metastatic colonization.

PubMed

Marturano-Kruik, Alessandro; Nava, Michele Maria; Yeager, Keith; Chramiec, Alan; Hao, Luke; Robinson, Samuel; Guo, Edward; Raimondi, Manuela Teresa; Vunjak-Novakovic, Gordana

2018-02-06

Eight out of 10 breast cancer patients die within 5 years after the primary tumor has spread to the bones. Tumor cells disseminated from the breast roam the vasculature, colonizing perivascular niches around blood capillaries. Slow flows support the niche maintenance by driving the oxygen, nutrients, and signaling factors from the blood into the interstitial tissue, while extracellular matrix, endothelial cells, and mesenchymal stem cells regulate metastatic homing. Here, we show the feasibility of developing a perfused bone perivascular niche-on-a-chip to investigate the progression and drug resistance of breast cancer cells colonizing the bone. The model is a functional human triculture with stable vascular networks within a 3D native bone matrix cultured on a microfluidic chip. Providing the niche-on-a-chip with controlled flow velocities, shear stresses, and oxygen gradients, we established a long-lasting, self-assembled vascular network without supplementation of angiogenic factors. We further show that human bone marrow-derived mesenchymal stem cells, which have undergone phenotypical transition toward perivascular cell lineages, support the formation of capillary-like structures lining the vascular lumen. Finally, breast cancer cells exposed to interstitial flow within the bone perivascular niche-on-a-chip persist in a slow-proliferative state associated with increased drug resistance. We propose that the bone perivascular niche-on-a-chip with interstitial flow promotes the formation of stable vasculature and mediates cancer cell colonization.

An automated microreactor for semi-continuous biosensor measurements.

PubMed

Buffi, Nina; Beggah, Siham; Truffer, Frederic; Geiser, Martial; van Lintel, Harald; Renaud, Philippe; van der Meer, Jan Roelof

2016-04-21

Living bacteria or yeast cells are frequently used as bioreporters for the detection of specific chemical analytes or conditions of sample toxicity. In particular, bacteria or yeast equipped with synthetic gene circuitry that allows the production of a reliable non-cognate signal (e.g., fluorescent protein or bioluminescence) in response to a defined target make robust and flexible analytical platforms. We report here how bacterial cells expressing a fluorescence reporter ("bactosensors"), which are mostly used for batch sample analysis, can be deployed for automated semi-continuous target analysis in a single concise biochip. Escherichia coli-based bactosensor cells were continuously grown in a 13 or 50 nanoliter-volume reactor on a two-layered polydimethylsiloxane-on-glass microfluidic chip. Physiologically active cells were directed from the nl-reactor to a dedicated sample exposure area, where they were concentrated and reacted in 40 minutes with the target chemical by localized emission of the fluorescent reporter signal. We demonstrate the functioning of the bactosensor-chip by the automated detection of 50 μgarsenite-As l(-1) in water on consecutive days and after a one-week constant operation. Best induction of the bactosensors of 6-9-fold to 50 μg l(-1) was found at an apparent dilution rate of 0.12 h(-1) in the 50 nl microreactor. The bactosensor chip principle could be widely applicable to construct automated monitoring devices for a variety of targets in different environments.

K6 linked polyubiquitylation of FADD by CHIP prevents death inducing signaling complex formation suppressing cell death.

PubMed

Seo, Jinho; Lee, Eun-Woo; Shin, Jihye; Seong, Daehyeon; Nam, Young Woo; Jeong, Manhyung; Lee, Seon-Hyeong; Lee, Cheolju; Song, Jaewhan

2018-05-23

Fas-associated death domain (FADD) is an adaptor protein recruiting complexes of caspase 8 to death ligand receptors to induce extrinsic apoptotic cell death in response to a TNF superfamily member. Although, formation of the complex of FADD and caspase 8 upon death stimuli has been studied in detail, posttranslational modifications fine-tuning these processes have yet to be identified. Here we revealed that K6-linked polyubiquitylation of FADD on lysines 149 and 153 mediated by C terminus HSC70-interacting protein (CHIP) plays an important role in preventing formation of the death inducing signaling complex (DISC), thus leading to the suppression of cell death. Cells depleted of CHIP showed higher sensitivity toward death ligands such as FasL and TRAIL, leading to upregulation of DISC formation composed of a death receptor, FADD, and caspase 8. CHIP was able to bind to FADD, induce K6-linked polyubiquitylation of FADD, and suppress DISC formation. By mass spectrometry, lysines 149 and 153 of FADD were found to be responsible for CHIP-mediated FADD ubiquitylation. FADD mutated at these sites was capable of more potent cell death induction as compared with the wild type and was no longer suppressed by CHIP. On the other hand, CHIP deficient in E3 ligase activity was not capable of suppressing FADD function and of FADD ubiquitylation. CHIP depletion in ME-180 cells induced significant sensitization of these cells toward TRAIL in xenograft analyses. These results imply that K6-linked ubiquitylation of FADD by CHIP is a crucial checkpoint in cytokine-dependent extrinsic apoptosis.

Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip.

PubMed

Khamenehfar, A; Beischlag, T V; Russell, P J; Ling, M T P; Nelson, C; Li, P C H

2015-11-01

Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients.

Development of a high-throughput Candida albicans biofilm chip.

PubMed

Srinivasan, Anand; Uppuluri, Priya; Lopez-Ribot, Jose; Ramasubramanian, Anand K

2011-04-22

We have developed a high-density microarray platform consisting of nano-biofilms of Candida albicans. A robotic microarrayer was used to print yeast cells of C. albicans encapsulated in a collagen matrix at a volume as low as 50 nL onto surface-modified microscope slides. Upon incubation, the cells grow into fully formed "nano-biofilms". The morphological and architectural complexity of these biofilms were evaluated by scanning electron and confocal scanning laser microscopy. The extent of biofilm formation was determined using a microarray scanner from changes in fluorescence intensities due to FUN 1 metabolic processing. This staining technique was also adapted for antifungal susceptibility testing, which demonstrated that, similar to regular biofilms, cells within the on-chip biofilms displayed elevated levels of resistance against antifungal agents (fluconazole and amphotericin B). Thus, results from structural analyses and antifungal susceptibility testing indicated that despite miniaturization, these biofilms display the typical phenotypic properties associated with the biofilm mode of growth. In its final format, the C. albicans biofilm chip (CaBChip) is composed of 768 equivalent and spatially distinct nano-biofilms on a single slide; multiple chips can be printed and processed simultaneously. Compared to current methods for the formation of microbial biofilms, namely the 96-well microtiter plate model, this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize labor intensive steps, and dramatically reduce assay costs. Such a chip should accelerate the antifungal drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously.

Transport, manipulation, and reaction of biological cells on-chip using electrokinetic effects.

PubMed

Li, P C; Harrison, D J

1997-04-15

A microfluidic system was fabricated on a glass chip to study mobilization of biological cells on-chip. Electroosmotic and/or electrophoretic pumping were used to drive the cell transport within a network of capillary channels. Whole cells such as Saccharomyces cerevisiae, canine erythrocyte, and Escherichia coli were employed in this work. Photographs are presented to illustrate how cells are selected and transported from one location to another within the capillary network, with velocities up to about 0.5 mm/s in capillaries with a 15- x 55-microns cross section. The mixing of canine erythrocytes with the lysing agent sodium dodecyl sulfate, at an intersection within the chip, was performed to demonstrate that cell selection and subsequent reaction can be accomplished within the microchip.

NanoTopoChip: High-throughput nanotopographical cell instruction.

PubMed

Hulshof, Frits F B; Zhao, Yiping; Vasilevich, Aliaksei; Beijer, Nick R M; de Boer, Meint; Papenburg, Bernke J; van Blitterswijk, Clemens; Stamatialis, Dimitrios; de Boer, Jan

2017-10-15

Surface topography is able to influence cell phenotype in numerous ways and offers opportunities to manipulate cells and tissues. In this work, we develop the Nano-TopoChip and study the cell instructive effects of nanoscale topographies. A combination of deep UV projection lithography and conventional lithography was used to fabricate a library of more than 1200 different defined nanotopographies. To illustrate the cell instructive effects of nanotopography, actin-RFP labeled U2OS osteosarcoma cells were cultured and imaged on the Nano-TopoChip. Automated image analysis shows that of many cell morphological parameters, cell spreading, cell orientation and actin morphology are mostly affected by the nanotopographies. Additionally, by using modeling, the changes of cell morphological parameters could by predicted by several feature shape parameters such as lateral size and spacing. This work overcomes the technological challenges of fabricating high quality defined nanoscale features on unprecedented large surface areas of a material relevant for tissue culture such as PS and the screening system is able to infer nanotopography - cell morphological parameter relationships. Our screening platform provides opportunities to identify and study the effect of nanotopography with beneficial properties for the culture of various cell types. The nanotopography of biomaterial surfaces can be modified to influence adhering cells with the aim to improve the performance of medical implants and tissue culture substrates. However, the necessary knowledge of the underlying mechanisms remains incomplete. One reason for this is the limited availability of high-resolution nanotopographies on relevant biomaterials, suitable to conduct systematic biological studies. The present study shows the fabrication of a library of nano-sized surface topographies with high fidelity. The potential of this library, called the 'NanoTopoChip' is shown in a proof of principle HTS study which demonstrates how cells are affected by nanotopographies. The large dataset, acquired by quantitative high-content imaging, allowed us to use predictive modeling to describe how feature dimensions affect cell morphology. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Injection by hydrostatic pressure in conjunction with electrokinetic force on a microfluidic chip.

PubMed

Gai, Hongwei; Yu, Linfen; Dai, Zhongpeng; Ma, Yinfa; Lin, Bingcheng

2004-06-01

A simple method was developed for injecting a sample on a cross-form microfluidic chip by means of hydrostatic pressure combined with electrokinetic forces. The hydrostatic pressure was generated simply by adjusting the liquid level in different reservoirs without any additional driven equipment such as a pump. Two dispensing strategies using a floating injection and a gated injection, coupled with hydrostatic pressure loading, were tested. The fluorescence observation verified the feasibility of hydrostatic pressure loading in the separation of a mixture of fluorescein sodium salt and fluorescein isothiocyanate. This method was proved to be effective in leading cells to a separation channel for single cell analysis.

Human Lung Small Airway-on-a-Chip Protocol.

PubMed

Benam, Kambez H; Mazur, Marc; Choe, Youngjae; Ferrante, Thomas C; Novak, Richard; Ingber, Donald E

2017-01-01

Organs-on-chips are microfluidic cell culture devices created using microchip manufacturing techniques that contain hollow microchannels lined by living cells, which recreate specialized tissue-tissue interfaces, physical microenvironments, and vascular perfusion necessary to recapitulate organ-level physiology in vitro. Here we describe a protocol for fabrication, culture, and operation of a human lung "small airway-on-a-chip," which contains a differentiated, mucociliary bronchiolar epithelium exposed to air and an underlying microvascular endothelium that experiences fluid flow. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin rigid porous membrane; this requires less than 1 day to complete. Next, primary human airway bronchiolar epithelial cells isolated from healthy normal donors or patients with respiratory disease are cultured on the porous membrane within one microchannel while lung microvascular endothelial cells are cultured on the opposite side of the same membrane in the second channel to create a mucociliated epithelium-endothelium interface; this process take about 4-6 weeks to complete. Finally, culture medium containing neutrophils isolated from fresh whole human blood are flowed through the microvascular channel of the device to enable real-time analysis of capture and recruitment of circulating leukocytes by endothelium under physiological shear; this step requires less than 1 day to complete. The small airway-on-a-chip represents a new microfluidic tool to model complex and dynamic inflammatory responses of healthy and diseased lungs in vitro.

Opto-fluidics based microscopy and flow cytometry on a cell phone for blood analysis.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2015-01-01

Blood analysis is one of the most important clinical tests for medical diagnosis. Flow cytometry and optical microscopy are widely used techniques to perform blood analysis and therefore cost-effective translation of these technologies to resource limited settings is critical for various global health as well as telemedicine applications. In this chapter, we review our recent progress on the integration of imaging flow cytometry and fluorescent microscopy on a cell phone using compact, light-weight and cost-effective opto-fluidic attachments integrated onto the camera module of a smartphone. In our cell-phone based opto-fluidic imaging cytometry design, fluorescently labeled cells are delivered into the imaging area using a disposable micro-fluidic chip that is positioned above the existing camera unit of the cell phone. Battery powered light-emitting diodes (LEDs) are butt-coupled to the sides of this micro-fluidic chip without any lenses, which effectively acts as a multimode slab waveguide, where the excitation light is guided to excite the fluorescent targets within the micro-fluidic chip. Since the excitation light propagates perpendicular to the detection path, an inexpensive plastic absorption filter is able to reject most of the scattered light and create a decent dark-field background for fluorescent imaging. With this excitation geometry, the cell-phone camera can record fluorescent movies of the particles/cells as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the solution under test. With a similar opto-fluidic design, we have recently demonstrated imaging and automated counting of stationary blood cells (e.g., labeled white blood cells or unlabeled red blood cells) loaded within a disposable cell counting chamber. We tested the performance of this cell-phone based imaging cytometry and blood analysis platform by measuring the density of red and white blood cells as well as hemoglobin concentration in human blood samples, which showed a good match to our measurement results obtained using a commercially available hematology analyzer. Such a cell-phone enabled opto-fluidics microscopy, flow cytometry, and blood analysis platform could be especially useful for various telemedicine applications in remote and resource-limited settings.

A microfluidic chip integrated with a high-density PDMS-based microfiltration membrane for rapid isolation and detection of circulating tumor cells.

PubMed

Fan, Xiaoyun; Jia, Chunping; Yang, Jun; Li, Gang; Mao, Hongju; Jin, Qinghui; Zhao, Jianlong

2015-09-15

Isolation of circulating tumor cells (CTCs) by size exclusion is a widely researched technique that offers the advantage of capturing tumor cells without reliance on cell surface expression markers. In this work, we report the development of a novel polydimethylsiloxane (PDMS) membrane filter-based microdevice for rapid and highly efficient isolation of CTCs from peripheral blood. A precise and highly porous PDMS microfilter was fabricated and integrated into the microfiltration chip by combining a sacrificial transferring film with a sandwich molding method. We achieved >90% recovery when isolating lung cancer cells from spiked blood samples, with a relatively high processing throughput of 10 mL/h. In contrast to existing CTC filtration systems, which rely on low-porosity track-etch filters or expensive lithography-based filters, our microfiltration chip does not require complex e-beam lithography or the reactive ion etching process, therefore it offers a low-cost alternative tool for highly efficient CTC enrichment and in situ analysis. Thus, this new microdevice has the potential for use in routine monitoring of cancer development and cancer therapy in a clinical setting. Copyright © 2015 Elsevier B.V. All rights reserved.

Automated reagent-dispensing system for microfluidic cell biology assays.

PubMed

Ly, Jimmy; Masterman-Smith, Michael; Ramakrishnan, Ravichandran; Sun, Jing; Kokubun, Brent; van Dam, R Michael

2013-12-01

Microscale systems that enable measurements of oncological phenomena at the single-cell level have a great capacity to improve therapeutic strategies and diagnostics. Such measurements can reveal unprecedented insights into cellular heterogeneity and its implications into the progression and treatment of complicated cellular disease processes such as those found in cancer. We describe a novel fluid-delivery platform to interface with low-cost microfluidic chips containing arrays of microchambers. Using multiple pairs of needles to aspirate and dispense reagents, the platform enables automated coating of chambers, loading of cells, and treatment with growth media or other agents (e.g., drugs, fixatives, membrane permeabilizers, washes, stains, etc.). The chips can be quantitatively assayed using standard fluorescence-based immunocytochemistry, microscopy, and image analysis tools, to determine, for example, drug response based on differences in protein expression and/or activation of cellular targets on an individual-cell level. In general, automation of fluid and cell handling increases repeatability, eliminates human error, and enables increased throughput, especially for sophisticated, multistep assays such as multiparameter quantitative immunocytochemistry. We report the design of the automated platform and compare several aspects of its performance to manually-loaded microfluidic chips.

High-throughput particle manipulation by hydrodynamic, electrokinetic, and dielectrophoretic effects in an integrated microfluidic chip

PubMed Central

Li, Shunbo; Li, Ming; Bougot-Robin, Kristelle; Cao, Wenbin; Yeung Yeung Chau, Irene; Li, Weihua; Wen, Weijia

2013-01-01

Integrating different steps on a chip for cell manipulations and sample preparation is of foremost importance to fully take advantage of microfluidic possibilities, and therefore make tests faster, cheaper and more accurate. We demonstrated particle manipulation in an integrated microfluidic device by applying hydrodynamic, electroosmotic (EO), electrophoretic (EP), and dielectrophoretic (DEP) forces. The process involves generation of fluid flow by pressure difference, particle trapping by DEP force, and particle redirect by EO and EP forces. Both DC and AC signals were applied, taking advantages of DC EP, EO and AC DEP for on-chip particle manipulation. Since different types of particles respond differently to these signals, variations of DC and AC signals are capable to handle complex and highly variable colloidal and biological samples. The proposed technique can operate in a high-throughput manner with thirteen independent channels in radial directions for enrichment and separation in microfluidic chip. We evaluated our approach by collecting Polystyrene particles, yeast cells, and E. coli bacteria, which respond differently to electric field gradient. Live and dead yeast cells were separated successfully, validating the capability of our device to separate highly similar cells. Our results showed that this technique could achieve fast pre-concentration of colloidal particles and cells and separation of cells depending on their vitality. Hydrodynamic, DC electrophoretic and DC electroosmotic forces were used together instead of syringe pump to achieve sufficient fluid flow and particle mobility for particle trapping and sorting. By eliminating bulky mechanical pumps, this new technique has wide applications for in situ detection and analysis. PMID:24404011

High-throughput particle manipulation by hydrodynamic, electrokinetic, and dielectrophoretic effects in an integrated microfluidic chip.

PubMed

Li, Shunbo; Li, Ming; Bougot-Robin, Kristelle; Cao, Wenbin; Yeung Yeung Chau, Irene; Li, Weihua; Wen, Weijia

2013-01-01

Integrating different steps on a chip for cell manipulations and sample preparation is of foremost importance to fully take advantage of microfluidic possibilities, and therefore make tests faster, cheaper and more accurate. We demonstrated particle manipulation in an integrated microfluidic device by applying hydrodynamic, electroosmotic (EO), electrophoretic (EP), and dielectrophoretic (DEP) forces. The process involves generation of fluid flow by pressure difference, particle trapping by DEP force, and particle redirect by EO and EP forces. Both DC and AC signals were applied, taking advantages of DC EP, EO and AC DEP for on-chip particle manipulation. Since different types of particles respond differently to these signals, variations of DC and AC signals are capable to handle complex and highly variable colloidal and biological samples. The proposed technique can operate in a high-throughput manner with thirteen independent channels in radial directions for enrichment and separation in microfluidic chip. We evaluated our approach by collecting Polystyrene particles, yeast cells, and E. coli bacteria, which respond differently to electric field gradient. Live and dead yeast cells were separated successfully, validating the capability of our device to separate highly similar cells. Our results showed that this technique could achieve fast pre-concentration of colloidal particles and cells and separation of cells depending on their vitality. Hydrodynamic, DC electrophoretic and DC electroosmotic forces were used together instead of syringe pump to achieve sufficient fluid flow and particle mobility for particle trapping and sorting. By eliminating bulky mechanical pumps, this new technique has wide applications for in situ detection and analysis.

Digital PCR on a SlipChip.

PubMed

Shen, Feng; Du, Wenbin; Kreutz, Jason E; Fok, Alice; Ismagilov, Rustem F

2010-10-21

This paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1280 reaction compartments (2.6 nL each) simultaneously. After thermal cycling, end-point fluorescence intensity was used to detect the presence of nucleic acid. Digital PCR on the SlipChip was tested quantitatively by using Staphylococcus aureus genomic DNA. As the concentration of the template DNA in the reaction mixture was diluted, the fraction of positive wells decreased as expected from the statistical analysis. No cross-contamination was observed during the experiments. At the extremes of the dynamic range of digital PCR the standard confidence interval determined using a normal approximation of the binomial distribution is not satisfactory. Therefore, statistical analysis based on the score method was used to establish these confidence intervals. The SlipChip provides a simple strategy to count nucleic acids by using PCR. It may find applications in research applications such as single cell analysis, prenatal diagnostics, and point-of-care diagnostics. SlipChip would become valuable for diagnostics, including applications in resource-limited areas after integration with isothermal nucleic acid amplification technologies and visual readout.

Isolation and mutational analysis of circulating tumor cells from lung cancer patients with magnetic sifters and biochips†

PubMed Central

Earhart, Christopher M.; Hughes, Casey E.; Gaster, Richard S.; Ooi, Chin Chun; Wilson, Robert J.; Zhou, Lisa Y.; Humke, Eric W.; Xu, Lingyun; Wong, Dawson J.; Willingham, Stephen B.; Schwartz, Erich J.; Weissman, Irving L.; Jeffrey, Stefanie S.; Neal, Joel W.; Rohatgi, Rajat; Wakelee, Heather A.; Wang, Shan X.

2014-01-01

Detection and characterization of circulating tumor cells (CTCs) may reveal insights into the diagnosis and treatment of malignant disease. Technologies for isolating CTCs developed thus far suffer from one or more limitations, such as low throughput, inability to release captured cells, and reliance on expensive instrumentation for enrichment or subsequent characterization. We report a continuing development of a magnetic separation device, the magnetic sifter, which is a miniature microfluidic chip with a dense array of magnetic pores. It offers high efficiency capture of tumor cells, labeled with magnetic nanoparticles, from whole blood with high throughput and efficient release of captured cells. For subsequent characterization of CTCs, an assay, using a protein chip with giant magnetoresistive nanosensors, has been implemented for mutational analysis of CTCs enriched with the magnetic sifter. The use of these magnetic technologies, which are separate devices, may lead the way to routine preparation and characterization of “liquid biopsies” from cancer patients. PMID:23969419

Microfabrication of human organs-on-chips.

PubMed

Huh, Dongeun; Kim, Hyun Jung; Fraser, Jacob P; Shea, Daniel E; Khan, Mohammed; Bahinski, Anthony; Hamilton, Geraldine A; Ingber, Donald E

2013-11-01

'Organs-on-chips' are microengineered biomimetic systems containing microfluidic channels lined by living human cells, which replicate key functional units of living organs to reconstitute integrated human organ-level pathophysiology in vitro. These microdevices can be used to test efficacy and toxicity of drugs and chemicals, and to create in vitro models of human disease. Thus, they potentially represent low-cost alternatives to conventional animal models for pharmaceutical, chemical and environmental applications. Here we describe a protocol for the fabrication, microengineering and operation of these microfluidic organ-on-chip systems. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin porous flexible membrane, along with two full-height, hollow vacuum chambers on either side; this requires ∼3.5 d to complete. To create a 'breathing' lung-on-a-chip that mimics the mechanically active alveolar-capillary interface of the living human lung, human alveolar epithelial cells and microvascular endothelial cells are cultured in the microdevice with physiological flow and cyclic suction applied to the side chambers to reproduce rhythmic breathing movements. We describe how this protocol can be easily adapted to develop other human organ chips, such as a gut-on-a-chip lined by human intestinal epithelial cells that experiences peristalsis-like motions and trickling fluid flow. Also, we discuss experimental techniques that can be used to analyze the cells in these organ-on-chip devices.

Neural Cell Chip Based Electrochemical Detection of Nanotoxicity

PubMed Central

Kafi, Md. Abdul; Cho, Hyeon-Yeol; Choi, Jeong Woo

2015-01-01

Development of a rapid, sensitive and cost-effective method for toxicity assessment of commonly used nanoparticles is urgently needed for the sustainable development of nanotechnology. A neural cell with high sensitivity and conductivity has become a potential candidate for a cell chip to investigate toxicity of environmental influences. A neural cell immobilized on a conductive surface has become a potential tool for the assessment of nanotoxicity based on electrochemical methods. The effective electrochemical monitoring largely depends on the adequate attachment of a neural cell on the chip surfaces. Recently, establishment of integrin receptor specific ligand molecules arginine-glycine-aspartic acid (RGD) or its several modifications RGD-Multi Armed Peptide terminated with cysteine (RGD-MAP-C), C(RGD)4 ensure farm attachment of neural cell on the electrode surfaces either in their two dimensional (dot) or three dimensional (rod or pillar) like nano-scale arrangement. A three dimensional RGD modified electrode surface has been proven to be more suitable for cell adhesion, proliferation, differentiation as well as electrochemical measurement. This review discusses fabrication as well as electrochemical measurements of neural cell chip with particular emphasis on their use for nanotoxicity assessments sequentially since inception to date. Successful monitoring of quantum dot (QD), graphene oxide (GO) and cosmetic compound toxicity using the newly developed neural cell chip were discussed here as a case study. This review recommended that a neural cell chip established on a nanostructured ligand modified conductive surface can be a potential tool for the toxicity assessments of newly developed nanomaterials prior to their use on biology or biomedical technologies. PMID:28347059

3D Printing of Organs-On-Chips

PubMed Central

Yi, Hee-Gyeong; Lee, Hyungseok; Cho, Dong-Woo

2017-01-01

Organ-on-a-chip engineering aims to create artificial living organs that mimic the complex and physiological responses of real organs, in order to test drugs by precisely manipulating the cells and their microenvironments. To achieve this, the artificial organs should to be microfabricated with an extracellular matrix (ECM) and various types of cells, and should recapitulate morphogenesis, cell differentiation, and functions according to the native organ. A promising strategy is 3D printing, which precisely controls the spatial distribution and layer-by-layer assembly of cells, ECMs, and other biomaterials. Owing to this unique advantage, integration of 3D printing into organ-on-a-chip engineering can facilitate the creation of micro-organs with heterogeneity, a desired 3D cellular arrangement, tissue-specific functions, or even cyclic movement within a microfluidic device. Moreover, fully 3D-printed organs-on-chips more easily incorporate other mechanical and electrical components with the chips, and can be commercialized via automated massive production. Herein, we discuss the recent advances and the potential of 3D cell-printing technology in engineering organs-on-chips, and provides the future perspectives of this technology to establish the highly reliable and useful drug-screening platforms. PMID:28952489

3D Printing of Organs-On-Chips.

PubMed

Yi, Hee-Gyeong; Lee, Hyungseok; Cho, Dong-Woo

2017-01-25

Organ-on-a-chip engineering aims to create artificial living organs that mimic the complex and physiological responses of real organs, in order to test drugs by precisely manipulating the cells and their microenvironments. To achieve this, the artificial organs should to be microfabricated with an extracellular matrix (ECM) and various types of cells, and should recapitulate morphogenesis, cell differentiation, and functions according to the native organ. A promising strategy is 3D printing, which precisely controls the spatial distribution and layer-by-layer assembly of cells, ECMs, and other biomaterials. Owing to this unique advantage, integration of 3D printing into organ-on-a-chip engineering can facilitate the creation of micro-organs with heterogeneity, a desired 3D cellular arrangement, tissue-specific functions, or even cyclic movement within a microfluidic device. Moreover, fully 3D-printed organs-on-chips more easily incorporate other mechanical and electrical components with the chips, and can be commercialized via automated massive production. Herein, we discuss the recent advances and the potential of 3D cell-printing technology in engineering organs-on-chips, and provides the future perspectives of this technology to establish the highly reliable and useful drug-screening platforms.

Computational analysis of integrated biosensing and shear flow in a microfluidic vascular model

NASA Astrophysics Data System (ADS)

Wong, Jeremy F.; Young, Edmond W. K.; Simmons, Craig A.

2017-11-01

Fluid flow and flow-induced shear stress are critical components of the vascular microenvironment commonly studied using microfluidic cell culture models. Microfluidic vascular models mimicking the physiological microenvironment also offer great potential for incorporating on-chip biomolecular detection. In spite of this potential, however, there are few examples of such functionality. Detection of biomolecules released by cells under flow-induced shear stress is a significant challenge due to severe sample dilution caused by the fluid flow used to generate the shear stress, frequently to the extent where the analyte is no longer detectable. In this work, we developed a computational model of a vascular microfluidic cell culture model that integrates physiological shear flow and on-chip monitoring of cell-secreted factors. Applicable to multilayer device configurations, the computational model was applied to a bilayer configuration, which has been used in numerous cell culture applications including vascular models. Guidelines were established that allow cells to be subjected to a wide range of physiological shear stress while ensuring optimal rapid transport of analyte to the biosensor surface and minimized biosensor response times. These guidelines therefore enable the development of microfluidic vascular models that integrate cell-secreted factor detection while addressing flow constraints imposed by physiological shear stress. Ultimately, this work will result in the addition of valuable functionality to microfluidic cell culture models that further fulfill their potential as labs-on-chips.

Dental Stem Cell Migration on Pulp Ceiling Cavities Filled with MTA, Dentin Chips, or Bio-Oss

PubMed Central

Lymperi, Stefania; Taraslia, Vasiliki; Tsatsoulis, Ioannis N.; Samara, Athina; Agrafioti, Anastasia; Anastasiadou, Ema; Kontakiotis, Evangelos

2015-01-01

MTA, Bio-Oss, and dentin chips have been successfully used in endodontics. The aim of this study was to assess the adhesion and migration of dental stem cells on human pulp ceiling cavities filled with these endodontic materials in an experimental model, which mimics the clinical conditions of regenerative endodontics. Cavities were formed, by a homemade mold, on untouched third molars, filled with endodontic materials, and observed with electron microscopy. Cells were seeded on cavities' surface and their morphology and number were analysed. The phenomenon of tropism was assessed in a migration assay. All three materials demonstrated appropriate microstructures for cell attachment. Cells grew on all reagents, but they showed a differential morphology. Moreover, variations were observed when comparing cells numbers on cavity's filling versus the surrounding dentine disc. The highest number of cells was recorded on dentin chips whereas the opposite was true for Bio-Oss. This was confirmed in the migration assay where a statistically significant lower number of cells migrated towards Bio-Oss as compared to MTA and dentin chips. This study highlights that MTA and dentin chips have a greater potential compared to Bio-Oss regarding the attraction of dental stem cells and are good candidates for bioengineered pulp regeneration. PMID:26146613

CHIP/Stub1 regulates the Warburg effect by promoting degradation of PKM2 in ovarian carcinoma.

PubMed

Shang, Y; He, J; Wang, Y; Feng, Q; Zhang, Y; Guo, J; Li, J; Li, S; Wang, Y; Yan, G; Ren, F; Shi, Y; Xu, J; Zeps, N; Zhai, Y; He, D; Chang, Z

2017-07-20

Tumor cells preferentially adopt aerobic glycolysis for their energy supply, a phenomenon known as the Warburg effect. It remains a matter of debate as to how the Warburg effect is regulated during tumor progression. Here, we show that CHIP (carboxyl terminus of Hsc70-interacting protein), a U-box E3 ligase, suppresses tumor progression in ovarian carcinomas by inhibiting aerobic glycolysis. While CHIP is downregulated in ovarian carcinoma, induced expression of CHIP results in significant inhibition of the tumor growth examined by in vitro and in vivo experiments. Reciprocally, depletion of CHIP leads to promotion of tumor growth. By a SiLAD proteomics analysis, we identified pyruvate kinase isoenzyme M2 (PKM2), a critical regulator of glycolysis in tumors, as a target that CHIP mediated for degradation. Accordingly, we show that CHIP regulates PKM2 protein stability and thereafter the energy metabolic processes. Depletion or knockout of CHIP increased the glycolytic products in both tumor and mouse embryonic fibroblast cells. Simultaneously, we observed that CHIP expression inversely correlated with PKM2 levels in human ovarian carcinomas. This study reveals a mechanism that the Warburg effect is regulated by CHIP through its function as an E3 ligase, which mediates the degradation of PKM2 during tumor progression. Our findings shed new light into understanding of ovarian carcinomas and may provide a new therapeutic strategy for ovarian cancer.

Hepatocyte spheroid arrays inside microwells connected with microchannels

PubMed Central

Fukuda, Junji; Nakazawa, Kohji

2011-01-01

Spheroid culture is a preferable cell culture approach for some cell types, including hepatocytes, as this type of culture often allows maintenance of organ-specific functions. In this study, we describe a spheroid microarray chip (SM chip) that allows stable immobilization of hepatocyte spheroids in microwells and that can be used to evaluate drug metabolism with high efficiency. The SM chip consists of 300-μm-diameter cylindrical wells with chemically modified bottom faces that form a 100-μm-diameter cell adhesion region surrounded by a nonadhesion region. Primary hepatocytes seeded onto this chip spontaneously formed spheroids of uniform diameter on the cell adhesion region in each microwell and these could be used for cytochrome P-450 fluorescence assays. A row of microwells could also be connected to a microchannel for simultaneous detection of different cytochrome P-450 enzyme activities on a single chip. The miniaturized features of this SM chip reduce the numbers of cells and the amounts of reagents required for assays. The detection of four cytochrome P-450 enzyme activities was demonstrated following induction by 3-methylcholantlene, with a sensitivity significantly higher than that in conventional monolayer culture. This microfabricated chip could therefore serve as a novel culture platform for various cell-based assays, including those used in drug screening, basic biological studies, and tissue engineering applications. PMID:21799712

E3 ligase CHIP and Hsc70 regulate Kv1.5 protein expression and function in mammalian cells.

PubMed

Li, Peili; Kurata, Yasutaka; Maharani, Nani; Mahati, Endang; Higaki, Katsumi; Hasegawa, Akira; Shirayoshi, Yasuaki; Yoshida, Akio; Kondo, Tatehito; Kurozawa, Youichi; Yamamoto, Kazuhiro; Ninomiya, Haruaki; Hisatome, Ichiro

2015-09-01

Kv1.5 confers ultra-rapid delayed-rectifier potassium channel current (IKur) which contributes to repolarization of the atrial action potential. Kv1.5 proteins, degraded via the ubiquitin-proteasome pathway, decreased in some atrial fibrillation patients. Carboxyl-terminus heat shock cognate 70-interacting protein (CHIP), an E3 ubiquitin ligase, is known to ubiquitinate short-lived proteins. Here, we investigated the roles of CHIP in Kv1.5 degradation to provide insights into the mechanisms of Kv1.5 decreases and treatments targeting Kv1.5 for atrial fibrillation. Coexpression of CHIP with Kv1.5 in HEK293 cells increased Kv1.5 protein ubiquitination and decreased the protein level. Immunofluorescence revealed decreases of Kv1.5 proteins in the endoplasmic reticulum and on the cell membrane. A siRNA against CHIP suppressed Kv1.5 protein ubiquitination and increased its protein level. CHIP mutants, lacking either the N-terminal tetratricopeptide region domain or the C-terminal U-box domain, failed to exert these effects on Kv1.5 proteins. Immunoprecipitation showed that CHIP formed complexes with Kv1.5 proteins and heat shock cognate protein 70 (Hsc70). Effects of Hsc70 on Kv1.5 were similar to CHIP by altering interaction of CHIP with Kv1.5 protein. Coexpression of CHIP and Hsc70 with Kv1.5 additionally enhanced Kv1.5 ubiquitination. Kv1.5 currents were decreased by overexpression of CHIP or Hsc70 but were increased by knockdown of CHIP or Hsc70 in HEK 293 cells stably expressing Kv1.5. These effects of CHIP and Hsc70 were also observed on endogenous Kv1.5 in HL-1 mouse cardiomyocytes, decreasing IKur and prolonging action potential duration. These results indicate that CHIP decreases the Kv1.5 protein level and functional channel by facilitating its degradation in concert with chaperone Hsc70. Copyright © 2015 Elsevier Ltd. All rights reserved.

A droplet-based microfluidic chip as a platform for leukemia cell lysate identification using surface-enhanced Raman scattering.

PubMed

Hassoun, Mohamed; Rüger, Jan; Kirchberger-Tolstik, Tatiana; Schie, Iwan W; Henkel, Thomas; Weber, Karina; Cialla-May, Dana; Krafft, Christoph; Popp, Jürgen

2018-01-01

A new approach is presented for cell lysate identification which uses SERS-active silver nanoparticles and a droplet-based microfluidic chip. Eighty-nanoliter droplets are generated by injecting silver nanoparticles, KCl as aggregation agent, and cell lysate containing cell constituents, such as nucleic acids, carbohydrates, metabolites, and proteins into a continuous flow of mineral oil. This platform enables accurate mixing of small volumes inside the meandering channels of the quartz chip and allows acquisition of thousands of SERS spectra with 785 nm excitation at an integration time of 1 s. Preparation of three batches of three leukemia cell lines demonstrated the experimental reproducibility. The main advantage of a high number of reproducible spectra is to apply statistics for large sample populations with robust classification results. A support vector machine with leave-one-batch-out cross-validation classified SERS spectra with sensitivities, specificities, and accuracies better than 99% to differentiate Jurkat, THP-1, and MONO-MAC-6 leukemia cell lysates. This approach is compared with previous published reports about Raman spectroscopy for leukemia detection, and an outlook is given for transfer to single cells. A quartz chip was designed for SERS at 785 nm excitation. Principal component analysis of SERS spectra clearly separates cell lysates using variations in band intensity ratios.

Axial-Centrifugal Compressor Program

DTIC Science & Technology

1975-10-01

chip detector, but they were not large enough to trigger the alarm circuit. These chips we-e analyzed as M50 bearing material, which was a positive...but an analysis of these particles indicated M50 bearing material and positively identified a thrust bearing problem. 50 ’ ! i VI Figure 18. Load Cel...load cell readout became erratic and the vehicle was shut down. An inspection showed that the aft bearing sump chip detector contained M50 bearing

Inhibition of the Hedgehog Signaling Pathway Depresses the Cigarette Smoke-Induced Malignant Transformation of 16HBE Cells on a Microfluidic Chip.

PubMed

Qin, Yong-Xin; Yang, Zhi-Hui; Du, Xiao-Hui; Zhao, Hui; Liu, Yuan-Bin; Guo, Zhe; Wang, Qi

2018-05-20

The hedgehog signaling system (HHS) plays an important role in the regulation of cell proliferation and differentiation during the embryonic phases. However, little is known about the involvement of HHS in the malignant transformation of cells. This study aimed to detect the role of HHS in the malignant transformation of human bronchial epithelial (16HBE) cells. In this study, two microfluidic chips were designed to investigate cigarette smoke extract (CSE)-induced malignant transformation of cells. Chip A contained a concentration gradient generator, while chip B had four cell chambers with a central channel. The 16HBE cells cultured in chip A were used to determine the optimal concentration of CSE for inducing malignant transformation. The 16HBE cells in chip B were cultured with 12.25% CSE (Group A), 12.25% CSE + 5 μmol/L cyclopamine (Group B), or normal complete medium as control for 8 months (Group C), to establish the in vitro lung inflammatory-cancer transformation model. The transformed cells were inoculated into 20 nude mice as cells alone (Group 1) or cells with cyclopamine (Group 2) for tumorigenesis testing. Expression of HHS proteins was detected by Western blot. Data were expressed as mean ± standard deviation. The t-test was used for paired samples, and the difference among groups was analyzed using a one-way analysis of variance. The optimal concentration of CSE was 12.25%. Expression of HHS proteins increased during the process of malignant transformation (Group B vs. Group A, F = 7.65, P < 0.05). After CSE exposure for 8 months, there were significant changes in cellular morphology, which allowed the transformed cells to grow into tumors in 40 days after being inoculated into nude mice. Cyclopamine could effectively depress the expression of HHS proteins (Group C vs. Group B, F = 6.47, P < 0.05) and prevent tumor growth in nude mice (Group 2 vs. Group 1, t = 31.59, P < 0.01). The activity of HHS is upregulated during the CSE-induced malignant transformation of 16HBE cells. Cyclopamine can effectively depress expression of HHS proteins in vitro and prevent tumor growth of the transformed cells in vivo.

Quantitative phase imaging characterization of tumor-associated blood vessel formation on a chip

NASA Astrophysics Data System (ADS)

Guo, Peng; Huang, Jing; Moses, Marsha A.

2018-02-01

Angiogenesis, the formation of new blood vessels from existing ones, is a biological process that has an essential role in solid tumor growth, development, and progression. Recent advances in Lab-on-a-Chip technology has created an opportunity for scientists to observe endothelial cell (EC) behaviors during the dynamic process of angiogenesis using a simple and economical in vitro platform that recapitulates in vivo blood vessel formation. Here, we use quantitative phase imaging (QPI) microscopy to continuously and non-invasively characterize the dynamic process of tumor cell-induced angiogenic sprout formation on a microfluidic chip. The live tumor cell-induced angiogenic sprouts are generated by multicellular endothelial sprouting into 3 dimensional (3D) Matrigel using human umbilical vein endothelial cells (HUVECs). By using QPI, we quantitatively measure a panel of cellular morphological and behavioral parameters of each individual EC participating in this sprouting. In this proof-of-principle study, we demonstrate that QPI is a powerful tool that can provide real-time quantitative analysis of biological processes in in vitro 3D biomimetic devices, which, in turn, can improve our understanding of the biology underlying functional tissue engineering.

Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics.

PubMed

Tung, Yi-Chung; Torisawa, Yu-suke; Futai, Nobuyuki; Takayama, Shuichi

2007-11-01

This paper describes a micro flow cytometer system designed for efficient and non-damaging analysis of samples with small numbers of precious cells. The system utilizes actuation of Braille-display pins for micro-scale fluid manipulation and a fluorescence microscope with a CCD camera for optical detection. The microfluidic chip is fully disposable and is composed of a polydimethylsiloxane (PDMS) slab with microchannel features sealed against a thin deformable PDMS membrane. The channels are designed with diffusers to alleviate pulsatile flow behaviors inherent in pin actuator-based peristaltic pumping schemes to maximize hydrodynamic focusing of samples with minimal disturbances in the laminar streams within the channel. A funnel connected to the microfluidic channel is designed for efficient loading of samples with small number of cells and is also positioned on the chip to prevent physical damages of the samples by the squeezing actions of Braille pins during actuation. The sample loading scheme was characterized by both computational fluidic dynamics (CFD) simulation and experimental observation. A fluorescein solution was first used for flow field investigation, followed by use of fluorescence beads with known relative intensities for optical detection performance calibration. Murine myoblast cells (C2C12) were exploited to investigate cell viability for the sample loading scheme of the device. Furthermore, human promyelocytic leukemia (HL60) cells stained by hypotonic DNA staining buffer were also tested in the system for cell cycle analysis. The ability to efficiently analyze cellular samples where the number of cells is small was demonstrated by analyzing cells from a single embryoid body derived from mouse embryonic stem cells. Consequently, the designed microfluidic device reported in this paper is promising for easy-to-use, small sample size flow cytometric analysis, and has potential to be further integrated with other Braille display-based microfluidic devices to facilitate a multi-functional lab-on-a-chip for mammalian cell manipulations.

Gene chips and arrays revealed: a primer on their power and their uses.

PubMed

Watson, S J; Akil, H

1999-03-01

This article provides an overview and general explanation of the rapidly developing area of gene chips and expression array technology. These are methods targeted at allowing the simultaneous study of thousands of genes or messenger RNAs under various physiological and pathological states. Their technical basis grows from the Human Genome Project. Both methods place DNA strands on glass computer chips (or microscope slides). Expression arrays start with complementary DNA (cDNA) clones derived from the EST data base, whereas Gene Chips synthesize oligonucleotides directly on the chip itself. Both are analyzed using image analysis systems, are capable of reading values from two different individuals at any one site, and can yield quantitative data for thousands of genes or mRNAs per slide. These methods promise to revolutionize molecular biology, cell biology, neuroscience and psychiatry. It is likely that this technology will radically open up our ability to study the actions and structure of the multiple genes involved in the complex genetics of brain disorders.

Cell identification using Raman spectroscopy in combination with optical trapping and microfluidics

NASA Astrophysics Data System (ADS)

Krafft, Christoph; Dochow, Sebastian; Beleites, Claudia; Popp, Jürgen

2014-03-01

Cell identification by Raman spectroscopy has evolved to be an attractive complement to established optical techniques. Raman activated cell sorting (RACS) offers prospects to complement the widely applied fluorescence activated cell sorting. RACS can be realized by combination with optical traps and microfluidic devices. The progress of RACS is reported for a cellular model system that can be found in peripheral blood of tumor patients. Lymphocytes and erythrocytes were extracted from blood samples. Breast carcinoma derived tumor cells (MCF-7, BT-20) and acute myeloid leukemia cells (OCI-AML3) were grown in cell cultures. First, Raman images were collected from dried cells on calcium fluoride slides. Support vector machines (SVM) classified 99.7% of the spectra to the correct cell type. Second, a 785 nm laser was used for optical trapping of single cells in aqueous buffer and for excitation of the Raman spectrum. SVM distinguished 1210 spectra of tumor and normal cells with a sensitivity of >99.7% and a specificity of >99.5%. Third, a microfluidic glass chip was designed to inject single cells, modify the flow speed, accommodate fibers of an optical trap and sort single cells after Raman based identification with 514 nm for excitation. Forth, the microfluidic chip was fabricated by quartz which improved cell identification results with 785 nm excitation. Here, partial least squares discriminant analysis gave classification rates of 98%. Finally, a Raman-on-chip approach was developed that integrates fibers for trapping, Raman excitation and signal detection in a single compact unit.

Engineered peptide-based nanobiomaterials for electrochemical cell chip.

PubMed

Kafi, Md Abdul; Cho, Hyeon-Yeol; Choi, Jeong-Woo

2016-01-01

Biomaterials having cell adhesion ability are considered to be integral part of a cell chip. A number of researches have been carried out to search for a suitable material for effective immobilization of cell on substrate. Engineered ECM materials or their components like collagen, Poly-l-Lysine (PLL), Arg-Gly-Asp (RGD) peptide have been extensively used for mammalian cell adhesion and proliferation with the aim of tissue regeneration or cell based sensing application. This review focuses on the various approaches for two- and three-dimensionally patterned nanostructures of a short peptide i.e. RGD peptide on chip surfaces together with their effects on cell behaviors and electrochemical measurements. Most of the study concluded with positive remarks on the well-oriented engineered RGD peptide over their homogenous thin film. The engineered RGD peptide not only influences cell adhesion, spreading and proliferation but also their periodic nano-arrays directly influence electrochemical measurements of the chips. The electrochemical signals found to be enhanced when RGD peptides were used in well-defined two-dimensional nano-arrays. The topographic alteration of three-dimensional structure of engineered RGD peptide was reported to be suitably contacted with the integrin receptors of cellular membrane which results indicated the enhanced cell-electrode adhesion and efficient electron exchange phenomenon. This enhanced electrochemical signal increases the sensitivity of the chip against the target analytes. Therefore, development of engineered cellular recognizable peptides and its 3D topological design for fabrication of cell chip will provide the synergetic effect on bio-affinity, sensitivity and accuracy for the in situ real-time monitoring of analytes.

Fast and sensitive detection of foodborne pathogen using electrochemical impedance analysis, urease catalysis and microfluidics.

PubMed

Chen, Qi; Wang, Dan; Cai, Gaozhe; Xiong, Yonghua; Li, Yuntao; Wang, Maohua; Huo, Huiling; Lin, Jianhan

2016-12-15

Early screening of pathogenic bacteria is a key to prevent and control of foodborne diseases. In this study, we developed a fast and sensitive bacteria detection method integrating electrochemical impedance analysis, urease catalysis with microfluidics and using Listeria as model. The Listeria cells, the anti-Listeria monoclonal antibodies modified magnetic nanoparticles (MNPs), and the anti-Listeria polyclonal antibodies and urease modified gold nanoparticles (AuNPs) were incubated in a fluidic separation chip with active mixing to form the MNP-Listeria-AuNP-urease sandwich complexes. The complexes were captured in the separation chip by applying a high gradient magnetic field, and the urea was injected to resuspend the complexes and hydrolyzed under the catalysis of the urease on the complexes into ammonium ions and carbonate ions, which were transported into a microfluidic detection chip with an interdigitated microelectrode for impedance measurement to determine the amount of the Listeria cells. The capture efficiency of the Listeria cells in the separation chip was ∼93% with a shorter time of 30min due to the faster immuno-reaction using the active magnetic mixing. The changes on both impedance magnitude and phase angle were demonstrated to be able to detect the Listeria cells as low as 1.6×10(2)CFU/mL. The detection time was reduced from original ∼2h to current ∼1h. The recoveries of the spiked lettuce samples ranged from 82.1% to 89.6%, indicating the applicability of this proposed biosensor. This microfluidic impedance biosensor has shown the potential for online, automatic and sensitive bacteria separation and detection. Copyright © 2016 Elsevier B.V. All rights reserved.

Fitting tissue chips and microphysiological systems into the grand scheme of medicine, biology, pharmacology, and toxicology.

PubMed

Watson, David E; Hunziker, Rosemarie; Wikswo, John P

2017-10-01

Microphysiological systems (MPS), which include engineered organoids (EOs), single organ/tissue chips (TCs), and multiple organs interconnected to create miniature in vitro models of human physiological systems, are rapidly becoming effective tools for drug development and the mechanistic understanding of tissue physiology and pathophysiology. The second MPS thematic issue of Experimental Biology and Medicine comprises 15 articles by scientists and engineers from the National Institutes of Health, the IQ Consortium, the Food and Drug Administration, and Environmental Protection Agency, an MPS company, and academia. Topics include the progress, challenges, and future of organs-on-chips, dissemination of TCs into Pharma, children's health protection, liver zonation, liver chips and their coupling to interconnected systems, gastrointestinal MPS, maturation of immature cardiomyocytes in a heart-on-a-chip, coculture of multiple cell types in a human skin construct, use of synthetic hydrogels to create EOs that form neural tissue models, the blood-brain barrier-on-a-chip, MPS models of coupled female reproductive organs, coupling MPS devices to create a body-on-a-chip, and the use of a microformulator to recapitulate endocrine circadian rhythms. While MPS hardware has been relatively stable since the last MPS thematic issue, there have been significant advances in cell sourcing, with increased reliance on human-induced pluripotent stem cells, and in characterization of the genetic and functional cell state in MPS bioreactors. There is growing appreciation of the need to minimize perfusate-to-cell-volume ratios and respect physiological scaling of coupled TCs. Questions asked by drug developers are followed by an analysis of the potential value, costs, and needs of Pharma. Of highest value and lowest switching costs may be the development of MPS disease models to aid in the discovery of disease mechanisms; novel compounds including probes, leads, and clinical candidates; and mechanism of action of drug candidates. Impact statement Microphysiological systems (MPS), which include engineered organoids and both individual and coupled organs-on-chips and tissue chips, are a rapidly growing topic of research that addresses the known limitations of conventional cellular monoculture on flat plastic - a well-perfected set of techniques that produces reliable, statistically significant results that may not adequately represent human biology and disease. As reviewed in this article and the others in this thematic issue, MPS research has made notable progress in the past three years in both cell sourcing and characterization. As the field matures, currently identified challenges are being addressed, and new ones are being recognized. Building upon investments by the Defense Advanced Research Projects Agency, National Institutes of Health, Food and Drug Administration, Defense Threat Reduction Agency, and Environmental Protection Agency of more than $200 million since 2012 and sizable corporate spending, academic and commercial players in the MPS community are demonstrating their ability to meet the translational challenges required to apply MPS technologies to accelerate drug development and advance toxicology.

Measurement of RBC agglutination with microscopic cell image analysis in a microchannel chip.

PubMed

Cho, Chi Hyun; Kim, Ju Yeon; Nyeck, Agnes E; Lim, Chae Seung; Hur, Dae Sung; Chung, Chanil; Chang, Jun Keun; An, Seong Soo A; Shin, Sehyun

2014-01-01

Since Landsteiner's discovery of ABO blood groups, RBC agglutination has been one of the most important immunohematologic techniques for ABO and RhD blood groupings. The conventional RBC agglutination grading system for RhD blood typings relies on macroscopic reading, followed by the assignment of a grade ranging from (-) to (4+) to the degree of red blood cells clumping. However, with the new scoring method introduced in this report, microscopically captured cell images of agglutinated RBCs, placed in a microchannel chip, are used for analysis. Indeed, the cell images' pixel number first allows the differentiation of agglutinated and non-agglutinated red blood cells. Finally, the ratio of agglutinated RBCs per total RBC counts (CRAT) from 90 captured images is then calculated. During the trial, it was observed that the agglutinated group's CRAT was significantly higher (3.77-0.003) than that of the normal control (0). Based on these facts, it was established that the microchannel method was more suitable for the discrimination between agglutinated RBCs and non-agglutinated RhD negative, and thus more reliable for the grading of RBCs agglutination than the conventional method.

Analysis of DNA-chip and antigen-chip data: studies of cancer, stem cells and autoimmune diseases

NASA Astrophysics Data System (ADS)

Domany, Eytan

2005-07-01

Biology has undergone a revolution during the past decade. Deciphering the human genome has opened new horizons, among which the advent of DNA microarrays has been perhaps the most significant. These miniature measuring devices report the levels at which tens of thousands of genes are expressed in a collection of cells of interest (such as tissue from a tumor). I describe here briefly this technology and present an example of how analysis of data obtained from such high throughput experiments provides insights of possible clinical and therapeutic relevance for Acute Lymphoblastic Leukemia. Next, I describe how gene expression data is used to deduce a new design principle, " Just In Case", used by stem cells. Finally I briefly review a different novel technology, of antigen chips, which provide a fingerprint of a subject's immune system and may become a predictive clinical tool. The work reviewed here was done in collaboration with numerous colleagues and students.

Expression of Pluripotency Genes in Chondrocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells

PubMed Central

Stelcer, Ewelina; Kulcenty, Katarzyna; Rucinski, Marcin; Jopek, Karol; Trzeciak, Tomasz; Richter, Magdalena; Wroblewska, Joanna P.; Suchorska, Wiktoria M.

2018-01-01

Human induced pluripotent stem cells (hiPSCs) constitute an important breakthrough in regenerative medicine, particularly in orthopedics, where more effective treatments are urgently needed. Despite the promise of hiPSCs only limited data on in vitro chondrogenic differentiation of hiPSCs are available. Therefore, we compared the gene expression profile of pluripotent genes in hiPSC-derived chondrocytes (ChiPS) to that of an hiPSC cell line created by our group (GPCCi001-A). The results are shown on heatmaps and plots and confirmed by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis. Unlike the ChiPS, our GPCCi001-A cells maintained their pluripotency state during long-term culture, thus demonstrating that this cell line was comprised of stable, fully pluripotent hiPSCs. Moreover, these chondrocyte-like cells not only presented features that are characteristic of chondrocytes, but they also lost their pluripotency, which is an important advantage in favor of using this cell line in future clinical studies. PMID:29439516

Chip-set for quality of service support in passive optical networks

NASA Astrophysics Data System (ADS)

Ringoot, Edwin; Hoebeke, Rudy; Slabbinck, B. Hans; Verhaert, Michel

1998-10-01

In this paper the design of a chip-set for QoS provisioning in ATM-based Passive Optical Networks is discussed. The implementation of a general-purpose switch chip on the Optical Network Unit is presented, with focus on the design of the cell scheduling and buffer management logic. The cell scheduling logic supports `colored' grants, priority jumping and weighted round-robin scheduling. The switch chip offers powerful buffer management capabilities enabling the efficient support of GFR and UBR services. Multicast forwarding is also supported. In addition, the architecture of a MAC controller chip developed for a SuperPON access network is introduced. In particular, the permit scheduling logic and its implementation on the Optical Line Termination will be discussed. The chip-set enables the efficient support of services with different service requirements on the SuperPON. The permit scheduling logic built into the MAC controller chip in combination with the cell scheduling and buffer management capabilities of the switch chip can be used by network operators to offer guaranteed service performance to delay sensitive services, and to efficiently and fairly distribute any spare capacity to delay insensitive services.

Micro-chromatin Immunoprecipation (μChIP) Protocol for Real-time PCR Analysis of a Limited Amount of Cells.

PubMed

Gillotin, Sébastien; Guillemot, François

2016-06-20

Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is an important strategy to study gene regulation. When availability of cells is limited, however, it can be useful to focus on specific genes to investigate in depth the role of transcription factors or histone marks. Unfortunately, performing ChIP experiments to study transcription factors' binding to DNA can be difficult when biological material is restricted. This protocol describes a robust method to perform μChIP for over-expressed or endogenous transcription factors using ~100,000 cells per ChIP experiment (Masserdotti et al ., 2015). We also describe optimization steps, which we think are critical for this protocol to work and which can be used to further reduce the number of cells.

Ubiquitin ligase CHIP functions as an oncogene and activates the AKT signaling pathway in prostate cancer.

PubMed

Cheng, Li; Zang, Jin; Dai, Han-Jue; Li, Feng; Guo, Feng

2018-07-01

Carboxyl terminus of Hsc-70-interacting protein (CHIP) is an E3 ubiquitin ligase that induces the ubiquitination and degradation of numerous tumor-associated proteins and serves as a suppressor or promoter in tumor progression. To date, the molecular mechanism of CHIP in prostate cancer remains unknown. Therefore, the present study investigated the biological function of CHIP in prostate cancer cells and obtained evidence that CHIP expression is upregulated in prostate cancer tissues. The CHIP vector was introduced into DU145 cancer cells and the cell biological behaviour was examined through a series of experiments, including cell growth, cell apoptosis and migration and invasion assays. The results indicated that the overexpression of CHIP in DU145 prostatic cancer cells promoted cell proliferation through activation of the protein kinase B (AKT) signaling pathway, which subsequently increased cyclin D1 protein levels and decreased p21 and p27 protein levels. The overexpression of CHIP significantly increased the migration and invasion of the DU145 cells, which is possible due to activation of the AKT signaling pathway and upregulation of vimentin. The expression level of CHIP was observed to be increased in human prostate cancer tissues compared with the adjacent normal tissue. Furthermore, the CHIP expression level exhibited a positively association with the Gleason score of the patents. These findings indicate that CHIP functions as an oncogene in prostate cancer.

Experimental and theoretical analysis of neuron-transistor hybrid electrical coupling: the relationships between the electro-anatomy of cultured Aplysia neurons and the recorded field potentials.

PubMed

Cohen, Ariel; Shappir, Joseph; Yitzchaik, Shlomo; Spira, Micha E

2006-12-15

Understanding the mechanisms that generate field potentials (FPs) by neurons grown on semiconductor chips is essential for implementing neuro-electronic devices. Earlier studies emphasized that FPs are generated by current flow between differentially expressed ion channels on the membranes facing the chip surface, and those facing the culture medium in electrically compact cells. Less is known, however, about the mechanisms that generate FPs by action potentials (APs) that propagate along typical non-isopotential neurons. Using Aplysia neurons cultured on floating gate-transistors, we found that the FPs generated by APs in cultured neurons are produced by current flow along neuronal compartments comprising the axon, cell body, and neurites, rather than by flow between the membrane facing the chip substrate and that facing the culture medium. We demonstrate that the FPs waveform generated by non-isopotential neurons largely depends on the morphology of the neuron.

Tumour-on-a-chip provides an optical window into nanoparticle tissue transport

NASA Astrophysics Data System (ADS)

Albanese, Alexandre; Lam, Alan K.; Sykes, Edward A.; Rocheleau, Jonathan V.; Chan, Warren C. W.

2013-10-01

Nanomaterials are used for numerous biomedical applications, but the selection of optimal properties for maximum delivery remains challenging. Thus, there is a significant interest in elucidating the nano-bio interactions underlying tissue accumulation. To date, researchers have relied on cell culture or animal models to study nano-bio interactions. However, cell cultures lack the complexity of biological tissues and animal models are prohibitively slow and expensive. Here we report a tumour-on-a-chip system where incorporation of tumour-like spheroids into a microfluidic channel permits real-time analysis of nanoparticle (NP) accumulation at physiological flow conditions. We show that penetration of NPs into the tissue is limited by their diameter and that retention can be improved by receptor targeting. NP transport is predominantly diffusion-limited with convection improving accumulation mostly at the tissue perimeter. A murine tumour model confirms these findings and demonstrates that the tumour-on-a-chip can be useful for screening optimal NP designs prior to in vivo studies.

A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays

NASA Astrophysics Data System (ADS)

Vergauwe, Nicolas; Witters, Daan; Ceyssens, Frederik; Vermeir, Steven; Verbruggen, Bert; Puers, Robert; Lammertyn, Jeroen

2011-05-01

Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assay's performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To demonstrate this potential, on-chip adhesion islands are fabricated to immobilize MCF-7 human breast cancer cells. Viability studies are performed to assess the functionalization efficiency.

Hydrodynamic lift for single cell manipulation in a femtosecond laser fabricated optofluidic chip

NASA Astrophysics Data System (ADS)

Bragheri, Francesca; Osellame, Roberto

2017-08-01

Single cell sorting based either on fluorescence or on mechanical properties has been exploited in the last years in microfluidic devices. Hydrodynamic focusing allows increasing the efficiency of theses devices by improving the matching between the region of optical analysis and that of cell flow. Here we present a very simple solution fabricated by femtosecond laser micromachining that exploits flow laminarity in microfluidic channels to easily lift the sample flowing position to the channel portion illuminated by the optical waveguides used for single cell trapping and analysis.

Positioning of the sensor cell on the sensing area using cell trapping pattern in incubation type planar patch clamp biosensor.

PubMed

Wang, Zhi-Hong; Takada, Noriko; Uno, Hidetaka; Ishizuka, Toru; Yawo, Hiromu; Urisu, Tsuneo

2012-08-01

Positioning the sensor cell on the micropore of the sensor chip and keeping it there during incubation are problematic tasks for incubation type planar patch clamp biosensors. To solve these problems, we formed on the Si sensor chip's surface a cell trapping pattern consisting of a lattice pattern with a round area 5 μm deep and with the micropore at the center of the round area. The surface of the sensor chip was coated with extra cellular matrix collagen IV, and HEK293 cells on which a chimera molecule of channel-rhodopsin-wide-receiver (ChR-WR) was expressed, were then seeded. We examined the effects of this cell trapping pattern on the biosensor's operation. In the case of a flat sensor chip without a cell trapping pattern, it took several days before the sensor cell covered the micropore and formed an almost confluent state. As a result, multi-cell layers easily formed and made channel current measurements impossible. On the other hand, the sensor chip with cell trapping pattern easily trapped cells in the round area, and formed the colony consisted of the cell monolayer covering the micropore. A laser (473 nm wavelength) induced channel current was observed from the whole cell arrangement formed using the nystatin perforation technique. The observed channel current characteristics matched measurements made by using a pipette patch clamp. Copyright © 2012 Elsevier B.V. All rights reserved.

Cancer stem-like cell related protein CD166 degrades through E3 ubiquitin ligase CHIP in head and neck cancer.

PubMed

Xiao, Meng; Yan, Ming; Zhang, Jianjun; Xu, Qin; Qi, Shengcai; Wang, Xu; Chen, Wantao

2017-04-01

Our previous studies have identified that CD166 works as a cancer stem-like cell (CSC) marker in epithelial cancers with a large repertoire of cellular functions. However, the post-translational regulatory mechanisms underlying CD166 turnover remain elusive. Several independent studies have reported that E3 ubiquitin ligase CHIP revealed significant biological effects through ubiquitin proteasome pathway on some kinds of malignant tumors. With analyzing the effects of CHIP expressions on stem-like cell populations, we found that CHIP represses CSC characteristics mainly targeting the CSC related protein CD166 in head and neck cancer (HNC). To investigate the role and relationship between CD166 and CHIP, HNC tissues and cell lines were used in this study. A significant negative correlation was observed between the expression levels of CHIP and CD166 in HNC patient samples. We also found that CHIP directly regulates the stability of CD166 protein through the ubiquitin proteasome system, which was also identified participating in the regulation of CSC behaviors in HNCs. Our findings demonstrate that CHIP-CD166-proteasome axis participates in regulating CSC properties in HNCs, suggesting that the regulation of CD166 by CHIP could provide new options for diagnosing and treating in the patients with HNCs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Perspective: Fabrication of integrated organ-on-a-chip via bioprinting.

PubMed

Yang, Qingzhen; Lian, Qin; Xu, Feng

2017-05-01

Organ-on-a-chip has emerged as a powerful platform with widespread applications in biomedical engineering, such as pathology studies and drug screening. However, the fabrication of organ-on-a-chip is still a challenging task due to its complexity. For an integrated organ-on-a-chip, it may contain four key elements, i.e., a microfluidic chip, live cells/microtissues that are cultured in this chip, components for stimulus loading to mature the microtissues, and sensors for results readout. Recently, bioprinting has been used for fabricating organ-on-a-chip as it enables the printing of multiple materials, including biocompatible materials and even live cells in a programmable manner with a high spatial resolution. Besides, all four elements for organ-on-a-chip could be printed in a single continuous procedure on one printer; in other words, the fabrication process is assembly free. In this paper, we discuss the recent advances of organ-on-a-chip fabrication by bioprinting. Light is shed on the printing strategies, materials, and biocompatibility. In addition, some specific bioprinted organs-on-chips are analyzed in detail. Because the bioprinted organ-on-a-chip is still in its early stage, significant efforts are still needed. Thus, the challenges presented together with possible solutions and future trends are also discussed.

An easy to assemble microfluidic perfusion device with a magnetic clamp

PubMed Central

Tkachenko, Eugene; Gutierrez, Edgar; Ginsberg, Mark H.; Groisman, Alex

2009-01-01

We have built and characterized a magnetic clamp for reversible sealing of PDMS microfluidic chips against cover glasses with cell cultures and a microfluidic chip for experiments on shear stress response of endothelial cells. The magnetic clamp exerts a reproducible uniform pressure on the microfluidic chip, achieving fast and reliable sealing for liquid pressures up to 40 kPa inside the chip with <10% deformations of microchannels and minimal variations of the substrate shear stress in perfusion flow. The microfluidic chip has 8 test regions with the substrate shear stress varying by a factor of 2 between each region, thus covering a 128-fold range from low venous to arterial. The perfusion is driven by differential pressure, which makes it possible to create pulsatile flows mimicking pulsing in the vasculature. The setup is tested by 15 – 40 hours perfusions over endothelial monolayers with shear stress in the range of 0.07 - 9 dyn/cm2. Excellent cell viability at all shear stresses and alignment of cells along the flow at high shear stresses are repeatedly observed. A scratch wound healing assay under a shear flow is demonstrated and cell migration velocities are measured. Transfection of cells with a fluorescent protein is performed, and migrating fluorescent cells are imaged at a high resolution under shear flow in real time. The magnetic clamp can be closed with minimal mechanical perturbation to cells on the substrate and used with a variety of microfluidic chips for experiments with adherent and non-adherent cells. PMID:19350090

A Versatile Microarray Platform for Capturing Rare Cells

NASA Astrophysics Data System (ADS)

Brinkmann, Falko; Hirtz, Michael; Haller, Anna; Gorges, Tobias M.; Vellekoop, Michael J.; Riethdorf, Sabine; Müller, Volkmar; Pantel, Klaus; Fuchs, Harald

2015-10-01

Analyses of rare events occurring at extremely low frequencies in body fluids are still challenging. We established a versatile microarray-based platform able to capture single target cells from large background populations. As use case we chose the challenging application of detecting circulating tumor cells (CTCs) - about one cell in a billion normal blood cells. After incubation with an antibody cocktail, targeted cells are extracted on a microarray in a microfluidic chip. The accessibility of our platform allows for subsequent recovery of targets for further analysis. The microarray facilitates exclusion of false positive capture events by co-localization allowing for detection without fluorescent labelling. Analyzing blood samples from cancer patients with our platform reached and partly outreached gold standard performance, demonstrating feasibility for clinical application. Clinical researchers free choice of antibody cocktail without need for altered chip manufacturing or incubation protocol, allows virtual arbitrary targeting of capture species and therefore wide spread applications in biomedical sciences.

Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer.

PubMed

Bhuripanyo, Karan; Wang, Yiyang; Liu, Xianpeng; Zhou, Li; Liu, Ruochuan; Duong, Duc; Zhao, Bo; Bi, Yingtao; Zhou, Han; Chen, Geng; Seyfried, Nicholas T; Chazin, Walter J; Kiyokawa, Hiroaki; Yin, Jun

2018-01-01

E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct "orthogonal UB transfer (OUT)" cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N -methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and β-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.

Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer

PubMed Central

Bhuripanyo, Karan; Wang, Yiyang; Liu, Xianpeng; Zhou, Li; Liu, Ruochuan; Duong, Duc; Zhao, Bo; Bi, Yingtao; Zhou, Han; Chen, Geng; Seyfried, Nicholas T.; Chazin, Walter J.; Kiyokawa, Hiroaki; Yin, Jun

2018-01-01

E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct “orthogonal UB transfer (OUT)” cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N-methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and β-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress. PMID:29326975

Whole-Cell Electrical Activity Under Direct Mechanical Stimulus by AFM Cantilever Using Planar Patch Clamp Chip Approach

PubMed Central

Upadhye, Kalpesh V.; Candiello, Joseph E.; Davidson, Lance A.; Lin, Hai

2011-01-01

Patch clamp is a powerful tool for studying the properties of ion-channels and cellular membrane. In recent years, planar patch clamp chips have been fabricated from various materials including glass, quartz, silicon, silicon nitride, polydimethyl-siloxane (PDMS), and silicon dioxide. Planar patch clamps have made automation of patch clamp recordings possible. However, most planar patch clamp chips have limitations when used in combination with other techniques. Furthermore, the fabrication methods used are often expensive and require specialized equipments. An improved design as well as fabrication and characterization of a silicon-based planar patch clamp chip are described in this report. Fabrication involves true batch fabrication processes that can be performed in most common microfabrication facilities using well established MEMS techniques. Our planar patch clamp chips can form giga-ohm seals with the cell plasma membrane with success rate comparable to existing patch clamp techniques. The chip permits whole-cell voltage clamp recordings on variety of cell types including Chinese Hamster Ovary (CHO) cells and pheochromocytoma (PC12) cells, for times longer than most available patch clamp chips. When combined with a custom microfluidics chamber, we demonstrate that it is possible to perfuse the extra-cellular as well as intra-cellular buffers. The chamber design allows integration of planar patch clamp with atomic force microscope (AFM). Using our planar patch clamp chip and microfluidics chamber, we have recorded whole-cell mechanosensitive (MS) currents produced by directly stimulating human keratinocyte (HaCaT) cells using an AFM cantilever. Our results reveal the spatial distribution of MS ion channels and temporal details of the responses from MS channels. The results show that planar patch clamp chips have great potential for multi-parametric high throughput studies of ion channel proteins. PMID:22174731

Prediction of metabolism-induced hepatotoxicity on three-dimensional hepatic cell culture and enzyme microarrays.

PubMed

Yu, Kyeong-Nam; Nadanaciva, Sashi; Rana, Payal; Lee, Dong Woo; Ku, Bosung; Roth, Alexander D; Dordick, Jonathan S; Will, Yvonne; Lee, Moo-Yeal

2018-03-01

Human liver contains various oxidative and conjugative enzymes that can convert nontoxic parent compounds to toxic metabolites or, conversely, toxic parent compounds to nontoxic metabolites. Unlike primary hepatocytes, which contain myriad drug-metabolizing enzymes (DMEs), but are difficult to culture and maintain physiological levels of DMEs, immortalized hepatic cell lines used in predictive toxicity assays are easy to culture, but lack the ability to metabolize compounds. To address this limitation and predict metabolism-induced hepatotoxicity in high-throughput, we developed an advanced miniaturized three-dimensional (3D) cell culture array (DataChip 2.0) and an advanced metabolizing enzyme microarray (MetaChip 2.0). The DataChip is a functionalized micropillar chip that supports the Hep3B human hepatoma cell line in a 3D microarray format. The MetaChip is a microwell chip containing immobilized DMEs found in the human liver. As a proof of concept for generating compound metabolites in situ on the chip and rapidly assessing their toxicity, 22 model compounds were dispensed into the MetaChip and sandwiched with the DataChip. The IC 50 values obtained from the chip platform were correlated with rat LD 50 values, human C max values, and drug-induced liver injury categories to predict adverse drug reactions in vivo. As a result, the platform had 100% sensitivity, 86% specificity, and 93% overall predictivity at optimum cutoffs of IC 50 and C max values. Therefore, the DataChip/MetaChip platform could be used as a high-throughput, early stage, microscale alternative to conventional in vitro multi-well plate platforms and provide a rapid and inexpensive assessment of metabolism-induced toxicity at early phases of drug development.

A volumetric meter chip for point-of-care quantitative detection of bovine catalase for food safety control.

PubMed

Cui, Xingye; Hu, Jie; Choi, Jane Ru; Huang, Yalin; Wang, Xuemin; Lu, Tian Jian; Xu, Feng

2016-09-07

A volumetric meter chip was developed for quantitative point-of-care (POC) analysis of bovine catalase, a bioindicator of bovine mastitis, in milk samples. The meter chip displays multiplexed quantitative results by presenting the distance of ink bar advancement that is detectable by the naked eye. The meter chip comprises a poly(methyl methacrylate) (PMMA) layer, a double-sided adhesive (DSA) layer and a glass slide layer fabricated by the laser-etching method, which is typically simple, rapid (∼3 min per chip), and cost effective (∼$0.2 per chip). Specially designed "U shape" reaction cells are covered by an adhesive tape that serves as an on-off switch, enabling the simple operation of the assay. As a proof of concept, we employed the developed meter chip for the quantification of bovine catalase in raw milk samples to detect catalase concentrations as low as 20 μg/mL. The meter chip has great potential to detect various target analytes for a wide range of POC applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Enrichment and Detection of Escherichia coli O157:H7 from Water Samples Using an Antibody Modified Microfluidic Chip

PubMed Central

Dharmasiri, Udara; Witek, Małgorzata A.; Adams, Andre A.; Osiri, John K.; Hupert, Mateusz L.; Bianchi, Thomas S.; Roelke, Daniel L.; Soper, Steven A.

2010-01-01

Low abundant (<100 cells mL-1) E. coli O157:H7 cells were isolated and enriched from environmental water samples using a microfluidic chip. The poly(methylmethacrylate), PMMA, chip contained 8 devices each equipped with 16 curvilinear high aspect ratio channels that were covalently decorated with polyclonal anti-O157 antibodies (pAb) and could search for rare cells through a pAb mediated process. The chip could process independently 8 different samples or one sample using 8 different parallel inputs to increase volume processing throughput. After cell enrichment, cells were released and enumerated using bench top real-time quantitative PCR, targeting genes which effectively discriminated the O157:H7 serotype from other non-pathogenic bacteria. The recovery of target cells from water samples was determined to be ~72%, and the limit-of-detection was found to be 6 colony forming units (cfu) using the slt1 gene as a reporter. We subsequently performed analysis of lake and waste water samples. The simplicity in manufacturing and ease of operation makes this device attractive for the selection of pathogenic species from a variety of water supplies suspected of containing bacterial pathogens at extremely low frequencies. PMID:20218574

Polymer coating on a micropillar chip for robust attachment of PuraMatrix peptide hydrogel for 3D hepatic cell culture.

PubMed

Roth, Alexander David; Lama, Pratap; Dunn, Stephen; Hong, Stephen; Lee, Moo-Yeal

2018-09-01

For better mimicking tissues in vivo and developing predictive cell models for high-throughput screening (HTS) of potential drug candidates, three-dimensional (3D) cell cultures have been performed in various hydrogels. In this study, we have investigated several polymer coating materials to robustly attach PuraMatrix peptide hydrogel on a micropillar chip for 3D culture of Hep3B human hepatic cells, which can be used as a tool for high-throughput assessment of compound hepatotoxicity. Among several amphiphilic polymers with maleic anhydride groups tested, 0.01% (w/v) poly(maleic anhydride-alt-1-octadecene) (PMA-OD) provided superior coating properties with no PuraMatrix spot detachment from the micropillar chip and no air bubble entrapment in a complementary microwell chip. To maintain Hep3B cell viability in PuraMatrix gel on the chip, gelation conditions were optimized in the presence of additional salts, at different seeding densities, and for growth medium washes. As a result, salts in growth media were sufficient for gelation, and relatively high cell seeding at 6 million cells/mL and two media washes for pH neutralization were required. With optimized 3D cell culture conditions, controlled gene expression and compound toxicity assessment were successfully demonstrated by using recombinant adenoviruses carrying genes for green and red fluorescent proteins as well as six model compounds. Overall, PuraMatrix hydrogel on the chip was suitable for 3D cell encapsulation, gene expression, and rapid toxicity assessment. Published by Elsevier B.V.

Biomimetic engineering of a generic cell-on-membrane architecture by microfluidic engraving for on-chip bioassays.

PubMed

Lee, Sang-Wook; Noh, Ji-Yoon; Park, Seung Chul; Chung, Jin-Ho; Lee, Byoungho; Lee, Sin-Doo

2012-05-22

We develop a biomimetic cell-on-membrane architecture in close-volume format which allows the interfacial biocompatibility and the reagent delivery capability for on-chip bioassays. The key concept lies in the microfluidic engraving of lipid membranes together with biological cells on a supported substrate with topographic patterns. The simultaneous engraving process of a different class of fluids is promoted by the front propagation of an air-water interface inside a flow-cell. This highly parallel, microfluidic cell-on-membrane approach opens a door to the natural biocompatibility in mimicking cellular stimuli-response behavior essential for diverse on-chip bioassays that can be precisely controlled in the spatial and temporal manner.

Nanoelectromechanical Chip (NELMEC) Combination of Nanoelectronics and Microfluidics to Diagnose Epithelial and Mesenchymal Circulating Tumor Cells from Leukocytes.

PubMed

Hosseini, Seied Ali; Abdolahad, Mohammad; Zanganeh, Somayeh; Dahmardeh, Mahyar; Gharooni, Milad; Abiri, Hamed; Alikhani, Alireza; Mohajerzadeh, Shams; Mashinchian, Omid

2016-02-17

An integrated nano-electromechanical chip (NELMEC) has been developed for the label-free distinguishing of both epithelial and mesenchymal circulating tumor cells (ECTCs and MCTCs, respectively) from white blood cells (WBCs). This nanoelectronic microfluidic chip fabricated by silicon micromachining can trap large single cells (>12 µm) at the opening of the analysis microchannel arrays. The nature of the captured cells is detected using silicon nanograss (SiNG) electrodes patterned at the entrance of the channels. There is an observable difference between the membrane capacitance of the ECTCs and MCTCs and that of WBCs (measured using SiNG electrodes), which is the key indication for our diagnosis. The NELMEC chip not only solves the problem of the size overlap between CTCs and WBCs but also detects MCTCs without the need for any markers or tagging processes, which has been an important problem in previously reported CTC detection systems. The great conductivity of the gold-coated SiNG nanocontacts as well as their safe penetration into the membrane of captured cells, facilitate a precise and direct signal extraction to distinguish the type of captured cell. The results achieved from epithelial (MCF-7) and mesenchymal (MDA-MB231) breast cancer cells circulated in unprocessed blood suggest the significant applications for these diagnostic abilities of NELMEC. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recent advancements in chemical luminescence-based lab-on-chip and microfluidic platforms for bioanalysis.

PubMed

Mirasoli, Mara; Guardigli, Massimo; Michelini, Elisa; Roda, Aldo

2014-01-01

Miniaturization of analytical procedures through microchips, lab-on-a-chip or micro total analysis systems is one of the most recent trends in chemical and biological analysis. These systems are designed to perform all the steps in an analytical procedure, with the advantages of low sample and reagent consumption, fast analysis, reduced costs, possibility of extra-laboratory application. A range of detection technologies have been employed in miniaturized analytical systems, but most applications relied on fluorescence and electrochemical detection. Chemical luminescence (which includes chemiluminescence, bioluminescence, and electrogenerated chemiluminescence) represents an alternative detection principle that offered comparable (or better) analytical performance and easier implementation in miniaturized analytical devices. Nevertheless, chemical luminescence-based ones represents only a small fraction of the microfluidic devices reported in the literature, and until now no review has been focused on these devices. Here we review the most relevant applications (since 2009) of miniaturized analytical devices based on chemical luminescence detection. After a brief overview of the main chemical luminescence systems and of the recent technological advancements regarding their implementation in miniaturized analytical devices, analytical applications are reviewed according to the nature of the device (microfluidic chips, microchip electrophoresis, lateral flow- and paper-based devices) and the type of application (micro-flow injection assays, enzyme assays, immunoassays, gene probe hybridization assays, cell assays, whole-cell biosensors). Copyright © 2013 Elsevier B.V. All rights reserved.

Sensor Access to the Cellular Microenvironment Using the Sensing Cell Culture Flask.

PubMed

Kieninger, Jochen; Tamari, Yaara; Enderle, Barbara; Jobst, Gerhard; Sandvik, Joe A; Pettersen, Erik O; Urban, Gerald A

2018-04-26

The Sensing Cell Culture Flask (SCCF) is a cell culture monitoring system accessing the cellular microenvironment in 2D cell culture using electrochemical microsensors. The system is based on microfabricated sensor chips embedded in standard cell culture flasks. Ideally, the sensor chips could be equipped with any electrochemical sensor. Its transparency allows optical inspection of the cells during measurement. The surface of the sensor chip is in-plane with the flask surface allowing undisturbed cell growth on the sensor chip. A custom developed rack system allows easy usage of multiple flasks in parallel within an incubator. The presented data demonstrates the application of the SCCF with brain tumor (T98G) and breast cancer (T-47D) cells. Amperometric oxygen sensors were used to monitor cellular respiration with different incubation conditions. Cellular acidification was accessed with potentiometric pH sensors using electrodeposited iridium oxide films. The system itself provides the foundation for electrochemical monitoring systems in 3D cell culture.

One-step fabrication of an organ-on-a-chip with spatial heterogeneity using a 3D bioprinting technology.

PubMed

Lee, Hyungseok; Cho, Dong-Woo

2016-07-05

Although various types of organs-on-chips have been introduced recently as tools for drug discovery, the current studies are limited in terms of fabrication methods. The fabrication methods currently available not only need a secondary cell-seeding process and result in severe protein absorption due to the material used, but also have difficulties in providing various cell types and extracellular matrix (ECM) environments for spatial heterogeneity in the organs-on-chips. Therefore, in this research, we introduce a novel 3D bioprinting method for organ-on-a-chip applications. With our novel 3D bioprinting method, it was possible to prepare an organ-on-a-chip in a simple one-step fabrication process. Furthermore, protein absorption on the printed platform was very low, which will lead to accurate measurement of metabolism and drug sensitivity. Moreover, heterotypic cell types and biomaterials were successfully used and positioned at the desired position for various organ-on-a-chip applications, which will promote full mimicry of the natural conditions of the organs. The liver organ was selected for the evaluation of the developed method, and liver function was shown to be significantly enhanced on the liver-on-a-chip, which was prepared by 3D bioprinting. Consequently, the results demonstrate that the suggested 3D bioprinting method is easier and more versatile for production of organs-on-chips.

Integrated microfluidic devices for combinatorial cell-based assays.

PubMed

Yu, Zeta Tak For; Kamei, Ken-ichiro; Takahashi, Hiroko; Shu, Chengyi Jenny; Wang, Xiaopu; He, George Wenfu; Silverman, Robert; Radu, Caius G; Witte, Owen N; Lee, Ki-Bum; Tseng, Hsian-Rong

2009-06-01

The development of miniaturized cell culture platforms for performing parallel cultures and combinatorial assays is important in cell biology from the single-cell level to the system level. In this paper we developed an integrated microfluidic cell-culture platform, Cell-microChip (Cell-microChip), for parallel analyses of the effects of microenvironmental cues (i.e., culture scaffolds) on different mammalian cells and their cellular responses to external stimuli. As a model study, we demonstrated the ability of culturing and assaying several mammalian cells, such as NIH 3T3 fibroblast, B16 melanoma and HeLa cell lines, in a parallel way. For functional assays, first we tested drug-induced apoptotic responses from different cell lines. As a second functional assay, we performed "on-chip" transfection of a reporter gene encoding an enhanced green fluorescent protein (EGFP) followed by live-cell imaging of transcriptional activation of cyclooxygenase 2 (Cox-2) expression. Collectively, our Cell-microChip approach demonstrated the capability to carry out parallel operations and the potential to further integrate advanced functions and applications in the broader space of combinatorial chemistry and biology.

Integrated microfluidic devices for combinatorial cell-based assays

PubMed Central

Yu, Zeta Tak For; Kamei, Ken-ichiro; Takahashi, Hiroko; Shu, Chengyi Jenny; Wang, Xiaopu; He, George Wenfu; Silverman, Robert

2010-01-01

The development of miniaturized cell culture platforms for performing parallel cultures and combinatorial assays is important in cell biology from the single-cell level to the system level. In this paper we developed an integrated microfluidic cell-culture platform, Cell-microChip (Cell-μChip), for parallel analyses of the effects of microenvir-onmental cues (i.e., culture scaffolds) on different mammalian cells and their cellular responses to external stimuli. As a model study, we demonstrated the ability of culturing and assaying several mammalian cells, such as NIH 3T3 fibro-blast, B16 melanoma and HeLa cell lines, in a parallel way. For functional assays, first we tested drug-induced apoptotic responses from different cell lines. As a second functional assay, we performed "on-chip" transfection of a reporter gene encoding an enhanced green fluorescent protein (EGFP) followed by live-cell imaging of transcriptional activation of cyclooxygenase 2 (Cox-2) expression. Collectively, our Cell-μChip approach demonstrated the capability to carry out parallel operations and the potential to further integrate advanced functions and applications in the broader space of combinatorial chemistry and biology. PMID:19130244

Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis.

PubMed

Zhang, Boyang; Montgomery, Miles; Chamberlain, M Dean; Ogawa, Shinichiro; Korolj, Anastasia; Pahnke, Aric; Wells, Laura A; Massé, Stéphane; Kim, Jihye; Reis, Lewis; Momen, Abdul; Nunes, Sara S; Wheeler, Aaron R; Nanthakumar, Kumaraswamy; Keller, Gordon; Sefton, Michael V; Radisic, Milica

2016-06-01

We report the fabrication of a scaffold (hereafter referred to as AngioChip) that supports the assembly of parenchymal cells on a mechanically tunable matrix surrounding a perfusable, branched, three-dimensional microchannel network coated with endothelial cells. The design of AngioChip decouples the material choices for the engineered vessel network and for cell seeding in the parenchyma, enabling extensive remodelling while maintaining an open-vessel lumen. The incorporation of nanopores and micro-holes in the vessel walls enhances permeability, and permits intercellular crosstalk and extravasation of monocytes and endothelial cells on biomolecular stimulation. We also show that vascularized hepatic tissues and cardiac tissues engineered by using AngioChips process clinically relevant drugs delivered through the vasculature, and that millimetre-thick cardiac tissues can be engineered in a scalable manner. Moreover, we demonstrate that AngioChip cardiac tissues implanted with direct surgical anastomosis to the femoral vessels of rat hindlimbs establish immediate blood perfusion.

Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis

NASA Astrophysics Data System (ADS)

Zhang, Boyang; Montgomery, Miles; Chamberlain, M. Dean; Ogawa, Shinichiro; Korolj, Anastasia; Pahnke, Aric; Wells, Laura A.; Massé, Stéphane; Kim, Jihye; Reis, Lewis; Momen, Abdul; Nunes, Sara S.; Wheeler, Aaron R.; Nanthakumar, Kumaraswamy; Keller, Gordon; Sefton, Michael V.; Radisic, Milica

2016-06-01

We report the fabrication of a scaffold (hereafter referred to as AngioChip) that supports the assembly of parenchymal cells on a mechanically tunable matrix surrounding a perfusable, branched, three-dimensional microchannel network coated with endothelial cells. The design of AngioChip decouples the material choices for the engineered vessel network and for cell seeding in the parenchyma, enabling extensive remodelling while maintaining an open-vessel lumen. The incorporation of nanopores and micro-holes in the vessel walls enhances permeability, and permits intercellular crosstalk and extravasation of monocytes and endothelial cells on biomolecular stimulation. We also show that vascularized hepatic tissues and cardiac tissues engineered by using AngioChips process clinically relevant drugs delivered through the vasculature, and that millimetre-thick cardiac tissues can be engineered in a scalable manner. Moreover, we demonstrate that AngioChip cardiac tissues implanted with direct surgical anastomosis to the femoral vessels of rat hindlimbs establish immediate blood perfusion.

Sorting cells by their density

PubMed Central

Norouzi, Nazila; Bhakta, Heran C.

2017-01-01

Sorting cells by their type is an important capability in biological research and medical diagnostics. However, most cell sorting techniques rely on labels or tags, which may have limited availability and specificity. Sorting different cell types by their different physical properties is an attractive alternative to labels because all cells intrinsically have these physical properties. But some physical properties, like cell size, vary significantly from cell to cell within a cell type; this makes it difficult to identify and sort cells based on their sizes alone. In this work we continuously sort different cells types by their density, a physical property with much lower cell-to-cell variation within a cell type (and therefore greater potential to discriminate different cell types) than other physical properties. We accomplish this using a 3D-printed microfluidic chip containing a horizontal flowing micron-scale density gradient. As cells flow through the chip, Earth’s gravity makes each cell move vertically to the point where the cell’s density matches the surrounding fluid’s density. When the horizontal channel then splits, cells with different densities are routed to different outlets. As a proof of concept, we use our density sorter chip to sort polymer microbeads by their material (polyethylene and polystyrene) and blood cells by their type (white blood cells and red blood cells). The chip enriches the fraction of white blood cells in a blood sample from 0.1% (in whole blood) to nearly 98% (in the output of the chip), a 1000x enrichment. Any researcher with access to a 3D printer can easily replicate our density sorter chip and use it in their own research using the design files provided as online Supporting Information. Additionally, researchers can simulate the performance of a density sorter chip in their own applications using the Python-based simulation software that accompanies this work. The simplicity, resolution, and throughput of this technique make it suitable for isolating even rare cell types in complex biological samples, in a wide variety of different research and clinical applications. PMID:28723908

A novel three-dimensional bone chip organ culture.

PubMed

Kuttenberger, Johannes; Polska, Elzbieta; Schaefer, Birgit M

2013-07-01

The objective of this study was to develop a 3D bone chip organ culture model. We aimed to collect in vitro evidence of the ability of vital bone chips to promote new bone formation. We developed a 3D in vitro hypoxic bone chip organ culture model. Histology of the bone chips was performed before and after culture and immunohistochemistry after 3-week culture. The 3D culture supernatants were tested for the presence of pro-angiogenic growth factors, TGFβ1, GADPH, bone alkaline phosphatase, osteocalcin, osteonectin, osteopontin, bone sialoprotein and collagen type I. Histology after culture revealed bone chips in a matrix of fibrin remnants and a fibrous-appearing matter. Collagen type I- and IV-positive structures were also identified. Cells could be seen on the surface of the bone chips, with spindle-shaped cells bridging the bone chip particles. Pro-angiogenic growth factors and TGFβ1were detected in the 3D cell culture supernatants. The transcripts for osteocalcin, bone sialoprotein and collagen type I were revealed only via PCR. Our results indicate that bone chips in our 3D organ culture remain vital and may stimulate the growth of a bone-forming matrix. The use of autogenous bone chips for oral and maxillofacial bone augmentation procedures is widespread in clinical practice. The rationale for this is that if bone chips remain vital in vivo, they could provide an environment promoting new bone formation through growth factors and cells. This 3D culture method is an essential tool for investigating the behaviour of bone chips.

An integrated cell culture lab on a chip: modular microdevices for cultivation of mammalian cells and delivery into microfluidic microdroplets.

PubMed

Hufnagel, Hansjörg; Huebner, Ansgar; Gülch, Carina; Güse, Katharina; Abell, Chris; Hollfelder, Florian

2009-06-07

We present a modular system of microfluidic PDMS devices designed to incorporate the steps necessary for cell biological assays based on mammalian tissue culture 'on-chip'. The methods described herein include the on-chip immobilization and culturing of cells as well as their manipulation by transfection. Assessment of cell viability by flow cytrometry suggests low attrition rates (<3%) and excellent growth properties in the device for up to 7 days for CHO-K1 cells. To demonstrate that key procedures from the repertoire of cell biology are possible in this format, transfection of a reporter gene (encoding green fluorescent protein) was carried out. The modular design enables efficient detachment and recollection of cells and allows assessment of the success of transfection achieved on-chip. The transfection levels (20%) are comparable to standard large scale procedures and more than 500 cells could be transfected. Finally, cells are transferred into microfluidic microdoplets, where in principle a wide range of subsequent assays can be carried out at the single cell level in droplet compartments. The procedures developed for this modular device layout further demonstrate that commonly used methods in cell biology involving mammalian cells can be reliably scaled down to allow single cell investigations in picolitre volumes.

On-chip microfluid induced by oscillation of microrobot for noncontact cell transportation

NASA Astrophysics Data System (ADS)

Feng, Lin; Liang, Shuzhang; Zhou, Xiangcong; Yang, Jianlei; Jiang, Yonggang; Zhang, Deyuan; Arai, Fumihito

2017-11-01

The importance of cell manipulation and cultivation is increasing rapidly in various fields, such as drug discovery, regenerative medicine, and investigation of new energy sources. This paper presents a method to transport cells in a microfluidic chip without contact. A local vortex was generated when high-frequency oscillation of a microtool was induced in a microfluidic chip. The vortex was controlled by tuning the tool's oscillation parameters, such as the oscillation amplitude and frequency. The cells were then transported in the chip based on the direction of the tool's movement, and their position, posture, and trajectories were controlled. Bovine oocyte manipulations, that is, transportation and rotation, were conducted to demonstrate the capability of the proposed method, without any contact by the microrobot with high-frequency oscillation.

A Cell Programmable Assay (CPA) chip.

PubMed

Ju, Jongil; Warrick, Jay; Beebe, David J

2010-08-21

This article describes two kinds of "Cell Programmable Assay" (CPA) chips that utilize passive pumping for the culture and autonomous staining of cells to simply common protocols. One is a single timer channel CPA (sCPA) chip that has one timer channel and one main channel containing a cell culture chamber. The sCPA is used to culture and stain cells using Hoechst nuclear staining dye (a 2 step staining process). The other is a dual timer channel CPA (dCPA) chip that has two timer channels and one main channel with a chamber for cell culture. The dCPA is used here to culture, fix, permeablize, and stain cells using DAPI. The additional timer channel of the dCPA chip allows for automation of 3 steps. The CPA chips were successfully evaluated using HEK 293 cells. In addition, we provide a simplified equation for tuning or redesigning CPA chips to meet the needs of a variety of protocols that may require different timings. The equation is easy to use as it only depends upon the dimensions of microchannel and the volume of the reagent drops. The sCPA and dCPA chips can be readily modified to apply to a wide variety of common cell culture methods and procedures.

Optical and electrical interfacing technologies for living cell bio-chips.

PubMed

Shacham-Diamand, Y; Belkin, S; Rishpon, J; Elad, T; Melamed, S; Biran, A; Yagur-Kroll, S; Almog, R; Daniel, R; Ben-Yoav, H; Rabner, A; Vernick, S; Elman, N; Popovtzer, R

2010-06-01

Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made by micro-system-technologies (MST). The cells are integrated, deposited or immersed in a media which is in contact with the chip. The cells behavior is monitored via electrical, electrochemical or optical methods. In this paper we describe such whole-cell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the biological component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the micro-electronics, micro-electro optics, micro-electro or magneto mechanics and micro-fluidics. The genetic response of the cells expresses proteins that generate: a. light by photo-luminescence or bioluminescence, b. electrochemical signal by interaction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that converts it to current or voltage amplifies and filters it. The resultant signal is analyzed and stored for further processing. In this paper we describe three examples of whole-cell bio chips, photo-luminescent, bioluminescent and electrochemical, which are based on the genetic response of genetically modified E. coli microbes integrated on a micro-fluidics MEMS platform. We describe the chip outline as well as the basic modeling scheme of such sensors. We discuss the highlights and problems of such system, from the point of view of micro-system-technology.

A nanoporous alumina microelectrode array for functional cell-chip coupling.

PubMed

Wesche, Manuel; Hüske, Martin; Yakushenko, Alexey; Brüggemann, Dorothea; Mayer, Dirk; Offenhäusser, Andreas; Wolfrum, Bernhard

2012-12-14

The design of electrode interfaces has a strong impact on cell-based bioelectronic applications. We present a new type of microelectrode array chip featuring a nanoporous alumina interface. The chip is fabricated in a combination of top-down and bottom-up processes using state-of-the-art clean room technology and self-assembled generation of nanopores by aluminum anodization. The electrode characteristics are investigated in phosphate buffered saline as well as under cell culture conditions. We show that the modified microelectrodes exhibit decreased impedance compared to planar microelectrodes, which is caused by a nanostructuring effect of the underlying gold during anodization. The stability and biocompatibility of the device are demonstrated by measuring action potentials from cardiomyocyte-like cells growing on top of the chip. Cross sections of the cell-surface interface reveal that the cell membrane seals the nanoporous alumina layer without bending into the sub-50 nm apertures. The nanoporous microelectrode array device may be used as a platform for combining extracellular recording of cell activity with stimulating topographical cues.

Covalent ISG15 conjugation to CHIP promotes its ubiquitin E3 ligase activity and inhibits lung cancer cell growth in response to type I interferon.

PubMed

Yoo, Lang; Yoon, A-Rum; Yun, Chae-Ok; Chung, Kwang Chul

2018-01-24

The carboxyl terminus of Hsp70-interacting protein (CHIP) acts as a ubiquitin E3 ligase and a link between the chaperones Hsp70/90 and the proteasome system, playing a vital role in maintaining protein homeostasis. CHIP regulates a number of proteins involved in a myriad of physiological and pathological processes, but the underlying mechanism of action via posttranslational modification has not been extensively explored. In this study, we investigated a novel modulatory mode of CHIP and its effect on CHIP enzymatic activity. ISG15, an ubiquitin-like modifier, is induced by type I interferon (IFN) stimulation and can be conjugated to target proteins (ISGylation). Here we demonstrated that CHIP may be a novel target of ISGylation in HEK293 cells stimulated with type I IFN. We also found that Lys143/144/145 and Lys287 residues in CHIP are important for and target residues of ISGylation. Moreover, ISGylation promotes the E3 ubiquitin ligase activity of CHIP, subsequently causing a decrease in levels of oncogenic c-Myc, one of its many ubiquitination targets, in A549 lung cancer cells and inhibiting A549 cell and tumor growth. In conclusion, the present study demonstrates that covalent ISG15 conjugation produces a novel CHIP regulatory mode that enhances the tumor-suppressive activity of CHIP, thereby contributing to the antitumor effect of type I IFN.

System-Level Biochip for Impedance Sensing and Programmable Manipulation of Bladder Cancer Cells

PubMed Central

Chuang, Cheng-Hsin; Huang, Yao-Wei; Wu, Yao-Tung

2011-01-01

This paper develops a dielectrophoretic (DEP) chip with multi-layer electrodes and a micro-cavity array for programmable manipulations of cells and impedance measurement. The DEP chip consists of an ITO top electrode, flow chamber, middle electrode on an SU-8 surface, micro-cavity arrays of SU-8 and distributed electrodes at the bottom of the micro-cavity. Impedance sensing of single cells could be performed as follows: firstly, cells were trapped in a micro-cavity array by negative DEP force provided by top and middle electrodes; then, the impedance measurement for discrimination of different stage of bladder cancer cells was accomplished by the middle and bottom electrodes. After impedance sensing, the individual releasing of trapped cells was achieved by negative DEP force using the top and bottom electrodes in order to collect the identified cells once more. Both cell manipulations and impedance measurement had been integrated within a system controlled by a PC-based LabVIEW program. In the experiments, two different stages of bladder cancer cell lines (grade III: T24 and grade II: TSGH8301) were utilized for the demonstration of programmable manipulation and impedance sensing; as the results show, the lower-grade bladder cancer cells (TSGH8301) possess higher impedance than the higher-grade ones (T24). In general, the multi-step manipulations of cells can be easily programmed by controlling the electrical signal in our design, which provides an excellent platform technology for lab-on-a-chip (LOC) or a micro-total-analysis-system (Micro TAS). PMID:22346685

Lab on chip microdevices for cellular mechanotransduction in urothelial cells

NASA Astrophysics Data System (ADS)

Maziz, A.; Guan, N.; Svennersten, K.; Hallén-Grufman, K.; Jager, Edwin W. H.

2016-04-01

Cellular mechanotransduction is crucial for physiological function in the lower urinary tract. The bladder is highly dependent on the ability to sense and process mechanical inputs, illustrated by the regulated filling and voiding of the bladder. However, the mechanisms by which the bladder integrates mechanical inputs, such as intravesicular pressure, and controls the smooth muscles, remain unknown. To date no tools exist that satisfactorily mimic in vitro the dynamic micromechanical events initiated e.g. by an emerging inflammatory process or a growing tumour mass in the urinary tract. More specifically, there is a need for tools to study these events on a single cell level or in a small population of cells. We have developed a micromechanical stimulation chip that can apply physiologically relevant mechanical stimuli to single cells to study mechanosensitive cells in the urinary tract. The chips comprise arrays of microactuators based on the electroactive polymer polypyrrole (PPy). PPy offers unique possibilities and is a good candidate to provide such physiological mechanical stimulation, since it is driven at low voltages, is biocompatible, and can be microfabricated. The PPy microactuators can provide mechanical stimulation at different strains and/or strain rates to single cells or clusters of cells, including controls, all integrated on one single chip, without the need to preprepare the cells. This paper reports initial results on the mechano-response of urothelial cells using the micromechanical stimulation chips. We show that urothelial cells are viable on our microdevices and do respond with intracellular Ca2+ increase when subjected to a micro-mechanical stimulation.

Quantitative measurements of intercellular adhesion between a macrophage and cancer cells using a cup-attached AFM chip.

PubMed

Kim, Hyonchol; Yamagishi, Ayana; Imaizumi, Miku; Onomura, Yui; Nagasaki, Akira; Miyagi, Yohei; Okada, Tomoko; Nakamura, Chikashi

2017-07-01

Intercellular adhesion between a macrophage and cancer cells was quantitatively measured using atomic force microscopy (AFM). Cup-shaped metal hemispheres were fabricated using polystyrene particles as a template, and a cup was attached to the apex of the AFM cantilever. The cup-attached AFM chip (cup-chip) approached a murine macrophage cell (J774.2), the cell was captured on the inner concave of the cup, and picked up by withdrawing the cup-chip from the substrate. The cell-attached chip was advanced towards a murine breast cancer cell (FP10SC2), and intercellular adhesion between the two cells was quantitatively measured. To compare cell adhesion strength, the work required to separate two adhered cells (separation work) was used as a parameter. Separation work was almost 2-fold larger between a J774.2 cell and FP10SC2 cell than between J774.2 cell and three additional different cancer cells (4T1E, MAT-LyLu, and U-2OS), two FP10SC2 cells, or two J774.2 cells. FP10SC2 was established from 4T1E as a highly metastatic cell line, indicates separation work increased as the malignancy of cancer cells became higher. One possible explanation of the strong adhesion of macrophages to cancer cells observed in this study is that the measurement condition mimicked the microenvironment of tumor-associated macrophages (TAMs) in vivo, and J774.2 cells strongly expressed CD204, which is a marker of TAMs. The results of the present study, which were obtained by measuring cell adhesion strength quantitatively, indicate that the fabricated cup-chip is a useful tool for measuring intercellular adhesion easily and quantitatively. Copyright © 2017 Elsevier B.V. All rights reserved.

3D Nanochannel Array Platform for High-throughput Cell Manipulation and Nano-electroporation

NASA Astrophysics Data System (ADS)

Chang, Lingqian

Electroporation is one of the most common non-viral methods for gene delivery. Recent progress in gene therapy has offered special opportunities to electroporation for in vitro and in vivo applications. However, conventional bulk electroporation (BEP) inevitably causes serious cell damage and stochastic transfection between cells. Microfluidic electroporation (MEP) has been claimed to provide benign single cell transfection for the last decade. Nevertheless, the intracellular transport in both MEP and BEP systems is highly diffusion-dominant, which prevents precise dose control and high uniformity. In this Ph.D. research, we developed a 3D nanochannel-electroporation (3D NEP) platform for mass cell transfection. A silicon-based nanochannel array (3D NEP) chip was designed and fabricated for cell manipulation and electroporation. The chip, designed as Z-directional microchannel - nanochannel array, was fabricated by clean room techniques including projection photolithography and deep reactive-ion etching (DRIE). The fabricated 3D NEP chip is capable of handling 40,000 cells per 1 cm2, up to 1 million per wafer (100 mm diameter). High-throughput cell manipulation technologies were investigated for precise alignment of individual cells to the nanochannel array, a key step for NEP to achieve dose control. We developed three techniques for cell trapping in this work. (1) Magnetic tweezers (MTs) were integrated on the chip to remotely control cells under a programmed magnetic field. (2) A positive dielectrophoresis (pDEP) power system was built as an alternative to trap cells onto the nanochannel array using DEP force. (3) A novel yet simple 'dipping-trap' method was used to rapidly trap cells onto a nanochannel array, aligned by a micro-cap array pattern on the 3D NEP chip, which eventually offered 70 - 90 % trapping efficiency and 90 % specificity. 3D NEP platforms were assembled for cell transfection based on the Si-based nanochannel array chip and cell manipulation techniques. Cells were patterned on the nanochannel array and collectively were electroporated in parallel, injected with cargo in Z-direction. Controlling the dose was demonstrated with the external pulse durations at high-throughput. The 'electrophoretic'- expedited delivery of large molecular weight plasmids were demonstrated with large numbers of primary cells simultaneously, which cannot be achieved in BEP and MEP. Two clinically valuable case studies were performed with our 3D NEP for living cell sensing / interrogation. (1) In the case of in vitro transfection of primary cardiomyocytes, we studied the dose-effects of miR-29 on mitochondrial changes and the suppression of the Mcl-1 gene in adult mouse cardiomyocytes by precisely controlling the miR-29 dose injected. (2) Glioma stem cells (GSCs), a type of cell hypothesized to be highly aggressive and to lead to the relapses of gliobastoma in human brain, was studied at single cell resolution on 3D NEP platform. The developed 3D NEP system moves towards clinically oriented and user-friendly tools for life science applications. The batch-treated cells with controlled dosage delivery provide a useful tool for single cell analysis. The pioneering experiments in this work have demonstrated the 3D NEP for the applications of cell reprogramming, adoptive immunotherapy, in vitro cardiomyocytes transfection and glioma stem cells study.

Self-digitization chip for single-cell genotyping of cancer-related mutations

PubMed Central

Monroe, Luke D.; Kreutz, Jason E.; Schneider, Thomas; Fujimoto, Bryant S.; Chiu, Daniel T.; Radich, Jerald P.; Paguirigan, Amy L.

2018-01-01

Cancer is a heterogeneous disease, and patient-level genetic assessments can guide therapy choice and impact prognosis. However, little is known about the impact of genetic variability within a tumor, intratumoral heterogeneity (ITH), on disease progression or outcome. Current approaches using bulk tumor specimens can suggest the presence of ITH, but only single-cell genetic methods have the resolution to describe the underlying clonal structures themselves. Current techniques tend to be labor and resource intensive and challenging to characterize with respect to sources of biological and technical variability. We have developed a platform using a microfluidic self-digitization chip to partition cells in stationary volumes for cell imaging and allele-specific PCR. Genotyping data from only confirmed single-cell volumes is obtained and subject to a variety of relevant quality control assessments such as allele dropout, false positive, and false negative rates. We demonstrate single-cell genotyping of the NPM1 type A mutation, an important prognostic indicator in acute myeloid leukemia, on single cells of the cell line OCI-AML3, describing a more complex zygosity distribution than would be predicted via bulk analysis. PMID:29718986

Self-digitization chip for single-cell genotyping of cancer-related mutations.

PubMed

Thompson, Alison M; Smith, Jordan L; Monroe, Luke D; Kreutz, Jason E; Schneider, Thomas; Fujimoto, Bryant S; Chiu, Daniel T; Radich, Jerald P; Paguirigan, Amy L

2018-01-01

Cancer is a heterogeneous disease, and patient-level genetic assessments can guide therapy choice and impact prognosis. However, little is known about the impact of genetic variability within a tumor, intratumoral heterogeneity (ITH), on disease progression or outcome. Current approaches using bulk tumor specimens can suggest the presence of ITH, but only single-cell genetic methods have the resolution to describe the underlying clonal structures themselves. Current techniques tend to be labor and resource intensive and challenging to characterize with respect to sources of biological and technical variability. We have developed a platform using a microfluidic self-digitization chip to partition cells in stationary volumes for cell imaging and allele-specific PCR. Genotyping data from only confirmed single-cell volumes is obtained and subject to a variety of relevant quality control assessments such as allele dropout, false positive, and false negative rates. We demonstrate single-cell genotyping of the NPM1 type A mutation, an important prognostic indicator in acute myeloid leukemia, on single cells of the cell line OCI-AML3, describing a more complex zygosity distribution than would be predicted via bulk analysis.

Single cell HaloChip assay on paper for point-of-care diagnosis.

PubMed

Ma, Liyuan; Qiao, Yong; Jones, Ross; Singh, Narendra; Su, Ming

2016-11-01

This article describes a paper-based low cost single cell HaloChip assay that can be used to assess drug- and radiation-induced DNA damage at point-of-care. Printing ink on paper effectively blocks fluorescence of paper materials, provides high affinity to charged polyelectrolytes, and prevents penetration of water in paper. After exposure to drug or ionizing radiation, cells are patterned on paper to create discrete and ordered single cell arrays, embedded inside an agarose gel, lysed with alkaline solution to allow damaged DNA fragments to diffuse out of nucleus cores, and form diffusing halos in the gel matrix. After staining DNA with a fluorescent dye, characteristic halos formed around cells, and the level of DNA damage can be quantified by determining sizes of halos and nucleus with an image processing program based on MATLAB. With its low fabrication cost and easy operation, this HaloChip on paper platform will be attractive to rapidly and accurately determine DNA damage for point-of-care evaluation of drug efficacy and radiation condition. Graphical Abstract Single cell HaloChip on paper.

Selective ligninolysis of wheat straw and wood chips by the white-rot fungus Lentinula edodes and its influence on in vitro rumen degradability.

PubMed

van Kuijk, Sandra J A; Del Río, José C; Rencoret, Jorge; Gutiérrez, Ana; Sonnenberg, Anton S M; Baars, Johan J P; Hendriks, Wouter H; Cone, John W

2016-01-01

The present work investigated the influence of lignin content and composition in the fungal treatment of lignocellulosic biomass in order to improve rumen degradability. Wheat straw and wood chips, differing in lignin composition, were treated with Lentinula edodes for 0, 2, 4, 8 and 12 wk and the changes occurring during fungal degradation were analyzed using pyrolysis-gas chromatography-mass spectrometry and detergent fiber analysis. L. edodes preferentially degraded lignin, with only limited cellulose degradation, in wheat straw and wood chips, leaving a substrate enriched in cellulose. Syringyl (S)-lignin units were preferentially degraded than guaiacyl (G)-lignin units, resulting in a decreased S/G ratio. A decreasing S/G ratio (wheat straw: r = -0.72, wood chips: r = -0.75) and selective lignin degradation (wheat straw: r = -0.69, wood chips: r = -0.88) were correlated with in vitro gas production (IVGP), a good indicator for rumen degradability. L. edodes treatment increased the IVGP of wheat straw and wood chips. Effects on IVGP were similar for wheat straw and wood chips indicating that lignin content and 3D-structure of cell walls influence in vitro rumen degradability more than lignin composition.

CHIP promotes thyroid cancer proliferation via activation of the MAPK and AKT pathways.

PubMed

Zhang, Li; Liu, Lianyong; He, Xiaohua; Shen, Yunling; Liu, Xuerong; Wei, Jing; Yu, Fang; Tian, Jianqing

2016-08-26

The carboxyl terminus of Hsp70-interacting protein (CHIP) is a U box-type ubiquitin ligase that plays crucial roles in various biological processes, including tumor progression. To date, the functional mechanism of CHIP in thyroid cancer remains unknown. Here, we obtained evidence of upregulation of CHIP in thyroid cancer tissues and cell lines. CHIP overexpression markedly enhanced thyroid cancer cell viability and colony formation in vitro and accelerated tumor growth in vivo. Conversely, CHIP knockdown impaired cell proliferation and tumor growth. Notably, CHIP promoted cell growth through activation of MAPK and AKT pathways, subsequently decreasing p27 and increasing cyclin D1 and p-FOXO3a expression. Our findings collectively indicate that CHIP functions as an oncogene in thyroid cancer, and is therefore a potential therapeutic target for this disease. Copyright © 2016 Elsevier Inc. All rights reserved.

A microfluidic microprocessor: controlling biomimetic containers and cells using hybrid integrated circuit/microfluidic chips.

PubMed

Issadore, David; Franke, Thomas; Brown, Keith A; Westervelt, Robert M

2010-11-07

We present an integrated platform for performing biological and chemical experiments on a chip based on standard CMOS technology. We have developed a hybrid integrated circuit (IC)/microfluidic chip that can simultaneously control thousands of living cells and pL volumes of fluid, enabling a wide variety of chemical and biological tasks. Taking inspiration from cellular biology, phospholipid bilayer vesicles are used as robust picolitre containers for reagents on the chip. The hybrid chip can be programmed to trap, move, and porate individual living cells and vesicles and fuse and deform vesicles using electric fields. The IC spatially patterns electric fields in a microfluidic chamber using 128 × 256 (32,768) 11 × 11 μm(2) metal pixels, each of which can be individually driven with a radio frequency (RF) voltage. The chip's basic functions can be combined in series to perform complex biological and chemical tasks and can be performed in parallel on the chip's many pixels for high-throughput operations. The hybrid chip operates in two distinct modes, defined by the frequency of the RF voltage applied to the pixels: Voltages at MHz frequencies are used to trap, move, and deform objects using dielectrophoresis and voltages at frequencies below 1 kHz are used for electroporation and electrofusion. This work represents an important step towards miniaturizing the complex chemical and biological experiments used for diagnostics and research onto automated and inexpensive chips.

Organs-on-a-chip: Current applications and consideration points for in vitro ADME-Tox studies.

PubMed

Ishida, Seiichi

2018-02-01

Assay systems using in vitro cultured cells are increasingly applied for evaluation of the efficacy, safety, and toxicity of drug candidates. In vitro cell-based assays have two main applications in the drug discovery process: searching for a compound that is effective against the target disease (seed investigation) and confirmation of safety during use of the identified compounds (safety assessment). Currently available in vitro cell-based assays have been designed to evaluate the efficacy and toxicity in single organs, but the in vivo pharmacokinetics and pharmacodynamics of the administered drug candidates have not been considered. Thus, an evaluation system that interconnects cell culture units, one of which has appropriate drug metabolism activities and the other assesses the efficacy and toxicity of compounds, is needed. Accordingly, the in vitro ADME-Tox culture system known as organs-on-a-chip has been proposed. In this review, after introducing the organs-on-a-chip system, the evaluation of enterohepatic circulation and the gut-liver axis relationship will be presented as an example of the application of the organs-on-a-chip system for ADME studies based on inter-organ network. Additionally, the functions required for the organs-on-a-chip system and the necessity of standardization of cells mounted on the chip system will be discussed. Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Mapping of transcription factor binding regions in mammalian cells by ChIP: Comparison of array- and sequencing-based technologies

PubMed Central

Euskirchen, Ghia M.; Rozowsky, Joel S.; Wei, Chia-Lin; Lee, Wah Heng; Zhang, Zhengdong D.; Hartman, Stephen; Emanuelsson, Olof; Stolc, Viktor; Weissman, Sherman; Gerstein, Mark B.; Ruan, Yijun; Snyder, Michael

2007-01-01

Recent progress in mapping transcription factor (TF) binding regions can largely be credited to chromatin immunoprecipitation (ChIP) technologies. We compared strategies for mapping TF binding regions in mammalian cells using two different ChIP schemes: ChIP with DNA microarray analysis (ChIP-chip) and ChIP with DNA sequencing (ChIP-PET). We first investigated parameters central to obtaining robust ChIP-chip data sets by analyzing STAT1 targets in the ENCODE regions of the human genome, and then compared ChIP-chip to ChIP-PET. We devised methods for scoring and comparing results among various tiling arrays and examined parameters such as DNA microarray format, oligonucleotide length, hybridization conditions, and the use of competitor Cot-1 DNA. The best performance was achieved with high-density oligonucleotide arrays, oligonucleotides ≥50 bases (b), the presence of competitor Cot-1 DNA and hybridizations conducted in microfluidics stations. When target identification was evaluated as a function of array number, 80%–86% of targets were identified with three or more arrays. Comparison of ChIP-chip with ChIP-PET revealed strong agreement for the highest ranked targets with less overlap for the low ranked targets. With advantages and disadvantages unique to each approach, we found that ChIP-chip and ChIP-PET are frequently complementary in their relative abilities to detect STAT1 targets for the lower ranked targets; each method detected validated targets that were missed by the other method. The most comprehensive list of STAT1 binding regions is obtained by merging results from ChIP-chip and ChIP-sequencing. Overall, this study provides information for robust identification, scoring, and validation of TF targets using ChIP-based technologies. PMID:17568005

The E3 ubiquitin ligase CHIP selectively regulates mutant epidermal growth factor receptor by ubiquitination and degradation.

PubMed

Chung, Chaeuk; Yoo, Geon; Kim, Tackhoon; Lee, Dahye; Lee, Choong-Sik; Cha, Hye Rim; Park, Yeon Hee; Moon, Jae Young; Jung, Sung Soo; Kim, Ju Ock; Lee, Jae Cheol; Kim, Sun Young; Park, Hee Sun; Park, Myoungrin; Park, Dong Il; Lim, Dae-Sik; Jang, Kang Won; Lee, Jeong Eun

2016-10-14

Somatic mutation in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) is a decisive factor for the therapeutic response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung adenocarcinoma. The stability of mutant EGFR is maintained by various regulators, including heat shock protein 90 (Hsp90). The C terminus of Hsc70-interacting protein (CHIP) is a Hsp70/Hsp90 co-chaperone and exhibits E3 ubiquitin ligase activity. The high-affinity Hsp90-CHIP complex recognizes and selectively regulates their client proteins. CHIP also works with its own E3 ligase activity independently of Hsp70/Hsp90. Here, we investigated the role of CHIP in regulating EGFR in lung adenocarcinoma and also evaluated the specificity of CHIP's effects on mutant EGFR. In HEK 293T cells transfected with either WT EGFR or EGFR mutants, the overexpression of CHIP selectively decreased the expression of certain EGFR mutants (G719S, L747_E749del A750P and L858R) but not WT EGFR. In a pull-down assay, CHIP selectively interacted with EGFR mutants and simultaneously induced their ubiquitination and proteasomal degradation. The expressions of mutant EGFR in PC9 and H1975 were diminished by CHIP, while the expression of WT EGFR in A549 was nearly not affected. In addition, CHIP overexpression inhibited cell proliferation and xenograft's tumor growth of EGFR mutant cell lines, but not WT EGFR cell lines. EGFR mutant specific ubiquitination by CHIP may provide a crucial regulating mechanism for EGFR in lung adenocarcinoma. Our results suggest that CHIP can be novel therapeutic target for overcoming the EGFR TKI resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

Direct quantification of transendothelial electrical resistance in organs-on-chips.

PubMed

van der Helm, Marinke W; Odijk, Mathieu; Frimat, Jean-Philippe; van der Meer, Andries D; Eijkel, Jan C T; van den Berg, Albert; Segerink, Loes I

2016-11-15

Measuring transendothelial or transepithelial electrical resistance (TEER) is a widely used method to monitor cellular barrier tightness in organs-on-chips. Unfortunately, integrated electrodes close to the cellular barrier hamper visual inspection of the cells or require specialized cleanroom processes to fabricate see-through electrodes. Out-of-view electrodes inserted into the chip's outlets are influenced by the fluid-filled microchannels with relatively high resistance. In this case, small changes in temperature or medium composition strongly affect the apparent TEER. To solve this, we propose a simple and universally applicable method to directly determine the TEER in microfluidic organs-on-chips without the need for integrated electrodes close to the cellular barrier. Using four electrodes inserted into two channels - two on each side of the porous membrane - and six different measurement configurations we can directly derive the isolated TEER independent of channel properties. We show that this method removes large variation of non-biological origin in chips filled with culture medium. Furthermore, we demonstrate the use of our method by quantifying the TEER of a monolayer of human hCMEC/D3 cerebral endothelial cells, mimicking the blood-brain barrier inside our microfluidic organ-on-chip device. We found stable TEER values of 22 Ω cm(2)±1.3 Ω cm(2) (average ± standard error of the mean of 4 chips), comparable to other TEER values reported for hCMEC/D3 cells in well-established Transwell systems. In conclusion, we demonstrate a simple and robust way to directly determine TEER that is applicable to any organ-on-chip device with two channels separated by a membrane. This enables stable and easily applicable TEER measurements without the need for specialized cleanroom processes and with visibility on the measured cell layer. Copyright © 2016 Elsevier B.V. All rights reserved.

Cancer-cells on a chip for label-free optic detection of secreted molecules

NASA Astrophysics Data System (ADS)

Berthuy, Ophélie I.; Blum, Loïc. J.; Marquette, Christophe A.

2015-05-01

To unravel cell complexity, living-cell chips have been developed that allow delivery of experimental stimuli but also measurement of the resulting cellular responses. We have been developing a new concept for multiplexed detection of biomolecules secreted by different cancer cells. In the present report, we are making the proof of concept of cell small populations (from 1 to 100 cells) spotting, culture and secretion detection on a gold surface. For that purpose, antibodies and different cell lines were spotted using a piezoelectric spotter. In order to keep the cells in a hydrated environment during the robotized micropipetting and to address different cell lines on a single chip, a biocompatible alginate polymer was used. This approach enables the encapsulation of the cell in a very small volume (30 nL), directly on the substrate and permits a precise control of the number of cells in each alginate bead. After 24h of culture, the adherent cells are ready for surface plasmon resonance imaging (SPRi) experimentation. To enable the detection of secreted proteins, various antibodies are immobilized in an organized manner on a SPRi sensor and permitted the multiplex detection of different proteins secreted by the different cultured cell lines. Evidence of the real-time detection will be presented for Prostate Specific Antigen (PSA) and β-2-microglobulin (B2M) secreted by prostate cancer cells following induction by dihydrotestosterone (DHT). Different kinetics for the two secreted proteins were then demonstrated and precisely determined using the chip. There is no doubt that our chip will, in a near future, be applied to more multiplexed and complex biological secretion systems for which kinetic data are at the moment not reachable using standard cellular biology tools.

Tumour-on-a-chip: microfluidic models of tumour morphology, growth and microenvironment

PubMed Central

Trubelja, Alen

2017-01-01

Cancer remains one of the leading causes of death, albeit enormous efforts to cure the disease. To overcome the major challenges in cancer therapy, we need to have a better understanding of the tumour microenvironment (TME), as well as a more effective means to screen anti-cancer drug leads; both can be achieved using advanced technologies, including the emerging tumour-on-a-chip technology. Here, we review the recent development of the tumour-on-a-chip technology, which integrates microfluidics, microfabrication, tissue engineering and biomaterials research, and offers new opportunities for building and applying functional three-dimensional in vitro human tumour models for oncology research, immunotherapy studies and drug screening. In particular, tumour-on-a-chip microdevices allow well-controlled microscopic studies of the interaction among tumour cells, immune cells and cells in the TME, of which simple tissue cultures and animal models are not amenable to do. The challenges in developing the next-generation tumour-on-a-chip technology are also discussed. PMID:28637915

Engineering of Neuron Growth and Enhancing Cell-Chip Communication via Mixed SAMs.

PubMed

Markov, Aleksandr; Maybeck, Vanessa; Wolf, Nikolaus; Mayer, Dirk; Offenhäusser, Andreas; Wördenweber, Roger

2018-06-06

The interface between cells and inorganic surfaces represents one of the key elements for bioelectronics experiments and applications ranging from cell cultures and bioelectronics devices to medical implants. In the present paper, we describe a way to tailor the biocompatibility of substrates in terms of cell growth and to significantly improve cell-chip communication, and we also demonstrate the reusability of the substrates for cell experiments. All these improvements are achieved by coating the substrates or chips with a self-assembled monolayer (SAM) consisting of a mixture of organic molecules, (3-aminopropyl)-triethoxysilane and (3-glycidyloxypropyl)-trimethoxysilane. By varying the ratio of these molecules, we are able to tune the cell density and live/dead ratios of rat cortical neurons cultured directly on the mixed SAM as well as neurons cultured on protein-coated SAMs. Furthermore, the use of the SAM leads to a significant improvement in cell-chip communications. Action potential signals of up to 9.4 ± 0.6 mV (signal-to-noise ratio up to 47) are obtained for HL-1 cells on microelectrode arrays. Finally, we demonstrate that the SAMs facilitate a reusability of the samples for all cell experiments with little re-processing.

Inhibition of formyl peptide receptor in high-grade astrocytoma by CHemotaxis Inhibitory Protein of S. aureus

PubMed Central

Boer, J C; Domanska, U M; Timmer-Bosscha, H; Boer, I G J; de Haas, C J C; Joseph, J V; Kruyt, F A E; de Vries, E G E; den Dunnen, W F A; van Strijp, J A G; Walenkamp, A M E

2013-01-01

Background: High-grade astrocytomas are malignant brain tumours that infiltrate the surrounding brain tissue and have a poor prognosis. Activation of formyl peptide receptor (FPR1) on the human astrocytoma cell line U87 promotes cell motility, growth and angiogenesis. We therefore investigated the FPR1 inhibitor, Chemotaxis Inhibitory Protein of S. aureus (CHIPS), as a potential anti-astrocytoma drug. Methods and results: FPR1 expression was studied immunohistochemically in astrocytomas WHO grades I–IV. With intracellular calcium mobilisation and migration assays, human ligands were tested for their ability to activate FPR1 on U87 cells and on a cell line derived from primary astrocytoma grade IV patient material. Thereafter, we selectively inhibited these ligand-induced responses of FPR1 with an anti-inflammatory compound called Chemotaxis Inhibitory Protein of S. aureus (CHIPS). U87 xenografts in NOD-SCID mice served to investigate the effects of CHIPS in vivo. FPR1 was expressed in 29 out of 32 (90%) of all grades of astrocytomas. Two human mitochondrial-derived formylated peptides, formyl-methionil-leucine-lysine-isoleucine-valine (fMLKLIV) and formyl-methionil-methionil-tyrosine-alanine-leucine-phenylalanine (fMMYALF), were potent activators of FPR1 on tumour cells. Ligand-induced responses of FPR1-expressing tumour cells could be inhibited with FPR1 inhibitor CHIPS. Treatment of tumour-bearing mice with CHIPS slightly reduced tumour growth and improved survival as compared to non-treated animals (P=0.0019). Conclusion: Targeting FPR1 with CHIPS reduces cell motility and tumour cell activation, and prolongs the survival of tumour-bearing mice. This strategy could be explored in future research to improve treatment results for astrocytoma patients. PMID:23322202

A portable optical reader and wall projector towards enumeration of bio-conjugated beads or cells

PubMed Central

McArdle, Niamh A.; Kendlin, Jane L.; O’Connell, Triona M.; Ducrée, Jens

2017-01-01

Measurement of the height of a packed column of cells or beads, which can be direclty related to the number of cells or beads present in a chamber, is an important step in a number of diagnostic assays. For example, haematocrit measurements may rapidly identify anemia or polycthemia. Recently, user-friendly and cost-efficient Lab-on-a-Chip devices have been developed towards isolating and counting cell sub-populations for diagnostic purposes. In this work, we present a low-cost optical module for estimating the filling level of packed magnetic beads within a Lab-on-a-Chip device. The module is compatible with a previously introduced, disposable microfluidic chip for rapid determination of CD4+ cell counts. The device is a simple optical microscope module is manufactured by 3D printing. An objective lens directly interrogates the height of packed beads which are efficiently isolated on the finger-actuated chip. Optionally, an inexpensive, battery-powered Light Emitting Diode may project a shadow of the microfluidic chip at approximately 50-fold magnification onto a nearby surface. The reader is calibrated with the filling levels of known concentrations of paramagnetic beads within the finger actuated chip. Results in direct and projector mode are compared to measurements from a conventional, inverted white-light microscope. All three read-out methods indicate a maximum variation of 6.5% between methods. PMID:29267367

Differential gene expression analysis in glioblastoma cells and normal human brain cells based on GEO database.

PubMed

Wang, Anping; Zhang, Guibin

2017-11-01

The differentially expressed genes between glioblastoma (GBM) cells and normal human brain cells were investigated to performed pathway analysis and protein interaction network analysis for the differentially expressed genes. GSE12657 and GSE42656 gene chips, which contain gene expression profile of GBM were obtained from Gene Expression Omniub (GEO) database of National Center for Biotechnology Information (NCBI). The 'limma' data packet in 'R' software was used to analyze the differentially expressed genes in the two gene chips, and gene integration was performed using 'RobustRankAggreg' package. Finally, pheatmap software was used for heatmap analysis and Cytoscape, DAVID, STRING and KOBAS were used for protein-protein interaction, Gene Ontology (GO) and KEGG analyses. As results: i) 702 differentially expressed genes were identified in GSE12657, among those genes, 548 were significantly upregulated and 154 were significantly downregulated (p<0.01, fold-change >1), and 1,854 differentially expressed genes were identified in GSE42656, among the genes, 1,068 were significantly upregulated and 786 were significantly downregulated (p<0.01, fold-change >1). A total of 167 differentially expressed genes including 100 upregulated genes and 67 downregulated genes were identified after gene integration, and the genes showed significantly different expression levels in GBM compared with normal human brain cells (p<0.05). ii) Interactions between the protein products of 101 differentially expressed genes were identified using STRING and expression network was established. A key gene, called CALM3, was identified by Cytoscape software. iii) GO enrichment analysis showed that differentially expressed genes were mainly enriched in 'neurotransmitter:sodium symporter activity' and 'neurotransmitter transporter activity', which can affect the activity of neurotransmitter transportation. KEGG pathway analysis showed that the differentially expressed genes were mainly enriched in 'protein processing in endoplasmic reticulum', which can affect protein processing in endoplasmic reticulum. The results showed that: i) 167 differentially expressed genes were identified from two gene chips after integration; and ii) protein interaction network was established, and GO and KEGG pathway analyses were successfully performed to identify and annotate the key gene, which provide new insights for the studies on GBN at gene level.

Carbon nanotubes for voltage reduction and throughput enhancement of electrical cell lysis on a lab-on-a-chip.

PubMed

Shahini, Mehdi; Yeow, John T W

2011-08-12

We report on the enhancement of electrical cell lysis using carbon nanotubes (CNTs). Electrical cell lysis systems are widely utilized in microchips as they are well suited to integration into lab-on-a-chip devices. However, cell lysis based on electrical mechanisms has high voltage requirements. Here, we demonstrate that by incorporating CNTs into microfluidic electrolysis systems, the required voltage for lysis is reduced by half and the lysis throughput at low voltages is improved by ten times, compared to non-CNT microchips. In our experiment, E. coli cells are lysed while passing through an electric field in a microchannel. Based on the lightning rod effect, the electric field strengthened at the tip of the CNTs enhances cell lysis at lower voltage and higher throughput. This approach enables easy integration of cell lysis with other on-chip high-throughput sample-preparation processes.

On-chip enucleation of an oocyte by untethered microrobots

NASA Astrophysics Data System (ADS)

Ichikawa, Akihiko; Sakuma, Shinya; Sugita, Masakuni; Shoda, Tatsuro; Tamakoshi, Takahiro; Akagi, Satoshi; Arai, Fumihito

2014-09-01

We propose a novel on-chip enucleation of an oocyte with zona pellucida by using a combination of untethered microrobots. To achieve enucleation within the closed space of a microfluidic chip, two microrobots, a microknife and a microgripper were integrated into the microfluidic chip. These microrobots were actuated by an external magnetic force produced by permanent magnets placed on the robotic stage. The tip of the microknife was designed by considering the biological geometric feature of an oocyte, i.e. the oocyte has a polar body in maturation stage II. Moreover, the microknife was fabricated by using grayscale lithography, which allows fabrication of three-dimensional microstructures. The microgripper has a gripping function that is independent of the driving mechanism. On-chip enucleation was demonstrated, and the enucleated oocytes are spherical, indicating that the cell membrane of the oocytes remained intact. To confirm successful enucleation using this method, we investigated the viability of oocytes after enucleation. The results show that the production rate, i.e. the ratio between the number of oocytes that reach the blastocyst stage and the number of bovine oocytes after nucleus transfer, is 100%. The technique will contribute to complex cell manipulation such as cell surgery in lab-on-a-chip devices.

Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip

PubMed Central

Musah, Samira; Mammoto, Akiko; Ferrante, Thomas C.; Jeanty, Sauveur S. F.; Hirano-Kobayashi, Mariko; Mammoto, Tadanori; Roberts, Kristen; Chung, Seyoon; Novak, Richard; Ingram, Miles; Fatanat-Didar, Tohid; Koshy, Sandeep; Weaver, James C.; Church, George M.; Ingber, Donald E.

2017-01-01

An in vitro model of the human kidney glomerulus — the major site of blood filtration — could facilitate drug discovery and illuminate kidney-disease mechanisms. Microfluidic organ-on-a-chip technology has been used to model the human proximal tubule, yet a kidney-glomerulus-on-a-chip has not been possible because of the lack of functional human podocytes — the cells that regulate selective permeability in the glomerulus. Here, we demonstrate an efficient (> 90%) and chemically defined method for directing the differentiation of human induced pluripotent stem (hiPS) cells into podocytes that express markers of the mature phenotype (nephrin+, WT1+, podocin+, Pax2−) and that exhibit primary and secondary foot processes. We also show that the hiPS-cell-derived podocytes produce glomerular basement-membrane collagen and recapitulate the natural tissue/tissue interface of the glomerulus, as well as the differential clearance of albumin and inulin, when co-cultured with human glomerular endothelial cells in an organ-on-a-chip microfluidic device. The glomerulus-on-a-chip also mimics adriamycin-induced albuminuria and podocyte injury. This in vitro model of human glomerular function with mature human podocytes may facilitate drug development and personalized-medicine applications. PMID:29038743

Tailoring microfluidic systems for organ-like cell culture applications using multiphysics simulations

NASA Astrophysics Data System (ADS)

Hagmeyer, Britta; Schütte, Julia; Böttger, Jan; Gebhardt, Rolf; Stelzle, Martin

2013-03-01

Replacing animal testing with in vitro cocultures of human cells is a long-term goal in pre-clinical drug tests used to gain reliable insight into drug-induced cell toxicity. However, current state-of-the-art 2D or 3D cell cultures aiming at mimicking human organs in vitro still lack organ-like morphology and perfusion and thus organ-like functions. To this end, microfluidic systems enable construction of cell culture devices which can be designed to more closely resemble the smallest functional unit of organs. Multiphysics simulations represent a powerful tool to study the various relevant physical phenomena and their impact on functionality inside microfluidic structures. This is particularly useful as it allows for assessment of system functions already during the design stage prior to actual chip fabrication. In the HepaChip®, dielectrophoretic forces are used to assemble human hepatocytes and human endothelial cells in liver sinusoid-like structures. Numerical simulations of flow distribution, shear stress, electrical fields and heat dissipation inside the cell assembly chambers as well as surface wetting and surface tension effects during filling of the microchannel network supported the design of this human-liver-on-chip microfluidic system for cell culture applications. Based on the device design resulting thereof, a prototype chip was injection-moulded in COP (cyclic olefin polymer). Functional hepatocyte and endothelial cell cocultures were established inside the HepaChip® showing excellent metabolic and secretory performance.

Stochastic Analysis of Antibody-antigen Binding in a Microfluidic Device

NASA Astrophysics Data System (ADS)

Adams, Shauna; Zhang, Cong; Zambrano, Harvey; Conlisk, A. T.

2012-11-01

Over the last decade, microfluidic ``Labs on a Chip'' (LOC) have evolved from a single microchannel to micro-total analysis systems (TAS) capable of integrating thousands of reaction vessels, conduits and valves-the contents of an entire chemical laboratory-on a single chip. These systems have several advantages in biomedical applications, including lower equipment and personnel costs, reduced power requirements, faster separations, and smaller sample and reagent volume requirements. Circulating tumor cells (CTC) are cancer cells found in the blood stream indicating the presence of a tumor in the body. We consider the population of magnetically tagged antibodies to be characterized by a collection of stochastic trajectories; the probability of finding an antibody at a given position is assumed to be defined by the Fokker-Planck equation. The first objective is to determine the probability that one or more magnetically labeled antibodies will assume a trajectory that is within the neighborhood of a given cancer cell. Once this occurs the binding process can be described using a deterministic analysis and the modeling of this process is the second objective of the paper. Supported by the NSF Nanoscale Science and Engineering center (NSEC) for the Affordable Nanoengineering of Polymeric Biomedical Devices EEC-0914790.

Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 μm CMOS chip.

PubMed

Huys, Roeland; Braeken, Dries; Jans, Danny; Stassen, Andim; Collaert, Nadine; Wouters, Jan; Loo, Josine; Severi, Simone; Vleugels, Frank; Callewaert, Geert; Verstreken, Kris; Bartic, Carmen; Eberle, Wolfgang

2012-04-07

To cope with the growing needs in research towards the understanding of cellular function and network dynamics, advanced micro-electrode arrays (MEAs) based on integrated complementary metal oxide semiconductor (CMOS) circuits have been increasingly reported. Although such arrays contain a large number of sensors for recording and/or stimulation, the size of the electrodes on these chips are often larger than a typical mammalian cell. Therefore, true single-cell recording and stimulation remains challenging. Single-cell resolution can be obtained by decreasing the size of the electrodes, which inherently increases the characteristic impedance and noise. Here, we present an array of 16,384 active sensors monolithically integrated on chip, realized in 0.18 μm CMOS technology for recording and stimulation of individual cells. Successful recording of electrical activity of cardiac cells with the chip, validated with intracellular whole-cell patch clamp recordings are presented, illustrating single-cell readout capability. Further, by applying a single-electrode stimulation protocol, we could pace individual cardiac cells, demonstrating single-cell addressability. This novel electrode array could help pave the way towards solving complex interactions of mammalian cellular networks. This journal is © The Royal Society of Chemistry 2012

Expression Profiling Smackdown: Human Transcriptome Array HTA 2.0 vs. RNA-Seq

PubMed Central

Palermo, Meghann; Driscoll, Heather; Tighe, Scott; Dragon, Julie; Bond, Jeff; Shukla, Arti; Vangala, Mahesh; Vincent, James; Hunter, Tim

2014-01-01

The advent of both microarray and massively parallel sequencing have revolutionized high-throughput analysis of the human transcriptome. Due to limitations in microarray technology, detecting and quantifying coding transcript isoforms, in addition to non-coding transcripts, has been challenging. As a result, RNA-Seq has been the preferred method for characterizing the full human transcriptome, until now. A new high-resolution array from Affymetrix, GeneChip Human Transcriptome Array 2.0 (HTA 2.0), has been designed to interrogate all transcript isoforms in the human transcriptome with >6 million probes targeting coding transcripts, exon-exon splice junctions, and non-coding transcripts. Here we compare expression results from GeneChip HTA 2.0 and RNA-Seq data using identical RNA extractions from three samples each of healthy human mesothelial cells in culture, LP9-C1, and healthy mesothelial cells treated with asbestos, LP9-A1. For GeneChip HTA 2.0 sample preparation, we chose to compare two target preparation methods, NuGEN Ovation Pico WTA V2 with the Encore Biotin Module versus Affymetrix's GeneChip WT PLUS with the WT Terminal Labeling Kit, on identical RNA extractions from both untreated and treated samples. These same RNA extractions were used for the RNA-Seq library preparation. All analyses were performed in Partek Genomics Suite 6.6. Expression profiles for control and asbestos-treated mesothelial cells prepared with NuGEN versus Affymetrix target preparation methods (GeneChip HTA 2.0) are compared to each other as well as to RNA-Seq results.

Chip-based magnetic solid phase microextraction coupled with ICP-MS for the determination of Cd and Se in HepG2 cells incubated with CdSe quantum dots.

PubMed

Yu, Xiaoxiao; Chen, Beibei; He, Man; Wang, Han; Hu, Bin

2018-03-01

The quantification of trace Cd and Se in cells incubated with CdSe quantum dots (QDs) is critical to investigate the cytotoxicity of CdSe QDs. In this work, a miniaturized platform, namely chip-based magnetic solid phase microextraction (MSPME) packing with sulfhydryl group functionalized magnetic nanoparticles, was fabricated and combined with inductively coupled plasma mass spectrometry (ICP-MS) for the determination of trace Cd and Se in cells. Under the optimized conditions, the limits of detection (LOD) of the developed chip-based MSPME-ICP-MS system are 2.2 and 21ngL -1 for Cd and Se, respectively. The proposed method is applied successfully to the analysis of total and released small molecular fraction of Cd and Se in Human hepatocellular carcinoma cells (HepG2 cells) incubated with CdSe QDs, and the recoveries for the spiked samples are in the range of 86.0-109%. This method shows great promise to analyze cell samples and the obtained results are instructive to explore the cytotoxicity mechanism of CdSe QDs in cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Fully Integrated Microfluidic Device for Direct Sample-to-Answer Genetic Analysis

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Grodzinski, Piotr

Integration of microfluidics technology with DNA microarrays enables building complete sample-to-answer systems that are useful in many applications such as clinic diagnostics. In this chapter, a fully integrated microfluidic device [1] that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and a DNA microarray sensor to perform DNA analysis of complex biological sample solutions is present. This device can perform on-chip sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis) of complex biological sample solutions (such as whole blood), polymerase chain reaction, DNA hybridization, and electrochemical detection. A few novel microfluidic techniques were developed and employed. A micromix-ing technique based on a cavitation microstreaming principle was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads. This technique was also employed to accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from ~mL whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Bone marrow mesenchymal stem cells ameliorate inflammatory factor-induced dysfunction of INS-1 cells on chip.

PubMed

Sun, Yu; Yao, Zhina; Lin, Peng; Hou, Xinguo; Chen, Li

2014-05-01

Using a microfluidic chip, we have investigated whether bone marrow mesenchymal stem cells (BM-MSCs) could ameliorate IL-1β/IFN-γ-induced dysfunction of INS-1 cells. BM-MSCs were obtained from diabetes mellitus patients and their cell surface antigen expression profiles were analyzed by flow cytometric. INS-1 cells were cocultured with BM-MSCs on a microfluidic chip with persistent perfusion of medium containing 1 ng/mL IL-1β and 2.5 U/mL IFN-γ for 72 h. BM-MSCs could partially rescue INS-1 cells from cytokine-induced dysfunction and ameliorate the expression of insulin and PDX-1 gene in INS-1 cells. Thus BM-MSCs can be viewed as a promising stem cell source to depress inflammatory factor-induced dysfunction of pancreatic β cells in diabetic patients. © 2014 International Federation for Cell Biology.

3D gut-liver chip with a PK model for prediction of first-pass metabolism.

PubMed

Lee, Dong Wook; Ha, Sang Keun; Choi, Inwook; Sung, Jong Hwan

2017-11-07

Accurate prediction of first-pass metabolism is essential for improving the time and cost efficiency of drug development process. Here, we have developed a microfluidic gut-liver co-culture chip that aims to reproduce the first-pass metabolism of oral drugs. This chip consists of two separate layers for gut (Caco-2) and liver (HepG2) cell lines, where cells can be co-cultured in both 2D and 3D forms. Both cell lines were maintained well in the chip, verified by confocal microscopy and measurement of hepatic enzyme activity. We investigated the PK profile of paracetamol in the chip, and corresponding PK model was constructed, which was used to predict PK profiles for different chip design parameters. Simulation results implied that a larger absorption surface area and a higher metabolic capacity are required to reproduce the in vivo PK profile of paracetamol more accurately. Our study suggests the possibility of reproducing the human PK profile on a chip, contributing to accurate prediction of pharmacological effect of drugs.

Stability of the cancer target DDIAS is regulated by the CHIP/HSP70 pathway in lung cancer cells

PubMed Central

Won, Kyoung-Jae; Im, Joo-Young; Kim, Bo-Kyung; Ban, Hyun Seung; Jung, Young-Jin; Jung, Kyeong Eun; Won, Misun

2017-01-01

DNA damage-induced apoptosis suppressor (DDIAS) rescues lung cancer cells from apoptosis in response to DNA damage. DDIAS is transcriptionally activated by NFATc1 and EGF-mediated ERK5/MEF2B, leading to cisplatin resistance and cell invasion. Therefore, DDIAS is suggested as a therapeutic target for lung cancer. Here, we report that DDIAS stability is regulated by E3 U-box ubiquitin ligase carboxyl terminus of HSP70-interacting protein (CHIP)-mediated proteasomal degradation. We first isolated CHIP as an interacting partner of DDIAS by yeast two-hybrid screening. CHIP physically associated with both the N- and C-terminal regions of DDIAS, targeting it for proteasomal degradation and reducing the DDIAS half-life. CHIP overexpression analyses indicated that the tetratrico peptide repeat (TPR) domain and the U-box are required for DDIAS ubiquitination. It is likely that HSP70-bound DDIAS is recruited to the CHIP E3 ligase via the TPR domain, suggesting DDIAS as a client protein of HSP70. In addition, CHIP overexpression in lung cancer cells expressing high DDIAS levels induced significant growth inhibition by enhancing DDIAS degradation. Furthermore, simultaneous CHIP overexpression and DNA damage agent treatment caused a substantial increase in the apoptosis of lung cancer cells. Taken together, these findings indicate that the stability of the DDIAS protein is regulated by CHIP/HSP70-mediated proteasomal degradation and that CHIP overexpression stimulates the apoptosis of lung cancer cells in response to DNA-damaging agents. PMID:28079882

Stability of the cancer target DDIAS is regulated by the CHIP/HSP70 pathway in lung cancer cells.

PubMed

Won, Kyoung-Jae; Im, Joo-Young; Kim, Bo-Kyung; Ban, Hyun Seung; Jung, Young-Jin; Jung, Kyeong Eun; Won, Misun

2017-01-12

DNA damage-induced apoptosis suppressor (DDIAS) rescues lung cancer cells from apoptosis in response to DNA damage. DDIAS is transcriptionally activated by NFATc1 and EGF-mediated ERK5/MEF2B, leading to cisplatin resistance and cell invasion. Therefore, DDIAS is suggested as a therapeutic target for lung cancer. Here, we report that DDIAS stability is regulated by E3 U-box ubiquitin ligase carboxyl terminus of HSP70-interacting protein (CHIP)-mediated proteasomal degradation. We first isolated CHIP as an interacting partner of DDIAS by yeast two-hybrid screening. CHIP physically associated with both the N- and C-terminal regions of DDIAS, targeting it for proteasomal degradation and reducing the DDIAS half-life. CHIP overexpression analyses indicated that the tetratrico peptide repeat (TPR) domain and the U-box are required for DDIAS ubiquitination. It is likely that HSP70-bound DDIAS is recruited to the CHIP E3 ligase via the TPR domain, suggesting DDIAS as a client protein of HSP70. In addition, CHIP overexpression in lung cancer cells expressing high DDIAS levels induced significant growth inhibition by enhancing DDIAS degradation. Furthermore, simultaneous CHIP overexpression and DNA damage agent treatment caused a substantial increase in the apoptosis of lung cancer cells. Taken together, these findings indicate that the stability of the DDIAS protein is regulated by CHIP/HSP70-mediated proteasomal degradation and that CHIP overexpression stimulates the apoptosis of lung cancer cells in response to DNA-damaging agents.

BAG2 Interferes with CHIP-Mediated Ubiquitination of HSP72.

PubMed

Schönbühler, Bianca; Schmitt, Verena; Huesmann, Heike; Kern, Andreas; Gamerdinger, Martin; Behl, Christian

2016-12-30

The maintenance of cellular proteostasis is dependent on molecular chaperones and protein degradation pathways. Chaperones facilitate protein folding, maturation, and degradation, and the particular fate of a misfolded protein is determined by the interaction of chaperones with co-chaperones. The co-factor CHIP (C-terminus of HSP70-inteacting protein, STUB1) ubiquitinates chaperone substrates and directs proteins to the cellular degradation systems. The activity of CHIP is regulated by two co-chaperones, BAG2 and HSPBP1, which are potent inhibitors of the E3 ubiquitin ligase activity. Here, we examined the functional correlation of HSP72, CHIP, and BAG2, employing human primary fibroblasts. We showed that HSP72 is a substrate of CHIP and that BAG2 efficiently prevented the ubiquitination of HSP72 in young cells as well as aged cells. Aging is associated with a decline in proteostasis and we observed increased protein levels of CHIP as well as BAG2 in senescent cells. Interestingly, the ubiquitination of HSP72 was strongly reduced during aging, which revealed that BAG2 functionally counteracted the increased levels of CHIP. Interestingly, HSPBP1 protein levels were down-regulated during aging. The data presented here demonstrates that the co-chaperone BAG2 influences HSP72 protein levels and is an important modulator of the ubiquitination activity of CHIP in young as well as aged cells.

Lensfree super-resolution holographic microscopy using wetting films on a chip

NASA Astrophysics Data System (ADS)

Mudanyali, Onur; Bishara, Waheb; Ozcan, Aydogan

2011-08-01

We investigate the use of wetting films to significantly improve the imaging performance of lensfree pixel super-resolution on-chip microscopy, achieving < 1 μm spatial resolution over a large imaging area of ~24 mm2. Formation of an ultra-thin wetting film over the specimen effectively creates a micro-lens effect over each object, which significantly improves the signal-to-noise-ratio and therefore the resolution of our lensfree images. We validate the performance of this approach through lensfree on-chip imaging of various objects having fine morphological features (with dimensions of e.g., ≤0.5 μm) such as Escherichia coli (E. coli), human sperm, Giardia lamblia trophozoites, polystyrene micro beads as well as red blood cells. These results are especially important for the development of highly sensitive field-portable microscopic analysis tools for resource limited settings.

Rat Bone Marrow-Derived Schwann-Like Cells Differentiated by the Optimal Inducers Combination on Microfluidic Chip and Their Functional Performance

PubMed Central

Lv, Decheng

2012-01-01

Numerous researches demonstrated the possibility of derivation of Schwann-like (SC-like) cells in vitro from bone marrow stromal cells (BMSCs). However, the concentration of the induce factors were different in those studies, especially for the critical factors forskolin (FSK) and β-heregulin (HRG). Here, we used a new and useful method to build an integrated microfluidic chip for rapid analyses of the optimal combination between the induce factors FSK and HRG. The microfluidic device was mainly composed of an upstream concentration gradient generator (CGG) and a downstream cell culture module. Rat BMSCs were cultured in the cell chambers for 11 days at the different concentrations of induce factors generated by CGG. The result of immunofluorescence staining on-chip showed that the group of 4.00 µM FSK and 250.00 ng/ml HRG presented an optimal effect to promote the derivation of SC-like cells. Moreover, the optimal SC-like cells obtained on-chip were further tested using DRG co-culture and ELISA to detect their functional performance. Our findings demonstrate that SC-like cells could be obtained with high efficiency and functional performance in the optimal inducers combination. PMID:22880114

Rat bone marrow-derived Schwann-like cells differentiated by the optimal inducers combination on microfluidic chip and their functional performance.

PubMed

Tian, Xiliang; Wang, Shouyu; Zhang, Zhen; Lv, Decheng

2012-01-01

Numerous researches demonstrated the possibility of derivation of Schwann-like (SC-like) cells in vitro from bone marrow stromal cells (BMSCs). However, the concentration of the induce factors were different in those studies, especially for the critical factors forskolin (FSK) and β-heregulin (HRG). Here, we used a new and useful method to build an integrated microfluidic chip for rapid analyses of the optimal combination between the induce factors FSK and HRG. The microfluidic device was mainly composed of an upstream concentration gradient generator (CGG) and a downstream cell culture module. Rat BMSCs were cultured in the cell chambers for 11 days at the different concentrations of induce factors generated by CGG. The result of immunofluorescence staining on-chip showed that the group of 4.00 µM FSK and 250.00 ng/ml HRG presented an optimal effect to promote the derivation of SC-like cells. Moreover, the optimal SC-like cells obtained on-chip were further tested using DRG co-culture and ELISA to detect their functional performance. Our findings demonstrate that SC-like cells could be obtained with high efficiency and functional performance in the optimal inducers combination.

Combined Dielectrophoresis and Impedance Systems for Bacteria Analysis in Microfluidic On-Chip Platforms

PubMed Central

Páez-Avilés, Cristina; Juanola-Feliu, Esteve; Punter-Villagrasa, Jaime; del Moral Zamora, Beatriz; Homs-Corbera, Antoni; Colomer-Farrarons, Jordi; Miribel-Català, Pere Lluís; Samitier, Josep

2016-01-01

Bacteria concentration and detection is time-consuming in regular microbiology procedures aimed to facilitate the detection and analysis of these cells at very low concentrations. Traditional methods are effective but often require several days to complete. This scenario results in low bioanalytical and diagnostic methodologies with associated increased costs and complexity. In recent years, the exploitation of the intrinsic electrical properties of cells has emerged as an appealing alternative approach for concentrating and detecting bacteria. The combination of dielectrophoresis (DEP) and impedance analysis (IA) in microfluidic on-chip platforms could be key to develop rapid, accurate, portable, simple-to-use and cost-effective microfluidic devices with a promising impact in medicine, public health, agricultural, food control and environmental areas. The present document reviews recent DEP and IA combined approaches and the latest relevant improvements focusing on bacteria concentration and detection, including selectivity, sensitivity, detection time, and conductivity variation enhancements. Furthermore, this review analyses future trends and challenges which need to be addressed in order to successfully commercialize these platforms resulting in an adequate social return of public-funded investments. PMID:27649201

Systematic analysis of CMOS-micromachined inductors with application to mixer matching circuits

NASA Astrophysics Data System (ADS)

Wu, Jerry Chun-Li

The growing demand for consumer voice and data communication systems and military communication applications has created a need for low-power, low-cost, high-performance radio-frequency (RF) front-end. To achieve this goal, bringing passive components, especially inductors, to silicon is imperative. On-chip passive components such as inductors and capacitors generally enhance the reliability and efficiency of silicon-integrated RF cells. They can provide circuit solutions with superior performance and contribute to a higher level of integration. With passive components on chip, there is a great opportunity to have transformers, filters, and matching networks on chip. However, inductors on silicon have a low quality factor (Q) due to both substrate and metal loss. This dissertation demonstrates the systematic analysis of inductors fabricated using standard complementary metal-oxide-semiconductor (CMOS) and micro-electro-mechanical (MEMS) system technologies. We report system-on-chip inductor modeling, simulation, and measurements of effective inductance and quality factors. In this analysis methodology, a number of systematic simulations are performed on regular and micromachined inductors with different parameters such as spiral topology, number of turns, outer diameter, thickness, and percentage of substrate removed by using micromachining technologies. Three different novel support structures of the micromachined spiral inductor are proposed, analyzed, and implemented for larger size suspended inductors. The sensitivity of the structure support and different degree of substrate etching by post-processing is illustrated. The results provide guidelines for the selection of inductor parameters, post-processing methodologies, and its spiral supports to meet the RF design specifications and the stability requirements for mobile communication. The proposed CMOS-micromachined inductor is used in a low cost-effective double-balanced Gilbert mixer with on-chip matching network. The integrated mixer inductor was implemented and tested to prove the concept.

CHIP regulates bone mass by targeting multiple TRAF family members in bone marrow stromal cells.

PubMed

Wang, Tingyu; Li, Shan; Yi, Dan; Zhou, Guang-Qian; Chang, Zhijie; Ma, Peter X; Xiao, Guozhi; Chen, Di

2018-01-01

Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that Chip deficient mice display bone loss phenotype due to increased osteoclast formation through enhancing TRAF6 activity in osteoclasts. In this study we provide novel evidence about the function of CHIP. We found that osteoblast differentiation and bone formation were also decreased in Chip KO mice. In bone marrow stromal (BMS) cells derived from Chip -/- mice, expression of a panel of osteoblast marker genes was significantly decreased. ALP activity and mineralized bone matrix formation were also reduced in Chip- deficient BMS cells. We also found that in addition to the regulation of TRAF6, CHIP also inhibits TNFα-induced NF-κB signaling through promoting TRAF2 and TRAF5 degradation. Specific deletion of Chip in BMS cells downregulated expression of osteoblast marker genes which could be reversed by the addition of NF-κB inhibitor. These results demonstrate that the osteopenic phenotype observed in Chip -/- mice was due to the combination of increased osteoclast formation and decreased osteoblast differentiation. Taken together, our findings indicate a significant role of CHIP in bone remodeling.

A versatile quantitation platform based on platinum nanoparticles incorporated volumetric bar-chart chip for highly sensitive assays.

PubMed

Wang, Yuzhen; Zhu, Guixian; Qi, Wenjin; Li, Ying; Song, Yujun

2016-11-15

Platinum nanoparticles incorporated volumetric bar-chart chip (PtNPs-V-Chip) is able to be used for point-of-care tests by providing quantitative and visualized readout without any assistance from instruments, data processing, or graphic plotting. To improve the sensitivity of PtNPs-V-Chip, hybridization chain reaction was employed in this quantitation platform for highly sensitive assays that can detect as low as 16 pM Ebola Virus DNA, 0.01ng/mL carcinoembryonic antigen (CEA), and the 10 HER2-expressing cancer cells. Based on this amplified strategy, a 100-fold decrease of detection limit was achieved for DNA by improving the number of platinum nanoparticle catalyst for the captured analyte. This quantitation platform can also distinguish single base mismatch of DNA hybridization and observe the concentration threshold of CEA. The new strategy lays the foundation for this quantitation platform to be applied in forensic analysis, biothreat detection, clinical diagnostics and drug screening. Copyright © 2016 Elsevier B.V. All rights reserved.

The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance

PubMed Central

Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L.; Abbas, Tarek; Larner, James M.

2017-01-01

Lung cancer resists radiation therapy, making it one of the deadliest forms of cancer. Here we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor p21 protein is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene (CDKN1A) in CHIP knockdown cells. Conversely, over-expression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone heat shock protein 70 (HSP70). These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity; thus, suggesting a novel strategy for the treatment of lung cancer. Implications The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. PMID:28232384

The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance.

PubMed

Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L; Abbas, Tarek; Larner, James M

2017-06-01

Lung cancer resists radiotherapy, making it one of the deadliest forms of cancer. Here, we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically, ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor, p21 protein, is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene ( CDKN1A) in CHIP knockdown cells. Conversely, overexpression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone HSP70. These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity, thus suggesting a novel strategy for the treatment of lung cancer. Implications: The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. Mol Cancer Res; 15(6); 651-9. ©2017 AACR . ©2017 American Association for Cancer Research.

Organ/body-on-a-chip based on microfluidic technology for drug discovery.

PubMed

Kimura, Hiroshi; Sakai, Yasuyuki; Fujii, Teruo

2018-02-01

Although animal experiments are indispensable for preclinical screening in the drug discovery process, various issues such as ethical considerations and species differences remain. To solve these issues, cell-based assays using human-derived cells have been actively pursued. However, it remains difficult to accurately predict drug efficacy, toxicity, and organs interactions, because cultivated cells often do not retain their original organ functions and morphologies in conventional in vitro cell culture systems. In the μTAS research field, which is a part of biochemical engineering, the technologies of organ-on-a-chip, based on microfluidic devices built using microfabrication, have been widely studied recently as a novel in vitro organ model. Since it is possible to physically and chemically mimic the in vitro environment by using microfluidic device technology, maintenance of cellular function and morphology, and replication of organ interactions can be realized using organ-on-a-chip devices. So far, functions of various organs and tissues, such as the lung, liver, kidney, and gut have been reproduced as in vitro models. Furthermore, a body-on-a-chip, integrating multi organ functions on a microfluidic device, has also been proposed for prediction of organ interactions. We herein provide a background of microfluidic systems, organ-on-a-chip, Body-on-a-chip technologies, and their challenges in the future. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Expanding the horizons for single-cell applications on lab-on-a-chip devices.

PubMed

Kim, Soo Hyeon; Fourmy, Dominique; Fujii, Teruo

2012-01-01

Stochastic events in gene expression, protein synthesis, and metabolite synthesis or degradation lead to cellular heterogeneity essential to life. In a tissue as we see in organs, there is strong heterogeneity among the constituting cells critical to its function. Thus, there exists a strong demand to develop new micro/nanosystems that would enable us to conduct single-cell analysis. This field is rapidly growing, as exemplified below with recent emerging technologies that now reveal sensitive single-cell "omics" analysis. We describe in the review some of the most promising technologies that will certainly transform our view of biology in the near future.

Intracellular protein determination using droplet-based immunoassays.

PubMed

Martino, Chiara; Zagnoni, Michele; Sandison, Mairi E; Chanasakulniyom, Mayuree; Pitt, Andrew R; Cooper, Jonathan M

2011-07-01

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ~50 pM to 1 μM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells.

Organ-on-a-chip: development and clinical prospects toward toxicity assessment with an emphasis on bone marrow.

PubMed

Kim, Jeehye; Lee, Hanna; Selimović, Šeila; Gauvin, Robert; Bae, Hojae

2015-05-01

Conventional approaches for toxicity evaluation of drugs and chemicals, such as animal tests, can be impractical due to the large experimental scale and the immunological differences between species. Organ-on-a-chip models have recently been recognized as a prominent alternative to conventional toxicity tests aiming to simulate the human in vivo physiology. This review focuses on the organ-on-a-chip applications for high-throughput screening of candidate drugs against toxicity, with a particular emphasis on bone-marrow-on-a-chip. Studies in which organ-on-a-chip models have been developed and utilized to maximize the efficiency and predictability in toxicity assessment are introduced. The potential of these devices to replace tests of acute systemic toxicity in animals, and the challenges that are inherent in simulating the human immune system are also discussed. As a promising approach to overcome the limitations, we further focus on an in-depth analysis of the development of bone-marrow-on-a-chip that is capable of simulating human immune responses against external stimuli due to the key roles of marrow in immune systems with hematopoietic activities. Owing to the complex interactions between hematopoietic stem cells and marrow microenvironments, precise control of both biochemical and physical niches that are critical in maintenance of hematopoiesis remains a key challenge. Thus, recently developed bone-marrow-on-a-chip models support immunogenicity and immunotoxicity testing in long-term cultivation with repeated antigen stimulation. In this review, we provide an overview of clinical studies that have been carried out on bone marrow transplants in patients with immune-related diseases and future aspects of clinical and pharmaceutical application of bone-marrow-on-a-chip.

Separation and dual detection of prostate cancer cells and protein biomarkers using a microchip device.

PubMed

Huang, Wanfeng; Chang, Chun-Li; Brault, Norman D; Gur, Onur; Wang, Zhe; Jalal, Shadia I; Low, Philip S; Ratliff, Timothy L; Pili, Roberto; Savran, Cagri A

2017-01-31

Current efforts for the detection of prostate cancer using only prostate specific antigen are not ideal and indicate a need to develop new assays - using multiple targets - that can more accurately stratify disease states. We previously introduced a device capable of the concurrent detection of cellular and molecular markers from a single sample fluid. Here, an improved design, which achieves affinity as well as size-based separation of captured targets using antibody-conjugated magnetic beads and a silicon chip containing micro-apertures, is presented. Upon injection of the sample, the integration of magnetic attraction with the micro-aperture chip permits larger cell-bead complexes to be isolated in an upper chamber with the smaller protein-bead complexes and remaining beads passing through the micro-apertures into the lower chamber. This enhances captured cell purity for on chip quantification, allows the separate retrieval of captured cells and proteins for downstream analysis, and enables higher bead concentrations for improved multiplexed ligand targeting. Using LNCaP cells and prostate specific membrane antigen (PSMA) to model prostate cancer, the device was able to detect 34 pM of spiked PSMA and achieve a cell capture efficiency of 93% from culture media. LNCaP cells and PSMA were then spiked into diluted healthy human blood to mimic a cancer patient. The device enabled the detection of spiked PSMA (relative to endogenous PSMA) while recovering 85-90% of LNCaP cells which illustrated the potential of new assays for the diagnosis of prostate cancer.

Suitability of shredded tyres as a substitute for a landfill leachate collection medium.

PubMed

Park, Jae K; Edil, Tuncer B; Kim, Jae Y; Huh, Mock; Lee, Sung Ho; Lee, Jung Jun

2003-06-01

A series of tests were conducted to investigate the fate of heavy metals and gasoline components in a simulated landfill, consisting of a 30 cm thick clay liner and a leachate collection layer containing tyres as well as in two test cells installed in a landfill. Arsenic, selenium, mercury, barium, and lead concentrations were lower while zinc concentration was higher in the tank containing tyre-chips than the tank without tyre-chips. When samples were filtered, however, concentrations of zinc as well as other inorganics were lower in the tank containing tyre-chips, indicating that metals in the leachate exposed to tyre-chips travel more slowly in a subsurface environment due to filtering effect. In a test cell study, arsenic, cobalt, lead and nickel concentrations were lower in the cell containing tyre-chips than in the cell without tyre-chips, except iron and zinc. Both tests indicate that some inorganic contaminants are sorbed to tyre-chips. Gasoline components were also significantly sorbed by tyre-chips in field cell tests. Although tyre-chips are known to leach organic and inorganic contaminants, concentrations in field conditions will be lower than the reported experimental results since the tests were performed under worst-case scenarios. If tyre-chips are used in areas where contamination levels are high, then they can be used as a sorbent for environmental clean-up.

A self-testing dynamic RAM chip

NASA Astrophysics Data System (ADS)

You, Y.; Hayes, J. P.

1985-02-01

A novel approach to making very large dynamic RAM chips self-testing is presented. It is based on two main concepts: on-chip generation of regular test sequences with very high fault coverage, and concurrent testing of storage-cell arrays to reduce overall testing time. The failure modes of a typical 64 K RAM employing one-transistor cells are analyzed to identify their test requirements. A comprehensive test generation algorithm that can be implemented with minimal modification to a standard cell layout is derived. The self-checking peripheral circuits necessary to implement this testing algorithm are described, and the self-testing RAM is briefly evaluated.

Microfluidic technologies for synthetic biology.

PubMed

Vinuselvi, Parisutham; Park, Seongyong; Kim, Minseok; Park, Jung Min; Kim, Taesung; Lee, Sung Kuk

2011-01-01

Microfluidic technologies have shown powerful abilities for reducing cost, time, and labor, and at the same time, for increasing accuracy, throughput, and performance in the analysis of biological and biochemical samples compared with the conventional, macroscale instruments. Synthetic biology is an emerging field of biology and has drawn much attraction due to its potential to create novel, functional biological parts and systems for special purposes. Since it is believed that the development of synthetic biology can be accelerated through the use of microfluidic technology, in this review work we focus our discussion on the latest microfluidic technologies that can provide unprecedented means in synthetic biology for dynamic profiling of gene expression/regulation with high resolution, highly sensitive on-chip and off-chip detection of metabolites, and whole-cell analysis.

Implementing oxygen control in chip-based cell and tissue culture systems.

PubMed

Oomen, Pieter E; Skolimowski, Maciej D; Verpoorte, Elisabeth

2016-09-21

Oxygen is essential in the energy metabolism of cells, as well as being an important regulatory parameter influencing cell differentiation and function. Interest in precise oxygen control for in vitro cultures of tissues and cells continues to grow, especially with the emergence of the organ-on-a-chip and the desire to emulate in vivo conditions. This was recently discussed in this journal in a Critical Review by Brennan et al. (Lab Chip (2014). DOI: ). Microfluidics can be used to introduce flow to facilitate nutrient supply to and waste removal from in vitro culture systems. Well-defined oxygen gradients can also be established. However, cells can quickly alter the oxygen balance in their vicinity. In this Tutorial Review, we expand on the Brennan paper to focus on the implementation of oxygen analysis in these systems to achieve continuous monitoring. Both electrochemical and optical approaches for the integration of oxygen monitoring in microfluidic tissue and cell culture systems will be discussed. Differences in oxygen requirements from one organ to the next are a challenging problem, as oxygen delivery is limited by its uptake into medium. Hence, we discuss the factors determining oxygen concentrations in solutions and consider the possible use of artificial oxygen carriers to increase dissolved oxygen concentrations. The selection of device material for applications requiring precise oxygen control is discussed in detail, focusing on oxygen permeability. Lastly, a variety of devices is presented, showing the diversity of approaches that can be employed to control and monitor oxygen concentrations in in vitro experiments.

Preservation of Cell Structure, Metabolism, and Biotransformation Activity of Liver-On-Chip Organ Models by Hypothermic Storage.

PubMed

Gröger, Marko; Dinger, Julia; Kiehntopf, Michael; Peters, Frank T; Rauen, Ursula; Mosig, Alexander S

2018-01-01

The liver is a central organ in the metabolization of nutrition, endogenous and exogenous substances, and xenobiotic drugs. The emerging organ-on-chip technology has paved the way to model essential liver functions as well as certain aspects of liver disease in vitro in liver-on-chip models. However, a broader use of this technology in biomedical research is limited by a lack of protocols that enable the short-term preservation of preassembled liver-on-chip models for stocking or delivery to researchers outside the bioengineering community. For the first time, this study tested the ability of hypothermic storage of liver-on-chip models to preserve cell viability, tissue morphology, metabolism and biotransformation activity. In a systematic study with different preservation solutions, liver-on-chip function can be preserved for up to 2 d using a derivative of the tissue preservation solution TiProtec, containing high chloride ion concentrations and the iron chelators LK614 and deferoxamine, supplemented with polyethylene glycol (PEG). Hypothermic storage in this solution represents a promising method to preserve liver-on-chip function for at least 2 d and allows an easier access to liver-on-chip technology and its versatile and flexible use in biomedical research. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Nano-Patch-Clamp Array: Microfabricated Glass Chips for High-Throughput Electrophysiology

NASA Astrophysics Data System (ADS)

Fertig, Niels

2003-03-01

Electrophysiology (i.e. patch clamping) remains the gold standard for pharmacological testing of putative ion channel active drugs (ICADs), but suffers from low throughput. A new ion channel screening technology based on microfabricated glass chip devices will be presented. The glass chips contain very fine apertures, which are used for whole-cell voltage clamp recordings as well as single channel recordings from mammalian cell lines. Chips containing multiple patch clamp wells will be used in a first bench-top device, which will allow perfusion and electrical readout of each well. This scalable technology will allow for automated, rapid and parallel screening on ion channel drug targets.

Simultaneous analysis of seven oligopeptides in microbial fuel cell by micro-fluidic chip with reflux injection mode.

PubMed

Wang, Wei; Wang, Zijian; Lin, Xiuli; Wang, ZongWen; Fu, FengFu

2012-10-15

In this work, a reflux injection mode for the cross form micro-fluidic chip was studied. This injection mode could flexibly control the length of sample plug from less than one channel width (<83 μm) to tens of channel widths (millimeter-sized) by adjusting the injection time. Namely, the separation resolution or sample detection sensitivity could be selectively improved by changing injection time. Composed of four steps, the reflux injection mode alleviated the electrophoretic sampling bias and prevented sample leakage successfully. On a micro-fluidic chip coupled with laser induced fluorescence (LIF) detector, the injection mode was applied to separate seven oligopeptides, namely GG, GL, RPP, KPV, VKK, WYD and YWS. All analytes were completely separated and detected within 12 min with detection limits of 25-625 nmol/L. At last, the proposed method had been successfully applied to detect oligopeptides consumed by bacillus licheniformis in anode chamber of microbial fuel cell (MFC) to study the effect of oligopeptides on the MFC running. Copyright © 2012 Elsevier B.V. All rights reserved.

Magnetic domain wall conduits for single cell applications.

PubMed

Donolato, M; Torti, A; Kostesha, N; Deryabina, M; Sogne, E; Vavassori, P; Hansen, M F; Bertacco, R

2011-09-07

The ability to trap, manipulate and release single cells on a surface is important both for fundamental studies of cellular processes and for the development of novel lab-on-chip miniaturized tools for biological and medical applications. In this paper we demonstrate how magnetic domain walls generated in micro- and nano-structures fabricated on a chip surface can be used to handle single yeast cells labeled with magnetic beads. In detail, first we show that the proposed approach maintains the microorganism viable, as proven by monitoring the division of labeled yeast cells trapped by domain walls over 16 hours. Moreover, we demonstrate the controlled transport and release of individual yeast cells via displacement and annihilation of individual domain walls in micro- and nano-sized magnetic structures. These results pave the way to the implementation of magnetic devices based on domain walls technology in lab-on-chip systems devoted to accurate individual cell trapping and manipulation.

A microfluidic approach to parallelized transcriptional profiling of single cells.

PubMed

Sun, Hao; Olsen, Timothy; Zhu, Jing; Tao, Jianguo; Ponnaiya, Brian; Amundson, Sally A; Brenner, David J; Lin, Qiao

2015-12-01

The ability to correlate single-cell genetic information with cellular phenotypes is of great importance to biology and medicine, as it holds the potential to gain insight into disease pathways that is unavailable from ensemble measurements. We present a microfluidic approach to parallelized, rapid, quantitative analysis of messenger RNA from single cells via RT-qPCR. The approach leverages an array of single-cell RT-qPCR analysis units formed by a set of parallel microchannels concurrently controlled by elastomeric pneumatic valves, thereby enabling parallelized handling and processing of single cells in a drastically simplified operation procedure using a relatively small number of microvalves. All steps for single-cell RT-qPCR, including cell isolation and immobilization, cell lysis, mRNA purification, reverse transcription and qPCR, are integrated on a single chip, eliminating the need for off-chip manual cell and reagent transfer and qPCR amplification as commonly used in existing approaches. Additionally, the approach incorporates optically transparent microfluidic components to allow monitoring of single-cell trapping without the need for molecular labeling that can potentially alter the targeted gene expression and utilizes a polycarbonate film as a barrier against evaporation to minimize the loss of reagents at elevated temperatures during the analysis. We demonstrate the utility of the approach by the transcriptional profiling for the induction of the cyclin-dependent kinase inhibitor 1a and the glyceraldehyde 3-phosphate dehydrogenase in single cells from the MCF-7 breast cancer cell line. Furthermore, the methyl methanesulfonate is employed to allow measurement of the expression of the genes in individual cells responding to a genotoxic stress.

On-Chip Biomedical Imaging

PubMed Central

Göröcs, Zoltán; Ozcan, Aydogan

2012-01-01

Lab-on-a-chip systems have been rapidly emerging to pave the way toward ultra-compact, efficient, mass producible and cost-effective biomedical research and diagnostic tools. Although such microfluidic and micro electromechanical systems achieved high levels of integration, and are capable of performing various important tasks on the same chip, such as cell culturing, sorting and staining, they still rely on conventional microscopes for their imaging needs. Recently several alternative on-chip optical imaging techniques have been introduced, which have the potential to substitute conventional microscopes for various lab-on-a-chip applications. Here we present a critical review of these recently emerging on-chip biomedical imaging modalities, including contact shadow imaging, lensfree holographic microscopy, fluorescent on-chip microscopy and lensfree optical tomography. PMID:23558399

In vitro study of the effects of ELF electric fields on gene expression in human epidermal cells.

PubMed

Collard, Jean-Francois; Mertens, Benjamin; Hinsenkamp, Maurice

2011-01-01

An acceleration of differentiation, at the expense of proliferation, is observed after exposure of various biological models to low frequency and low amplitude electric and electromagnetic fields. Following these results showing significant modifications, we try to identify the biological mechanism involved at the cell level through microarray screening. For this study, we use epidermis cultures harvested from human abdominoplasty. Two platinum electrodes are used to apply the electric signal. The gene expressions of 38,500 well-characterized human genes are analyzed using Affymetrix(®) microarray U133 Plus 2.0 chips. The protocol is repeated on three different patients. After three periods of exposure, a total of 24 chips have been processed. After the application of ELF electric fields, the microarray analysis confirms a modification of the gene expression of epidermis cells. Particularly, four up-regulated genes (DKK1, TXNRD1, ATF3, and MME) and one down-regulated gene (MACF1) are involved in the regulation of proliferation and differentiation. Expression of these five genes was also confirmed by real-time rtPCR in all samples used for microarray analysis. These results corroborate an acceleration of cell differentiation at the expense of cell proliferation. © 2010 Wiley-Liss, Inc.

Micro-array isolation of circulating tumor cells (CTCs): the droplet biopsy chip

NASA Astrophysics Data System (ADS)

Panchapakesan, B.

2017-08-01

We present a new method for circulating tumor cell capture based on micro-array isolation from droplets. Called droplet biopsy, our technique uses a 76-element array of carbon nanotube devices functionalized with anti-EpCAM and antiHer2 antibodies for immunocapture of spiked breast cancer cells in the blood. This droplet biopsy chip can enable capture of CTCs based on both positive and negative selection strategy. Negative selection is achieved through depletion of contaminating leukocytes through the differential settling of blood into layers. We report 55%-100% cancer cell capture yield in this first droplet biopsy chip study. The droplet biopsy is an enabling idea where one can capture CTCs based on multiple biomarkers in a single blood sample.

Modeling Hematopoiesis and Responses to Radiation Countermeasures in a Bone Marrow-on-a-Chip.

PubMed

Torisawa, Yu-Suke; Mammoto, Tadanori; Jiang, Elisabeth; Jiang, Amanda; Mammoto, Akiko; Watters, Alexander L; Bahinski, Anthony; Ingber, Donald E

2016-05-01

Studies on hematopoiesis currently rely on animal models because in vitro culture methods do not accurately recapitulate complex bone marrow physiology. We recently described a bone marrow-on-a-chip microfluidic device that enables the culture of living hematopoietic bone marrow and mimics radiation toxicity in vitro. In the present study, we used this microdevice to demonstrate continuous blood cell production in vitro and model bone marrow responses to potential radiation countermeasure drugs. The device maintained mouse hematopoietic stem and progenitor cells in normal proportions for at least 2 weeks in culture. Increases in the number of leukocytes and red blood cells into the microfluidic circulation also could be detected over time, and addition of erythropoietin induced a significant increase in erythrocyte production. Exposure of the bone marrow chip to gamma radiation resulted in reduction of leukocyte production, and treatment of the chips with two potential therapeutics, granulocyte-colony stimulating factor or bactericidal/permeability-increasing protein (BPI), induced significant increases in the number of hematopoietic stem cells and myeloid cells in the fluidic outflow. In contrast, BPI was not found to have any effect when analyzed using static marrow cultures, even though it has been previously shown to accelerate recovery from radiation-induced toxicity in vivo. These findings demonstrate the potential value of the bone marrow-on-a-chip for modeling blood cell production, monitoring responses to hematopoiesis-modulating drugs, and testing radiation countermeasures in vitro.

Fabrication and characterization of microfabricated on-chip microelectrochemical cell for biosensing applications

NASA Astrophysics Data System (ADS)

Said, N. A. Mohd; Twomey, K.; Herzog, G.; Ogurtsov, V. I.

2017-03-01

The fabrication of on-chip microelectrochemical cell on Si wafer by means of photolithography is described here. The single on-chip microelectrochemical cell device has dimensions of 100 × 380 mm with integrated Pt counter electrode (CE), Ag/AgCl reference electrode (RE) and gold microelectrode array of 500 nm recess depth as the working electrode (WE). Two geometries of electrode array were implemented, band and disc, with fixed diameter/width of 10 µm; and varied centre-to-centre spacing (d) and number of electrodes (N) in the array. The on-chip microelectrochemical cell structure has been designed to facilitate further WE biomodifications. Firstly, the developed microelectrochemical cell does not require packaging hence reducing the production cost and time. Secondly, the working electrode (WE) on the microelectrochemical cell is positioned towards the end of the chip enabling modification of the working electrode surface to be carried out for surface bio-functionalisation without affecting both the RE and CE surface conditions. The developed on-chip microelectrochemical cell was examined with scanning electron microscopy (SEM) and characterised by two electrochemical techniques. Both cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed in 1 mM ferrocenecarboxylic acid (FCA) in 0.01 M phosphate buffered saline (PBS) solution at pH7.4. Electrochemical experiments showed that in the case of halving the interspacing distance of the microdisc WE array (50 nm instead of 100 nm), the voltammogram shifted from a steady-state CV (feature of hemispherical diffusion) to an inclined peak-shaped CV (feature of linear diffusion) albeit the arrays had the same surface area. In terms of EIS it was also found that linear diffusion dominates the surface instead of hemispherical diffusion once the interspacing distance was reduced, supporting the fact that closely packed arrays may behave like a macroelectrode

Wideband Fully-Programmable Dual-Mode CMOS Analogue Front-End for Electrical Impedance Spectroscopy

PubMed Central

Valente, Virgilio; Demosthenous, Andreas

2016-01-01

This paper presents a multi-channel dual-mode CMOS analogue front-end (AFE) for electrochemical and bioimpedance analysis. Current-mode and voltage-mode readouts, integrated on the same chip, can provide an adaptable platform to correlate single-cell biosensor studies with large-scale tissue or organ analysis for real-time cancer detection, imaging and characterization. The chip, implemented in a 180-nm CMOS technology, combines two current-readout (CR) channels and four voltage-readout (VR) channels suitable for both bipolar and tetrapolar electrical impedance spectroscopy (EIS) analysis. Each VR channel occupies an area of 0.48 mm2, is capable of an operational bandwidth of 8 MHz and a linear gain in the range between −6 dB and 42 dB. The gain of the CR channel can be set to 10 kΩ, 50 kΩ or 100 kΩ and is capable of 80-dB dynamic range, with a very linear response for input currents between 10 nA and 100 μA. Each CR channel occupies an area of 0.21 mm2. The chip consumes between 530 μA and 690 μA per channel and operates from a 1.8-V supply. The chip was used to measure the impedance of capacitive interdigitated electrodes in saline solution. Measurements show close matching with results obtained using a commercial impedance analyser. The chip will be part of a fully flexible and configurable fully-integrated dual-mode EIS system for impedance sensors and bioimpedance analysis. PMID:27463721

Proteomic analysis of single mammalian cells enabled by microfluidic nanodroplet sample preparation and ultrasensitive nanoLC-MS.

PubMed

Zhu, Ying; Clair, Geremy; Chrisler, William; Shen, Yufeng; Zhao, Rui; Shukla, Anil; Moore, Ronald; Misra, Ravi; Pryhuber, Gloria; Smith, Richard; Ansong, Charles; Kelly, Ryan T

2018-05-24

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analysed by ultrasensitive nanoLC-MS. An average of ~670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Downstream targets of HOXB4 in a cell line model of primitive hematopoietic progenitor cells.

PubMed

Lee, Han M; Zhang, Hui; Schulz, Vincent; Tuck, David P; Forget, Bernard G

2010-08-05

Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell self-renewal and expansion ex vivo and in vivo. To investigate the downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line EML. Two genome-wide analytical techniques were used: RNA expression profiling using microarrays and chromatin immunoprecipitation (ChIP)-chip. RNA expression profiling revealed that 465 gene transcripts were differentially expressed in KLS (c-Kit(+), Lin(-), Sca-1(+))-EML cells that overexpressed HOXB4 (KLS-EML-HOXB4) compared with control KLS-EML cells that were transduced with vector alone. In particular, erythroid-specific gene transcripts were observed to be highly down-regulated in KLS-EML-HOXB4 cells. ChIP-chip analysis revealed that the promoter region for 1910 genes, such as CD34, Sox4, and B220, were occupied by HOXB4 in KLS-EML-HOXB4 cells. Side-by-side comparison of the ChIP-chip and RNA expression profiling datasets provided correlative information and identified Gp49a and Laptm4b as candidate "stemness-related" genes. Both genes were highly ranked in both dataset lists and have been previously shown to be preferentially expressed in hematopoietic stem cells and down-regulated in mature hematopoietic cells, thus making them attractive candidates for future functional studies in hematopoietic cells.

1-Million droplet array with wide-field fluorescence imaging for digital PCR.

PubMed

Hatch, Andrew C; Fisher, Jeffrey S; Tovar, Armando R; Hsieh, Albert T; Lin, Robert; Pentoney, Stephen L; Yang, David L; Lee, Abraham P

2011-11-21

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.

CHIP is a novel tumor suppressor in pancreatic cancer and inhibits tumor growth through targeting EGFR

PubMed Central

Wang, Tianxiao; Yang, Jingxuan; Xu, Jianwei; Li, Jian; Cao, Zhe; Zhou, Li; You, Lei; Shu, Hong; Lu, Zhaohui; Li, Huihua; Li, Min; Zhang, Taiping; Zhao, Yupei

2014-01-01

Carboxyl terminus of heat shock protein 70-interacting protein (CHIP) is an E3 ubiquitin ligase that is involved in protein quality control and mediates several tumor-related proteins in many cancers, but the function of CHIP in pancreatic cancer is not known. Here we show that CHIP interacts and ubiquitinates epidermal growth factor receptor (EGFR) for proteasome-mediated degradation in pancreatic cancer cells, thereby inhibiting the activation of EGFR downstream pathways. CHIP suppressed cell proliferation, anchor-independent growth, invasion and migration, as well as enhanced apoptosis induced by erlotinib in vitro and in vivo. The expression of CHIP was decreased in pancreatic cancer tissues or sera. Low CHIP expression in tumor tissues was correlated with tumor differentiation and shorter overall survival. These observations indicate that CHIP serves as a novel tumor suppressor by down-regulating EGFR pathway in pancreatic cancer cells, decreased expression of CHIP was associated with poor prognosis in pancreatic cancer. PMID:24722501

Enzyme catalysis-electrophoresis titration for multiplex enzymatic assay via moving reaction boundary chip.

PubMed

Zhong, Ran; Xie, Haiyang; Kong, Fanzhi; Zhang, Qiang; Jahan, Sharmin; Xiao, Hua; Fan, Liuyin; Cao, Chengxi

2016-09-21

In this work, we developed the concept of enzyme catalysis-electrophoresis titration (EC-ET) under ideal conditions, the theory of EC-ET for multiplex enzymatic assay (MEA), and a related method based on a moving reaction boundary (MRB) chip with a collateral channel and cell phone imaging. As a proof of principle, the model enzymes horseradish peroxidase (HRP), laccase and myeloperoxidase (MPO) were chosen for the tests of the EC-ET model. The experiments revealed that the EC-ET model could be achieved via coupling EC with ET within a MRB chip; particularly the MEA analyses of catalysis rate, maximum rate, activity, Km and Kcat could be conducted via a single run of the EC-ET chip, systemically demonstrating the validity of the EC-ET theory. Moreover, the developed method had these merits: (i) two orders of magnitude higher sensitivity than a fluorescence microplate reader, (ii) simplicity and low cost, and (iii) fairly rapid (30 min incubation, 20 s imaging) analysis, fair stability (<5.0% RSD) and accuracy, thus validating the EC-ET method. Finally, the developed EC-ET method was used for the clinical assay of MPO activity in blood samples; the values of MPO activity detected via the EC-ET chip were in agreement with those obtained by a traditional fluorescence microplate reader, indicating the applicability of the EC-ET method. The work opens a window for the development of enzymatic research, enzyme assay, immunoassay, and point-of-care testing as well as titration, one of the oldest methods of analysis, based on a simple chip.

Microfluidic Purification and Concentration of Malignant Pleural Effusions for Improved Molecular and Cytomorphological Diagnostics

PubMed Central

Go, Derek E.; Talati, Ish; Ying, Yong; Rao, Jianyu; Kulkarni, Rajan P.; Di Carlo, Dino

2013-01-01

Evaluation of pleural fluids for metastatic cells is a key component of diagnostic cytopathology. However, a large background of smaller leukocytes and/or erythrocytes can make accurate diagnosis difficult and reduce specificity in identification of mutations of interest for targeted anti-cancer therapies. Here, we describe an automated microfluidic system (Centrifuge Chip) which employs microscale vortices for the size-based isolation and concentration of cancer cells and mesothelial cells from a background of blood cells. We are able to process non-diluted pleural fluids at 6 mL/min and enrich target cells significantly over the background; we achieved improved purity in all patient samples analyzed. The resulting isolated and viable cells are readily available for immunostaining, cytological analysis, and detection of gene mutations. To demonstrate the utility towards aiding companion diagnostics, we also show improved detection accuracy of KRAS gene mutations in lung cancer cells processed using the Centrifuge Chip, leading to an increase in the area under the curve (AUC) of the receiver operating characteristic from 0.90 to 0.99. The Centrifuge Chip allows for rapid concentration and processing of large volumes of bodily fluid samples for improved cytological diagnosis and purification of cells of interest for genetic testing, which will be helpful for enhancing diagnostic accuracy. PMID:24205153

Capture of mesothelioma cells with 'universal' CTC-chip.

PubMed

Yoneda, Kazue; Chikaishi, Yasuhiro; Kuwata, Taiji; Ohnaga, Takashi; Tanaka, Fumihiro

2018-02-01

Malignant mesothelioma (MM) is a highly aggressive malignant tumor, predominantly associated with job-related exposure to asbestos. Development of effective and non-invasive modalities for diagnosis is an important issue in occupational medicine. Circulating tumor cells (CTCs), which are tumor cells that are shed from primary tumors and circulate in the peripheral blood, may be detected at an earlier stage than malignant tumors, and detection of CTCs may provide a novel insight into the diagnosis of MM. In a previous study evaluating clinical utility of CTCs, detected with a widely used system 'CellSearch', the authors indicated a significant however insufficient capability in the diagnosis of MM, suggesting need for a more sensitive system. Accordingly, the authors developed a novel microfluidic system to capture CTCs (CTC-chip), and demonstrated that the CTC-chip effectively captured MM cells (ACC-MESO-4) spiked in the blood by conjugating an anti-podoplanin antibody. The results of the present study demonstrated that the CTC-chip coated with the anti-podoplanin antibody captured another MM cell (ACC-MESO-1). However, the capture efficiencies were lower than those for ACC-MESO-4. In addition, an anti-mesothelin antibody was used to capture CTCs, however the CTC-chip coated with the anti-mesothelin antibody failed to effectively capture MM cells, possibly due to low mesothelin expression. Overall, the CTC-chip may capture specific types of CTCs by conjugating any antibody against an antigen expressed on CTCs, and may be a useful system for the diagnosis of malignant tumors, including MM.

Open-access and multi-directional electroosmotic flow chip for positioning heterotypic cells.

PubMed

Terao, Kyohei; Kitazawa, Yuko; Yokokawa, Ryuji; Okonogi, Atsuhito; Kotera, Hidetoshi

2011-04-21

We propose a novel method of cell positioning using electroosmotic flow (EOF) to analyze cell-cell interactions. The EOF chip has an open-to-air configuration, is equipped with four electrodes to induce multi-directional EOF, and allows access of tools for liquid handling and of physical probes for cell measurements. Evaluation of the flow within this chip indicated that it controlled hydrodynamic transport of cells, in terms of both speed and direction. We also evaluated cell viability after EOF application and determined appropriate conditions for cell positioning. Two cells were successively positioned in pocket-like microstructures, one in each micropocket, by controlling the EOF direction. As an experimental demonstration, we observed contact interactions between two individual cells through gap junction channels. The EOF chip should provide ways to elucidate various cell-cell interactions between heterotypic cells.

Microfluidic antibody arrays for simultaneous cell separation and stimulus.

PubMed

Liu, Yan; Germain, Todd; Pappas, Dimitri

2014-12-01

A microfluidic chip containing stamped antibody arrays was developed for simultaneous cell separation and drug testing. Poly(dimethyl siloxane) (PDMS) stamping was used to deposit antibodies in a microfluidic channel, forming discrete cell-capture regions on the surface. Cell mixtures were then introduced, resulting in the separation of cells when specific antibodies were used. Anti-CD19 antibody regions resulted in 94 % capture purity for CD19+ Ramos cells. An antibody that captures multiple cell types, for example anti-CD71, can also be used to capture several cell types simultaneously. Cells could also be loaded onto the arrays with spatial control using laminar streams. Both Ramos B cells and HuT 78 T cells were isolated in the chip and exposed to staurosporine in the same channel. Both cell lines had similar responses to the drug, with 2-10 % of cells remaining viable after 20 h of drug treatment, depending on cell type. The chip can also be used to analyze the efficacy of antibody therapy against cancer cells. Anti-CD95 was deposited on the surface and used for simultaneous cell capture and apoptosis induction via the extrinsic pathway. Cells captured on anti-CD95 surfaces had significant viability loss (15 % viability after 24 h) when compared with a control anti-CD71 antibody (81 % viability after 24 h). This chip can be used for a variety of cell separation and/or drug testing studies, enabling researchers to isolate cells and test them against different anti-cancer compounds and to follow cell response using fluorescence or other readout methods.

[AFM fishing of proteins under impulse electric field].

PubMed

Ivanov, Yu D; Pleshakova, T O; Malsagova, K A; Kaysheva, A L; Kopylov, A T; Izotov, A A; Tatur, V Yu; Vesnin, S G; Ivanova, N D; Ziborov, V S; Archakov, A I

2016-05-01

A combination of (atomic force microscopy)-based fishing (AFM-fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat surface of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM-fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM-chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM-fishing has been demonstrated using detection of cytochrome b5 protein. Selection of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell.

On-chip cell sorting via patterned magnetic traps

NASA Astrophysics Data System (ADS)

Byvank, Tom; Prikockis, Michael; Chen, Aaron; Miller, Brandon; Chalmers, Jeffrey; Sooryakumar, Ratnasingham

2015-03-01

Due to their importance in research for the diagnosis and treatment of cancer, numerous schemes have been developed to sort rare cell populations, e.g., circulating tumor cells (CTCs), from a larger ensemble of cells. Here, we improve upon a previously developed microfluidic device (Lab Chip 13, 1172, (2013)) to increase throughput and sorting purity of magnetically labeled cells. The separation mechanism involves controlling magnetic forces by manipulating the magnetic domain structures of embedded permalloy microdisks with weak external fields. These forces move labeled cells from the input flow stream into an adjacent buffer flow stream. Such magnetically activated transfer separates the magnetic entities from their non-magnetic counterparts as the two flow streams split apart and move toward their respective outputs. Purity of the magnetic output is modulated by the withdrawal rate of the non-magnetic output relative to the inputs. A proof of concept shows that CTCs from metastatic breast cancer patients can be sorted, recovered from the device, and confirmed as CTCs using separate immunofluorescence staining and analysis. With further optimizations, the channel could become a useful device for high purity final sorting of enriched patient cell samples.

From bioseparation to artificial micro-organs: microfluidic chip based particle manipulation techniques

NASA Astrophysics Data System (ADS)

Stelzle, Martin

2010-02-01

Microfluidic device technology provides unique physical phenomena which are not available in the macroscopic world. These may be exploited towards a diverse array of applications in biotechnology and biomedicine ranging from bioseparation of particulate samples to the assembly of cells into structures that resemble the smallest functional unit of an organ. In this paper a general overview of chip-based particle manipulation and separation is given. In the state of the art electric, magnetic, optical and gravitational field effects are utilized. Also, mechanical obstacles often in combination with force fields and laminar flow are employed to achieve separation of particles or molecules. In addition, three applications based on dielectrophoretic forces for particle manipulation in microfluidic systems are discussed in more detail. Firstly, a virus assay is demonstrated. There, antibody-loaded microbeads are used to bind virus particles from a sample and subsequently are accumulated to form a pico-liter sized aggregate located at a predefined position in the chip thus enabling highly sensitive fluorescence detection. Secondly, subcellular fractionation of mitochondria from cell homogenate yields pure samples as was demonstrated by Western Blot and 2D PAGE analysis. Robust long-term operation with complex cell homogenate samples while avoiding electrode fouling is achieved by a set of dedicated technical means. Finally, a chip intended for the dielectrophoretic assembly of hepatocytes and endothelial cells into a structure resembling a liver sinusoid is presented. Such "artificial micro organs" are envisioned as substance screening test systems providing significantly higher predictability with respect to the in vivo response towards a substance under test.

Organ-On-A-Chip Platforms: A Convergence of Advanced Materials, Cells, and Microscale Technologies.

PubMed

Ahadian, Samad; Civitarese, Robert; Bannerman, Dawn; Mohammadi, Mohammad Hossein; Lu, Rick; Wang, Erika; Davenport-Huyer, Locke; Lai, Ben; Zhang, Boyang; Zhao, Yimu; Mandla, Serena; Korolj, Anastasia; Radisic, Milica

2018-01-01

Significant advances in biomaterials, stem cell biology, and microscale technologies have enabled the fabrication of biologically relevant tissues and organs. Such tissues and organs, referred to as organ-on-a-chip (OOC) platforms, have emerged as a powerful tool in tissue analysis and disease modeling for biological and pharmacological applications. A variety of biomaterials are used in tissue fabrication providing multiple biological, structural, and mechanical cues in the regulation of cell behavior and tissue morphogenesis. Cells derived from humans enable the fabrication of personalized OOC platforms. Microscale technologies are specifically helpful in providing physiological microenvironments for tissues and organs. In this review, biomaterials, cells, and microscale technologies are described as essential components to construct OOC platforms. The latest developments in OOC platforms (e.g., liver, skeletal muscle, cardiac, cancer, lung, skin, bone, and brain) are then discussed as functional tools in simulating human physiology and metabolism. Future perspectives and major challenges in the development of OOC platforms toward accelerating clinical studies of drug discovery are finally highlighted. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of Her2-overexpressing cancer cells using keyhole shaped chamber array employing a magnetic droplet-handling system.

PubMed

Okochi, Mina; Koike, Shinji; Tanaka, Masayoshi; Honda, Hiroyuki

2017-07-15

An on-chip gene expression analysis compartmentalized in droplets was developed for detection of cancer cells at a single-cell level. The chip consists of a keyhole-shaped reaction chamber with hydrophobic modification employing a magnetic bead-droplet-handling system with a gate for bead separation. Using three kinds of water-based droplets in oil, a droplet with sample cells, a lysis buffer with magnetic beads, and RT-PCR buffer, parallel magnetic manipulation and fusion of droplets were performed using a magnet-handling device containing small external magnet patterns in an array. The actuation with the magnet offers a simple system for droplet manipulation that allows separation and fusion of droplets containing magnetic beads. After reverse transcription and amplification by thermal cycling, fluorescence was obtained for detection of overexpressing genes. For clinical detection of gastric cancer cells in peritoneal washing, the Her2-overexpressing gastric cancer cells spiked within normal cells was detected by gene expression analysis of droplets containing an average of 2.5 cells. Our developed droplet-based cancer detection system manipulated by external magnetic force without pumps or valves offers a simple and flexible set-up for transcriptional detection of cancer cells, and will be greatly advantageous for less-invasive clinical diagnosis and prognostic prediction. Copyright © 2016 Elsevier B.V. All rights reserved.

Microwell Arrays for Studying Many Individual Cells

NASA Technical Reports Server (NTRS)

Folch, Albert; Kosar, Turgut Fettah

2009-01-01

"Laboratory-on-a-chip" devices that enable the simultaneous culturing and interrogation of many individual living cells have been invented. Each such device includes a silicon nitride-coated silicon chip containing an array of micromachined wells sized so that each well can contain one cell in contact or proximity with a patch clamp or other suitable single-cell-interrogating device. At the bottom of each well is a hole, typically 0.5 m wide, that connects the well with one of many channels in a microfluidic network formed in a layer of poly(dimethylsiloxane) on the underside of the chip. The microfluidic network makes it possible to address wells (and, thus, cells) individually to supply them with selected biochemicals. The microfluidic channels also provide electrical contact to the bottoms of the wells.

Microfluidic cell chips for high-throughput drug screening

PubMed Central

Chi, Chun-Wei; Ahmed, AH Rezwanuddin; Dereli-Korkut, Zeynep; Wang, Sihong

2016-01-01

The current state of screening methods for drug discovery is still riddled with several inefficiencies. Although some widely used high-throughput screening platforms may enhance the drug screening process, their cost and oversimplification of cell–drug interactions pose a translational difficulty. Microfluidic cell-chips resolve many issues found in conventional HTS technology, providing benefits such as reduced sample quantity and integration of 3D cell culture physically more representative of the physiological/pathological microenvironment. In this review, we introduce the advantages of microfluidic devices in drug screening, and outline the critical factors which influence device design, highlighting recent innovations and advances in the field including a summary of commercialization efforts on microfluidic cell chips. Future perspectives of microfluidic cell devices are also provided based on considerations of present technological limitations and translational barriers. PMID:27071838

Integrated hybrid polystyrene-polydimethylsiloxane device for monitoring cellular release with microchip electrophoresis and electrochemical detection

PubMed Central

Johnson, Alicia S.; Mehl, Benjamin T.; Martin, R. Scott

2015-01-01

In this work, a polystyrene (PS)-polydimethylsiloxane (PDMS) hybrid device was developed to enable the integration of cell culture with analysis by microchip electrophoresis and electrochemical detection. It is shown that this approach combines the fundamental advantages of PDMS devices (the ability to integrate pumps and valves) and PS devices (the ability to permanently embed fluidic tubing and electrodes). The embedded fused-silica capillary enables high temporal resolution measurements from off-chip cell culture dishes and the embedded electrodes provide close to real-time analysis of small molecule neurotransmitters. A novel surface treatment for improved (reversible) adhesion between PS and PDMS is described using a chlorotrimethylsilane stamping method. It is demonstrated that a Pd decoupler is efficient at handling the high current (and cathodic hydrogen production) resulting from use of high ionic strength buffers needed for cellular analysis; thus allowing an electrophoretic separation and in-channel detection. The separation of norepinephrine (NE) and dopamine (DA) in highly conductive biological buffers was optimized using a mixed surfactant system. This PS-PDMS hybrid device integrates multiple processes including continuous sampling from a cell culture dish, on-chip pump and valving technologies, microchip electrophoresis, and electrochemical detection to monitor neurotransmitter release from PC 12 cells. PMID:25663849

CHIP involves in non-small cell lung cancer prognosis through VEGF pathway.

PubMed

Tingting, Qian; Jiao, Wang; Qingfeng, Wang; Yancheng, Liu; Shijun, Y U; Zhaoqi, Wang; Dongmei, Sun; ShiLong, Wang

2016-10-01

CHIP (c-terminal Hsp70-interacting protein) is an E3 ligase playing vital roles in various cancers. The VEGF pathway has become an important therapeutic target in non-small cell lung cancer (NSCLC). However, little is known about the role of CHIP and the relationship between CHIP and VEGF-VEGFR2 (VEGF receptor 2) pathway in NSCLC. In this study we aimed to investigate the clinical function of CHIP in NSCLC and explore the relevant regulatory mechanism. QRT-PCR was performed to detect CHIP expression in NSCLC tissues. The association of CHIP expression and clinical parameters was analyzed using the Chi-square test. Kaplan- Meier and Cox analyses were performed to identify the role of CHIP in the prognosis of NSCLC patients. ELISA test was used to detect the VEGF secretion of NSCLC cells and western blot were used to detected the protein expression of VEGFR2 in NSCLC cells. and the results revealed that CHIP expression was decreased in NSCLC tissues and significantly correlated with clinical stages, lymph node metastasis and distant metastasis (P<0.05). Moreover, Kaplan-Meier and Cox regression analyses showed that patients with negative expression of CHIP had a shorter survival time and CHIP could be an independent prognostic biomarker. In addition, ELISA tests showed that CHIP negatively regulated the secretion level of VEGF. Furthermore, western blot assay indicated that the VEGFR2 protein level was reduced after CHIP over-expression. Taken together, our findings demonstrate for the first time that CHIP may serve as a promising prognostic biomarker for NSCLC patients and it may be involved in NSCLC angiogenesis through regulating VEGF secretion and expression of VEGFR2. Copyright © 2016. Published by Elsevier Masson SAS.

High-voltage solar-cell chip

NASA Technical Reports Server (NTRS)

Kapoor, V. J.; Valco, G. J.; Skebe, G. G.; Evans, J. C., Jr.

1985-01-01

Integrated circuit technology has been successfully applied to the design and fabrication of 0.5 x 0.5-cm planar multijunction solar-cell chips. Each of these solar cells consisted of six voltage-generating unit cells monolithically connected in series and fabricated on a 75-micron-thick, p-type, single crystal, silicon substrate. A contact photolithic process employing five photomask levels together with a standard microelectronics batch-processing technique were used to construct the solar-cell chip. The open-circuit voltage increased rapidly with increasing illumination up to 5 AM1 suns where it began to saturate at the sum of the individual unit-cell voltages at a maximum of 3.0 V. A short-circuit current density per unit cell of 240 mA/sq cm was observed at 10 AM1 suns.

Application of Microchip for Biomarker Analysis

NASA Astrophysics Data System (ADS)

Kataoka, Masatoshi; Yatsushiro, Shouki; Yamamura, Shouhei; Abe, Hiroko

Microchip technologies have received considerable attention, due to their competitive advantages, especially in regards to reduced sample and reagent consumption, analysis time, and easy operation. This approach has been successfully used to analyze DNA, amino acids, proteins, and carbohydrates. In the present study, we showed the potential of microchip technologies for the biomarker analysis, blood carbohydrate analysis on microchip electrophoresis, quantitative analysis of protein with antigen-antibody reaction on microchip, and the detection of malaria-infected erythrocyte with a cell microarray chip.

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system

PubMed Central

Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori; Fujii, Teruo; Laurell, Thomas

2017-01-01

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice. PMID:28425472

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system.

PubMed

Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori; Fujii, Teruo; Laurell, Thomas

2017-04-20

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice.

Transient deformation of a droplet near a microfluidic constriction: A quantitative analysis

NASA Astrophysics Data System (ADS)

Trégouët, Corentin; Salez, Thomas; Monteux, Cécile; Reyssat, Mathilde

2018-05-01

We report on experiments that consist of deforming a collection of monodisperse droplets produced by a microfluidic chip through a flow-focusing device. We show that a proper numerical modeling of the flow is necessary to access the stress applied by the latter on the droplet along its trajectory through the chip. This crucial step enables the full integration of the differential equation governing the dynamical deformation, and consequently the robust measurement of the interfacial tension by fitting the experiments with the calculated deformation. Our study thus demonstrates the feasibility of quantitative in situ rheology in microfluidic flows involving, e.g., droplets, capsules, or cells.

Histone deacetylase inhibitors suppress ABO transcription in vitro, leading to reduced expression of the antigens.

PubMed

Takahashi, Yoichiro; Kubo, Rieko; Sano, Rie; Nakajima, Tamiko; Takahashi, Keiko; Kobayashi, Momoko; Handa, Hiroshi; Tsukada, Junichi; Kominato, Yoshihiko

2017-03-01

The ABO system is of fundamental importance in the fields of transfusion and transplantation and has apparent associations with certain diseases, including cardiovascular disorders. ABO expression is reduced in the late phase of erythroid differentiation in vitro, whereas histone deacetylase inhibitors (HDACIs) are known to promote cell differentiation. Therefore, whether or not HDACIs could reduce the amount of ABO transcripts and A or B antigens is an intriguing issue. Quantitative polymerase chain reactions were carried out for the ABO transcripts in erythroid-lineage K562 and epithelial-lineage KATOIII cells after incubation with HDACIs, such as sodium butyrate, panobinostat, vorinostat, and sodium valproate. Flow cytometric analysis was conducted to evaluate the amounts of antigen in KATOIII cells treated with panobinostat. Quantitative chromatin immunoprecipitation (ChIP) assays and luciferase assays were performed on both cell types to examine the mechanisms of ABO suppression. HDACIs reduced the ABO transcripts in both K562 and KATOIII cells, with panobinostat exerting the most significant effect. Flow cytometric analysis demonstrated a decrease in B-antigen expression on panobinostat-treated KATOIII cells. ChIP assays indicated that panobinostat altered the modification of histones in the transcriptional regulatory regions of ABO, and luciferase assays demonstrated reduced activity of these elements. ABO transcription seems to be regulated by an epigenetic mechanism. Panobinostat appears to suppress ABO transcription, reducing the amount of antigens on the surface of cultured cells. © 2016 AABB.

A perforated CMOS microchip for immobilization and activity monitoring of electrogenic cells

NASA Astrophysics Data System (ADS)

Greve, F.; Lichtenberg, J.; Kirstein, K.-U.; Frey, U.; Perriard, J.-C.; Hierlemann, A.

2007-03-01

CMOS-based microelectrode systems offer decisive advantages over conventional micro-electrode arrays, which include the possibility to perform on-chip signal conditioning or to efficiently use larger numbers of electrodes to obtain statistically relevant data, e.g., in pharmacological drug screening. A larger number of electrodes can only be realized with the help of on-chip multiplexing and readout schemes, which require integrated electronics. Another fundamental issue in performing high-fidelity recordings from electrogenic cells is a good electrical coupling between the cells and the microelectrodes, in particular, since the recorded extracellular signals are in the range of only 10-1000 µV. In this paper we present the first CMOS microelectrode system with integrated micromechanical cell-placement features fabricated in a commercial CMOS process with subsequent post-CMOS bulk micromachining. This new microdevice aims at enabling the precise placement of single cells in the center of the electrodes to ensure an efficient use of the available electrodes, even for low-density cell cultures. Small through-chip holes have been generated at the metal-electrode sites by using a combination of bulk micromachining and reactive-ion etching. These holes act as orifices so that cell immobilization can be achieved by means of pneumatic anchoring. The chip additionally hosts integrated circuitry, i.e., multiplexers to select the respective readout electrodes, an amplifier with selectable gain (2×, 10×, 100×), and a high-pass filter (100 Hz cut-off). In this paper we show that electrical signals from most of the electrodes can be recorded, even in low-density cultures of neonatal rat cardiomyocytes, by using perforated metal electrodes and by applying a small underpressure from the backside of the chip. The measurements evidenced that, in most cases, about 90% of the electrodes were covered with single cells, approximately 4% were covered with more than one cell due to clustering and approximately 6% were not covered with any cell, mostly as a consequence of orifice clogging. After 4 days of culturing, the cells were still in place on the electrodes so that the cell electrical activity could be measured using the on-chip circuitry. Measured signal amplitudes were in the range of 500-700 µV, while the input-referred noise of the readout was below 15 µVrms (100 Hz-4 kHz bandwidth). We report on the development and fabrication of this new cell-biological tool and present first results collected during the characterization and evaluation of the chip. The recordings of electrical potentials of neonatal rat cardiomyocytes after several days in vitro, which, on the one hand, were conventionally cultured (no pneumatic anchoring) and, on the other hand, were anchored and immobilized, will be detailed.

Design, development, fabrication and delivery of register and multiplexer units. [CMOS monolithic chip development

NASA Technical Reports Server (NTRS)

Feller, A.; Lombardi, T.

1978-01-01

Several approaches for implementing the register and multiplexer unit into two CMOS monolithic chip types were evaluated. The CMOS standard cell array technique was selected and implemented. Using this design automation technology, two LSI CMOS arrays were designed, fabricated, packaged, and tested for proper static, functional, and dynamic operation. One of the chip types, multiplexer register type 1, is fabricated on a 0.143 x 0.123 inch chip. It uses nine standard cell types for a total of 54 standard cells. This involves more than 350 transistors and has the functional equivalent of 111 gates. The second chip, multiplexer register type 2, is housed on a 0.12 x 0.12 inch die. It uses 13 standard cell types, for a total of 42 standard cells. It contains more than 300 transistors, the functional equivalent of 112 gates. All of the hermetically sealed units were initially screened for proper functional operation. The static leakage and the dynamic leakage were measured. Dynamic measurements were made and recorded. At 10 V, 14 megabit shifting rates were measured on multiplexer register type 1. At 5 V these units shifted data at a 6.6 MHz rate. The units were designed to operate over the 3 to 15 V operating range and over a temperature range of -55 to 125 C.

Coplanar electrode microfluidic chip enabling accurate sheathless impedance cytometry.

PubMed

De Ninno, Adele; Errico, Vito; Bertani, Francesca Romana; Businaro, Luca; Bisegna, Paolo; Caselli, Federica

2017-03-14

Microfluidic impedance cytometry offers a simple non-invasive method for single-cell analysis. Coplanar electrode chips are especially attractive due to ease of fabrication, yielding miniaturized, reproducible, and ultimately low-cost devices. However, their accuracy is challenged by the dependence of the measured signal on particle trajectory within the interrogation volume, that manifests itself as an error in the estimated particle size, unless any kind of focusing system is used. In this paper, we present an original five-electrode coplanar chip enabling accurate particle sizing without the need for focusing. The chip layout is designed to provide a peculiar signal shape from which a new metric correlating with particle trajectory can be extracted. This metric is exploited to correct the estimated size of polystyrene beads of 5.2, 6 and 7 μm nominal diameter, reaching coefficient of variations lower than the manufacturers' quoted values. The potential impact of the proposed device in the field of life sciences is demonstrated with an application to Saccharomyces cerevisiae yeast.

Trehalose Improves Human Fibroblast Deficits in a New CHIP-Mutation Related Ataxia

PubMed Central

Casarejos, Maria Jose; Perucho, Juan; López-Sendón, Jose Luis; García de Yébenes, Justo; Bettencourt, Conceição; Gómez, Ana; Ruiz, Carolina; Heutink, Peter; Rizzu, Patrizia; Mena, Maria Angeles

2014-01-01

In this work we investigate the role of CHIP in a new CHIP-mutation related ataxia and the therapeutic potential of trehalose. The patient's fibroblasts with a new form of hereditary ataxia, related to STUB1 gene (CHIP) mutations, and three age and sex-matched controls were treated with epoxomicin and trehalose. The effects on cell death, protein misfolding and proteostasis were evaluated. Recent studies have revealed that mutations in STUB-1 gene lead to a growing list of molecular defects as deregulation of protein quality, inhibition of proteasome, cell death, decreased autophagy and alteration in CHIP and HSP70 levels. In this CHIP-mutant patient fibroblasts the inhibition of proteasome with epoxomicin induced severe pathophysiological age-associated changes, cell death and protein ubiquitination. Additionally, treatment with epoxomicin produced a dose-dependent increase in the number of cleaved caspase-3 positive cells. However, co-treatment with trehalose, a disaccharide of glucose present in a wide variety of organisms and known as a autophagy enhancer, reduced these pathological events. Trehalose application also increased CHIP and HSP70 expression and GSH free radical levels. Furthermore, trehalose augmented macro and chaperone mediated autophagy (CMA), rising the levels of LC3, LAMP2, CD63 and increasing the expression of Beclin-1 and Atg5-Atg12. Trehalose treatment in addition increased the percentage of immunoreactive cells to HSC70 and LAMP2 and reduced the autophagic substrate, p62. Although this is an individual case based on only one patient and the statistical comparisons are not valid between controls and patient, the low variability among controls and the obvious differences with this patient allow us to conclude that trehalose, through its autophagy activation capacity, anti-aggregation properties, anti-oxidative effects and lack of toxicity, could be very promising for the treatment of CHIP-mutation related ataxia, and possibly a wide spectrum of neurodegenerative disorders related to protein disconformation. PMID:25259530

MUC1 Functions as a Oncogene by Targeting the Nucleus of Human Breast Cancer Cells

DTIC Science & Technology

2006-09-01

chromatin immunoprecipitation (ChIP) assays were performed on the p53 proximal promoter (PP; -118 to +14) with an anti-MUC1-C antibody (Fig. 3A). MUC1...assays were performed using anti-MUC1-C and anti-KLF4 antibodies . Analysis of MCF-7 and ZR-75-1 cells showed that anti-KLF4 precipitates the PE21 region...performed using anti-MUC1-C, anti-HDAC1 and HDAC3 antibodies . Analysis of MCF-7 and ZR-75-1 cells showed that anti-HDAC1 precipitates the PE21 region after

Controlling Differentiation of Stem Cells for Developing Personalized Organ-on-Chip Platforms.

PubMed

Geraili, Armin; Jafari, Parya; Hassani, Mohsen Sheikh; Araghi, Behnaz Heidary; Mohammadi, Mohammad Hossein; Ghafari, Amir Mohammad; Tamrin, Sara Hasanpour; Modarres, Hassan Pezeshgi; Kolahchi, Ahmad Rezaei; Ahadian, Samad; Sanati-Nezhad, Amir

2018-01-01

Organ-on-chip (OOC) platforms have attracted attentions of pharmaceutical companies as powerful tools for screening of existing drugs and development of new drug candidates. OOCs have primarily used human cell lines or primary cells to develop biomimetic tissue models. However, the ability of human stem cells in unlimited self-renewal and differentiation into multiple lineages has made them attractive for OOCs. The microfluidic technology has enabled precise control of stem cell differentiation using soluble factors, biophysical cues, and electromagnetic signals. This study discusses different tissue- and organ-on-chip platforms (i.e., skin, brain, blood-brain barrier, bone marrow, heart, liver, lung, tumor, and vascular), with an emphasis on the critical role of stem cells in the synthesis of complex tissues. This study further recaps the design, fabrication, high-throughput performance, and improved functionality of stem-cell-based OOCs, technical challenges, obstacles against implementing their potential applications, and future perspectives related to different experimental platforms. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microfluidic Technologies for Synthetic Biology

PubMed Central

Vinuselvi, Parisutham; Park, Seongyong; Kim, Minseok; Park, Jung Min; Kim, Taesung; Lee, Sung Kuk

2011-01-01

Microfluidic technologies have shown powerful abilities for reducing cost, time, and labor, and at the same time, for increasing accuracy, throughput, and performance in the analysis of biological and biochemical samples compared with the conventional, macroscale instruments. Synthetic biology is an emerging field of biology and has drawn much attraction due to its potential to create novel, functional biological parts and systems for special purposes. Since it is believed that the development of synthetic biology can be accelerated through the use of microfluidic technology, in this review work we focus our discussion on the latest microfluidic technologies that can provide unprecedented means in synthetic biology for dynamic profiling of gene expression/regulation with high resolution, highly sensitive on-chip and off-chip detection of metabolites, and whole-cell analysis. PMID:21747695

Single-cell codetection of metabolic activity, intracellular functional proteins, and genetic mutations from rare circulating tumor cells.

PubMed

Zhang, Yu; Tang, Yin; Sun, Shuai; Wang, Zhihua; Wu, Wenjun; Zhao, Xiaodong; Czajkowsky, Daniel M; Li, Yan; Tian, Jianhui; Xu, Ling; Wei, Wei; Deng, Yuliang; Shi, Qihui

2015-10-06

The high glucose uptake and activation of oncogenic signaling pathways in cancer cells has long made these features, together with the mutational spectrum, prime diagnostic targets of circulating tumor cells (CTCs). Further, an ability to characterize these properties at a single cell resolution is widely believed to be essential, as the known extensive heterogeneity in CTCs can obscure important correlations in data obtained from cell population-based methods. However, to date, it has not been possible to quantitatively measure metabolic, proteomic, and genetic data from a single CTC. Here we report a microchip-based approach that allows for the codetection of glucose uptake, intracellular functional proteins, and genetic mutations at the single-cell level from rare tumor cells. The microchip contains thousands of nanoliter grooves (nanowells) that isolate individual CTCs and allow for the assessment of their glucose uptake via imaging of a fluorescent glucose analog, quantification of a panel of intracellular signaling proteins using a miniaturized antibody barcode microarray, and retrieval of the individual cell nuclei for subsequent off-chip genome amplification and sequencing. This approach integrates molecular-scale information on the metabolic, proteomic, and genetic status of single cells and permits the inference of associations between genetic signatures, energy consumption, and phosphoproteins oncogenic signaling activities in CTCs isolated from blood samples of patients. Importantly, this microchip chip-based approach achieves this multidimensional molecular analysis with minimal cell loss (<20%), which is the bottleneck of the rare cell analysis.

Route to one-step microstructure mold fabrication for PDMS microfluidic chip

NASA Astrophysics Data System (ADS)

Lv, Xiaoqing; Geng, Zhaoxin; Fan, Zhiyuan; Wang, Shicai; Su, Yue; Fang, Weihao; Pei, Weihua; Chen, Hongda

2018-04-01

The microstructure mold fabrication for PDMS microfluidic chip remains complex and time-consuming process requiring special equipment and protocols: photolithography and etching. Thus, a rapid and cost-effective method is highly needed. Comparing with the traditional microfluidic chip fabricating process based on the micro-electromechanical system (MEMS), this method is simple and easy to implement, and the whole fabrication process only requires 1-2 h. Different size of microstructure from 100 to 1000 μm was fabricated, and used to culture four kinds of breast cancer cell lines. Cell viability and morphology was assessed when they were cultured in the micro straight channels, micro square holes and the bonding PDMS-glass microfluidic chip. The experimental results indicate that the microfluidic chip is good and meet the experimental requirements. This method can greatly reduce the process time and cost of the microfluidic chip, and provide a simple and effective way for the structure design and in the field of biological microfabrications and microfluidic chips.

Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

NASA Astrophysics Data System (ADS)

Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan

2018-03-01

EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.

Nanostructure-enhanced surface plasmon resonance imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Špašková, Barbora; Lynn, Nicholas S.; Slabý, Jiří Bocková, Markéta; Homola, Jiří

2017-06-01

There remains a need for the multiplexed detection of biomolecules at extremely low concentrations in fields of medical diagnostics, food safety, and security. Surface plasmon resonance imaging is an established biosensing approach in which the measurement of the intensity of light across a sensor chip is correlated with the amount of target biomolecules captured by the respective areas on the chip. In this work, we present a new approach for this method allowing for enhanced bioanalytical performance via the introduction of nanostructured sensing chip and polarization contrast measurement, which enable the exploitation of both amplitude and phase properties of plasmonic resonances on the nanostructures. Here we will discuss a complex theoretical analysis of the sensor performance, whereby we investigate aspects related to both the optical performance as well as the transport of the analyte molecules to the functionalized surfaces. This analysis accounts for the geometrical parameters of the nanostructured sensing surface, the properties of functional coatings, and parameters related to the detection assay. Based on the results of the theoretical analysis, we fabricated sensing chips comprised of arrays of gold nanoparticles (by electron-beam lithography), which were modified by a biofunctional coating to allow for the selective capturing of the target biomolecules in the regions with high sensitivity. In addition, we developed a compact optical reader with an integrated microfluidic cell, allowing for the measurement from 50 independent sensing channels. The performance of this biosensor is demonstrated through the sensitive detection of short oligonucleotides down to the low picomolar level.

CHIP mediates down-regulation of nucleobindin-1 in preosteoblast cell line models.

PubMed

Xue, Fuying; Wu, Yanping; Zhao, Xinghui; Zhao, Taoran; Meng, Ying; Zhao, Zhanzhong; Guo, Junwei; Chen, Wei

2016-08-01

Nucleobindin-1 (NUCB1), also known as Calnuc, is a highly conserved, multifunctional protein widely expressed in tissues and cells. It contains two EF-hand motifs which have been shown to play a crucial role in binding Ca(2+) ions. In this study, we applied comparative two-dimensional gel electrophoresis to characterize differentially expressed proteins in HA-CHIP over-expressed and endogenous CHIP depleted MC3T3-E1 stable cell lines, identifying NUCB1 as a novel CHIP/Stub1 targeted protein. NUCB1 interacts with and is down-regulated by CHIP by both proteasomal dependent and independent pathways, suggesting that CHIP-mediated down-regulation of nucleobindin-1 might play a role in osteoblast differentiation. The chaperone protein Hsp70 was found to be important for CHIP and NUCB1 interaction as well as CHIP-mediated NUCB1 down-regulation. Our findings provide new insights into understanding the stability regulation of NUCB1. Copyright © 2016 Elsevier Inc. All rights reserved.

Model-based approach for design verification and co-optimization of catastrophic and parametric-related defects due to systematic manufacturing variations

NASA Astrophysics Data System (ADS)

Perry, Dan; Nakamoto, Mark; Verghese, Nishath; Hurat, Philippe; Rouse, Rich

2007-03-01

Model-based hotspot detection and silicon-aware parametric analysis help designers optimize their chips for yield, area and performance without the high cost of applying foundries' recommended design rules. This set of DFM/ recommended rules is primarily litho-driven, but cannot guarantee a manufacturable design without imposing overly restrictive design requirements. This rule-based methodology of making design decisions based on idealized polygons that no longer represent what is on silicon needs to be replaced. Using model-based simulation of the lithography, OPC, RET and etch effects, followed by electrical evaluation of the resulting shapes, leads to a more realistic and accurate analysis. This analysis can be used to evaluate intelligent design trade-offs and identify potential failures due to systematic manufacturing defects during the design phase. The successful DFM design methodology consists of three parts: 1. Achieve a more aggressive layout through limited usage of litho-related recommended design rules. A 10% to 15% area reduction is achieved by using more aggressive design rules. DFM/recommended design rules are used only if there is no impact on cell size. 2. Identify and fix hotspots using a model-based layout printability checker. Model-based litho and etch simulation are done at the cell level to identify hotspots. Violations of recommended rules may cause additional hotspots, which are then fixed. The resulting design is ready for step 3. 3. Improve timing accuracy with a process-aware parametric analysis tool for transistors and interconnect. Contours of diffusion, poly and metal layers are used for parametric analysis. In this paper, we show the results of this physical and electrical DFM methodology at Qualcomm. We describe how Qualcomm was able to develop more aggressive cell designs that yielded a 10% to 15% area reduction using this methodology. Model-based shape simulation was employed during library development to validate architecture choices and to optimize cell layout. At the physical verification stage, the shape simulator was run at full-chip level to identify and fix residual hotspots on interconnect layers, on poly or metal 1 due to interaction between adjacent cells, or on metal 1 due to interaction between routing (via and via cover) and cell geometry. To determine an appropriate electrical DFM solution, Qualcomm developed an experiment to examine various electrical effects. After reporting the silicon results of this experiment, which showed sizeable delay variations due to lithography-related systematic effects, we also explain how contours of diffusion, poly and metal can be used for silicon-aware parametric analysis of transistors and interconnect at the cell-, block- and chip-level.

Numerical and experimental evaluation of microfluidic sorting devices.

PubMed

Taylor, Jay K; Ren, Carolyn L; Stubley, G D

2008-01-01

The development of lab-on-a-chip devices calls for the isolation or separation of specific bioparticles or cells. The design of a miniaturized cell-sorting device for handheld operation must follow the strict parameters associated with lab-on-a-chip technology. The limitations include applied voltage, high efficiency of cell-separation, reliability, size, flow control, and cost, among others. Currently used designs have achieved successful levels of cell isolation; however, further improvements in the microfluidic chip design are important to incorporate into larger systems. This study evaluates specific design modifications that contribute to the reduction of required applied potential aiming for developing portable devices, improved operation reliability by minimizing induced pressure disturbance when electrokinetic pumping is employed, and improved flow control by incorporating directing streams achieving dynamic sorting and counting. The chip designs fabricated in glass and polymeric materials include asymmetric channel widths for sample focusing, nonuniform channel depth for minimizing induced pressure disturbance, directing streams to assist particle flow control, and online filters for reducing channel blockage. Fluorescence-based visualization experimental results of electrokinetic focusing, flow field phenomena, and dynamic sorting demonstrate the advantages of the chip design. Numerical simulations in COMSOL are validated by the experimental data and used to investigate the effects of channel geometry and fluid properties on the flow field.

The recent development and applications of fluidic channels by 3D printing.

PubMed

Zhou, Yufeng

2017-10-18

The technology of "Lab-on-a-Chip" allows the synthesis and analysis of chemicals and biological substance within a portable or handheld device. The 3D printed structures enable precise control of various geometries. The combination of these two technologies in recent years makes a significant progress. The current approaches of 3D printing, such as stereolithography, polyjet, and fused deposition modeling, are introduced. Their manufacture specifications, such as surface roughness, resolution, replication fidelity, cost, and fabrication time, are compared with each other. Finally, novel application of 3D printed channel in biology are reviewed, including pathogenic bacteria detection using magnetic nanoparticle clusters in a helical microchannel, cell stimulation by 3D chemical gradients, perfused functional vascular channels, 3D tissue construct, organ-on-a-chip, and miniaturized fluidic "reactionware" devices for chemical syntheses. Overall, the 3D printed fluidic chip is becoming a powerful tool in the both medical and chemical industries.

Highly sensitive detection and mutational analysis of lung cancer circulating tumor cells using integrated combined immunomagnetic beads with a droplet digital PCR chip.

PubMed

Gao, Wanlei; Huang, Ting; Yuan, Haojun; Yang, Jun; Jin, Qinghui; Jia, Chunping; Mao, Guoxin; Zhao, Jianlong

2018-08-01

Circulating tumor cells (CTCs) have become an important biomarker for liquid biopsy to monitor tumor progression and indicate response to therapies. Many epithelial cellular adhesion molecule (EpCAM) dependent CTC isolation methods have been developed, which have a limitation for low EpCAM expressed tumor cells. In an effort to overcome these drawbacks, we developed combined immunomagnetic beads (EpCAM, Mucin1 and epidermal growth factor receptor) to sensitively isolate CTCs for immunofluorescence analysis and genetic characterization. With this combined immunomagnetic beads, 93.35% H446 cells from spiked blood sample can be recovered. We were able to detect CTCs in 127 among 143 patients included in the study (88.8%). Some CTC clusters were captured with the combined magnetic beads system. In 17 of them, CTCs after chemotherapy significantly decreased compared to that before chemotherapy (4.42 (± 3.94) vs. 12 (± 7)/mL, P = 0.002). For subsequent genetic characterization of CTCs, 2 of 6 samples, using a droplet digital PCR (ddPCR) chip, have detectable EGFR L858R mutation in the cells enriched with the combined immunomagnetic beads. In conclusion, this method integrating the combined immunomagnetic beads and the ddPCR chip for CTCs detection can be of potential application in terms of diagnosis, therapeutic evaluation and personalized medicine in lung cancer. Copyright © 2018 Elsevier B.V. All rights reserved.

Microfluidic liquid-air dual-gradient chip for synergic effect bio-evaluation of air pollutant.

PubMed

Liu, Xian-Jun; Hu, Shan-Wen; Xu, Bi-Yi; Zhao, Ge; Li, Xiang; Xie, Fu-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

2018-05-15

In this paper, a novel prototype liquid-air dual gradient chip is introduced, which has paved the way for effective synergic effect bio-evaluation of air pollutant. The chip is composed of an array of the agarose liquid-air interfaces, top air gradient layer and bottom liquid gradient layer. The novel agarose liquid-air interface allows for non-biased exposure of cells to all the substances in the air and diffusive interactions with the liquid phase; while the dual liquid-air gradient provides powerful screening abilities, which well reduced errors, saved time and cost from repeated experiment. Coupling the two functions, the chip subsequently facilitates synergic effect evaluation of both liquid and air factors on cells. Here cigarette smoke was taken as the model air pollutant, and its strong synergic effects with inflammatory level of A549 lung cancer cells on their fate were successfully quantified for the first time. These results well testified that the proposed dual-gradient chip is powerful and indispensable for bio-evaluation of air pollutant. Copyright © 2018 Elsevier B.V. All rights reserved.

A novel miniaturized PCR multi-reactor array fabricated using flip-chip bonding techniques

NASA Astrophysics Data System (ADS)

Zou, Zhi-Qing; Chen, Xiang; Jin, Qing-Hui; Yang, Meng-Su; Zhao, Jian-Long

2005-08-01

This paper describes a novel miniaturized multi-chamber array capable of high throughput polymerase chain reaction (PCR). The structure of the proposed device is verified by using finite element analysis (FEA) to optimize the thermal performance, and then implemented on a glass-silicon substrate using a standard MEMS process and post-processing. Thermal analysis simulation and verification of each reactor cell is equipped with integrated Pt temperature sensors and heaters at the bottom of the reaction chamber for real-time accurate temperature sensing and control. The micro-chambers are thermally separated from each other, and can be controlled independently. The multi-chip array was packaged on a printed circuit board (PCB) substrate using a conductive polymer flip-chip bonding technique, which enables effective heat dissipation and suppresses thermal crosstalk between the chambers. The designed system has successfully demonstrated a temperature fluctuation of ±0.5 °C during thermal multiplexing of up to 2 × 2 chambers, a full speed of 30 min for 30 cycle PCR, as well as the capability of controlling each chamber digitally and independently.

Comprehensive Study of Microgel Electrode for On-Chip Electrophoretic Cell Sorting

NASA Astrophysics Data System (ADS)

Akihiro Hattori,; Kenji Yasuda,

2010-06-01

We have developed an on-chip cell sorting system and microgel electrode for applying electrostatic force in microfluidic pathways in the chip. The advantages of agarose electrodes are 1) current-driven electrostatic force generation, 2) stability against pH change and chemicals, and 3) no bubble formation caused by electrolysis. We examined the carrier ion type and concentration dependence of microgel electrode impedance, and found that CoCl2 has less than 1/10 of the impedance from NaCl, and the reduction of the impedance of NaCl gel electrode was plateaued at 0.5 M. The structure control of the microgel electrode exploiting the surface tension of sol-state agarose was also introduced. The addition of 1% (w/v) trehalose into the microgel electrode allowed the frozen storage of the microgel electrode chip. The experimental results demonstrate the potential of our system and microgel electrode for practical applications in microfluidic chips.

A scalable self-priming fractal branching microchannel net chip for digital PCR.

PubMed

Zhu, Qiangyuan; Xu, Yanan; Qiu, Lin; Ma, Congcong; Yu, Bingwen; Song, Qi; Jin, Wei; Jin, Qinhan; Liu, Jinyu; Mu, Ying

2017-05-02

As an absolute quantification method at the single-molecule level, digital PCR has been widely used in many bioresearch fields, such as next generation sequencing, single cell analysis, gene editing detection and so on. However, existing digital PCR methods still have some disadvantages, including high cost, sample loss, and complicated operation. In this work, we develop an exquisite scalable self-priming fractal branching microchannel net digital PCR chip. This chip with a special design inspired by natural fractal-tree systems has an even distribution and 100% compartmentalization of the sample without any sample loss, which is not available in existing chip-based digital PCR methods. A special 10 nm nano-waterproof layer was created to prevent the solution from evaporating. A vacuum pre-packaging method called self-priming reagent introduction is used to passively drive the reagent flow into the microchannel nets, so that this chip can realize sequential reagent loading and isolation within a couple of minutes, which is very suitable for point-of-care detection. When the number of positive microwells stays in the range of 100 to 4000, the relative uncertainty is below 5%, which means that one panel can detect an average of 101 to 15 374 molecules by the Poisson distribution. This chip is proved to have an excellent ability for single molecule detection and quantification of low expression of hHF-MSC stem cell markers. Due to its potential for high throughput, high density, low cost, lack of sample and reagent loss, self-priming even compartmentalization and simple operation, we envision that this device will significantly expand and extend the application range of digital PCR involving rare samples, liquid biopsy detection and point-of-care detection with higher sensitivity and accuracy.

nDEP-driven cell patterning and bottom-up construction of cell aggregates using a new bioelectronic chip.

PubMed

Menad, S; Franqueville, L; Haddour, N; Buret, F; Frenea-Robin, M

2015-04-01

Creating cell aggregates of controlled size and shape and patterning cells on substrates using a bottom-up approach constitutes important challenges for tissue-engineering applications and studies of cell-cell interactions. In this paper, we report nDEP (negative dielectrophoresis) driven assembly of cells as compact aggregates or onto defined areas using a new bioelectronic chip. This chip is composed of a quadripolar electrode array obtained using coplanar electrodes partially covered with a thin, micropatterned PDMS membrane. This thin PDMS layer was coated with poly-L-lysine and played the role of adhesive substrate for cell patterning. For the formation of detachable cell aggregates, the PDMS was not pretreated and cells were simply immobilized into assemblies maintained by cell-cell adhesion after the electric field removal. Cell viability after exposition to DEP buffer was also assessed, as well as cell spreading activity following DEP-driven assembly. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing

NASA Astrophysics Data System (ADS)

Riba, J.; Gleichmann, T.; Zimmermann, S.; Zengerle, R.; Koltay, P.

2016-09-01

The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry.

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples.

PubMed

Rehimi, Rizwan; Bartusel, Michaela; Solinas, Francesca; Altmüller, Janine; Rada-Iglesias, Alvaro

2017-08-29

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 10 4 - 5 x 10 5 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence.

PubMed

Mazzatti, Dawn J; Pawelec, Graham; Longdin, Robin; Powell, Jonathan R; Forsey, Rosalyn J

2007-06-05

The adaptive immune response requires waves of T-cell clonal expansion on contact with pathogen and elimination after clearance of the source of antigen. However, lifelong persistent infections with common viruses cause chronic antigenic stimulation which takes its toll on adaptive immunity in late life. Chronic antigenic stress results in deregulation of the T-cell response and accumulation of anergic cells. Longitudinal studies of the elderly show that this impacts on survival. Identifying the nature of the defects in chronically-stimulated T-cells and protein bio-markers of these dysfunctional cells would help to understand age-associated compromised T-cell function (immunosenescence) and facilitate the development of targeted intervention strategies.The purpose of this work was to use surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to analyse proteins associated with T-cell senescence in order to identify potential bio-markers. Clonal populations of T-cells isolated from elderly octogenarian and centenarian donors were grown in vitro until senescence, and early passage and late passage (pre-senescent) cells were analysed using SELDI-TOF-MS ProteinChip arrays. Discriminant analysis identified several protein or peptide peaks in the region of 14.5-16.5 kDa that were associated with T-cell clone senescence. Human profilin-1, a ubiquitous protein associated with actin remodelling and cellular motility was unambiguously identified. Altered expression of profilin-1 in senescent T-cell clones was confirmed by Western blot analysis. Due to the proposed roles of profilin-1 in cellular survival, cytoskeleton remodelling, motility, and proliferation, it is hypothesised that differential expression of profilin-1 in ageing may contribute directly to immunosenescence.

Single-cell trapping and selective treatment via co-flow within a microfluidic platform.

PubMed

Benavente-Babace, A; Gallego-Pérez, D; Hansford, D J; Arana, S; Pérez-Lorenzo, E; Mujika, M

2014-11-15

Lab on a chip (LOC) systems provide interesting and low-cost solutions for key studies and applications in the biomedical field. Along with microfluidics, these microdevices make single-cell manipulation possible with high spatial and temporal resolution. In this work we have designed, fabricated and characterized a versatile and inexpensive microfluidic platform for on-chip selective single-cell trapping and treatment using laminar co-flow. The combination of co-existing laminar flow manipulation and hydrodynamic single-cell trapping for selective treatment offers a cost-effective solution for studying the effect of novel drugs on single-cells. The operation of the whole system is experimentally simple, highly adaptable and requires no specific equipment. As a proof of concept, a cytotoxicity study of ethanol in isolated hepatocytes is presented. The developed microfluidic platform controlled by means of co-flow is an attractive and multipurpose solution for the study of new substances of high interest in cell biology research. In addition, this platform will pave the way for the study of cell behavior under dynamic and controllable fluidic conditions providing information at the individual cell level. Thus, this analysis device could also hold a great potential to easily use the trapped cells as sensing elements expanding its functionalities as a cell-based biosensor with single-cell resolution. Copyright © 2014 Elsevier B.V. All rights reserved.

A mini-microscope for in situ monitoring of cells.

PubMed

Kim, Sang Bok; Koo, Kyo-in; Bae, Hojae; Dokmeci, Mehmet R; Hamilton, Geraldine A; Bahinski, Anthony; Kim, Sun Min; Ingber, Donald E; Khademhosseini, Ali

2012-10-21

A mini-microscope was developed for in situ monitoring of cells by modifying off-the-shelf components of a commercial webcam. The mini-microscope consists of a CMOS imaging module, a small plastic lens and a white LED illumination source. The CMOS imaging module was connected to a laptop computer through a USB port for image acquisition and analysis. Due to its compact size, 8 × 10 × 9 cm, the present microscope is portable and can easily fit inside a conventional incubator, and enables real-time monitoring of cellular behaviour. Moreover, the mini-microscope can be used for imaging cells in conventional cell culture flasks, such as Petri dishes and multi-well plates. To demonstrate the operation of the mini-microscope, we monitored the cellular migration of mouse 3T3 fibroblasts in a scratch assay in medium containing three different concentrations of fetal bovine serum (5, 10, and 20%) and demonstrated differential responses depending on serum levels. In addition, we seeded embryonic stem cells inside poly(ethylene glycol) microwells and monitored the formation of stem cell aggregates in real time using the mini-microscope. Furthermore, we also combined a lab-on-a-chip microfluidic device for microdroplet generation and analysis with the mini-microscope and observed the formation of droplets under different flow conditions. Given its cost effectiveness, robust imaging and portability, the presented platform may be useful for a range of applications for real-time cellular imaging using lab-on-a-chip devices at low cost.

Microfluidic Cultivation and Laser Tweezers Raman Spectroscopy of E. coli under Antibiotic Stress

PubMed Central

Pilát, Zdeněk; Bernatová, Silvie; Ježek, Jan; Kirchhoff, Johanna; Tannert, Astrid; Samek, Ota; Zemánek, Pavel

2018-01-01

Analyzing the cells in various body fluids can greatly deepen the understanding of the mechanisms governing the cellular physiology. Due to the variability of physiological and metabolic states, it is important to be able to perform such studies on individual cells. Therefore, we developed an optofluidic system in which we precisely manipulated and monitored individual cells of Escherichia coli. We tested optical micromanipulation in a microfluidic chamber chip by transferring individual bacteria into the chambers. We then subjected the cells in the chambers to antibiotic cefotaxime and we observed the changes by using time-lapse microscopy. Separately, we used laser tweezers Raman spectroscopy (LTRS) in a different micro-chamber chip to manipulate and analyze individual cefotaxime-treated E. coli cells. Additionally, we performed conventional Raman micro-spectroscopic measurements of E. coli cells in a micro-chamber. We found observable changes in the cellular morphology (cell elongation) and in Raman spectra, which were consistent with other recently published observations. The principal component analysis (PCA) of Raman data distinguished between the cefotaxime treated cells and control. We tested the capabilities of the optofluidic system and found it to be a reliable and versatile solution for this class of microbiological experiments. PMID:29783713

Chromatin Immunoprecipitation in Early Mouse Embryos.

PubMed

García-González, Estela G; Roque-Ramirez, Bladimir; Palma-Flores, Carlos; Hernández-Hernández, J Manuel

2018-01-01

Epigenetic regulation is achieved at many levels by different factors such as tissue-specific transcription factors, members of the basal transcriptional apparatus, chromatin-binding proteins, and noncoding RNAs. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method that allows elucidating gene regulation at the molecular level by assessing if chromatin modifications or proteins are present at a specific locus. Initially, the majority of ChIP experiments were performed on cultured cell lines and more recently this technique has been adapted to a variety of tissues in different model organisms. Using ChIP on mouse embryos, it is possible to document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development and to get biological meaning from observations made on tissue culture analyses. We describe here a ChIP protocol on freshly isolated mouse embryonic somites for in vivo analysis of muscle specific transcription factor binding on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.

Numerical simulation of isolation of cancer cells in a microfluidic chip

NASA Astrophysics Data System (ADS)

Djukic, T.; Topalovic, M.; Filipovic, N.

2015-08-01

Cancer is a disease that is characterized by the uncontrolled increase of numbers of cells. Circulating tumour cells (CTCs) are separated from the primary tumor, circulate in the bloodstream and form metastases. Circulating tumor cells can be identified in the blood of a patient by taking a blood sample. Microfluidic chips are a new technique that is used to isolate these cells from the blood sample. In this paper a numerical model is presented that is able to simulate the motion of individual cells through a microfluidic chip. The proposed numerical model gives very valuable insight into the processes happening within a microfluidic chip. The accuracy of the proposed model is compared with experimental results. The experimental setup that is described in literature is used to create identical geometrical domains and define simulation parameters. A good agreement of experimental and numerical results demonstrates that the proposed model can be successfully used to simulate complex behaviour of CTCs inside microfluidic chips.

Chip-based three-dimensional cell culture in perfused micro-bioreactors.

PubMed

Gottwald, Eric; Lahni, Brigitte; Thiele, David; Giselbrecht, Stefan; Welle, Alexander; Weibezahn, Karl-Friedrich

2008-05-21

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermo-forming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 micron (h x w x l) or round recesses with diameters of 300 micron and a depth of 300 micron (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed sterile circulation loop that, in the simplest configuration, is additionally comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

An Analysis of the Effects of Chip-groove Geometry on Machining Performance Using Finite Element Methods

NASA Astrophysics Data System (ADS)

Ee, K. C.; Dillon, O. W.; Jawahir, I. S.

2004-06-01

This paper discusses the influence of major chip-groove parameters of a cutting tool on the chip formation process in orthogonal machining using finite element (FE) methods. In the FE formulation, a thermal elastic-viscoplastic material model is used together with a modified Johnson-Cook material law for the flow stress. The chip back-flow angle and the chip up-curl radius are calculated for a range of cutting conditions by varying the chip-groove parameters. The analysis provides greater understanding of the effectiveness of chip-groove configurations and points a way to correlate cutting conditions with tool-wear when machining with a grooved cutting tool.

A primary battery-on-a-chip using monolayer graphene.

PubMed

Iost, Rodrigo M; Crespilho, Frank N; Kern, Klaus; Balasubramanian, Kannan

2016-06-14

We present here a bottom-up approach for realizing on-chip on-demand batteries starting out with chemical vapor deposition-grown graphene. Single graphene monolayers contacted by electrode lines on a silicon chip serve as electrodes. The anode and cathode are realized by electrodeposition of zinc and copper respectively onto graphene, leading to the realization of a miniature graphene-based Daniell cell on a chip. The electrolyte is housed partly in a gel and partly in liquid form in an on-chip enclosure molded using a 3d printer or made out of poly(dimethylsiloxane). The realized batteries provide a stable voltage (∼1.1 V) for many hours and exhibit capacities as high as 15 μAh, providing enough power to operate a pocket calculator. The realized batteries show promise for deployment as on-chip power sources for autonomous systems in lab-on-a-chip or biomedical applications.

A primary battery-on-a-chip using monolayer graphene

NASA Astrophysics Data System (ADS)

Iost, Rodrigo M.; Crespilho, Frank N.; Kern, Klaus; Balasubramanian, Kannan

2016-07-01

We present here a bottom-up approach for realizing on-chip on-demand batteries starting out with chemical vapor deposition-grown graphene. Single graphene monolayers contacted by electrode lines on a silicon chip serve as electrodes. The anode and cathode are realized by electrodeposition of zinc and copper respectively onto graphene, leading to the realization of a miniature graphene-based Daniell cell on a chip. The electrolyte is housed partly in a gel and partly in liquid form in an on-chip enclosure molded using a 3d printer or made out of poly(dimethylsiloxane). The realized batteries provide a stable voltage (∼1.1 V) for many hours and exhibit capacities as high as 15 μAh, providing enough power to operate a pocket calculator. The realized batteries show promise for deployment as on-chip power sources for autonomous systems in lab-on-a-chip or biomedical applications.

On-chip clearing of arrays of 3-D cell cultures and micro-tissues.

PubMed

Grist, S M; Nasseri, S S; Poon, T; Roskelley, C; Cheung, K C

2016-07-01

Three-dimensional (3-D) cell cultures are beneficial models for mimicking the complexities of in vivo tissues, especially in tumour studies where transport limitations can complicate response to cancer drugs. 3-D optical microscopy techniques are less involved than traditional embedding and sectioning, but are impeded by optical scattering properties of the tissues. Confocal and even two-photon microscopy limit sample imaging to approximately 100-200 μm depth, which is insufficient to image hypoxic spheroid cores. Optical clearing methods have permitted high-depth imaging of tissues without physical sectioning, but they are difficult to implement for smaller 3-D cultures due to sample loss in solution exchange. In this work, we demonstrate a microfluidic platform for high-throughput on-chip optical clearing of breast cancer spheroids using the SeeDB, Clear(T2), and ScaleSQ clearing methods. Although all three methods are able to effectively clear the spheroids, we find that SeeDB and ScaleSQ more effectively clear the sample than Clear(T2); however, SeeDB induces green autofluorescence while ScaleS causes sample expansion. Our unique on-chip implementation permits clearing arrays of 3-D cultures using perfusion while monitoring the 3-D cultures throughout the process, enabling visualization of the clearing endpoint as well as monitoring of transient changes that could induce image artefacts. Our microfluidic device is compatible with on-chip 3-D cell culture, permitting the use of on-chip clearing at the endpoint after monitoring the same spheroids during their culture. This on-chip method has the potential to improve readout from 3-D cultures, facilitating their use in cell-based assays for high-content drug screening and other applications.

miR-764-5p promotes osteoblast differentiation through inhibition of CHIP/STUB1 expression.

PubMed

Guo, Junwei; Ren, Fangli; Wang, Yinyin; Li, Shan; Gao, Zhengrong; Wang, Xiaoyan; Ning, Hongxiu; Wu, Jianguo; Li, Yi; Wang, Zhao; Chim, Shek Man; Xu, Jiake; Chang, Zhijie

2012-07-01

Differentiation of committed precursor cells into the osteoblast lineage is tightly regulated by several factors, including Runx2 and BMP2. We previously reported that C terminus of Hsc70-interacting protein/STIP1 homology and U-Box containing protein 1 (CHIP/STUB1) negatively regulated osteoblast differentiation through promoting Runx2 protein degradation. However, how CHIP is regulated during osteoblast differentiation remains unknown. In this study, we found that miR-764-5p is up-expressed during the osteoblast differentiation in calvarial and osteoblast progenitor cells, coupled with down-expression of CHIP protein. We observed that forced expression or inhibition of miR-764-5p decreased or increased the CHIP protein level through affecting its translation by targeting the 3'-UTR region. Perturbation of miR-764-5p resulted in altered differentiation fate of osteoblast progenitor cells and the role of miR-764-5p was reversed by overexpression of CHIP, whereas depletion of CHIP impaired the effect of miR-764-5p. Our data showed that miR-764-5p positively regulates osteoblast differentiation from osteoblast progenitor cells by repressing the translation of CHIP protein. Copyright © 2012 American Society for Bone and Mineral Research.

A monolithic glass chip for active single-cell sorting based on mechanical phenotyping.

PubMed

Faigle, Christoph; Lautenschläger, Franziska; Whyte, Graeme; Homewood, Philip; Martín-Badosa, Estela; Guck, Jochen

2015-03-07

The mechanical properties of biological cells have long been considered as inherent markers of biological function and disease. However, the screening and active sorting of heterogeneous populations based on serial single-cell mechanical measurements has not been demonstrated. Here we present a novel monolithic glass chip for combined fluorescence detection and mechanical phenotyping using an optical stretcher. A new design and manufacturing process, involving the bonding of two asymmetrically etched glass plates, combines exact optical fiber alignment, low laser damage threshold and high imaging quality with the possibility of several microfluidic inlet and outlet channels. We show the utility of such a custom-built optical stretcher glass chip by measuring and sorting single cells in a heterogeneous population based on their different mechanical properties and verify sorting accuracy by simultaneous fluorescence detection. This offers new possibilities of exact characterization and sorting of small populations based on rheological properties for biological and biomedical applications.

Mesenchymal Stem Cells for Vascular Target Discovery in Breast Cancer-Associated Angiogenesis

DTIC Science & Technology

2005-09-01

demonstrating this marker as demonstrated by flow cytometry . These GFP+ MSCs were subsequently analyzed for expression of commonly reported markers of...phenotypically and genotypically analyzed by flow cytometry and gene chip analysis, respectively. We have also shown that MSCs can then be stimulated to...positive MSCs retrieved by collagenase digestion of the Matrigel plug and sorted by flow cytometry . Sorting of these retrieved cells based on co-expression

Aurora Kinase A Promotes AR Degradation via the E3 Ligase CHIP.

PubMed

Sarkar, Sukumar; Brautigan, David L; Larner, James M

2017-08-01

Reducing the levels of the androgen receptor (AR) is one of the most viable approaches to combat castration-resistant prostate cancer. Previously, we observed that proteasomal-dependent degradation of AR in response to 2-methoxyestradiol (2-ME) depends primarily on the E3 ligase C-terminus of HSP70-interacting protein (STUB1/CHIP). Here, 2-ME stimulation activates CHIP by phosphorylation via Aurora kinase A (AURKA). Aurora A kinase inhibitors and RNAi knockdown of Aurora A transcript selectively blocked CHIP phosphorylation and AR degradation. Aurora A kinase is activated by 2-ME in the S-phase as well as during mitosis, and phosphorylates CHIP at S273. Prostate cancer cells expressing an S273A mutant of CHIP have attenuated AR degradation upon 2-ME treatment compared with cells expressing wild-type CHIP, supporting the idea that CHIP phosphorylation by Aurora A activates its E3 ligase activity for the AR. These results reveal a novel 2-ME→Aurora A→CHIP→AR pathway that promotes AR degradation via the proteasome that may offer novel therapeutic opportunities for prostate cancer. Mol Cancer Res; 15(8); 1063-72. ©2017 AACR . ©2017 American Association for Cancer Research.

Pyrolysis of ground pine chip and ground pellet particles

DOE PAGES

Rezaei, Hamid; Yazdanpanah, Fahimeh; Lim, C. Jim; ...

2016-08-04

In addition to particle size, biomass density influences heat and mass transfer rates during the thermal treatment processes. In this research, thermal behaviour of ground pine chip particles and ground pine pellet particles in the range of 0.25–5 mm was investigated. A single particle from ground pellets was almost 3 to 4 times denser than a single particle from ground chips at a similar size and volume of particle. Temperature was ramped up from room temperature (~25 °C) to 600 °C with heating rates of 10, 20, 30, and 50 °C/min. Pellet particles took 25–88 % longer time to drymore » than the chip particles. Microscopic examination of 3 mm and larger chip particles showed cracks during drying. No cracks were observed for pellet particles. The mass loss due to treatment at temperatures higher than 200 °C was about 80% both for chip and pellet particles. It took 4 min for chip and pellet particles to lose roughly 63% of their dry mass at a heating rate of 50 °C/min. The SEM structural analysis showed enlarged pores and cracks in cell walls of the pyrolyzed wood chips. As a result, these pores were not observed in pyrolyzed pellet particles.« less

Indium tin oxide based chip for optical and electrochemical characterization of protein-cell interaction

NASA Astrophysics Data System (ADS)

Choi, Yong Hyun; Min, Junhong; Cho, Sungbo

2015-06-01

Analysis on the interaction between proteins and cells is required for understanding the cellular behaviour and response. In this article, we characterized the adhesion and growth of 293/GFP cells on fetal bovine serum (FBS) coated indium tin oxide (ITO) electrode. Using optical and electrochemical measurement, it was able to detect the adsorption of the protein on the surface of the ITO electrode dependent on the concentration of the protein in the immersing solution or the immersing time. An increase in the amount of the adsorbed serum protein resulted in a decrease in anodic peak current and an increase in the charge transfer resistance extracted from the equivalent circuit fitting analysis. More cells adhered and proliferated on the ITO electrode which was pre-immersed in FBS medium rather than bare electrode. The effect of the FBS on cell behaviors was reflected in the impedance monitoring of cells at 21.5 kHz.

Microfluidic chip integrated with flexible PDMS-based electrochemical cytosensor for dynamic analysis of drug-induced apoptosis on HeLa cells.

PubMed

Cao, Jun-Tao; Zhu, Ying-Di; Rana, Rohit Kumar; Zhu, Jun-Jie

2014-01-15

A novel microfluidic platform integrated with a flexible PDMS-based electrochemical cytosensor was developed for real-time monitoring of the proliferation and apoptosis of HeLa cells. The PDMS-gold film, which had a conductive smooth surface and was semi-transparent, facilitated electrochemical measurements and optical microscope observations. We observed distinct increases and decreases in peak current intensity, corresponding to cell proliferation in culture medium and apoptosis in the presence of an anticancer drug, respectively. This electrochemical analysis method permitted real-time, label-free monitoring of cell behavior, and the electrochemical results were confirmed with optical microscopy. The flexible microfluidic electrochemical platform presented here is suitable for on-site monitoring of cell behavior in microenvironments. © 2013 Elsevier B.V. All rights reserved.

Loss of the Nuclear Pool of Ubiquitin Ligase CHIP/STUB1 in Breast Cancer Unleashes the MZF1-Cathepsin Pro-oncogenic Program.

PubMed

Luan, Haitao; Mohapatra, Bhopal; Bielecki, Timothy A; Mushtaq, Insha; Mirza, Sameer; Jennings, Tameka A; Clubb, Robert J; An, Wei; Ahmed, Dena; El-Ansari, Rokaya; Storck, Matthew D; Mishra, Nitish K; Guda, Chittibabu; Sheinin, Yuri M; Meza, Jane L; Raja, Srikumar; Rakha, Emad A; Band, Vimla; Band, Hamid

2018-05-15

CHIP/STUB1 ubiquitin ligase is a negative co-chaperone for HSP90/HSC70, and its expression is reduced or lost in several cancers, including breast cancer. Using an extensive and well-annotated breast cancer tissue collection, we identified the loss of nuclear but not cytoplasmic CHIP to predict more aggressive tumorigenesis and shorter patient survival, with loss of CHIP in two thirds of ErbB2 + and triple-negative breast cancers (TNBC) and in one third of ER + breast cancers. Reduced CHIP expression was seen in breast cancer patient-derived xenograft tumors and in ErbB2 + and TNBC cell lines. Ectopic CHIP expression in ErbB2 + lines suppressed in vitro oncogenic traits and in vivo xenograft tumor growth. An unbiased screen for CHIP-regulated nuclear transcription factors identified many candidates whose DNA-binding activity was up- or downregulated by CHIP. We characterized myeloid zinc finger 1 (MZF1) as a CHIP target, given its recently identified role as a positive regulator of cathepsin B/L (CTSB/L)-mediated tumor cell invasion downstream of ErbB2. We show that CHIP negatively regulates CTSB/L expression in ErbB2 + and other breast cancer cell lines. CTSB inhibition abrogates invasion and matrix degradation in vitro and halts ErbB2 + breast cancer cell line xenograft growth. We conclude that loss of CHIP remodels the cellular transcriptome to unleash critical pro-oncogenic pathways, such as the matrix-degrading enzymes of the cathepsin family, whose components can provide new therapeutic opportunities in breast and other cancers with loss of CHIP expression. Significance: These findings reveal a novel targetable pathway of breast oncogenesis unleashed by the loss of tumor suppressor ubiquitin ligase CHIP/STUB1. Cancer Res; 78(10); 2524-35. ©2018 AACR . ©2018 American Association for Cancer Research.

Quantification of cellular penetrative forces using lab-on-a-chip technology and finite element modeling

PubMed Central

Sanati Nezhad, Amir; Naghavi, Mahsa; Packirisamy, Muthukumaran; Bhat, Rama; Geitmann, Anja

2013-01-01

Tip-growing cells have the unique property of invading living tissues and abiotic growth matrices. To do so, they exert significant penetrative forces. In plant and fungal cells, these forces are generated by the hydrostatic turgor pressure. Using the TipChip, a microfluidic lab-on-a-chip device developed for tip-growing cells, we tested the ability to exert penetrative forces generated in pollen tubes, the fastest-growing plant cells. The tubes were guided to grow through microscopic gaps made of elastic polydimethylsiloxane material. Based on the deformation of the gaps, the force exerted by the elongating tubes to permit passage was determined using finite element methods. The data revealed that increasing mechanical impedance was met by the pollen tubes through modulation of the cell wall compliance and, thus, a change in the force acting on the obstacle. Tubes that successfully passed a narrow gap frequently burst, raising questions about the sperm discharge mechanism in the flowering plants. PMID:23630253

The characterization of four gene expression analysis in circulating tumor cells made by Multiplex-PCR from the AdnaTest kit on the lab-on-a-chip Agilent DNA 1000 platform.

PubMed

Škereňová, Markéta; Mikulová, Veronika; Čapoun, Otakar; Zima, Tomáš

2016-01-01

Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analysis as a part of circulating tumour cell (CTC) detection technique. Despite the common use of this kit a complex analysis of the results from a long-term project is still missing. A commercially available Agilent DNA 1000 kit was used as a final step in the CTC detection (AdnaTest) for the determination of the presence of PCR fragments generated by Multiplex PCR. Data from 30 prostate cancer patients obtained during two years of research were analyzed to determine the trueness and precision of the PCR fragment size determination. Additional experiments were performed to demonstrate the precision (repeatability, reproducibility) and robustness of PCR fragment concentration determination. The trueness and precision of the size determination was below 3% and 2% respectively. The repeatability of the concentration determination was below 15%. The difference in concentration determination increases when Multiplex-PCR/storage step is added between the two measurements of one sample. The characteristics established in our study are in concordance with the manufacturer's specifications established for a ladder as a sample. However, the concentration determination may vary depending on chip preparation, sample storage and concentration. The 15% variation of concentration determination repeatability was shown to be partly proportional and can be suppressed by proper normalization.

Stiffness-independent highly efficient on-chip extraction of cell-laden hydrogel microcapsules from oil emulsion into aqueous solution by dielectrophoresis

PubMed Central

Huang, Haishui; Sun, Mingrui; Heisler-Taylor, Tyler; Kiourti, Asimina; Volakis, John; Lafyatis, Gregory

2015-01-01

A dielectrophoresis (DEP)-based method is reported to achieve highly efficient on-chip extraction of cell-laden microcapsules of any stiffness from oil into aqueous solution. The hydrogel microcapsules can be extracted into the aqueous solution by DEP and interfacial tension (IFT) forces with no trapped oil while the encapsulated cells are free from the electrical damages due to the Faraday cage effect. PMID:26297051

Microfluidics for cell-based high throughput screening platforms - A review.

PubMed

Du, Guansheng; Fang, Qun; den Toonder, Jaap M J

2016-01-15

In the last decades, the basic techniques of microfluidics for the study of cells such as cell culture, cell separation, and cell lysis, have been well developed. Based on cell handling techniques, microfluidics has been widely applied in the field of PCR (Polymerase Chain Reaction), immunoassays, organ-on-chip, stem cell research, and analysis and identification of circulating tumor cells. As a major step in drug discovery, high-throughput screening allows rapid analysis of thousands of chemical, biochemical, genetic or pharmacological tests in parallel. In this review, we summarize the application of microfluidics in cell-based high throughput screening. The screening methods mentioned in this paper include approaches using the perfusion flow mode, the droplet mode, and the microarray mode. We also discuss the future development of microfluidic based high throughput screening platform for drug discovery. Copyright © 2015 Elsevier B.V. All rights reserved.

Transcript Profiling of Two Alfalfa Genotypes with Contrasting Cell Wall Composition in Stems Using a Cross-Species Platform: Optimizing Analysis by Masking Biased Probes

USDA-ARS?s Scientific Manuscript database

The stem cell walls of alfalfa [Medicago sativa (L.) ssp. sativa] genotype 252 have high cellulose and lignin concentrations, while stem cell walls of genotype 1283 have low cellulose and lignin concentrations. The GeneChip® Medicago Genome Array, developed for Medicago truncatula, is a suitable pla...

Microfluidic perfusion culture chip providing different strengths of shear stress for analysis of vascular endothelial function.

PubMed

Hattori, Koji; Munehira, Yoichi; Kobayashi, Hideki; Satoh, Taku; Sugiura, Shinji; Kanamori, Toshiyuki

2014-09-01

We developed a microfluidic perfusion cell culture chip that provides three different shear stress strengths and a large cell culture area for the analysis of vascular endothelial functions. The microfluidic network was composed of shallow flow-control channels of three different depths and deep cell culture channels. The flow-control channels with high fluidic resistances created shear stress strengths ranging from 1.0 to 10.0 dyn/cm(2) in the cell culture channels. The large surface area of the culture channels enabled cultivation of a large number (approximately 6.0 × 10(5)) of cells. We cultured human umbilical vein endothelial cells (HUVECs) and evaluated the changes in cellular morphology and gene expression in response to applied shear stress. The HUVECs were aligned in the direction of flow when exposed to a shear stress of 10.0 dyn/cm(2). Compared with conditions of no shear stress, endothelial nitric oxide synthase mRNA expression increased by 50% and thrombomodulin mRNA expression increased by 8-fold under a shear stress of 9.5 dyn/cm(2). Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Digital Microfluidics for Manipulation and Analysis of a Single Cell.

PubMed

He, Jie-Long; Chen, An-Te; Lee, Jyong-Huei; Fan, Shih-Kang

2015-09-15

The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed.

Digital Microfluidics for Manipulation and Analysis of a Single Cell

PubMed Central

He, Jie-Long; Chen, An-Te; Lee, Jyong-Huei; Fan, Shih-Kang

2015-01-01

The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed. PMID:26389890

A mini-microscope for in situ monitoring of cells†‡

PubMed Central

Kim, Sang Bok; Koo, Kyo-in; Bae, Hojae; Dokmeci, Mehmet R.; Hamilton, Geraldine A.; Bahinski, Anthony; Kim, Sun Min; Ingber, Donald E.

2013-01-01

A mini-microscope was developed for in situ monitoring of cells by modifying off-the-shelf components of a commercial webcam. The mini-microscope consists of a CMOS imaging module, a small plastic lens and a white LED illumination source. The CMOS imaging module was connected to a laptop computer through a USB port for image acquisition and analysis. Due to its compact size, 8 × 10 × 9 cm, the present microscope is portable and can easily fit inside a conventional incubator, and enables real-time monitoring of cellular behaviour. Moreover, the mini-microscope can be used for imaging cells in conventional cell culture flasks, such as Petri dishes and multi-well plates. To demonstrate the operation of the mini-microscope, we monitored the cellular migration of mouse 3T3 fibroblasts in a scratch assay in medium containing three different concentrations of fetal bovine serum (5, 10, and 20%) and demonstrated differential responses depending on serum levels. In addition, we seeded embryonic stem cells inside poly(ethylene glycol) microwells and monitored the formation of stem cell aggregates in real time using the mini-microscope. Furthermore, we also combined a lab-on-a-chip microfluidic device for microdroplet generation and analysis with the mini-microscope and observed the formation of droplets under different flow conditions. Given its cost effectiveness, robust imaging and portability, the presented platform may be useful for a range of applications for real-time cellular imaging using lab-on-a-chip devices at low cost. PMID:22911426

Characterizing Rat PNS Electrophysiological Response to Electrical Stimulation Using in vitro Chip-Based Human Investigational Platform (iCHIP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khani, Joshua; Prescod, Lindsay; Enright, Heather

Ex vivo systems and organ-on-a-chip technology offer an unprecedented approach to modeling the inner workings of the human body. The ultimate goal of LLNL’s in vitro Chip-based Human Investigational Platform (iCHIP) is to integrate multiple organ tissue cultures using microfluidic channels, multi-electrode arrays (MEA), and other biosensors in order to effectively simulate and study the responses and interactions of the major organs to chemical and physical stimulation. In this study, we focused on the peripheral nervous system (PNS) component of the iCHIP system. Specifically we sought to expound on prior research investigating the electrophysiological response of rat dorsal root ganglionmore » cells (rDRGs) to chemical exposures, such as capsaicin. Our aim was to establish a protocol for electrical stimulation using the iCHIP device that would reliably elicit a characteristic response in rDRGs. By varying the parameters for both the stimulation properties – amplitude, phase width, phase shape, and stimulation/ return configuration – and the culture conditions – day in vitro and neural cell types - we were able to make several key observations and uncover a potential convention with a minimal number of devices tested. Future work will seek to establish a standard protocol for human DRGs in the iCHIP which will afford a portable, rapid method for determining the effects of toxins and novel therapeutics on the PNS.« less

Advances in pulmonary therapy and drug development: Lung tissue engineering to lung-on-a-chip.

PubMed

Doryab, Ali; Amoabediny, Ghassem; Salehi-Najafabadi, Amir

2016-01-01

Lung disease is one of the major causes of death, and the rate of pulmonary diseases has been increasing for decades. Although lung transplantation is the only treatment for majority of patients, this method has been limited due to lack of donors. Therefore, recently, attentions have increased to some new strategies with the aid of tissue engineering and microfluidics techniques not only for the functional analysis, but also for drug screening. In fact, in tissue engineering, the engineered tissue is able to grow by using the patient's own cells without intervention in the immune system. On the other hand, microfluidics devices are applied in order to evaluate drug screenings, function analysis and toxicity. This article reviews new advances in lung tissue engineering and lung-on-a-chip. Furthermore, future directions, difficulties and drawbacks of pulmonary therapy in these areas are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Dual-Color Fluorescence Imaging of EpCAM and EGFR in Breast Cancer Cells with a Bull's Eye-Type Plasmonic Chip.

PubMed

Izumi, Shota; Yamamura, Shohei; Hayashi, Naoko; Toma, Mana; Tawa, Keiko

2017-12-19

Surface plasmon field-enhanced fluorescence microscopic observation of a live breast cancer cell was performed with a plasmonic chip. Two cell lines, MDA-MB-231 and Michigan Cancer Foundation-7 (MCF-7), were selected as breast cancer cells, with two kinds of membrane protein, epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR), observed in both cells. The membrane proteins are surface markers used to differentiate and classify breast cancer cells. EGFR and EpCAM were detected with Alexa Fluor ® 488-labeled anti-EGFR antibody (488-EGFR) and allophycocyanin (APC)-labeled anti-EpCAM antibody (APC-EpCAM), respectively. In MDA-MB231 cells, three-fold plus or minus one and seven-fold plus or minus two brighter fluorescence of 488-EGFR were observed on the 480-nm pitch and the 400-nm pitch compared with that on a glass slide. Results show the 400-nm pitch is useful. Dual-color fluorescence of 488-EGFR and APC-EpCAM in MDA-MB231 was clearly observed with seven-fold plus or minus two and nine-fold plus or minus three, respectively, on the 400-nm pitch pattern of a plasmonic chip. Therefore, the 400-nm pitch contributed to the dual-color fluorescence enhancement for these wavelengths. An optimal grating pitch of a plasmonic chip improved a fluorescence image of membrane proteins with the help of the surface plasmon-enhanced field.

Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines.

PubMed

Gulec, Cagri; Coban, Neslihan; Ozsait-Selcuk, Bilge; Sirma-Ekmekci, Sema; Yildirim, Ozlem; Erginel-Unaltuna, Nihan

2017-04-01

ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses. Copyright © 2017 Elsevier Inc. All rights reserved.

Optoelectronic tweezers integrated with lensfree holographic microscopy for wide-field interactive cell and particle manipulation on a chip.

PubMed

Huang, Kuo-Wei; Su, Ting-Wei; Ozcan, Aydogan; Chiou, Pei-Yu

2013-06-21

We demonstrate an optoelectronic tweezer (OET) coupled to a lensfree holographic microscope for real-time interactive manipulation of cells and micro-particles over a large field-of-view (FOV). This integrated platform can record the holographic images of cells and particles over the entire active area of a CCD sensor array, perform digital image reconstruction to identify target cells, dynamically track the positions of cells and particles, and project light beams to trigger light-induced dielectrophoretic forces to pattern and sort cells on a chip. OET technology has been previously shown to be capable of performing parallel single cell manipulation over a large area. However, its throughput has been bottlenecked by the number of cells that can be imaged within the limited FOV of a conventional microscope objective lens. Integrating lensfree holographic imaging with OET solves this fundamental FOV barrier, while also creating a compact on-chip cell/particle manipulation platform. Using this unique platform, we have successfully demonstrated real-time interactive manipulation of thousands of single cells and micro-particles over an ultra-large area of e.g., 240 mm(2) (i.e. 17.96 mm × 13.52 mm).

Numerical Simulation and Performance Optimization of a Magnetophoretic Bio-separation chip

NASA Astrophysics Data System (ADS)

Golozar, Matin; Darabi, Jeff; Molki, Majid

Separation of micro/nanoparticles is important in biomedicine and biotechnology. This research presents the modeling and optimization of a magnetophoretic bio-separation chip for the isolation of biomaterials, such as circulating tumor cells (CTCs) from the peripheral blood. The chip consists of a continuous flow through microfluidic channels that contains locally engineered magnetic field gradients. The high gradient magnetic field produced by the magnets is spatially non-uniform and gives rise to an attractive force on magnetic particles that move through the flow channel. The computational model takes into account the magnetic and fluidic forces as well as the effect of the volume fraction of particles on the continuous phase. The model is used to investigate the effect of two-way particle-fluid coupling on both the capture efficiency and the flow pattern in the separation chip. The results show that the microfluidic device has the capability of separating CTCs from their native environment. Additionally, a parametric study is performed to investigate the effects of the channel height, substrate thickness, magnetic bead size, bioparticle size, and the number of beads per cell on the cell separation performance.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Branch, Shirmir D.; Lines, Amanda M.; Lynch, John

The electrochemical and spectroelectrochemical applications of an optically transparent thin film electrode chip are investigated. The working electrode is composed of indium tin oxide (ITO); the counter and quasi-reference electrodes are composed of platinum. The stability of the platinum quasi-reference electrode is modified by coating it with a planar, solid state Ag/AgCl layer. The Ag/AgCl reference is characterized with scanning electron microscopy and energy-dispersive X-ray spectroscopy. Open circuit potential measurements indicate that the potential of the planar Ag/AgCl electrode varies a maximum of 20 mV over four days. Cyclic voltammetry measurements show that the electrode chip is comparable to amore » standard electrochemical cell. Randles-Sevcik analysis of 10 mM K3[Fe(CN)6] in 0.1 M KCl using the electrode chip shows a diffusion coefficient of 1.59 × 10-6 cm2/s, in comparison to the standard electrochemical cell value of 2.38 × 10-6 cm2/s. By using the electrode chip in an optically transparent thin layer electrode (OTTLE), the spectroelectrochemical modulation of [Ru(bpy)3]2+ florescence was demonstrated, achieving a detection limit of 36 nM.« less

A Cost-Effective Fluorescence Mini-Microscope with Adjustable Magnifications for Biomedical Applications

PubMed Central

Zhang, Yu Shrike; Ribas, João; Nadhman, Akhtar; Aleman, Julio; Selimović, Šeila; Lesher-Perez, Sasha Cai; Wang, Ting; Manoharan, Vijayan; Shin, Su-Ryon; Damilano, Alessia; Annabi, Nasim; Dokmeci, Mehmet Remzi; Takayama, Shuichi; Khademhosseini, Ali

2015-01-01

We have designed and fabricated a miniature microscope from off-the-shelf components and webcam, with built-in fluorescence capability for biomedical applications. The mini-microscope was able to detect both biochemical parameters such as cell/tissue viability (e.g. Live/Dead assay), and biophysical properties of the microenvironment such as oxygen levels in microfabricated tissues based on an oxygen-sensitive fluorescent dye. This mini-microscope has adjustable magnifications from 8-60X, achieves a resolution as high as <2 μm, and possesses a long working distance of 4.5 mm (at a magnification of 8X). The mini-microscope was able to chronologically monitor cell migration and analyze beating of microfluidic liver and cardiac bioreactors in real time, respectively. The mini-microscope system is cheap, and its modularity allows convenient integration with a wide variety of pre-existing platforms including but not limited to, cell culture plates, microfluidic devices, and organs-on-a-chip systems. Therefore, we envision its widespread applications in cell biology, tissue engineering, biosensing, microfluidics, and organs-on-chips, which can potentially replace conventional bench-top microscopy where long-term in situ and large-scale imaging/analysis is required. PMID:26282117

A cost-effective fluorescence mini-microscope for biomedical applications.

PubMed

Zhang, Yu Shrike; Ribas, João; Nadhman, Akhtar; Aleman, Julio; Selimović, Šeila; Lesher-Perez, Sasha Cai; Wang, Ting; Manoharan, Vijayan; Shin, Su-Ryon; Damilano, Alessia; Annabi, Nasim; Dokmeci, Mehmet Remzi; Takayama, Shuichi; Khademhosseini, Ali

2015-01-01

We have designed and fabricated a miniature microscope from off-the-shelf components and a webcam, with built-in fluorescence capability for biomedical applications. The mini-microscope was able to detect both biochemical parameters, such as cell/tissue viability (e.g. live/dead assay), and biophysical properties of the microenvironment such as oxygen levels in microfabricated tissues based on an oxygen-sensitive fluorescent dye. This mini-microscope has adjustable magnifications from 8-60×, achieves a resolution as high as <2 μm, and possesses a long working distance of 4.5 mm (at a magnification of 8×). The mini-microscope was able to chronologically monitor cell migration and analyze beating of microfluidic liver and cardiac bioreactors in real time, respectively. The mini-microscope system is cheap, and its modularity allows convenient integration with a wide variety of pre-existing platforms including, but not limited to, cell culture plates, microfluidic devices, and organs-on-a-chip systems. Therefore, we envision its widespread application in cell biology, tissue engineering, biosensing, microfluidics, and organs-on-chips, which can potentially replace conventional bench-top microscopy where long-term in situ and large-scale imaging/analysis is required.

Stand-Sit Microchip for High-Throughput, Multiplexed Analysis of Single Cancer Cells.

PubMed

Ramirez, Lisa; Herschkowitz, Jason I; Wang, Jun

2016-09-01

Cellular heterogeneity in function and response to therapeutics has been a major challenge in cancer treatment. The complex nature of tumor systems calls for the development of advanced multiplexed single-cell tools that can address the heterogeneity issue. However, to date such tools are only available in a laboratory setting and don't have the portability to meet the needs in point-of-care cancer diagnostics. Towards that application, we have developed a portable single-cell system that is comprised of a microchip and an adjustable clamp, so on-chip operation only needs pipetting and adjusting of clamping force. Up to 10 proteins can be quantitated from each cell with hundreds of single-cell assays performed in parallel from one chip operation. We validated the technology and analyzed the oncogenic signatures of cancer stem cells by quantitating both aldehyde dehydrogenase (ALDH) activities and 5 signaling proteins in single MDA-MB-231 breast cancer cells. The technology has also been used to investigate the PI3K pathway activities of brain cancer cells expressing mutant epidermal growth factor receptor (EGFR) after drug intervention targeting EGFR signaling. Our portable single-cell system will potentially have broad application in the preclinical and clinical settings for cancer diagnosis in the future.

A pilot study of gene expression analysis in workers with hand-arm vibration syndrome.

PubMed

Maeda, Setsuo; Yu, Xiaozhong; Wang, Rui-Sheng; Sakakibara, Hisataka

2008-04-01

The purpose of this pilot study was to examine differences in gene expressions by cDNA microarray analysis of hand-arm vibration syndrome (HAVS) patients. Vein blood samples were collected and total RNA was extracted. All blood samples were obtained in the morning in one visit after a standard light breakfast. We performed microarray analysis with the labeled cDNA prepared by reverse transcription from RNA samples, using the Human CHIP version 1 (DNA Chip Research Inc, Yokohama, Japan). There are 2,976 genes on the chip, and these genes were selected from a cDNA library prepared with human peripheral white blood cells (WBC). Different gene levels between the HAVS patients and controls, and between groups of HAVS with different levels of symptoms, were indicated by the randomized variance model. The most up-regulated genes were analyzed for their possible functions and association with the occurrence of HAVS. From the results of this pilot study, although the results were obtained a limited number of subjects, it would appear that cDNA microarray analysis of HAVS patients has potential as a new objective method of HAVS diagnosis. Further research is needed to examine the gene expression with increased numbers of patients at different stages of HAVS.

Stiffness-Independent Highly Efficient On-Chip Extraction of Cell-Laden Hydrogel Microcapsules from Oil Emulsion into Aqueous Solution by Dielectrophoresis.

PubMed

Huang, Haishui; Sun, Mingrui; Heisler-Taylor, Tyler; Kiourti, Asimina; Volakis, John; Lafyatis, Gregory; He, Xiaoming

2015-10-28

A dielectrophoresis (DEP)-based method achieves highly efficient on-chip extraction of cell-laden microcapsules of any stiffness from oil into aqueous solution. The hydrogel microcapsules can be extracted into the aqueous solution by DEP and interfacial tension forces with no trapped oil, while the encapsulated cells are free from electrical damage due to the Faraday cage effect. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Promotion of osteoblast differentiation in 3D biomaterial micro-chip arrays comprising fibronectin-coated poly(methyl methacrylate) polycarbonate.

PubMed

Altmann, Brigitte; Steinberg, Thorsten; Giselbrecht, Stefan; Gottwald, Eric; Tomakidi, Pascal; Bächle-Haas, Maria; Kohal, Ralf-Joachim

2011-12-01

Due to the architecture of solid body tissues including bone, three-dimensional (3D) in vitro microenvironments appear favorable, since herein cell growth proceeds under more physiological conditions compared to conventional 2D systems. In the present study we show that a 3D microenvironment comprising a fibronectin-coated PMMA/PC-based micro-chip promotes differentiation of primary human osteoblasts as reflected by the densely-packed 3D bone cell aggregates and expression of biomarkers indicating osteoblast differentiation. Morphogenesis and fluorescence dye-based live/dead staining revealed homogenous cell coverage of the microcavities of the chip array, whereat cells showed high viability up to 14 days. Moreover, Azur II staining proved formation of uniform sized multilayered aggregates, exhibiting progressive intracellular deposition of extracellular bone matrix constituents comprising fibronectin, osteocalcin and osteonectin from day 7 on. Compared to 2D monolayers, osteoblasts grown in the 3D chip environment displayed differential mostly higher gene expression for osteocalcin, osteonectin, and alkaline phosphatase, while collagen type I remained fairly constant in both culture environments. Our results indicate that the 3D microenvironment, based on the PMMA biomaterial chip array promotes osteoblast differentiation, and hereby renders a promising tool for tissue-specific in vitro preconditioning of osteoblasts designated for clinically-oriented bone augmentation or regeneration. Copyright © 2011 Elsevier Ltd. All rights reserved.

Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells.

PubMed

Kuriyama, Kentaro; Shintaku, Hirofumi; Santiago, Juan G

2015-07-01

There is a substantial need for simultaneous analyses of RNA and DNA from individual single cells. Such analysis provides unique evidence of cell-to-cell differences and the correlation between gene expression and genomic mutation in highly heterogeneous cell populations. We present a novel microfluidic system that leverages isotachophoresis to fractionate and isolate cytoplasmic RNA and genomic DNA (gDNA) from single cells. The system uniquely enables independent, sequence-specific analyses of these critical markers. Our system uses a microfluidic chip with a simple geometry and four end-channel electrodes, and completes the entire process in <5 min, including lysis, purification, fractionation, and delivery to DNA and RNA output reservoirs, each containing high quality and purity aliquots with no measurable cross-contamination of cytoplasmic RNA versus gDNA. We demonstrate our system with simultaneous, sequence-specific quantitation using off-chip RT-qPCR and qPCR for simultaneous cytoplasmic RNA and gDNA analyses, respectively. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Design of a front-end integrated circuit for 3D acoustic imaging using 2D CMUT arrays.

PubMed

Ciçek, Ihsan; Bozkurt, Ayhan; Karaman, Mustafa

2005-12-01

Integration of front-end electronics with 2D capacitive micromachined ultrasonic transducer (CMUT) arrays has been a challenging issue due to the small element size and large channel count. We present design and verification of a front-end drive-readout integrated circuit for 3D ultrasonic imaging using 2D CMUT arrays. The circuit cell dedicated to a single CMUT array element consists of a high-voltage pulser and a low-noise readout amplifier. To analyze the circuit cell together with the CMUT element, we developed an electrical CMUT model with parameters derived through finite element analysis, and performed both the pre- and postlayout verification. An experimental chip consisting of 4 X 4 array of the designed circuit cells, each cell occupying a 200 X 200 microm2 area, was formed for the initial test studies and scheduled for fabrication in 0.8 microm, 50 V CMOS technology. The designed circuit is suitable for integration with CMUT arrays through flip-chip bonding and the CMUT-on-CMOS process.

Helicase dependent OnChip-amplification and its use in multiplex pathogen detection.

PubMed

Andresen, Dennie; von Nickisch-Rosenegk, Markus; Bier, Frank F

2009-05-01

The need for fast, specific and sensitive multiparametric detection methods is an ever growing demand in molecular diagnostics. Here we report on a newly developed method, the helicase dependent OnChip amplification (OnChip-HDA). This approach integrates the analysis and detection in one single reaction thus leading to time and cost savings in multiparametric analysis. HDA is an isothermal amplification method that is not depending on thermocycling as known from PCR due to the helicases' ability to unwind DNA double-strands. We have combined the HDA with microarray based detection, making it suitable for multiplex detection. As an example we used the OnChip HDA in single and multiplex amplifications for the detection of the two pathogens N. gonorrhoeae and S. aureus directly on surface bound primers. We have successfully shown the OnChip-HDA and applied it for single- and duplex-detection of the pathogens N. gonorrhoeae and S. aureus. We have developed a new method, the OnChip-HDA for the multiplex detection of pathogens. Its simplicity in reaction setup and potential for miniaturization and multiparametric analysis is advantageous for the integration in miniaturized Lab on Chip systems, e.g. needed in point of care diagnostics.

Human Gut-On-A-Chip Supports Polarized Infection of Coxsackie B1 Virus In Vitro

PubMed Central

Papafragkou, Efstathia; Weaver, James C.; Ferrante, Thomas C.; Bahinski, Anthony; Elkins, Christopher A.; Kulka, Michael; Ingber, Donald E.

2017-01-01

Analysis of enterovirus infection is difficult in animals because they express different virus receptors than humans, and static cell culture systems do not reproduce the physical complexity of the human intestinal epithelium. Here, using coxsackievirus B1 (CVB1) as a prototype enterovirus strain, we demonstrate that human enterovirus infection, replication and infectious virus production can be analyzed in vitro in a human Gut-on-a-Chip microfluidic device that supports culture of highly differentiated human villus intestinal epithelium under conditions of fluid flow and peristalsis-like motions. When CVB1 was introduced into the epithelium-lined intestinal lumen of the device, virions entered the epithelium, replicated inside the cells producing detectable cytopathic effects (CPEs), and both infectious virions and inflammatory cytokines were released in a polarized manner from the cell apex, as they could be detected in the effluent from the epithelial microchannel. When the virus was introduced via a basal route of infection (by inoculating virus into fluid flowing through a parallel lower ‘vascular’ channel separated from the epithelial channel by a porous membrane), significantly lower viral titers, decreased CPEs, and delayed caspase-3 activation were observed; however, cytokines continued to be secreted apically. The presence of continuous fluid flow through the epithelial lumen also resulted in production of a gradient of CPEs consistent with the flow direction. Thus, the human Gut-on-a-Chip may provide a suitable in vitro model for enteric virus infection and for investigating mechanisms of enterovirus pathogenesis. PMID:28146569

Integrated potentiometric detector for use in chip-based flow cells

PubMed

Tantra; Manz

2000-07-01

A new kind of potentiometric chip sensor for ion-selective electrodes (ISE) based on a solvent polymeric membrane is described. The chip sensor is designed to trap the organic cocktail inside the chip and to permit sample solution to flow past the membrane. The design allows the sensor to overcome technical problems of ruggedness and would therefore be ideal for industrial processes. The sensor performance for a Ba2+-ISE membrane based on a Vogtle ionophore showed electrochemical behavior similar to that observed in conventional electrodes and microelectrode arrangements.

Analysis of tumoral spheres growing in a multichamber microfluidic device.

PubMed

Belgorosky, Denise; Fernández-Cabada, Tamara; Peñaherrera-Pazmiño, Ana Belén; Langle, Yanina; Booth, Ross; Bhansali, Shekhar; Pérez, Maximiliano S; Eiján, Ana María; Lerner, Betiana

2018-09-01

Lab on a Chip (LOC) farming systems have emerged as a powerful tool for single cell studies combined with a non-adherent cell culture substrate and single cell capture chips for the study of single cell derived tumor spheres. Cancer is characterized by its cellular heterogeneity where only a small population of cancer stem cells (CSCs) are responsible for tumor metastases and recurrences. Thus, the in vitro strategy to the formation of a single cell-derived sphere is an attractive alternative to identify CSCs. In this study, we test the effectiveness of microdevices for analysis of heterogeneity within CSC populations and its interaction with different components of the extracellular matrix. CSC could be identify using specific markers related to its pluripotency and self-renewal characteristics such as the transcription factor Oct-4 or the surface protein CD44. The results confirm the usefulness of LOC as an effective method for quantification of CSC, through the formation of spheres under conditions of low adhesion or growing on components of the extracellular matrix. The device used is also a good alternative for evaluating the individual growth of each sphere and further identification of these CSC markers by immunofluorescence. In conclusion, LOC devices have not only the already known advantages, but they are also a promising tool since they use small amounts of reagents and are under specific culture parameters. LOC devices could be considered as a novel technology to be used as a complement or replacement of traditional studies on culture plates. © 2018 Wiley Periodicals, Inc.

Divergent Binding and Transactivation by Two Related Steroid Receptors at the Same Response Element*

PubMed Central

Tesikova, Martina; Dezitter, Xavier; Nenseth, Hatice Z.; Klokk, Tove I.; Mueller, Florian; Hager, Gordon L.; Saatcioglu, Fahri

2016-01-01

Transcription factor (TF) recruitment to chromatin is central to activation of transcription. TF-chromatin interactions are highly dynamic, which are evaluated by recovery half time (t1/2) in seconds, determined by fluorescence recovery experiments in living cells, and chromatin immunoprecipitation (ChIP) analysis, measured in minutes. These two states are related: the larger the t1/2, the longer the ChIP occupancy resulting in increased transcription. Here we present data showing that this relationship does not always hold. We found that histone deacetylase inhibitors (HDACis) significantly increased t1/2 of green fluorescent protein (GFP) fused androgen receptor (AR) on a tandem array of positive hormone response elements (HREs) in chromatin. This resulted in increased ChIP signal of GFP-AR. Unexpectedly, however, transcription was inhibited. In contrast, the GFP-fused glucocorticoid receptor (GR), acting through the same HREs, displayed a profile consistent with current models. We provide evidence that these differences are mediated, at least in part, by HDACs. Our results provide insight into TF action in living cells and show that very closely related TFs may trigger significantly divergent outcomes at the same REs. PMID:27056330

Research highlights: Microtechnologies for engineering the cellular environment.

PubMed

Tseng, Peter; Kunze, Anja; Kittur, Harsha; Di Carlo, Dino

2014-04-07

In this issue we highlight recent microtechnology-enabled approaches to control the physical and biomolecular environment around cells: (1) developing micropatterned surfaces to quantify cell affinity choices between two adhesive patterns, (2) controlling topographical cues to align cells and improve reprogramming to a pluripotent state, and (3) controlling gradients of biomolecules to maintain pluripotency in embryonic stem cells. Quantitative readouts of cell-surface affinity in environments with several cues should open up avenues in tissue engineering where self-assembly of complex multi-cellular structures is possible by precisely engineering relative adhesive cues in three dimensional constructs. Methods of simple and local epigenetic modification of chromatin structure with microtopography and biomolecular gradients should also be of use in regenerative medicine, as well as in high-throughput quantitative analysis of external signals that impact and can be used to control cells. Overall, approaches to engineer the cellular environment will continue to be an area of further growth in the microfluidic and lab on a chip community, as the scale of the technologies seamlessly matches that of biological systems. However, because of regulations and other complexities with tissue engineered therapies, these micro-engineering approaches will likely first impact organ-on-a-chip technologies that are poised to improve drug discovery pipelines.

An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking

PubMed Central

Hoffman, Elizabeth A.; Zaidi, Hussain; Shetty, Savera J.; Bekiranov, Stefan; Auble, David T.

2018-01-01

Formaldehyde crosslinking is widely used in combination with chromatin immunoprecipitation (ChIP) to measure the locations along DNA and relative levels of transcription factor (TF)-DNA interactions in vivo. However, the measurements that are typically made do not provide unambiguous information about the dynamic properties of these interactions. We have developed a method to estimate binding kinetic parameters from time-dependent formaldehyde crosslinking data, called crosslinking kinetics (CLK) analysis. Cultures of yeast cells are crosslinked with formaldehyde for various periods of time, yielding the relative ChIP signal at particular loci. We fit the data using the mass-action CLK model to extract kinetic parameters of the TF-chromatin interaction, including the on- and off-rates and crosslinking rate. From the on- and off-rate we obtain the occupancy and residence time. The following protocol is the second iteration of this method, CLKv2, updated with improved crosslinking and quenching conditions, more information about crosslinking rates, and systematic procedures for modeling the observed kinetic regimes. CLKv2 analysis has been applied to investigate the binding behavior of the TATA-binding protein (TBP), and a selected subset of other TFs. The protocol was developed using yeast cells, but may be applicable to cells from other organisms as well. PMID:29682595

Multilayer based lab-on-a-chip-systems for substance testing

NASA Astrophysics Data System (ADS)

Sonntag, Frank; Grünzner, Stefan; Schmieder, Florian; Busek, Mathias; Klotzbach, Udo; Franke, Volker

2015-03-01

An integrated technology chain for laser-microstructuring and bonding of polymer foils for fast, flexible and low-cost manufacturing of multilayer lab-on-a-chip devices especially for complex cell and tissue culture applications, which provides pulsatile fluid flow within physiological ranges at low media-to-cells ratio, was developed and established. Initially the microfluidic system is constructively divided into individual layers which are formed by separate foils or plates. Based on the functional boundary conditions and the necessary properties of each layer the corresponding foils and plates are chosen. In the third step the foils and plates are laser microstructured and functionalized from both sides. In the fourth and last manufacturing step the multiple plates and foils are joined using thermal diffusion bonding. Membranes for pneumatically driven valves and micropumps where bonded via chemical surface modification. Based on the established lab-on-a-chip platform for perfused cell-based assays, a multilayer microfluidic system with two parallel connected cell culture chambers was successfully implemented.

Microwell arrays with nanoholes

NASA Technical Reports Server (NTRS)

Kosar, Turgut Fettah (Inventor); Folch, Albert (Inventor)

2009-01-01

A device for conducting parallel analysis or manipulation of multiple cells or biomolecules is disclosed. In one embodiment, the device comprises a silicon chip with a microwell, and at least one membrane suspended at the bottom opening of the microwell. The suspended portion of the membrane defines a nanohole that provides access to the material on the other side of the membrane.

Evaluation of forest thinning materials for TMP production

Treesearch

John H. Klungness; Roland Gleisner; Doreen Mann; Karen L. Scallon; J.Y. Zhu; Eric G. Horn; Louis L. Edwards

2005-01-01

We used SilviScan analysis and tracheid measurement to evaluate the effect of suppressed growth on the fundamental properties of wood fiber. Suppressed growth reduced cell tracheid length, but the high content of mature wood may translate into longer fibers overall. In pilot-scale refining experiments, blending 25% chips from small-diameter trees (SMD) with 75% mill...

A multichip aVLSI system emulating orientation selectivity of primary visual cortical cells.

PubMed

Shimonomura, Kazuhiro; Yagi, Tetsuya

2005-07-01

In this paper, we designed and fabricated a multichip neuromorphic analog very large scale integrated (aVLSI) system, which emulates the orientation selective response of the simple cell in the primary visual cortex. The system consists of a silicon retina and an orientation chip. An image, which is filtered by a concentric center-surround (CS) antagonistic receptive field of the silicon retina, is transferred to the orientation chip. The image transfer from the silicon retina to the orientation chip is carried out with analog signals. The orientation chip selectively aggregates multiple pixels of the silicon retina, mimicking the feedforward model proposed by Hubel and Wiesel. The chip provides the orientation-selective (OS) outputs which are tuned to 0 degrees, 60 degrees, and 120 degrees. The feed-forward aggregation reduces the fixed pattern noise that is due to the mismatch of the transistors in the orientation chip. The spatial properties of the orientation selective response were examined in terms of the adjustable parameters of the chip, i.e., the number of aggregated pixels and size of the receptive field of the silicon retina. The multichip aVLSI architecture used in the present study can be applied to implement higher order cells such as the complex cell of the primary visual cortex.

Cyclin-dependent kinase 5-mediated phosphorylation of CHIP promotes the tAIF-dependent death pathway in rotenone-treated cortical neurons.

PubMed

Kim, Chiho; Lee, Juhyung; Ko, Yeon Uk; Oh, Young J

2018-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. Its dysregulation has been implicated in various neurodegenerative diseases. We previously reported that phosphorylation of the C-terminus of the Hsc70-interacting protein (CHIP) by Cdk5 promotes truncated apoptosis-inducing factor (tAIF)-mediated neuronal death induced by oxidative stress. Here, we determined whether this Cdk5-dependent cell death signaling pathway is present in experimental models of Parkinson's disease. First, we showed that rotenone activates Cdk5 in primary cultures of cortical neurons and causes tAIF-dependent neuronal cell death. This event was attenuated by negative regulation of endogenous Cdk5 activity by the pharmacological Cdk5 inhibitor, roscovitine, or by lentiviral knockdown of Cdk5. Cdk5 phosphorylates CHIP at Ser20 in rotenone-treated neurons. Consequently, overexpression of CHIP S20A , but not CHIP WT , attenuates tAIF-induced cell death in rotenone-treated cortical neurons. Taken together, these results indicate that phosphorylation of CHIP at Ser20 by Cdk5 activation inhibits CHIP-mediated tAIF degradation, thereby contributing to tAIF-induced neuronal cell death following rotenone treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Phosphorylation of CHIP at Ser20 by Cdk5 promotes tAIF-mediated neuronal death

PubMed Central

Kim, C; Yun, N; Lee, J; Youdim, M B H; Ju, C; Kim, W-K; Han, P-L; Oh, Y J

2016-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase and its dysregulation is implicated in neurodegenerative diseases. Likewise, C-terminus of Hsc70-interacting protein (CHIP) is linked to neurological disorders, serving as an E3 ubiquitin ligase for targeting damaged or toxic proteins for proteasomal degradation. Here, we demonstrate that CHIP is a novel substrate for Cdk5. Cdk5 phosphorylates CHIP at Ser20 via direct binding to a highly charged domain of CHIP. Co-immunoprecipitation and ubiquitination assays reveal that Cdk5-mediated phosphorylation disrupts the interaction between CHIP and truncated apoptosis-inducing factor (tAIF) without affecting CHIP's E3 ligase activity, resulting in the inhibition of CHIP-mediated degradation of tAIF. Lentiviral transduction assay shows that knockdown of Cdk5 or overexpression of CHIPS20A, but not CHIPWT, attenuates tAIF-mediated neuronal cell death induced by hydrogen peroxide. Thus, we conclude that Cdk5-mediated phosphorylation of CHIP negatively regulates its neuroprotective function, thereby contributing to neuronal cell death progression following neurotoxic stimuli. PMID:26206088

Detection of viability of micro-algae cells by optofluidic hologram pattern.

PubMed

Wang, Junsheng; Yu, Xiaomei; Wang, Yanjuan; Pan, Xinxiang; Li, Dongqing

2018-03-01

A rapid detection of micro-algae activity is critical for analysis of ship ballast water. A new method for detecting micro-algae activity based on lens-free optofluidic holographic imaging is presented in this paper. A compact lens-free optofluidic holographic imaging device was developed. This device is mainly composed of a light source, a small through-hole, a light propagation module, a microfluidic chip, and an image acquisition and processing module. The excited light from the light source passes through a small hole to reach the surface of the micro-algae cells in the microfluidic chip, and a holographic image is formed by the diffraction light of surface of micro-algae cells. The relation between the characteristics in the hologram pattern and the activity of micro-algae cells was investigated by using this device. The characteristics of the hologram pattern were extracted to represent the activity of micro-algae cells. To demonstrate the accuracy of the presented method and device, four species of micro-algae cells were employed as the test samples and the comparison experiments between the alive and dead cells of four species of micro-algae were conducted. The results show that the developed method and device can determine live/dead microalgae cells accurately.

Sample flow switching techniques on microfluidic chips.

PubMed

Pan, Yu-Jen; Lin, Jin-Jie; Luo, Win-Jet; Yang, Ruey-Jen

2006-02-15

This paper presents an experimental investigation into electrokinetically focused flow injection for bio-analytical applications. A novel microfluidic device for microfluidic sample handling is presented. The microfluidic chip is fabricated on glass substrates using conventional photolithographic and chemical etching processes and is bonded using a high-temperature fusion method. The proposed valve-less device is capable not only of directing a single sample flow to a specified output port, but also of driving multiple samples to separate outlet channels or even to a single outlet to facilitate sample mixing. The experimental results confirm that the sample flow can be electrokinetically pre-focused into a narrow stream and guided to the desired outlet port by means of a simple control voltage model. The microchip presented within this paper has considerable potential for use in a variety of applications, including high-throughput chemical analysis, cell fusion, fraction collection, sample mixing, and many other applications within the micro-total-analysis systems field.

Isolation of cells for selective treatment and analysis using a magnetic microfluidic chip.

PubMed

Yassine, O; Gooneratne, C P; Abu Smara, D; Li, F; Mohammed, H; Merzaban, J; Kosel, J

2014-05-01

This study describes the development and testing of a magnetic microfluidic chip (MMC) for trapping and isolating cells tagged with superparamagnetic beads (SPBs) in a microfluidic environment for selective treatment and analysis. The trapping and isolation are done in two separate steps; first, the trapping of the tagged cells in a main channel is achieved by soft ferromagnetic disks and second, the transportation of the cells into side chambers for isolation is executed by tapered conductive paths made of Gold (Au). Numerical simulations were performed to analyze the magnetic flux and force distributions of the disks and conducting paths, for trapping and transporting SPBs. The MMC was fabricated using standard microfabrication processes. Experiments were performed with E. coli (K12 strand) tagged with 2.8 μm SPBs. The results showed that E. coli can be separated from a sample solution by trapping them at the disk sites, and then isolated into chambers by transporting them along the tapered conducting paths. Once the E. coli was trapped inside the side chambers, two selective treatments were performed. In one chamber, a solution with minimal nutrition content was added and, in another chamber, a solution with essential nutrition was added. The results showed that the growth of bacteria cultured in the second chamber containing nutrient was significantly higher, demonstrating that the E. coli was not affected by the magnetically driven transportation and the feasibility of performing different treatments on selectively isolated cells on a single microfluidic platform.

Clinical Significance of an HPV DNA Chip Test with Emphasis on HPV-16 and/or HPV-18 Detection in Korean Gynecological Patients.

PubMed

Yeo, Min-Kyung; Lee, Ahwon; Hur, Soo Young; Park, Jong Sup

2016-07-01

Human papillomavirus (HPV) is a major risk factor for cervical cancer. We evaluated the clinical significance of the HPV DNA chip genotyping assay (MyHPV chip, Mygene Co.) compared with the Hybrid Capture 2 (HC2) chemiluminescent nucleic acid hybridization kit (Digene Corp.) in 867 patients. The concordance rate between the MyHPV chip and HC2 was 79.4% (kappa coefficient, κ = 0.55). The sensitivity and specificity of both HPV tests were very similar (approximately 85% and 50%, respectively). The addition of HPV result (either MyHPV chip or HC2) to cytology improved the sensitivity (95%, each) but reduced the specificity (approximately 30%, each) compared with the HPV test or cytology alone. Based on the MyHPV chip results, the odds ratio (OR) for ≥ high-grade squamous intraepithelial lesions (HSILs) was 9.9 in the HPV-16/18 (+) group and 3.7 in the non-16/18 high-risk (HR)-HPV (+) group. Based on the HC2 results, the OR for ≥ HSILs was 5.9 in the HR-HPV (+) group. When considering only patients with cytological diagnoses of "negative for intraepithelial lesion or malignancy" and "atypical squamous cell or atypical glandular cell," based on the MyHPV chip results, the ORs for ≥ HSILs were 6.8 and 11.7, respectively, in the HPV-16/18 (+) group. The sensitivity and specificity of the MyHPV chip test are similar to the HC2. Detecting HPV-16/18 with an HPV DNA chip test, which is commonly used in many Asian countries, is useful in assessing the risk of high-grade cervical lesions.

Meniscal repair using engineered tissue.

PubMed

Peretti, G M; Caruso, E M; Randolph, M A; Zaleske, D J

2001-03-01

In this study, devitalized meniscal tissue pre-seeded with viable cultured chondrocytes was used to repair a bucket-handle incision in meniscal tissue transplanted to nude mice. Lamb knee menisci were devitalized by cyclic freezing and thawing. Chips measuring four by two by one-half millimeters were cut from this devitalized tissue to serve as scaffolds. These chips were then cultured either with or without viable allogeneic lamb chondrocytes. From the inner third of the devitalized meniscal tissue, rectangles were also cut approximately 8 x 6 mm. A 4 mm bucket-handle type incision was made in these blocks. The previously prepared chips either with (experimental group) or without viable chondrocytes (control group) were positioned into the incisions and secured with suture. Further control groups included blocks of devitalized menisci with incisions into which no chips were positioned and either closed with suture or left open with no suture. Specimens were transplanted to subcutaneous pouches of nude mice for 14 weeks. After 14 weeks, seven of eight experimental specimens (chips with viable chondrocytes) demonstrated bridging of the incision assessed by gross inspection and manual distraction. All the control groups were markedly different from the experimental group in that the incision remained grossly visible. Histological analysis was consistent with the differences apparent at the gross level. Only the experimental specimens (chips with viable chondrocytes) with gross bridging demonstrated obliteration of the interface between incision and scaffold. None of the control specimens revealed any cells or tissue filling the incision. Tissue engineering using scaffolds and viable cells may have an application in meniscal repair in vivo.

Automated, Ultra-Sterile Solid Sample Handling and Analysis on a Chip

NASA Technical Reports Server (NTRS)

Mora, Maria F.; Stockton, Amanda M.; Willis, Peter A.

2013-01-01

There are no existing ultra-sterile lab-on-a-chip systems that can accept solid samples and perform complete chemical analyses without human intervention. The proposed solution is to demonstrate completely automated lab-on-a-chip manipulation of powdered solid samples, followed by on-chip liquid extraction and chemical analysis. This technology utilizes a newly invented glass micro-device for solid manipulation, which mates with existing lab-on-a-chip instrumentation. Devices are fabricated in a Class 10 cleanroom at the JPL MicroDevices Lab, and are plasma-cleaned before and after assembly. Solid samples enter the device through a drilled hole in the top. Existing micro-pumping technology is used to transfer milligrams of powdered sample into an extraction chamber where it is mixed with liquids to extract organic material. Subsequent chemical analysis is performed using portable microchip capillary electrophoresis systems (CE). These instruments have been used for ultra-highly sensitive (parts-per-trillion, pptr) analysis of organic compounds including amines, amino acids, aldehydes, ketones, carboxylic acids, and thiols. Fully autonomous amino acid analyses in liquids were demonstrated; however, to date there have been no reports of completely automated analysis of solid samples on chip. This approach utilizes an existing portable instrument that houses optics, high-voltage power supplies, and solenoids for fully autonomous microfluidic sample processing and CE analysis with laser-induced fluorescence (LIF) detection. Furthermore, the entire system can be sterilized and placed in a cleanroom environment for analyzing samples returned from extraterrestrial targets, if desired. This is an entirely new capability never demonstrated before. The ability to manipulate solid samples, coupled with lab-on-a-chip analysis technology, will enable ultraclean and ultrasensitive end-to-end analysis of samples that is orders of magnitude more sensitive than the ppb goal given in the Science Instruments.

Quantitative impedimetric monitoring of cell migration under the stimulation of cytokine or anti-cancer drug in a microfluidic chip

PubMed Central

Xiao, Xia; Lei, Kin Fong; Huang, Chia-Hao

2015-01-01

Cell migration is a cellular response and results in various biological processes such as cancer metastasis, that is, the primary cause of death for cancer patients. Quantitative investigation of the correlation between cell migration and extracellular stimulation is essential for developing effective therapeutic strategies for controlling invasive cancer cells. The conventional method to determine cell migration rate based on comparison of successive images may not be an objective approach. In this work, a microfluidic chip embedded with measurement electrodes has been developed to quantitatively monitor the cell migration activity based on the impedimetric measurement technique. A no-damage wound was constructed by microfluidic phenomenon and cell migration activity under the stimulation of cytokine and an anti-cancer drug, i.e., interleukin-6 and doxorubicin, were, respectively, investigated. Impedance measurement was concurrently performed during the cell migration process. The impedance change was directly correlated to the cell migration activity; therefore, the migration rate could be calculated. In addition, a good match was found between impedance measurement and conventional imaging analysis. But the impedimetric measurement technique provides an objective and quantitative measurement. Based on our technique, cell migration rates were calculated to be 8.5, 19.1, and 34.9 μm/h under the stimulation of cytokine at concentrations of 0 (control), 5, and 10 ng/ml. This technique has high potential to be developed into a powerful analytical platform for cancer research. PMID:26180566

E3 ubiquitin ligase CHIP interacts with C-type lectin-like receptor CLEC-2 and promotes its ubiquitin-proteasome degradation.

PubMed

Shao, Miaomiao; Li, Lili; Song, Shushu; Wu, Weicheng; Peng, Peike; Yang, Caiting; Zhang, Mingming; Duan, Fangfang; Jia, Dongwei; Zhang, Jie; Wu, Hao; Zhao, Ran; Wang, Lan; Ruan, Yuanyuan; Gu, Jianxin

2016-10-01

C-type lectin-like receptor 2 (CLEC-2) was originally identified as a member of non-classical C-type lectin-like receptors in platelets and immune cells. Activation of CLEC-2 is involved in thrombus formation, lymphatic/blood vessel separation, platelet-mediated tumor metastasis and immune response. Nevertheless, the regulation of CLEC-2 expression is little understood. In this study, we identified that the C terminus of Hsc70-interacting protein (CHIP) interacted with CLEC-2 by mass spectrometry analysis, and CHIP decreased the protein expression of CLEC-2 through lysine-48-linked ubiquitination and proteasomal degradation. Deleted and point mutation also revealed that CHIP controlled CLEC-2 protein expression via both tetratricopeptide repeats (TPR) domain and Ubox domain in a HSP70/90-independent manner. Moreover, reduced CHIP expression was associated with decreased CLEC-2 polyubiquitination and increased CLEC-2 protein levels in PMA-induced differentiation of THP-1 monocytes into macrophages. These results indicate that CLEC-2 is the target substrate of E3 ubiquitin ligase CHIP, and suggest that the CHIP/CLEC-2 axis may play an important role in the modulation of immune response. Copyright © 2016 Elsevier Inc. All rights reserved.

Multilayer-based lab-on-a-chip systems for perfused cell-based assays

NASA Astrophysics Data System (ADS)

Klotzbach, Udo; Sonntag, Frank; Grünzner, Stefan; Busek, Mathias; Schmieder, Florian; Franke, Volker

2014-12-01

A novel integrated technology chain of laser-microstructured multilayer foils for fast, flexible, and low-cost manufacturing of lab-on-a-chip devices especially for complex cell and tissue culture applications, which provides pulsatile fluid flow within physiological ranges at low media-to-cells ratio, was developed and established. Initially the microfluidic system is constructively divided into individual layers, which are formed by separate foils or plates. Based on the functional boundary conditions and the necessary properties of each layer, their corresponding foils and plates are chosen. In the third step, the foils and plates are laser microstructured and functionalized from both sides. In the fourth and last manufacturing step, the multiple plates and foils are joined using different bonding techniques like adhesive bonding, welding, etc. This multilayer technology together with pneumatically driven micropumps and valves permits the manufacturing of fluidic structures and perfusion systems, which spread out above multiple planes. Based on the established lab-on-a-chip platform for perfused cell-based assays, a multilayer microfluidic system with two parallel connected cell culture chambers was successfully implemented.

Electrochemical monitoring-on-chip (E-MoC) of HIV-infection in presence of cocaine and therapeutics.

PubMed

Kaushik, Ajeet; Vabbina, Phani Kiran; Atluri, Venkata; Shah, Pratikkumar; Vashist, Arti; Jayant, Rahul Dev; Yandart, Adriana; Nair, Madhavan

2016-12-15

Electrochemical monitoring-on-chip (E-MoC)-based approach for rapid assessment of human immunodeficiency virus (HIV)-infection in the presence of cocaine (Coc) and specific drugs namely i.e., tenofovir (Tef), rimcazole (RA) is demonstrated here, for the first time, using electrochemical impedance spectroscopy (EIS). An in-vitro primary human astrocytes (HA) model was developed using a cultureware chip (CC, used for E-MoC) for HIV-infection, Coc exposure and treatment with anti-HIV drug i.e., Tef, and Coc antagonist i.e., RA. The charge transfer resistance (Rct) value of each CC well varies with respect to infection and treatment demonstrated highly responsive sensitivity of developed chip. The results of E-MoC, a proof-of-the concept, suggested that HIV-infection progression due to Coc ingestion and therapeutic effects of highly specific drugs are measurable on the basis of cell electrophysiology. Though, this work needs various molecular biology-based optimizations to promote this technology as an analytical tool for the rapid assessment of HIV-infection in a patient to manage HIV diseases for timely diagnosis. The presented study is based on using CNS cells and efforts are being made to perform this method using peripheral cells such as monocytes derived dendritic cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Bench-Top Fabrication of an All-PDMS Microfluidic Electrochemical Cell Sensor Integrating Micro/Nanostructured Electrodes.

PubMed

Saem, Sokunthearath; Zhu, Yujie; Luu, Helen; Moran-Mirabal, Jose

2017-03-31

In recent years, efforts in the development of lab-on-a-chip (LoC) devices for point-of-care (PoC) applications have increased to bring affordable, portable, and sensitive diagnostics to the patients' bedside. To reach this goal, research has shifted from using traditional microfabrication methods to more versatile, rapid, and low-cost options. This work focuses on the benchtop fabrication of a highly sensitive, fully transparent, and flexible poly (dimethylsiloxane) (PDMS) microfluidic (μF) electrochemical cell sensor. The μF device encapsulates 3D structured gold and platinum electrodes, fabricated using a shape-memory polymer shrinking method, which are used to set up an on-chip electrochemical cell. The PDMS to PDMS-structured electrode bonding protocol to fabricate the μF chip was optimized and found to have sufficient bond strength to withstand up to 100 mL/min flow rates. The sensing capabilities of the on-chip electrochemical cell were demonstrated by using cyclic voltammetry to monitor the adhesion of murine 3T3 fibroblasts in the presence of a redox reporter. The charge transfer across the working electrode was reduced upon cell adhesion, which was used as the detection mechanism, and allowed the detection of as few as 24 cells. The effective utilization of simple and low cost bench-top fabrication methods could accelerate the prototyping and development of LoC technologies and bring PoC diagnostics and personalized medicine to the patients' bedside.

Bench-Top Fabrication of an All-PDMS Microfluidic Electrochemical Cell Sensor Integrating Micro/Nanostructured Electrodes

PubMed Central

Saem, Sokunthearath; Zhu, Yujie; Luu, Helen; Moran-Mirabal, Jose

2017-01-01

In recent years, efforts in the development of lab-on-a-chip (LoC) devices for point-of-care (PoC) applications have increased to bring affordable, portable, and sensitive diagnostics to the patients’ bedside. To reach this goal, research has shifted from using traditional microfabrication methods to more versatile, rapid, and low-cost options. This work focuses on the benchtop fabrication of a highly sensitive, fully transparent, and flexible poly (dimethylsiloxane) (PDMS) microfluidic (μF) electrochemical cell sensor. The μF device encapsulates 3D structured gold and platinum electrodes, fabricated using a shape-memory polymer shrinking method, which are used to set up an on-chip electrochemical cell. The PDMS to PDMS-structured electrode bonding protocol to fabricate the μF chip was optimized and found to have sufficient bond strength to withstand up to 100 mL/min flow rates. The sensing capabilities of the on-chip electrochemical cell were demonstrated by using cyclic voltammetry to monitor the adhesion of murine 3T3 fibroblasts in the presence of a redox reporter. The charge transfer across the working electrode was reduced upon cell adhesion, which was used as the detection mechanism, and allowed the detection of as few as 24 cells. The effective utilization of simple and low cost bench-top fabrication methods could accelerate the prototyping and development of LoC technologies and bring PoC diagnostics and personalized medicine to the patients’ bedside. PMID:28362329

Parallel manipulation of individual magnetic microbeads for lab-on-a-chip applications

NASA Astrophysics Data System (ADS)

Peng, Zhengchun

Many scientists and engineers are turning to lab-on-a-chip systems for faster and cheaper analysis of chemical reactions and biomolecular interactions. A common approach that facilitates the handling of reagents and biomolecules in these systems utilizes micro/nano beads as the solid carrier. Physical manipulation, such as assembly, transport, sorting, and tweezing, of beads on a chip represents an essential step for fully utilizing their potentials in a wide spectrum of bead-based analysis. Previous work demonstrated manipulation of either an ensemble of beads without individual control, or single beads but lacks the capability for parallel operation. Parallel manipulation of individual beads is required to meet the demand for high-throughput and location-specific analysis. In this work, we introduced two methods for parallel manipulation of individual magnetic microbeads, which can serve as effective lab-on-a-chip platforms and/or efficient analytic tools. The first method employs arrays of soft ferromagnetic patterns fabricated inside a microfluidic channel and subjected to an external magnetic field. We demonstrated that the system can be used to assemble individual beads (1-3 mum) from a flow of suspended beads into a regular array on the chip, hence improving the integrated electrochemical detection of biomolecules bound to the bead surface. By rotating the external field, the assembled microbeads can be remotely controlled with synchronized, high-speed circular motion around individual soft magnets on the chip. We employed this manipulation mode for efficient sample mixing in continuous microflow. Furthermore, we discovered a simple but effective way of transporting the microbeads on the chip by varying the strength of the local bias field within a revolution of the external field. In addition, selective transport of microbeads with different size was realized, providing a platform for effective on-chip sample separation and offering the potential for multiplexing capability. The second method integrates magnetic and dielectrophoretic manipulations of the same microbeads. The device combines tapered conducting wires and fingered electrodes to generate desirable magnetic and electric fields, respectively. By externally programming the magnetic attraction and dielectrophoretic repulsion forces, out-of-plane oscillation of the microbeads across the channel height was realized. This manipulation mode can facilitate the interaction between the beads with multiple layers of sample fluid inside the channel. We further demonstrated the tweezing of microbeads in liquid with high spatial resolutions, i.e., from submicrometer to nanometer range, by fine-tuning the net force from magnetic attraction and dielectrophoretic repulsion of the beads. The highresolution control of the out-of-plane motion of the microbeads led to the invention of massively parallel biomolecular tweezers. We believe the maturation of bead-based microtweezers will revolutionize the state-of-art tools currently used for single cell and single molecule studies.

System Integration - A Major Step toward Lab on a Chip

PubMed Central

2011-01-01

Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications. PMID:21612614

Fully solution-processed organic light-emitting electrochemical cells (OLEC) with inkjet-printed micro-lenses for disposable lab-on-chip applications at ambient conditions

NASA Astrophysics Data System (ADS)

Shu, Zhe; Pabst, Oliver; Beckert, Erik; Eberhardt, Ramona; Tünnermann, Andreas

2016-02-01

Microfluidic lab-on-chip devices can be used for chemical and biological analyses such as DNA tests or environmental monitoring. Such devices integrate most of the basic functionalities needed for scientific analysis on a microfluidic chip. When using such devices, cost and space-intensive lab equipment is no longer necessary. However, in order to make a monolithic and cost-efficient/disposable microfluidic sensing device, direct integration of the excitation light source for fluorescent sensing is often required. To achieve this, we introduce a fully solution processable deviation of OLEDs, organic light-emitting electrochemical cells (OLECs), as a low-cost excitation light source for a disposable microfluidic sensing platform. By mixing metal ions and a solid electrolyte with light-emitting polymers as active materials, an in-situ doping and in-situ PN-junction can be generated within a three layer sandwich device. Thanks to this doping effect, work function adaptation is not necessary and air-stable electrode can be used. An ambient manufacturing process for fully solution-processed OLECs is presented, which consist of a spin-coated blue light-emitting polymer plus dopants on an ITO cathode and an inkjet-printed PEDOT:PSS transparent top anode. A fully transparent blue OLEC is able to obtain light intensity > 2500 cd/m2 under pulsed driving mode and maintain stable after 1000 cycles, which fulfils requirements for simple fluorescent on-chip sensing applications. However, because of the large refractive index difference between substrates and air, about 80% of emitted light is trapped inside the device. Therefore, inkjet printed micro-lenses on the rear side are introduced here to further increase light-emitting brightness.

Stress analysis of ultra-thin silicon chip-on-foil electronic assembly under bending

NASA Astrophysics Data System (ADS)

Wacker, Nicoleta; Richter, Harald; Hoang, Tu; Gazdzicki, Pawel; Schulze, Mathias; Angelopoulos, Evangelos A.; Hassan, Mahadi-Ul; Burghartz, Joachim N.

2014-09-01

In this paper we investigate the bending-induced uniaxial stress at the top of ultra-thin (thickness \\leqslant 20 μm) single-crystal silicon (Si) chips adhesively attached with the aid of an epoxy glue to soft polymeric substrate through combined theoretical and experimental methods. Stress is first determined analytically and numerically using dedicated models. The theoretical results are validated experimentally through piezoresistive measurements performed on complementary metal-oxide-semiconductor (CMOS) transistors built on specially designed chips, and through micro-Raman spectroscopy investigation. Stress analysis of strained ultra-thin chips with CMOS circuitry is crucial, not only for the accurate evaluation of the piezoresistive behavior of the built-in devices and circuits, but also for reliability and deformability analysis. The results reveal an uneven bending-induced stress distribution at the top of the Si-chip that decreases from the central area towards the chip's edges along the bending direction, and increases towards the other edges. Near these edges, stress can reach very high values, facilitating the emergence of cracks causing ultimate chip failure.

Microfluidic CODES: a scalable multiplexed electronic sensor for orthogonal detection of particles in microfluidic channels.

PubMed

Liu, Ruxiu; Wang, Ningquan; Kamili, Farhan; Sarioglu, A Fatih

2016-04-21

Numerous biophysical and biochemical assays rely on spatial manipulation of particles/cells as they are processed on lab-on-a-chip devices. Analysis of spatially distributed particles on these devices typically requires microscopy negating the cost and size advantages of microfluidic assays. In this paper, we introduce a scalable electronic sensor technology, called microfluidic CODES, that utilizes resistive pulse sensing to orthogonally detect particles in multiple microfluidic channels from a single electrical output. Combining the techniques from telecommunications and microfluidics, we route three coplanar electrodes on a glass substrate to create multiple Coulter counters producing distinct orthogonal digital codes when they detect particles. We specifically design a digital code set using the mathematical principles of Code Division Multiple Access (CDMA) telecommunication networks and can decode signals from different microfluidic channels with >90% accuracy through computation even if these signals overlap. As a proof of principle, we use this technology to detect human ovarian cancer cells in four different microfluidic channels fabricated using soft lithography. Microfluidic CODES offers a simple, all-electronic interface that is well suited to create integrated, low-cost lab-on-a-chip devices for cell- or particle-based assays in resource-limited settings.

Fabrication and characterization of SPR chips with the modified bovine serum albumin

NASA Astrophysics Data System (ADS)

Chen, Xing; Zhang, Lu-lu; Cui, Da-fu

2016-03-01

A facile surface plasmon resonance (SPR) chip is developed for small molecule determination and analysis. The SPR chip was prepared based on a self assembling principle, in which the modified bovine serum albumin (BSA) was directly self-assembled onto the bare gold surface. The surface morphology of the chip with the modified BSA was investigated by atomic force microscopy (AFM) and its optical properties were characterized. The surface binding capacity of the bare facile SPR chip with a uniform morphology is 8 times of that of the bare control SPR chip. Based on the experiments of immune reaction between cortisol antibody and cortisol derivative, the sensitivity of the facile SPR chip with the modified BSA is much higher than that of the control SPR chip with the un-modified BSA. The facile SPR chip has been successfully used to detect small molecules. The lowest detection limit is 5 ng/mL with a linear range of 5—100 ng/mL for cortisol analysis. The novel facile SPR chip can also be applied to detect other small molecules.

Deployable Laboratory Applications of Nano- and Bio-Technology (Applications de nanotechnologie et biotechnologie destinees a un laboratoire deployable)

DTIC Science & Technology

2014-10-01

applications of present nano-/ bio -technology include advanced health and fitness monitoring, high-resolution imaging, new environmental sensor platforms...others areas where nano-/ bio -technology development is needed: • Sensors : Diagnostic and detection kits (gene-chips, protein-chips, lab-on-chips, etc...studies on chemo- bio nano- sensors , ultra-sensitive biochips (“lab-on-a-chip” and “cells-on-chips” devices) have been prepared for routine medical

CHIP Regulates Aquaporin-2 Quality Control and Body Water Homeostasis.

PubMed

Wu, Qi; Moeller, Hanne B; Stevens, Donté A; Sanchez-Hodge, Rebekah; Childers, Gabrielle; Kortenoeven, Marleen L A; Cheng, Lei; Rosenbaek, Lena L; Rubel, Carrie; Patterson, Cam; Pisitkun, Trairak; Schisler, Jonathan C; Fenton, Robert A

2018-03-01

The importance of the kidney distal convoluted tubule (DCT) and cortical collecting duct (CCD) is highlighted by various water and electrolyte disorders that arise when the unique transport properties of these segments are disturbed. Despite this critical role, little is known about which proteins have a regulatory role in these cells and how these cells can be regulated by individual physiologic stimuli. By combining proteomics, bioinformatics, and cell biology approaches, we found that the E3 ubiquitin ligase CHIP is highly expressed throughout the collecting duct; is modulated in abundance by vasopressin; interacts with aquaporin-2 (AQP2), Hsp70, and Hsc70; and can directly ubiquitylate the water channel AQP2 in vitro shRNA knockdown of CHIP in CCD cells increased AQP2 protein t 1/2 and reduced AQP2 ubiquitylation, resulting in greater levels of AQP2 and phosphorylated AQP2. CHIP knockdown increased the plasma membrane abundance of AQP2 in these cells. Compared with wild-type controls, CHIP knockout mice or novel CRISPR/Cas9 mice without CHIP E3 ligase activity had greater AQP2 abundance and altered renal water handling, with decreased water intake and urine volume, alongside higher urine osmolality. We did not observe significant changes in other water- or sodium-transporting proteins in the gene-modified mice. In summary, these results suggest that CHIP regulates AQP2 and subsequently, renal water handling. Copyright © 2018 by the American Society of Nephrology.

C-MOS array design techniques

NASA Technical Reports Server (NTRS)

Feller, A.

1978-01-01

The entire complement of standard cells and components, except for the set-reset flip-flop, was completed. Two levels of checking were performed on each device. Logic cells and topological layout are described. All the related computer programs were coded and one level of debugging was completed. The logic for the test chip was modified and updated. This test chip served as the first test vehicle to exercise the standard cell complementary MOS(C-MOS) automatic artwork generation capability.

Microengineered physiological biomimicry: organs-on-chips.

PubMed

Huh, Dongeun; Torisawa, Yu-suke; Hamilton, Geraldine A; Kim, Hyun Jung; Ingber, Donald E

2012-06-21

Microscale engineering technologies provide unprecedented opportunities to create cell culture microenvironments that go beyond current three-dimensional in vitro models by recapitulating the critical tissue-tissue interfaces, spatiotemporal chemical gradients, and dynamic mechanical microenvironments of living organs. Here we review recent advances in this field made over the past two years that are focused on the development of 'Organs-on-Chips' in which living cells are cultured within microfluidic devices that have been microengineered to reconstitute tissue arrangements observed in living organs in order to study physiology in an organ-specific context and to develop specialized in vitro disease models. We discuss the potential of organs-on-chips as alternatives to conventional cell culture models and animal testing for pharmaceutical and toxicology applications. We also explore challenges that lie ahead if this field is to fulfil its promise to transform the future of drug development and chemical safety testing.

3-D readout-electronics packaging for high-bandwidth massively paralleled imager

DOEpatents

Kwiatkowski, Kris; Lyke, James

2007-12-18

Dense, massively parallel signal processing electronics are co-packaged behind associated sensor pixels. Microchips containing a linear or bilinear arrangement of photo-sensors, together with associated complex electronics, are integrated into a simple 3-D structure (a "mirror cube"). An array of photo-sensitive cells are disposed on a stacked CMOS chip's surface at a 45.degree. angle from light reflecting mirror surfaces formed on a neighboring CMOS chip surface. Image processing electronics are held within the stacked CMOS chip layers. Electrical connections couple each of said stacked CMOS chip layers and a distribution grid, the connections for distributing power and signals to components associated with each stacked CSMO chip layer.

Silicon ball grid array chip carrier

DOEpatents

Palmer, David W.; Gassman, Richard A.; Chu, Dahwey

2000-01-01

A ball-grid-array integrated circuit (IC) chip carrier formed from a silicon substrate is disclosed. The silicon ball-grid-array chip carrier is of particular use with ICs having peripheral bond pads which can be reconfigured to a ball-grid-array. The use of a semiconductor substrate such as silicon for forming the ball-grid-array chip carrier allows the chip carrier to be fabricated on an IC process line with, at least in part, standard IC processes. Additionally, the silicon chip carrier can include components such as transistors, resistors, capacitors, inductors and sensors to form a "smart" chip carrier which can provide added functionality and testability to one or more ICs mounted on the chip carrier. Types of functionality that can be provided on the "smart" chip carrier include boundary-scan cells, built-in test structures, signal conditioning circuitry, power conditioning circuitry, and a reconfiguration capability. The "smart" chip carrier can also be used to form specialized or application-specific ICs (ASICs) from conventional ICs. Types of sensors that can be included on the silicon ball-grid-array chip carrier include temperature sensors, pressure sensors, stress sensors, inertia or acceleration sensors, and/or chemical sensors. These sensors can be fabricated by IC processes and can include microelectromechanical (MEM) devices.

Temperature regulation during ultrasonic manipulation for long-term cell handling in a microfluidic chip

NASA Astrophysics Data System (ADS)

Svennebring, J.; Manneberg, O.; Wiklund, M.

2007-12-01

We demonstrate simultaneous micromanipulation and temperature regulation by the use of ultrasonic standing wave technology in a microfluidic chip. The system is based on a microfabricated silicon structure sandwiched between two glass layers, and an external ultrasonic transducer using a refractive wedge placed on top of the chip for efficient coupling of ultrasound into the microchannel. The chip is fully transparent and compatible with any kind of high-resolution optical microscopy. The temperature regulation method uses calibration data of the temperature increase due to the ultrasonic actuation for determining the temperature of the surrounding air and microscope table, controlled by a warm-air heating unit and a heatable mounting frame. The heating methods are independent of each other, resulting in a flexible choice of ultrasonic actuation voltage and flow rate for different cell and particle manipulation purposes. Our results indicate that it is possible to perform stable temperature regulation with an accuracy of the order of ±0.1 °C around any physiologically relevant temperature (e.g., 37 °C) with high temporal stability and repeatability. The purpose is to use ultrasound for long-term cell and/or particle handling in a microfluidic chip while controlling and maintaining the biocompatibility of the system.

Modeling and analysis of the chip formation and transient cutting force during elliptical vibration cutting process

NASA Astrophysics Data System (ADS)

Lin, Jieqiong; Guan, Liang; Lu, Mingming; Han, Jinguo; Kan, Yudi

2017-12-01

In traditional diamond cutting, the cutting force is usually large and it will affect tool life and machining quality. Elliptical vibration cutting (EVC) as one of the ultra-precision machining technologies has a lot of advantages, such as reduces cutting force, extend tool life and so on. It's difficult to predict the transient cutting force of EVC due to its unique elliptical motion trajectory. Study on chip formation will helpfully to predict cutting force. The geometric feature of chip has important effects on cutting force, however, few scholars have studied the chip formation. In order to investigate the time-varying cutting force of EVC, the geometric feature model of chip is established based on analysis of chip formation, and the effects of cutting parameters on the geometric feature of chip are analyzed. To predict transient force quickly and effectively, the geometric feature of chip is introduced into the cutting force model. The calculated results show that the error between the predicted cutting force in this paper and that in the literature is less than 2%, which proves its feasibility.

Tilted pillar array fabrication by the combination of proton beam writing and soft lithography for microfluidic cell capture Part 2: Image sequence analysis based evaluation and biological application.

PubMed

Járvás, Gábor; Varga, Tamás; Szigeti, Márton; Hajba, László; Fürjes, Péter; Rajta, István; Guttman, András

2018-02-01

As a continuation of our previously published work, this paper presents a detailed evaluation of a microfabricated cell capture device utilizing a doubly tilted micropillar array. The device was fabricated using a novel hybrid technology based on the combination of proton beam writing and conventional lithography techniques. Tilted pillars offer unique flow characteristics and support enhanced fluidic interaction for improved immunoaffinity based cell capture. The performance of the microdevice was evaluated by an image sequence analysis based in-house developed single-cell tracking system. Individual cell tracking allowed in-depth analysis of the cell-chip surface interaction mechanism from hydrodynamic point of view. Simulation results were validated by using the hybrid device and the optimized surface functionalization procedure. Finally, the cell capture capability of this new generation microdevice was demonstrated by efficiently arresting cells from a HT29 cell-line suspension. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid determination of cell mass and density using digitally controlled electric field in a microfluidic chip.

PubMed

Zhao, Yuliang; Lai, Hok Sum Sam; Zhang, Guanglie; Lee, Gwo-Bin; Li, Wen Jung

2014-11-21

The density of a single cell is a fundamental property of cells. Cells in the same cycle phase have similar volume, but the differences in their mass and density could elucidate each cell's physiological state. Here we report a novel technique to rapidly measure the density and mass of a single cell using an optically induced electrokinetics (OEK) microfluidic platform. Presently, single cellular mass and density measurement devices require a complicated fabrication process and their output is not scalable, i.e., it is extremely difficult to measure the mass and density of a large quantity of cells rapidly. The technique reported here operates on a principle combining sedimentation theory, computer vision, and microparticle manipulation techniques in an OEK microfluidic platform. We will show in this paper that this technique enables the measurement of single-cell volume, density, and mass rapidly and accurately in a repeatable manner. The technique is also scalable - it allows simultaneous measurement of volume, density, and mass of multiple cells. Essentially, a simple time-controlled projected light pattern is used to illuminate the selected area on the OEK microfluidic chip that contains cells to lift the cells to a particular height above the chip's surface. Then, the cells are allowed to "free fall" to the chip's surface, with competing buoyancy, gravitational, and fluidic drag forces acting on the cells. By using a computer vision algorithm to accurately track the motion of the cells and then relate the cells' motion trajectory to sedimentation theory, the volume, mass, and density of each cell can be rapidly determined. A theoretical model of micro-sized spheres settling towards an infinite plane in a microfluidic environment is first derived and validated experimentally using standard micropolystyrene beads to demonstrate the viability and accuracy of this new technique. Next, we show that the yeast cell volume, mass, and density could be rapidly determined using this technology, with results comparable to those using the existing method suspended microchannel resonator.

Hydrogel-based three-dimensional cell culture for organ-on-a-chip applications.

PubMed

Lee, Seung Hwan; Shim, Kyu Young; Kim, Bumsang; Sung, Jong Hwan

2017-05-01

Recent studies have reported that three-dimensionally cultured cells have more physiologically relevant functions than two-dimensionally cultured cells. Cells are three-dimensionally surrounded by the extracellular matrix (ECM) in complex in vivo microenvironments and interact with the ECM and neighboring cells. Therefore, replicating the ECM environment is key to the successful cell culture models. Various natural and synthetic hydrogels have been used to mimic ECM environments based on their physical, chemical, and biological characteristics, such as biocompatibility, biodegradability, and biochemical functional groups. Because of these characteristics, hydrogels have been combined with microtechnologies and used in organ-on-a-chip applications to more closely recapitulate the in vivo microenvironment. Therefore, appropriate hydrogels should be selected depending on the cell types and applications. The porosity of the selected hydrogel should be controlled to facilitate the movement of nutrients and oxygen. In this review, we describe various types of hydrogels, external stimulation-based gelation of hydrogels, and control of their porosity. Then, we introduce applications of hydrogels for organ-on-a-chip. Last, we also discuss the challenges of hydrogel-based three-dimensional cell culture techniques and propose future directions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:580-589, 2017. © 2017 American Institute of Chemical Engineers.

Survey of critical failure events in on-chip interconnect by fault tree analysis

NASA Astrophysics Data System (ADS)

Yokogawa, Shinji; Kunii, Kyousuke

2018-07-01

In this paper, a framework based on reliability physics is proposed for adopting fault tree analysis (FTA) to the on-chip interconnect system of a semiconductor. By integrating expert knowledge and experience regarding the possibilities of failure on basic events, critical issues of on-chip interconnect reliability will be evaluated by FTA. In particular, FTA is used to identify the minimal cut sets with high risk priority. Critical events affecting the on-chip interconnect reliability are identified and discussed from the viewpoint of long-term reliability assessment. The moisture impact is evaluated as an external event.

Plasma micro-nanotextured polymeric micromixer for DNA purification with high efficiency and dynamic range.

PubMed

Kastania, Athina S; Tsougeni, Katerina; Papadakis, George; Gizeli, Electra; Kokkoris, George; Tserepi, Angeliki; Gogolides, Evangelos

2016-10-26

We present a polymeric microfluidic chip capable of purifying DNA through solid phase extraction. It is designed to be used as a module of an integrated Lab-on-chip platform for pathogen detection, but it can also be used as a stand-alone device. The microfluidic channels are oxygen plasma micro-nanotextured, i.e. randomly roughened in the micro-nano scale, a process creating high surface area as well as high density of carboxyl groups (COOH). The COOH groups together with a buffer that contains polyethylene glycol (PEG), NaCl and ethanol are able to bind DNA on the microchannel surface. The chip design incorporates a mixer so that sample and buffer can be efficiently mixed on chip under continuous flow. DNA is subsequently eluted in water. The chip is able to isolate DNA with high recovery efficiency (96± 11%) in an extremely large dynamic range of prepurified Salmonella DNA as well as from Salmonella cell lysates that correspond to a range of 5 to 1.9 × 10 8  cells (0.263 fg to 2 × 500 ng). The chip was evaluated via absorbance measurements, polymerase chain reaction (PCR), and gel electrophoresis. Copyright © 2016 Elsevier B.V. All rights reserved.

The MicroActive project: automatic detection of disease-related molecular cell activity

NASA Astrophysics Data System (ADS)

Furuberg, Liv; Mielnik, Michal; Johansen, Ib-Rune; Voitel, Jörg; Gulliksen, Anja; Solli, Lars; Karlsen, Frank; Bayer, Tobias; Schönfeld, Friedhelm; Drese, Klaus; Keegan, Helen; Martin, Cara; O'Leary, John; Riegger, Lutz; Koltay, Peter

2007-05-01

The aim of the MicroActive project is to develop an instrument for molecular diagnostics. The instrument will first be tested for patient screening for a group of viruses causing cervical cancer. Two disposable polymer chips with reagents stored on-chip will be inserted into the instrument for each patient sample. The first chip performs sample preparation of the epithelial cervical cells while mRNA amplification and fluorescent detection takes place in the second chip. More than 10 different virus markers will be analysed in one chip. We report results on sub-functions of the amplification chip. The sample is split into smaller droplets, and the droplets move in parallel channels containing different dried reagents for the different analyses. We report experimental results on parallel droplet movement control using one external pump only, combined with hydrophobic valves. Valve burst pressures are controlled by geometry. We show droplet control using valves with burst pressures between 800 and 4500 Pa. We also monitored the re-hydration times for two necessary dried reagents. After sample insertion, uniform concentration of the reagents in the droplet was reached after respectively 60 s and 10 min. These times are acceptable for successful amplification. Finally we have shown positive amplification of HPV type 16 using dried enzymes stored in micro chambers.

Characterization of radiation effects in 65 nm digital circuits with the DRAD digital radiation test chip

NASA Astrophysics Data System (ADS)

Jara Casas, L. M.; Ceresa, D.; Kulis, S.; Miryala, S.; Christiansen, J.; Francisco, R.; Gnani, D.

2017-02-01

A Digital RADiation (DRAD) test chip has been specifically designed to study the impact of Total Ionizing Dose (TID) (<1 Grad) and Single Event Upset (SEU) on digital logic gates in a 65 nm CMOS technology. Nine different versions of standard cell libraries are studied in this chip, basically differing in the device dimensions, Vt flavor and layout of the device. Each library has eighteen test structures specifically designed to characterize delay degradation and power consumption of the standard cells. For SEU study, a dedicated test structure based on a shift register is designed for each library. TID results up to 500 Mrad are reported.

Design and Construction of a Multi-Organ Microfluidic Chip Mimicking the in vivo Microenvironment of Lung Cancer Metastasis.

PubMed

Xu, Zhiyun; Li, Encheng; Guo, Zhe; Yu, Ruofei; Hao, Hualong; Xu, Yitong; Sun, Zhao; Li, Xiancheng; Lyu, Jianxin; Wang, Qi

2016-10-05

Metastasis is a complex pathophysiological process. As the main cause of cancer mortality in humans it represents a serious challenge to both basic researchers and clinicians. Here we report the design and construction of a multi-organ microfluidic chip that closely mimics the in vivo microenvironment of lung cancer metastasis. This multi-organs-on-a-chip includes an upstream "lung" and three downstream "distant organs", with three polydimethylsiloxane (PDMS) layers and two thin PDMS microporous membranes bonded to form three parallel microchannels. Bronchial epithelial, lung cancer, microvascular endothelial, mononuclear, and fibroblast cells were grown separated by the biomembrane in upstream "lung", while astrocytes, osteocytes, and hepatocytes were grown in distant chambers, to mimic lung cancer cell metastasis to the brain, bone, and liver. After culture in this system, lung cancer cells formed a "tumor mass", showed epithelial-mesenchymal transition (with altered expression of E-cadherin, N-cadherin, Snail1, and Snail2) and invasive capacity. A549 cells co-cultured with astrocytes overexpressed CXCR4 protein, indicating damage of astrocytes after cancer cell metastasis to the brain. Osteocytes overexpressed RANKL protein indicates damage of osteocytes after cancer cell metastasis to the bone, and hepatocytes overexpressed AFP protein indicates damage to hepatocytes after cancer cell metastasis to the liver. Finally, in vivo imaging of cancer growth and metastasis in a nude mice model validated the performance of metastasis in the organs-on-chip system. This system provides a useful tool to mimic the in vivo microenvironment of cancer metastasis and to investigate cell-cell interactions during metastasis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gulec, Cagri, E-mail: cagri.gulec@gmail.com; Coban, Neslihan, E-mail: neslic@istanbul.edu.tr; Ozsait-Selcuk, Bilge, E-mail: ozsaitb@istanbul.edu.tr

ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analysesmore » of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses.« less

Microfluidic Biosensing Systems Using Magnetic Nanoparticles

PubMed Central

Giouroudi, Ioanna; Keplinger, Franz

2013-01-01

In recent years, there has been rapidly growing interest in developing hand held, sensitive and cost-effective on-chip biosensing systems that directly translate the presence of certain bioanalytes (e.g., biomolecules, cells and viruses) into an electronic signal. The impressive and rapid progress in micro- and nanotechnology as well as in biotechnology enables the integration of a variety of analytical functions in a single chip. All necessary sample handling and analysis steps are then performed within the chip. Microfluidic systems for biomedical analysis usually consist of a set of units, which guarantees the manipulation, detection and recognition of bioanalytes in a reliable and flexible manner. Additionally, the use of magnetic fields for performing the aforementioned tasks has been steadily gaining interest. This is because magnetic fields can be well tuned and applied either externally or from a directly integrated solution in the biosensing system. In combination with these applied magnetic fields, magnetic nanoparticles are utilized. Some of the merits of magnetic nanoparticles are the possibility of manipulating them inside microfluidic channels by utilizing high gradient magnetic fields, their detection by integrated magnetic microsensors, and their flexibility due to functionalization by means of surface modification and specific binding. Their multi-functionality is what makes them ideal candidates as the active component in miniaturized on-chip biosensing systems. In this review, focus will be given to the type of biosening systems that use microfluidics in combination with magnetoresistive sensors and detect the presence of bioanalyte tagged with magnetic nanoparticles. PMID:24022689

Application of an improved magnetic immunosorbent in an Ephesia chip designed for circulating tumor cell capture.

PubMed

Svobodova, Zuzana; Kucerova, Jana; Autebert, Julien; Horak, Daniel; Bruckova, Lenka; Viovy, Jean-Louis; Bilkova, Zuzana

2014-02-01

In this study, we describe a particular step in developing a microfluidic device for capture and detection of circulating tumor cells-specifically the preparation of an immunosorbent for implementation into the separation chip. We highlight some of the most important specifics connected with superparamegnetic microspheres for microfluidic purposes. Factors such as nonspecific adsorption on microfluidic channels, interactions with model cell lines, and tendency to aggregation were investigated. Poly(glycidyl methacrylate) microspheres with carboxyl groups were employed for this purpose. To address the aforementioned challenges, the microspheres were coated with hydrazide-PEG-hydrazide, and subsequently anti-epithelial cell adhesion molecule (EpCAM) antibody was immobilized. The prepared anti-EpCAM immunosorbent was pretested using model cell lines with differing EpCAM density (MCF7, SKBR3, A549, and Raji) in a batchwise arrangement. Finally, the entire system was implemented and studied in an Ephesia chip and an evaluation was performed by the MCF7 cell line. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Opening of K+ channels by capacitive stimulation from silicon chip

NASA Astrophysics Data System (ADS)

Ulbrich, M. H.; Fromherz, P.

2005-10-01

The development of stable neuroelectronic systems requires a stimulation of nerve cells from semiconductor devices without electrochemical effects at the electrolyte/solid interface and without damage of the cell membrane. The interaction must rely on a reversible opening of voltage-gated ion channels by capacitive coupling. In a proof-of-principle experiment, we demonstrate that Kv1.3 potassium channels expressed in HEK293 cells can be opened from an electrolyte/oxide/silicon (EOS) capacitor. A sufficient strength of electrical coupling is achieved by insulating silicon with a thin film of TiO2 to achieve a high capacitance and by removing NaCl from the electrolyte to enhance the resistance of the cell-chip contact. When a decaying voltage ramp is applied to the EOS capacitor, an outward current through the attached cell membrane is observed that is specific for Kv1.3 channels. An open probability up to fifty percent is estimated by comparison with a numerical simulation of the cell-chip contact.

Selective isolation of magnetic nanoparticle-mediated heterogeneity subpopulation of circulating tumor cells using magnetic gradient based microfluidic system.

PubMed

Kwak, Bongseop; Lee, Jaehun; Lee, Dongkyu; Lee, Kangho; Kwon, Ohwon; Kang, Shinwon; Kim, Youngwoo

2017-02-15

Relocation mechanisms of the circulating tumor cells (CTCs) from the primary site to the secondary site through the blood vessel network cause tumor metastasis. Despite of the importance to diagnose the cancer metastasis by CTCs, still it is formidable challenge to use in the clinical purpose because of the rarity and the heterogeneity of CTCs in the cancer patient's peripheral blood sample. In this study we have developed magnetic force gradient based microfluidic chip (Mag-Gradient Chip) for isolating the total number of CTCs in the sample and characterizing the state of CTCs simultaneously with respect to the epithelial cell adhesion molecule (EpCAM) expression level. We have synthesized magnetic nanoparticles (MNPs) using hydrothermal method and functionalized anti-EpCAM on their surface for the specific binding with CTCs. The Mag-Gradient Chip designed to isolate and classify the CTCs by isolating at the different location in the chip using magnetic force differences depending on the EpCAM expression level. We observed 95.7% of EpCAM positive and 79.3% of EpCAM negative CTCs isolated in the Mag-Gradient Chip. At the same time, the 71.3% of isolated EpCAM positive CTCs were isolated at the first half area whereas the 76.9% of EpCAM negative CTCs were collected at the latter half area. The Mag-Gradient Chip can isolate the 3ml of heterogeneous CTCs sample in 1h with high isolating yield. The EpCAM expression level dose not means essential condition of the metastatic CTCs, but the Mag-Gradient Chip can shorten the date to diagnose the cancer metastasis in clinic. Copyright © 2016 Elsevier B.V. All rights reserved.

Microfluidic size separation of cells and particles using a swinging bucket centrifuge.

PubMed

Yeo, Joo Chuan; Wang, Zhiping; Lim, Chwee Teck

2015-09-01

Biomolecular separation is crucial for downstream analysis. Separation technique mainly relies on centrifugal sedimentation. However, minuscule sample volume separation and extraction is difficult with conventional centrifuge. Furthermore, conventional centrifuge requires density gradient centrifugation which is laborious and time-consuming. To overcome this challenge, we present a novel size-selective bioparticles separation microfluidic chip on a swinging bucket minifuge. Size separation is achieved using passive pressure driven centrifugal fluid flows coupled with centrifugal force acting on the particles within the microfluidic chip. By adopting centrifugal microfluidics on a swinging bucket rotor, we achieved over 95% efficiency in separating mixed 20 μm and 2 μm colloidal dispersions from its liquid medium. Furthermore, by manipulating the hydrodynamic resistance, we performed size separation of mixed microbeads, achieving size efficiency of up to 90%. To further validate our device utility, we loaded spiked whole blood with MCF-7 cells into our microfluidic device and subjected it to centrifugal force for a mere duration of 10 s, thereby achieving a separation efficiency of over 75%. Overall, our centrifugal microfluidic device enables extremely rapid and label-free enrichment of different sized cells and particles with high efficiency.

Microfluidic size separation of cells and particles using a swinging bucket centrifuge

PubMed Central

Yeo, Joo Chuan; Wang, Zhiping; Lim, Chwee Teck

2015-01-01

Biomolecular separation is crucial for downstream analysis. Separation technique mainly relies on centrifugal sedimentation. However, minuscule sample volume separation and extraction is difficult with conventional centrifuge. Furthermore, conventional centrifuge requires density gradient centrifugation which is laborious and time-consuming. To overcome this challenge, we present a novel size-selective bioparticles separation microfluidic chip on a swinging bucket minifuge. Size separation is achieved using passive pressure driven centrifugal fluid flows coupled with centrifugal force acting on the particles within the microfluidic chip. By adopting centrifugal microfluidics on a swinging bucket rotor, we achieved over 95% efficiency in separating mixed 20 μm and 2 μm colloidal dispersions from its liquid medium. Furthermore, by manipulating the hydrodynamic resistance, we performed size separation of mixed microbeads, achieving size efficiency of up to 90%. To further validate our device utility, we loaded spiked whole blood with MCF-7 cells into our microfluidic device and subjected it to centrifugal force for a mere duration of 10 s, thereby achieving a separation efficiency of over 75%. Overall, our centrifugal microfluidic device enables extremely rapid and label-free enrichment of different sized cells and particles with high efficiency. PMID:26487900

Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP*

PubMed Central

Narayan, Vikram; Pion, Emmanuelle; Landré, Vivien; Müller, Petr; Ball, Kathryn L.

2011-01-01

Characteristically for a regulatory protein, the IRF-1 tumor suppressor turns over rapidly with a half-life of between 20–40 min. This allows IRF-1 to reach new steady state protein levels swiftly in response to changing environmental conditions. Whereas CHIP (C terminus of Hsc70-interacting protein), appears to chaperone IRF-1 in unstressed cells, formation of a stable IRF-1·CHIP complex is seen under specific stress conditions. Complex formation, in heat- or heavy metal-treated cells, is accompanied by a decrease in IRF-1 steady state levels and an increase in IRF-1 ubiquitination. CHIP binds directly to an intrinsically disordered domain in the central region of IRF-1 (residues 106–140), and this site is sufficient to form a stable complex with CHIP in cells and to compete in trans with full-length IRF-1, leading to a reduction in its ubiquitination. The study reveals a complex relationship between CHIP and IRF-1 and highlights the role that direct binding or “docking” of CHIP to its substrate(s) can play in its mechanism of action as an E3 ligase. PMID:20947504

Detection of solder bump defects on a flip chip using vibration analysis

NASA Astrophysics Data System (ADS)

Liu, Junchao; Shi, Tielin; Xia, Qi; Liao, Guanglan

2012-03-01

Flip chips are widely used in microelectronics packaging owing to the high demand of integration in IC fabrication. Solder bump defects on flip chips are difficult to detect, because the solder bumps are obscured by the chip and substrate. In this paper a nondestructive detection method combining ultrasonic excitation with vibration analysis is presented for detecting missing solder bumps, which is a typical defect in flip chip packaging. The flip chip analytical model is revised by considering the influence of spring mass on mechanical energy of the system. This revised model is then applied to estimate the flip chip resonance frequencies. We use an integrated signal generator and power amplifier together with an air-coupled ultrasonic transducer to excite the flip chips. The vibrations are measured by a laser scanning vibrometer to detect the resonance frequencies. A sensitivity coefficient is proposed to select the sensitive resonance frequency order for defect detection. Finite element simulation is also implemented for further investigation. The results of analytical computation, experiment, and simulation prove the efficacy of the revised flip chip analytical model and verify the effectiveness of this detection method. Therefore, it may provide a guide for the improvement and innovation of the flip chip on-line inspection systems.

A High-Resolution Minimicroscope System for Wireless Real-Time Monitoring.

PubMed

Wang, Zongjie; Boddeda, Akash; Parker, Benjamin; Samanipour, Roya; Ghosh, Sanjoy; Menard, Frederic; Kim, Keekyoung

2018-07-01

Compact, cost-effective, and high-performance microscope that enables the real-time imaging of cells and lab-on-a-chip devices is highly demanded for cell biology and biomedical engineering. This paper aims to present the design and application of an inexpensive wireless minimicroscope with resolution up to 2592 × 1944 pixels and speed up to 90 f/s. The minimicroscope system was built on a commercial embedded system (Raspberry Pi). We modified a camera module and adopted an inverse dual lens system to obtain the clear field of view and appropriate magnification for tens of micrometer objects. The system was capable of capturing time-lapse images and transferring image data wirelessly. The entire system can be operated wirelessly and cordlessly in a conventional cell culturing incubator. The developed minimicroscope was used to monitor the attachment and proliferation of NIH-3T3 and HEK 293 cells inside an incubator for 50 h. In addition, the minimicroscope was used to monitor a droplet generation process in a microfluidic device. The high-quality images captured by the minimicroscope enabled us an automated analysis of experimental parameters. The successful applications prove the great potential of the developed minimicroscope for monitoring various biological samples and microfluidic devices. This paper presents the design of a high-resolution minimicroscope system that enables the wireless real-time imaging of cells inside the incubator. This system has been verified to be a useful tool to obtain high-quality images and videos for the automated quantitative analysis of biological samples and lab-on-a-chip devices in the long term.

UW VLSI chip tester

NASA Astrophysics Data System (ADS)

McKenzie, Neil

1989-12-01

We present a design for a low-cost, functional VLSI chip tester. It is based on the Apple MacIntosh II personal computer. It tests chips that have up to 128 pins. All pin drivers of the tester are bidirectional; each pin is programmed independently as an input or an output. The tester can test both static and dynamic chips. Rudimentary speed testing is provided. Chips are tested by executing C programs written by the user. A software library is provided for program development. Tests run under both the Mac Operating System and A/UX. The design is implemented using Xilinx Logic Cell Arrays. Price/performance tradeoffs are discussed.

Microfluidic-Based Robotic Sampling System for Radioactive Solutions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jack D. Law; Julia L. Tripp; Tara E. Smith

A novel microfluidic based robotic sampling system has been developed for sampling and analysis of liquid solutions in nuclear processes. This system couples the use of a microfluidic sample chip with a robotic system designed to allow remote, automated sampling of process solutions in-cell and facilitates direct coupling of the microfluidic sample chip with analytical instrumentation. This system provides the capability for near real time analysis, reduces analytical waste, and minimizes the potential for personnel exposure associated with traditional sampling methods. A prototype sampling system was designed, built and tested. System testing demonstrated operability of the microfluidic based sample systemmore » and identified system modifications to optimize performance.« less

A brief review on microfluidic platforms for hormones detection.

PubMed

Ozhikandathil, Jayan; Badilescu, Simona; Packirisamy, Muthukumaran

2017-01-01

Lab-on-chip technology is attracting great interest due to its potential as miniaturized devices that can automate and integrate many sample-handling steps, minimize consumption of reagent and samples, have short processing time and enable multiplexed analysis. Microfluidic devices have demonstrated their potential for a broad range of applications in life sciences, including point-of-care diagnostics and personalized medicine, based on the routine diagnosis of levels of hormones, cancer markers, and various metabolic products in blood, serum, etc. Microfluidics offers an adaptable platform that can facilitate cell culture as well as monitor their activity and control the cellular environment. Signaling molecules released from cells such as neurotransmitters and hormones are important in assessing the health of cells and the effect of drugs on their functions. In this review, we provide an insight into the state-of-art applications of microfluidics for monitoring of hormones released by cells. In our works, we have demonstrated efficient detection methods for bovine growth hormones using nano and microphotonics integrated microfluidics devices. The bovine growth hormone can be used as a growth promoter in dairy farming to enhance the milk and meat production. In the recent years, a few attempts have been reported on developing very sensitive, fast and low-cost methods of detection of bovine growth hormone using micro devices. This paper reviews the current state-of-art of detection and analysis of hormone using integrated optical micro and nanofluidics systems. In addition, the paper also focuses on various lab-on-a-chip technologies reported recently, and their benefits for screening growth hormones in milk.

Vitamin K2 downregulates the expression of fibroblast growth factor receptor 3 in human hepatocellular carcinoma cells.

PubMed

Cao, Ke; Liu, Weidong; Nakamura, Hideji; Enomoto, Hirayuki; Yamamoto, Teruhisa; Saito, Masaki; Imanishi, Hiroyasu; Shimomura, Soji; Cao, Peiguo; Nishiguchi, Shuhei

2009-11-01

Vitamin K2 exerts an antitumor activity on human hepatocellular carcinoma (HCC), however, its inhibitory mechanism has not yet been clarified. This study was designed to identify the attractive target molecule of vitamin K2 and shed some light on its effects on fibroblast growth factor receptor (FGFR)3 in HCC cells. The changes in the gene expression of HuH-7 after vitamin K2 treatment were evaluated by a DNA chip analysis. The mRNA and protein levels of FGFR were evaluated by semiquantitative reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and western blot analysis. The promoter activity of the FGFR3 gene was measured by a dual-luciferase assay. The DNA chip analysis revealed different inhibitory rates of gene expression of FGFR3 (60.6%) and FGFR1 (19.4%) after vitamin K2 treatment. Vitamin K2 suppresses the proliferation of HuH-7 in a dose-dependent manner and its inhibitory rate reached approximately 61.8% at the dose of 30 microM. FGFR3 mRNA was significantly reduced based on semiquantitative RT-PCR and decreased 61.5% by a real-time PCR method after vitamin K2 treatment, but FGFR1 mRNA was not. The level of FGFR3 protein was also reduced by vitamin K2 treatment. The luciferase assay demonstrated that vitamin K2 significantly suppressed the promoter activity of FGFR3. Furthermore, the FGFR3-ERK1/2 signaling pathway was suppressed by vitamin K2 treatment. These findings suggest that vitamin K2 may suppress the proliferation of HCC cells through the downregulation of the FGFR3 expression. The transcriptional suppression of FGFR3 may be a novel mechanism of the vitamin K2 action for HCC cells.

A Label-Free Microfluidic Biosensor for Activity Detection of Single Microalgae Cells Based on Chlorophyll Fluorescence

PubMed Central

Wang, Junsheng; Sun, Jinyang; Song, Yongxin; Xu, Yongyi; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

2013-01-01

Detection of living microalgae cells is very important for ballast water treatment and analysis. Chlorophyll fluorescence is an indicator of photosynthetic activity and hence the living status of plant cells. In this paper, we developed a novel microfluidic biosensor system that can quickly and accurately detect the viability of single microalgae cells based on chlorophyll fluorescence. The system is composed of a laser diode as an excitation light source, a photodiode detector, a signal analysis circuit, and a microfluidic chip as a microalgae cell transportation platform. To demonstrate the utility of this system, six different living and dead algae samples (Karenia mikimotoi Hansen, Chlorella vulgaris, Nitzschia closterium, Platymonas subcordiformis, Pyramidomonas delicatula and Dunaliella salina) were tested. The developed biosensor can distinguish clearly between the living microalgae cells and the dead microalgae cells. The smallest microalgae cells that can be detected by using this biosensor are 3 μm ones. Even smaller microalgae cells could be detected by increasing the excitation light power. The developed microfluidic biosensor has great potential for in situ ballast water analysis. PMID:24287532

Programming Cell Adhesion for On-Chip Sequential Boolean Logic Functions.

PubMed

Qu, Xiangmeng; Wang, Shaopeng; Ge, Zhilei; Wang, Jianbang; Yao, Guangbao; Li, Jiang; Zuo, Xiaolei; Shi, Jiye; Song, Shiping; Wang, Lihua; Li, Li; Pei, Hao; Fan, Chunhai

2017-08-02

Programmable remodelling of cell surfaces enables high-precision regulation of cell behavior. In this work, we developed in vitro constructed DNA-based chemical reaction networks (CRNs) to program on-chip cell adhesion. We found that the RGD-functionalized DNA CRNs are entirely noninvasive when interfaced with the fluidic mosaic membrane of living cells. DNA toehold with different lengths could tunably alter the release kinetics of cells, which shows rapid release in minutes with the use of a 6-base toehold. We further demonstrated the realization of Boolean logic functions by using DNA strand displacement reactions, which include multi-input and sequential cell logic gates (AND, OR, XOR, and AND-OR). This study provides a highly generic tool for self-organization of biological systems.

Endocrine system on chip for a diabetes treatment model.

PubMed

Nguyen, Dao Thi Thuy; van Noort, Danny; Jeong, In-Kyung; Park, Sungsu

2017-02-21

The endocrine system is a collection of glands producing hormones which, among others, regulates metabolism, growth and development. One important group of endocrine diseases is diabetes, which is caused by a deficiency or diminished effectiveness of endogenous insulin. By using a microfluidic perfused 3D cell-culture chip, we developed an 'endocrine system on chip' to potentially be able to screen drugs for the treatment of diabetes by measuring insulin release over time. Insulin-secreting β-cells are located in the pancreas, while L-cells, located in the small intestines, stimulate insulin secretion. Thus, we constructed a co-culture of intestinal-pancreatic cells to measure the effect of glucose on the production of glucagon-like peptide-1 (GLP-1) from the L-cell line (GLUTag) and insulin from the pancreatic β-cell line (INS-1). After three days of culture, both cell lines formed aggregates, exhibited 3D cell morphology, and showed good viability (>95%). We separately measured the dynamic profile of GLP-1 and insulin release at glucose concentrations of 0.5 and 20 mM, as well as the combined effect of GLP-1 on insulin production at these glucose concentrations. In response to glucose stimuli, GLUTag and INS-1 cells produced higher amounts of GLP-1 and insulin, respectively, compared to a static 2D cell culture. INS-1 combined with GLUTag cells exhibited an even higher insulin production in response to glucose stimulation. At higher glucose concentrations, the diabetes model on chip showed faster saturation of the insulin level. Our results suggest that the endocrine system developed in this study is a useful tool for observing dynamical changes in endocrine hormones (GLP-1 and insulin) in a glucose-dependent environment. Moreover, it can potentially be used to screen GLP-1 analogues and natural insulin and GLP-1 stimulants for diabetes treatment.

Towards autonomous lab-on-a-chip devices for cell phone biosensing.

PubMed

Comina, Germán; Suska, Anke; Filippini, Daniel

2016-03-15

Modern cell phones are a ubiquitous resource with a residual capacity to accommodate chemical sensing and biosensing capabilities. From the different approaches explored to capitalize on such resource, the use of autonomous disposable lab-on-a-chip (LOC) devices-conceived as only accessories to complement cell phones-underscores the possibility to entirely retain cell phones' ubiquity for distributed biosensing. The technology and principles exploited for autonomous LOC devices are here selected and reviewed focusing on their potential to serve cell phone readout configurations. Together with this requirement, the central aspects of cell phones' resources that determine their potential for analytical detection are examined. The conversion of these LOC concepts into universal architectures that are readable on unaccessorized phones is discussed within this context. Copyright © 2015 Elsevier B.V. All rights reserved.

Deep Artificial Neural Networks and Neuromorphic Chips for Big Data Analysis: Pharmaceutical and Bioinformatics Applications.

PubMed

Pastur-Romay, Lucas Antón; Cedrón, Francisco; Pazos, Alejandro; Porto-Pazos, Ana Belén

2016-08-11

Over the past decade, Deep Artificial Neural Networks (DNNs) have become the state-of-the-art algorithms in Machine Learning (ML), speech recognition, computer vision, natural language processing and many other tasks. This was made possible by the advancement in Big Data, Deep Learning (DL) and drastically increased chip processing abilities, especially general-purpose graphical processing units (GPGPUs). All this has created a growing interest in making the most of the potential offered by DNNs in almost every field. An overview of the main architectures of DNNs, and their usefulness in Pharmacology and Bioinformatics are presented in this work. The featured applications are: drug design, virtual screening (VS), Quantitative Structure-Activity Relationship (QSAR) research, protein structure prediction and genomics (and other omics) data mining. The future need of neuromorphic hardware for DNNs is also discussed, and the two most advanced chips are reviewed: IBM TrueNorth and SpiNNaker. In addition, this review points out the importance of considering not only neurons, as DNNs and neuromorphic chips should also include glial cells, given the proven importance of astrocytes, a type of glial cell which contributes to information processing in the brain. The Deep Artificial Neuron-Astrocyte Networks (DANAN) could overcome the difficulties in architecture design, learning process and scalability of the current ML methods.

Deep Artificial Neural Networks and Neuromorphic Chips for Big Data Analysis: Pharmaceutical and Bioinformatics Applications

PubMed Central

Pastur-Romay, Lucas Antón; Cedrón, Francisco; Pazos, Alejandro; Porto-Pazos, Ana Belén

2016-01-01

Over the past decade, Deep Artificial Neural Networks (DNNs) have become the state-of-the-art algorithms in Machine Learning (ML), speech recognition, computer vision, natural language processing and many other tasks. This was made possible by the advancement in Big Data, Deep Learning (DL) and drastically increased chip processing abilities, especially general-purpose graphical processing units (GPGPUs). All this has created a growing interest in making the most of the potential offered by DNNs in almost every field. An overview of the main architectures of DNNs, and their usefulness in Pharmacology and Bioinformatics are presented in this work. The featured applications are: drug design, virtual screening (VS), Quantitative Structure–Activity Relationship (QSAR) research, protein structure prediction and genomics (and other omics) data mining. The future need of neuromorphic hardware for DNNs is also discussed, and the two most advanced chips are reviewed: IBM TrueNorth and SpiNNaker. In addition, this review points out the importance of considering not only neurons, as DNNs and neuromorphic chips should also include glial cells, given the proven importance of astrocytes, a type of glial cell which contributes to information processing in the brain. The Deep Artificial Neuron–Astrocyte Networks (DANAN) could overcome the difficulties in architecture design, learning process and scalability of the current ML methods. PMID:27529225

Associative Pattern Recognition In Analog VLSI Circuits

NASA Technical Reports Server (NTRS)

Tawel, Raoul

1995-01-01

Winner-take-all circuit selects best-match stored pattern. Prototype cascadable very-large-scale integrated (VLSI) circuit chips built and tested to demonstrate concept of electronic associative pattern recognition. Based on low-power, sub-threshold analog complementary oxide/semiconductor (CMOS) VLSI circuitry, each chip can store 128 sets (vectors) of 16 analog values (vector components), vectors representing known patterns as diverse as spectra, histograms, graphs, or brightnesses of pixels in images. Chips exploit parallel nature of vector quantization architecture to implement highly parallel processing in relatively simple computational cells. Through collective action, cells classify input pattern in fraction of microsecond while consuming power of few microwatts.

A Single-Chip Solar Energy Harvesting IC Using Integrated Photodiodes for Biomedical Implant Applications.

PubMed

Chen, Zhiyuan; Law, Man-Kay; Mak, Pui-In; Martins, Rui P

2017-02-01

In this paper, an ultra-compact single-chip solar energy harvesting IC using on-chip solar cell for biomedical implant applications is presented. By employing an on-chip charge pump with parallel connected photodiodes, a 3.5 × efficiency improvement can be achieved when compared with the conventional stacked photodiode approach to boost the harvested voltage while preserving a single-chip solution. A photodiode-assisted dual startup circuit (PDSC) is also proposed to improve the area efficiency and increase the startup speed by 77%. By employing an auxiliary charge pump (AQP) using zero threshold voltage (ZVT) devices in parallel with the main charge pump, a low startup voltage of 0.25 V is obtained while minimizing the reversion loss. A 4 V in gate drive voltage is utilized to reduce the conduction loss. Systematic charge pump and solar cell area optimization is also introduced to improve the energy harvesting efficiency. The proposed system is implemented in a standard 0.18- [Formula: see text] CMOS technology and occupies an active area of 1.54 [Formula: see text]. Measurement results show that the on-chip charge pump can achieve a maximum efficiency of 67%. With an incident power of 1.22 [Formula: see text] from a halogen light source, the proposed energy harvesting IC can deliver an output power of 1.65 [Formula: see text] at 64% charge pump efficiency. The chip prototype is also verified using in-vitro experiment.

The Role of the Co-Chaperone, CHIP, in Androgen-Independent Prostate Cancer

DTIC Science & Technology

2012-02-01

LNCap C4-2 but not in LNCap cells. Figure 9:. CHIP gene expression induced SASH1 gene expression in LNCaP cells but not in LNCap Tsai and LNCap...PCR 16 Gene Product (Gene) C4-2 Cells LNCaP Cells LNCaP Tsai Cells RhoE (RND3) Increased Unchanged Increased SASH1 Unchanged Brief Increase

Clinical validation of an ultra high-throughput spiral microfluidics for the detection and enrichment of viable circulating tumor cells.

PubMed

Khoo, Bee Luan; Warkiani, Majid Ebrahimi; Tan, Daniel Shao-Weng; Bhagat, Ali Asgar S; Irwin, Darryl; Lau, Dawn Pingxi; Lim, Alvin S T; Lim, Kiat Hon; Krisna, Sai Sakktee; Lim, Wan-Teck; Yap, Yoon Sim; Lee, Soo Chin; Soo, Ross A; Han, Jongyoon; Lim, Chwee Teck

2014-01-01

Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation. Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12-1275 CTCs/ml; Lung cancer samples: 10-1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples. We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.

Advances of lab-on-a-chip in isolation, detection and post-processing of circulating tumour cells.

PubMed

Yu, Ling; Ng, Shu Rui; Xu, Yang; Dong, Hua; Wang, Ying Jun; Li, Chang Ming

2013-08-21

Circulating tumour cells (CTCs) are shed by primary tumours and are found in the peripheral blood of patients with metastatic cancers. Recent studies have shown that the number of CTCs corresponds with disease severity and prognosis. Therefore, detection and further functional analysis of CTCs are important for biomedical science, early diagnosis of cancer metastasis and tracking treatment efficacy in cancer patients, especially in point-of-care applications. Over the last few years, there has been an increasing shift towards not only capturing and detecting these rare cells, but also ensuring their viability for post-processing, such as cell culture and genetic analysis. High throughput lab-on-a-chip (LOC) has been fuelled up to process and analyse heterogeneous real patient samples while gaining profound insights for cancer biology. In this review, we highlight how miniaturisation strategies together with nanotechnologies have been used to advance LOC for capturing, separating, enriching and detecting different CTCs efficiently, while meeting the challenges of cell viability, high throughput multiplex or single-cell detection and post-processing. We begin this survey with an introduction to CTC biology, followed by description of the use of various materials, microstructures and nanostructures for design of LOC to achieve miniaturisation, as well as how various CTC capture or separation strategies can enhance cell capture and enrichment efficiencies, purity and viability. The significant progress of various nanotechnologies-based detection techniques to achieve high sensitivities and low detection limits for viable CTCs and/or to enable CTC post-processing are presented and the fundamental insights are also discussed. Finally, the challenges and perspectives of the technologies are enumerated.

Droplet Microfluidics for Compartmentalized Cell Lysis and Extension of DNA from Single-Cells

NASA Astrophysics Data System (ADS)

Zimny, Philip; Juncker, David; Reisner, Walter

Current single cell DNA analysis methods suffer from (i) bias introduced by the need for molecular amplification and (ii) limited ability to sequence repetitive elements, resulting in (iii) an inability to obtain information regarding long range genomic features. Recent efforts to circumvent these limitations rely on techniques for sensing single molecules of DNA extracted from single-cells. Here we demonstrate a droplet microfluidic approach for encapsulation and biochemical processing of single-cells inside alginate microparticles. In our approach, single-cells are first packaged inside the alginate microparticles followed by cell lysis, DNA purification, and labeling steps performed off-chip inside this microparticle system. The alginate microparticles are then introduced inside a micro/nanofluidic system where the alginate is broken down via a chelating buffer, releasing long DNA molecules which are then extended inside nanofluidic channels for analysis via standard mapping protocols.

Online oxygen monitoring using integrated inkjet-printed sensors in a liver-on-a-chip system.

PubMed

Moya, A; Ortega-Ribera, M; Guimerà, X; Sowade, E; Zea, M; Illa, X; Ramon, E; Villa, R; Gracia-Sancho, J; Gabriel, G

2018-06-12

The demand for real-time monitoring of cell functions and cell conditions has dramatically increased with the emergence of organ-on-a-chip (OOC) systems. However, the incorporation of co-cultures and microfluidic channels in OOC systems increases their biological complexity and therefore makes the analysis and monitoring of analytical parameters inside the device more difficult. In this work, we present an approach to integrate multiple sensors in an extremely thin, porous and delicate membrane inside a liver-on-a-chip device. Specifically, three electrochemical dissolved oxygen (DO) sensors were inkjet-printed along the microfluidic channel allowing local online monitoring of oxygen concentrations. This approach demonstrates the existence of an oxygen gradient up to 17.5% for rat hepatocytes and 32.5% for human hepatocytes along the bottom channel. Such gradients are considered crucial for the appearance of zonation of the liver. Inkjet printing (IJP) was the selected technology as it allows drop on demand material deposition compatible with delicate substrates, as used in this study, which cannot withstand temperatures higher than 130 °C. For the deposition of uniform gold and silver conductive inks on the porous membrane, a primer layer using SU-8 dielectric material was used to seal the porosity of the membrane at defined areas, with the aim of building a uniform sensor device. As a proof-of-concept, experiments with cell cultures of primary human and rat hepatocytes were performed, and oxygen consumption rate was stimulated with carbonyl-cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), accelerating the basal respiration of 0.23 ± 0.07 nmol s-1/106 cells up to 5.95 ± 0.67 nmol s-1/106 cells s for rat cells and the basal respiration of 0.17 ± 0.10 nmol s-1/106 cells by up to 10.62 ± 1.15 nmol s-1/106 cells for human cells, with higher oxygen consumption of the cells seeded at the outflow zone. These results demonstrate that the approach of printing sensors inside an OOC has tremendous potential because IJP is a feasible technique for the integration of different sensors for evaluating metabolic activity of cells, and overcomes one of the major challenges still remaining on how to tap the full potential of OOC systems.

Lab-on-a-chip platforms for quantification of multicellular interactions in bone remodeling.

PubMed

George, Estee L; Truesdell, Sharon L; York, Spencer L; Saunders, Marnie M

2018-04-01

Researchers have been using lab-on-a-chip systems to isolate factors for study, simulate laboratory analysis and model cellular, tissue and organ level processes. The technology is increasing rapidly, but the bone field has been slow to keep pace. Novel models are needed that have the power and flexibility to investigate the elegant and synchronous multicellular interactions that occur in normal bone turnover and in disease states in which remodeling is implicated. By removing temporal and spatial limitations and enabling quantification of functional outcomes, the platforms should provide unique environments that are more biomimetic than single cell type systems while minimizing complex systemic effects of in vivo models. This manuscript details the development and characterization of lab-on-a-chip platforms for stimulating osteocytes and quantifying bone remodeling. Our platforms provide the foundation for a model that can be used to investigate remodeling interactions as a whole or as a standard mechanotransduction tool by which isolated activity can be quantified as a function of load. Copyright © 2018 Elsevier Inc. All rights reserved.

Self-priming compartmentalization digital LAMP for point-of-care.

PubMed

Zhu, Qiangyuan; Gao, Yibo; Yu, Bingwen; Ren, Hao; Qiu, Lin; Han, Sihai; Jin, Wei; Jin, Qinhan; Mu, Ying

2012-11-21

Digital nucleic acid amplification provides unprecedented opportunities for absolute nucleic acid quantification by counting of single molecules. This technique is useful for molecular genetic analysis in cancer, stem cell, bacterial, non-invasive prenatal diagnosis in which many biologists are interested. This paper describes a self-priming compartmentalization (SPC) microfluidic chip platform for performing digital loop-mediated amplification (LAMP). The energy for the pumping is pre-stored in the degassed bulk PDMS by exploiting the high gas solubility of PDMS; therefore, no additional structures other than channels and reservoirs are required. The sample and oil are sequentially sucked into the channels, and the pressure difference of gas dissolved in PDMS allows sample self-compartmentalization without the need for further chip manipulation such as with pneumatic microvalves and control systems, and so on. The SPC digital LAMP chip can be used like a 384-well plate, so, the world-to-chip fluidic interconnections are avoided. The microfluidic chip contains 4 separate panels, each panel contains 1200 independent 6 nL chambers and can be used to detect 4 samples simultaneously. Digital LAMP on the microfluidic chip was tested quantitatively by using β-actin DNA from humans. The self-priming compartmentalization behavior is roughly predictable using a two-dimensional model. The uniformity of compartmentalization was analyzed by fluorescent intensity and fraction of volume. The results showed that the feasibility and flexibility of the microfluidic chip platform for amplifying single nucleic acid molecules in different chambers made by diluting and distributing sample solutions. The SPC chip has the potential to meet the requirements of a general laboratory: power-free, valve-free, operating at isothermal temperature, inexpensive, sensitive, economizing labour time and reagents. The disposable analytical devices with appropriate air-tight packaging should be useful for point-of-care, and enabling it to become one of the common tools for biology research, especially, in point-of-care testing.

ChAMP: updated methylation analysis pipeline for Illumina BeadChips.

PubMed

Tian, Yuan; Morris, Tiffany J; Webster, Amy P; Yang, Zhen; Beck, Stephan; Feber, Andrew; Teschendorff, Andrew E

2017-12-15

The Illumina Infinium HumanMethylationEPIC BeadChip is the new platform for high-throughput DNA methylation analysis, effectively doubling the coverage compared to the older 450 K array. Here we present a significantly updated and improved version of the Bioconductor package ChAMP, which can be used to analyze EPIC and 450k data. Many enhanced functionalities have been added, including correction for cell-type heterogeneity, network analysis and a series of interactive graphical user interfaces. ChAMP is a BioC package available from https://bioconductor.org/packages/release/bioc/html/ChAMP.html. a.teschendorff@ucl.ac.uk or s.beck@ucl.ac.uk or a.feber@ucl.ac.uk. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Design and Characterization of a Sensorized Microfluidic Cell-Culture System with Electro-Thermal Micro-Pumps and Sensors for Cell Adhesion, Oxygen, and pH on a Glass Chip.

PubMed

Bonk, Sebastian M; Stubbe, Marco; Buehler, Sebastian M; Tautorat, Carsten; Baumann, Werner; Klinkenberg, Ernst-Dieter; Gimsa, Jan

2015-07-30

We combined a multi-sensor glass-chip with a microfluidic channel grid for the characterization of cellular behavior. The grid was imprinted in poly-dimethyl-siloxane. Mouse-embryonal/fetal calvaria fibroblasts (MC3T3-E1) were used as a model system. Thin-film platinum (Pt) sensors for respiration (amperometric oxygen electrode), acidification (potentiometric pH electrodes) and cell adhesion (interdigitated-electrodes structures, IDES) allowed us to monitor cell-physiological parameters as well as the cell-spreading behavior. Two on-chip electro-thermal micro-pumps (ETμPs) permitted the induction of medium flow in the system, e.g., for medium mixing and drug delivery. The glass-wafer technology ensured the microscopic observability of the on-chip cell culture. Connecting Pt structures were passivated by a 1.2 μm layer of silicon nitride (Si3N4). Thin Si3N4 layers (20 nm or 60 nm) were used as the sensitive material of the pH electrodes. These electrodes showed a linear behavior in the pH range from 4 to 9, with a sensitivity of up to 39 mV per pH step. The oxygen sensors were circular Pt electrodes with a sensor area of 78.5 μm(2). Their sensitivity was 100 pA per 1% oxygen increase in the range from 0% to 21% oxygen (air saturated). Two different IDES geometries with 30- and 50-μm finger spacings showed comparable sensitivities in detecting the proliferation rate of MC3T3 cells. These cells were cultured for 11 days in vitro to test the biocompatibility, microfluidics and electric sensors of our system under standard laboratory conditions.

Improved hydrophilic interaction chromatography LC/MS of heparinoids using a chip with postcolumn makeup flow.

PubMed

Staples, Gregory O; Naimy, Hicham; Yin, Hongfeng; Kileen, Kevin; Kraiczek, Karsten; Costello, Catherine E; Zaia, Joseph

2010-01-15

Heparan sulfate (HS) and heparin are linear, heterogeneous carbohydrates of the glycosaminoglycan (GAG) family that are modified by N-acetylation, N-sulfation, O-sulfation, and uronic acid epimerization. HS interacts with growth factors in the extracellular matrix, thereby modulating signaling pathways that govern cell growth, development, differentiation, proliferation, and adhesion. High-performance liquid chromatography (HPLC)-chip-based hydrophilic interaction liquid chromatography/mass spectrometry has emerged as a method for analyzing the domain structure of GAGs. However, analysis of highly sulfated GAG structures decasaccharide or larger in size has been limited by spray instability in the negative-ion mode. This report demonstrates that addition of postcolumn makeup flow to the amide-HPLC-chip configuration permits robust and reproducible analysis of extended GAG domains (up to degree of polymerization 18) from HS and heparin. This platform provides quantitative information regarding the oligosaccharide profile, degree of sulfation, and nonreducing chain termini. It is expected that this technology will enable quantitative, comparative glycomics profiling of extended GAG oligosaccharide domains of functional interest.

Allergen screening bioassays: recent developments in lab-on-a-chip and lab-on-a-disc systems.

PubMed

Ho, Ho-pui; Lau, Pui-man; Kwok, Ho-chin; Wu, Shu-yuen; Gao, Minghui; Cheung, Anthony Ka-lun; Chen, Qiulan; Wang, Guanghui; Kwan, Yiu-wa; Wong, Chun-kwok; Kong, Siu-kai

2014-01-01

Allergies occur when a person's immune system mounts an abnormal response with or without IgE to a normally harmless substance called an allergen. The standard skin-prick test introduces suspected allergens into the skin with lancets in order to trigger allergic reactions. This test is annoying and sometimes life threatening. New tools such as lab-on-a-chip and lab-on-a-disc, which rely on microfabrication, are designed for allergy testing. These systems provide benefits such as short analysis times, enhanced sensitivity, simplified procedures, minimal consumption of sample and reagents and low cost. This article gives a summary of these systems. In particular, a cell-based assay detecting both the IgE- and non-IgE-type triggers through the study of degranulation in a centrifugal microfluidic system is highlighted.

Analysis of Photonic Networks for a Chip Multiprocessor Using Scientific Applications

DTIC Science & Technology

2009-05-01

Analysis of Photonic Networks for a Chip Multiprocessor Using Scientific Applications Gilbert Hendry†, Shoaib Kamil‡?, Aleksandr Biberman†, Johnnie...electronic networks -on-chip warrants investigating real application traces on functionally compa- rable photonic and electronic network designs. We... network can achieve 75× improvement in energy ef- ficiency for synthetic benchmarks and up to 37× improve- ment for real scientific applications

[Fabrications of a poly (methyl methacrylate) (PMMA) microfluidic chip-based DNA analysis device].

PubMed

Du, Xiao-Guang

2009-12-01

A DNA analysis device based on poly(methyl methacrylate) (PMMA) microfluidic chips was developed. A PMMA chip with cross microchannels was fabricated by a simple hot embossing. Microchannels were modified with a static adsorptive coating method using 2% hydroxyethyl cellulose. A high-voltage power unit, variable in the range 0-1 800 V, was used for on-chip DNA sample injection and gel electrophoretic separation. High speed, high resolution DNA analysis was obtained with the home-built PMMA chip in a sieving matrix containing 2% hydroxyethyl cellulose with a blue intercalating dye, TO-PRO-3 (TP3), by using diode laser induced fluorescence detection based on optical fibers with a 670 nm long-pass filter. The DNA analysis device was applied for the separation of phiX-174/HaeIII DNA digest sample with 11 fragments ranging from 72 to 1 353 bp. A separation efficiency of 1.14 x 10(6) plates/m was obtained for the 603 bp fragments, while the R of 271/281 bp fragments was 1.2. The device was characterized by simple design, low cost for fabrication and operation, reusable PMMA chips, and good reproducibility. A portable microfluidic device for DNA analysis can be developed for clinical diagnosis and disease screening.

Microphysiological modeling of the reproductive tract: a fertile endeavor.

PubMed

Eddie, Sharon L; Kim, J Julie; Woodruff, Teresa K; Burdette, Joanna E

2014-09-01

Preclinical toxicity testing in animal models is a cornerstone of the drug development process, yet it is often unable to predict adverse effects and tolerability issues in human subjects. Species-specific responses to investigational drugs have led researchers to utilize human tissues and cells to better estimate human toxicity. Unfortunately, human cell-derived models are imperfect because toxicity is assessed in isolation, removed from the normal physiologic microenvironment. Microphysiological modeling often referred to as 'organ-on-a-chip' or 'human-on-a-chip' places human tissue into a microfluidic system that mimics the complexity of human in vivo physiology, thereby allowing for toxicity testing on several cell types, tissues, and organs within a more biologically relevant environment. Here we describe important concepts when developing a repro-on-a-chip model. The development of female and male reproductive microfluidic systems is critical to sex-based in vitro toxicity and drug testing. This review addresses the biological and physiological aspects of the male and female reproductive systems in vivo and what should be considered when designing a microphysiological human-on-a-chip model. Additionally, interactions between the reproductive tract and other systems are explored, focusing on the impact of factors and hormones produced by the reproductive tract and disease pathophysiology. © 2014 by the Society for Experimental Biology and Medicine.

μOrgano: A Lego®-Like Plug & Play System for Modular Multi-Organ-Chips.

PubMed

Loskill, Peter; Marcus, Sivan G; Mathur, Anurag; Reese, Willie Mae; Healy, Kevin E

2015-01-01

Human organ-on-a-chip systems for drug screening have evolved as feasible alternatives to animal models, which are unreliable, expensive, and at times erroneous. While chips featuring single organs can be of great use for both pharmaceutical testing and basic organ-level studies, the huge potential of the organ-on-a-chip technology is revealed by connecting multiple organs on one chip to create a single integrated system for sophisticated fundamental biological studies and devising therapies for disease. Furthermore, since most organ-on-a-chip systems require special protocols with organ-specific media for the differentiation and maturation of the tissues, multi-organ systems will need to be temporally customizable and flexible in terms of the time point of connection of the individual organ units. We present a customizable Lego®-like plug & play system, μOrgano, which enables initial individual culture of single organ-on-a-chip systems and subsequent connection to create integrated multi-organ microphysiological systems. As a proof of concept, the μOrgano system was used to connect multiple heart chips in series with excellent cell viability and spontaneously physiological beat rates.

μOrgano: A Lego®-Like Plug & Play System for Modular Multi-Organ-Chips

PubMed Central

Loskill, Peter; Marcus, Sivan G.; Mathur, Anurag; Reese, Willie Mae; Healy, Kevin E.

2015-01-01

Human organ-on-a-chip systems for drug screening have evolved as feasible alternatives to animal models, which are unreliable, expensive, and at times erroneous. While chips featuring single organs can be of great use for both pharmaceutical testing and basic organ-level studies, the huge potential of the organ-on-a-chip technology is revealed by connecting multiple organs on one chip to create a single integrated system for sophisticated fundamental biological studies and devising therapies for disease. Furthermore, since most organ-on-a-chip systems require special protocols with organ-specific media for the differentiation and maturation of the tissues, multi-organ systems will need to be temporally customizable and flexible in terms of the time point of connection of the individual organ units. We present a customizable Lego®-like plug & play system, μOrgano, which enables initial individual culture of single organ-on-a-chip systems and subsequent connection to create integrated multi-organ microphysiological systems. As a proof of concept, the μOrgano system was used to connect multiple heart chips in series with excellent cell viability and spontaneously physiological beat rates. PMID:26440672

Microarray analysis of laser capture microdissected-anulus cells from the human intervertebral disc.

PubMed

Gruber, Helen E; Mougeot, Jean-Luc; Hoelscher, Gretchen; Ingram, Jane A; Hanley, Edward N

2007-05-15

Five Thompson Grade I/II discs (Group 1), 7 Grade III discs (Group 2), and 3 Grade IV discs (Group IV) were studied here in a project approved by the authors' Human Subjects Institutional Review Board. Our objective was to use laser capture microdissection (LCM) to harvest cells from the human anulus and to derive gene expression profiles using microarray analysis. Appropriate gene expression is essential in the intervertebral disc for maintenance of extracellular matrix (ECM), ECM remodeling, and maintenance of a viable disc cell population. During disc degeneration, cell numbers drop, making gene expression studies challenging. LCM was used to harvest cells from paraffin-embedded sections of human anulus tissue. Gene profiling used Affymetrix GeneChip Human X3P arrays. ANOVA and SAM permutation analysis were applied to dCHIP normalized, filtered, and log-transformed gene expression data ( approximately 33,500 probes), and data analyzed to identify genes that were significantly differentially expressed between the 3 groups. We identified 47 genes that were significantly differentially expressed between the 3 groups (P < 0.001 and lowest q values). Compared with the healthiest discs (Grade I/II), 13 genes were up-regulated and 19 down-regulated in both the Grade III and the Grade IV discs. Genes with biologic significance regulated during degeneration involved cell senescence, low cell division rates, hypoxia-related genes, heat-shock protein 70 interacting protein, neuropilin 2, and interleukin-23p19 (interleukin-12 family). Results expand our understanding of disc aging and degeneration and show that LCM is a valuable technique that can be used to collect mRNA amounts adequate for microarray analysis from the sparse cell population of the human anulus.

Investigation of type-I interferon dysregulation by arenaviruses : a multidisciplinary approach.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kozina, Carol L.; Moorman, Matthew Wallace; Branda, Catherine

2011-09-01

This report provides a detailed overview of the work performed for project number 130781, 'A Systems Biology Approach to Understanding Viral Hemorrhagic Fever Pathogenesis.' We report progress in five key areas: single cell isolation devices and control systems, fluorescent cytokine and transcription factor reporters, on-chip viral infection assays, molecular virology analysis of Arenavirus nucleoprotein structure-function, and development of computational tools to predict virus-host protein interactions. Although a great deal of work remains from that begun here, we have developed several novel single cell analysis tools and knowledge of Arenavirus biology that will facilitate and inform future publications and funding proposals.

3D printing of biomimetic microstructures for cancer cell migration.

PubMed

Huang, Tina Qing; Qu, Xin; Liu, Justin; Chen, Shaochen

2014-02-01

To understand the physical behavior and migration of cancer cells, a 3D in vitro micro-chip in hydrogel was created using 3D projection printing. The micro-chip has a honeycomb branched structure, aiming to mimic 3D vascular morphology to test, monitor, and analyze differences in the behavior of cancer cells (i.e. HeLa) vs. non-cancerous cell lines (i.e. 10 T1/2). The 3D Projection Printing system can fabricate complex structures in seconds from user-created designs. The fabricated microstructures have three different channel widths of 25, 45, and 120 microns wide to reflect a range of blood vessel diameters. HeLa and 10 T1/2 cells seeded within the micro-chip were then analyzed for morphology and cell migration speed. 10 T1/2 cells exhibited greater changes in morphology due to channel size width than HeLa cells; however, channel width had a limited effect on 10 T1/2 cell migration while HeLa cancer cell migration increased as channel width decreased. This physiologically relevant 3D cancer tissue model has the potential to be a powerful tool for future drug discoveries and cancer migration studies.

Development of a versatile lab-on-a-chip enzyme assay platform for pathogen detection in CBRNE scenarios

NASA Astrophysics Data System (ADS)

Klemm, Richard; Schattschneider, Sebastian; Jahn, Tobias; Hlawatsch, Nadine; Julich, Sandra; Becker, Holger; Gärtner, Claudia

2013-05-01

The ability to integrate complete assays on a microfluidic chip helps to greatly simplify instrument requirements and allows the use of lab-on-a-chip technology in the field. A core application for such field-portable systems is the detection of pathogens in a CBRNE scenario such as permanent monitoring of airborne pathogens, e.g. in metro stations or hospitals etc. As one assay methodology for the pathogen identification, enzymatic assays were chosen. In order evaluate different detection strategies, the realized on-chip enzyme assay module has been designed as a general platform chip. In all application cases, the assays are based on immobilized probes located in microfluidic channels. Therefore a microfluidic chip was realized containing a set of three individually addressable channels, not only for detection of the sample itself also to have a set of references for a quantitative analysis. It furthermore includes two turning valves and a waste container for clear and sealed storage of potential pathogenic liquids to avoid contamination of the environment. All liquids remain in the chip and can be disposed of in proper way subsequently to the analysis. The chip design includes four inlet ports consisting of one sample port (Luer interface) and three mini Luer interfaces for fluidic support of e.g. washing buffer, substrate and enzyme solution. The sample can be applied via a special, sealable sampling vessel with integrated female Luer interface. Thereby also pre-anaytical contamination of the environment can be provided. Other reagents that are required for analysis will be stored off chip.

Precision lens assembly with alignment turning system

NASA Astrophysics Data System (ADS)

Ho, Cheng-Fang; Huang, Chien-Yao; Lin, Yi-Hao; Kuo, Hui-Jean; Kuo, Ching-Hsiang; Hsu, Wei-Yao; Chen, Fong-Zhi

2017-10-01

The poker chip assembly with high precision lens barrels is widely applied to ultra-high performance optical system. ITRC applies the poker chip assembly technology to the high numerical aperture objective lenses and lithography projection lenses because of its high efficiency assembly process. In order to achieve high precision lens cell for poker chip assembly, an alignment turning system (ATS) is developed. The ATS includes measurement, alignment and turning modules. The measurement module is equipped with a non-contact displacement sensor (NCDS) and an autocollimator (ACM). The NCDS and ACM are used to measure centration errors of the top and the bottom surface of a lens respectively; then the amount of adjustment of displacement and tilt with respect to the rotational axis of the turning machine for the alignment module can be determined. After measurement, alignment and turning processes on the ATS, the centration error of a lens cell with 200 mm in diameter can be controlled within 10 arcsec. Furthermore, a poker chip assembly lens cell with three sub-cells is demonstrated, each sub-cells are measured and accomplished with alignment and turning processes. The lens assembly test for five times by each three technicians; the average transmission centration error of assembly lens is 12.45 arcsec. The results show that ATS can achieve high assembly efficiency for precision optical systems.

High Voltage Dielectrophoretic and Magnetophoretic Hybrid Integrated Circuit / Microfluidic Chip.

PubMed

Issadore, David; Franke, Thomas; Brown, Keith A; Hunt, Thomas P; Westervelt, Robert M

2009-12-01

A hybrid integrated circuit (IC) / microfluidic chip is presented that independently and simultaneously traps and moves microscopic objects suspended in fluid using both electric and magnetic fields. This hybrid chip controls the location of dielectric objects, such as living cells and drops of fluid, on a 60 × 61 array of pixels that are 30 × 38 μm(2) in size, each of which can be individually addressed with a 50 V peak-to-peak, DC to 10 MHz radio frequency voltage. These high voltage pixels produce electric fields above the chip's surface with a magnitude , resulting in strong dielectrophoresis (DEP) forces . Underneath the array of DEP pixels there is a magnetic matrix that consists of two perpendicular sets of 60 metal wires running across the chip. Each wire can be sourced with 120 mA to trap and move magnetically susceptible objects using magnetophoresis (MP). The DEP pixel array and magnetic matrix can be used simultaneously to apply forces to microscopic objects, such as living cells or lipid vesicles, that are tagged with magnetic nanoparticles. The capabilities of the hybrid IC / microfluidic chip demonstrated in this paper provide important building blocks for a platform for biological and chemical applications.

3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs.

PubMed

Knowlton, Stephanie; Yu, Chu Hsiang; Ersoy, Fulya; Emadi, Sharareh; Khademhosseini, Ali; Tasoglu, Savas

2016-06-20

Three-dimensional (3D) printing offers potential to fabricate high-throughput and low-cost fabrication of microfluidic devices as a promising alternative to traditional techniques which enables efficient design iterations in the development stage. In this study, we demonstrate a single-step fabrication of a 3D transparent microfluidic chip using two alternative techniques: a stereolithography-based desktop 3D printer and a two-step fabrication using an industrial 3D printer based on polyjet technology. This method, compared to conventional fabrication using relatively expensive materials and labor-intensive processes, presents a low-cost, rapid prototyping technique to print functional 3D microfluidic chips. We enhance the capabilities of 3D-printed microfluidic devices by coupling 3D cell encapsulation and spatial patterning within photocrosslinkable gelatin methacryloyl (GelMA). The platform presented here serves as a 3D culture environment for long-term cell culture and growth. Furthermore, we have demonstrated the ability to print complex 3D microfluidic channels to create predictable and controllable fluid flow regimes. Here, we demonstrate the novel use of 3D-printed microfluidic chips as controllable 3D cell culture environments, advancing the applicability of 3D printing to engineering physiological systems for future applications in bioengineering.

A hydrophilic polymer based microfluidic system with planar patch clamp electrode array for electrophysiological measurement from cells.

PubMed

Xu, Baojian; Ye, WeiWei; Zhang, Yu; Shi, JingYu; Chan, ChunYu; Yao, XiaoQiang; Yang, Mo

2014-03-15

This paper presents a microfluidic planar patch clamp system based on a hydrophilic polymer poly(ethylene glycol) diacrylate (PEGDA) for whole cell current recording. The whole chip is fabricated by UV-assisted molding method for both microfluidic channel structure and planar electrode partition. This hydrophilic patch clamp chip has demonstrated a relatively high gigaseal success rate of 44% without surface modification compared with PDMS based patch clamp devices. This chip also shows a capability of rapid intracellular and extracellular solution exchange with high stability of gigaseals. The capillary flow kinetic experiments demonstrate that the flow rates of PEGDA microfluidic channels are around two orders of magnitude greater than those for PDMS-glass channels with the same channel dimensions. This hydrophilic polymer based patch clamp chips have significant advantages over current PDMS elastomer based systems such as no need for surface modification, much higher success rate of cell gigaseals and rapid solution exchange with stable cell gigaseals. Our results indicate the potential of these devices to serve as useful tools for pharmaceutical screening and biosensing tasks. © 2013 Elsevier B.V. All rights reserved.

Automated Microfluidic Instrument for Label-Free and High-Throughput Cell Separation.

PubMed

Zhang, Xinjie; Zhu, Zhixian; Xiang, Nan; Long, Feifei; Ni, Zhonghua

2018-03-20

Microfluidic technologies for cell separation were reported frequently in recent years. However, a compact microfluidic instrument enabling thoroughly automated cell separation is still rarely reported until today due to the difficult hybrid between the macrosized fluidic control system and the microsized microfluidic device. In this work, we propose a novel and automated microfluidic instrument to realize size-based separation of cancer cells in a label-free and high-throughput manner. Briefly, the instrument is equipped with a fully integrated microfluidic device and a set of robust fluid-driven and control units, and the instrument functions of precise fluid infusion and high-throughput cell separation are guaranteed by a flow regulatory chip and two cell separation chips which are the key components of the microfluidic device. With optimized control programs, the instrument is successfully applied to automatically sort human breast adenocarcinoma cell line MCF-7 from 5 mL of diluted human blood with a high recovery ratio of ∼85% within a rapid processing time of ∼23 min. We envision that our microfluidic instrument will be potentially useful in many biomedical applications, especially cell separation, enrichment, and concentration for the purpose of cell culture and analysis.

Optic nerve signals in a neuromorphic chip II: Testing and results.

PubMed

Zaghloul, Kareem A; Boahen, Kwabena

2004-04-01

Seeking to match the brain's computational efficiency, we draw inspiration from its neural circuits. To model the four main output (ganglion) cell types found in the retina, we morphed outer and inner retina circuits into a 96 x 60-photoreceptor, 3.5 x 3.3 mm2, 0.35 microm-CMOS chip. Our retinomorphic chip produces spike trains for 3600 ganglion cells (GCs), and consumes 62.7 mW at 45 spikes/s/GC. This chip, which is the first silicon retina to successfully model inner retina circuitry, approaches the spatial density of the retina. We present experimental measurements showing that the chip's subthreshold current-mode circuits realize luminance adaptation, bandpass spatiotemporal filtering, temporal adaptation and contrast gain control. The four different GC outputs produced by our chip encode light onset or offset in a sustained or transient fashion, producing a quadrature-like representation. The retinomorphic chip's circuit design is described in a companion paper [Zaghloul and Boahen (2004)].

Using the developed cross-flow filtration chip for collecting blood plasma under high flow rate condition and applying the immunoglobulin E detection

NASA Astrophysics Data System (ADS)

Yeh, Chia-Hsien; Hung, Chia-Wei; Wu, Chun-Han; Lin, Yu-Cheng

2014-09-01

This paper presents a cross-flow filtration chip for separating blood cells (white blood cells, red blood cells, and platelets) and obtaining blood plasma from human blood. Our strategy is to flow the sample solution in parallel to the membrane, which can generate a parallel shear stress to remove the clogging microparticles on the membrane, so the pure sample solution is obtained in the reservoir. The cross-flow filtration chip includes a cross-flow layer, a Ni-Pd alloy micro-porous membrane, and a reservoir layer. The three layers are packaged in a polymethylmethacrylate (PMMA) frame to create the cross-flow filtration chip. Various dilutions of the blood sample (original, 2 × , 3 × , 5 × , and 10×), pore sizes with different diameters (1 µm, 2 µm, 4 µm, 7 µm, and 10 µm), and different flow rates (1 mL/min, 3 mL/min, 5 mL/min, 7 mL/min, and 10 mL/min) are tested to determine their effects on filtration percentage. The best filtration percentage is 96.2% when the dilution of the blood sample is 10 × , the diameter of pore size of a Ni-Pd alloy micro-porous membrane is 2 µm, and the flow rate is 10 mL/min. Finally, for the clinical tests of the immunoglobulin E (IgE) concentration, the cross-flow filtration chip is used to filter the blood of the allergy patients to obtain the blood plasma. This filtered blood plasma is compared with that obtained using the conventional centrifugation based on the enzyme-linked immunosorbent assay. The results reveal that these two blood separation methods have similar detection trends. The proposed filtration chip has the advantages of low cost, short filtration time, and easy operation and thus can be applied to the separation of microparticles, cells, bacteria, and blood.

Adhesion strength of a living cell to various substrates measured using a cup-attached atomic force microscopy chip

NASA Astrophysics Data System (ADS)

Kim, Hyonchol; Ishibashi, Kenta; Matsuo, Kosuke; Kira, Atsushi; Onomura, Yui; Okada, Tomoko; Nakamura, Chikashi

2018-03-01

Cell adhesion strengths to various substrates were quantitatively measured using atomic force microscopy (AFM). A cup-shaped metal hemisphere was attached to the apex of the AFM cantilever, the “cup-chip” approached a cell (FP10SC2) to pick it up, the captured cell approached any one of six different substrates [gold (Au), nickel (Ni), bovine serum albumin (BSA), an amino group (NH2), poly(tetrafluoroethylene) (PTFE), and structured PTFE (sPTFE)], and the cell adhesion strength at the initial contact period was evaluated by detaching the cell from the substrate. The results obtained showed that the force needed to detach the cell from the NH2 substrate was more than 3-fold larger than that of metal substrates (Au and Ni), more than 15-fold larger than that of biochemically treated substrates (BSA), and more than 20-fold larger than that of hydrophobic substrates (PTFE and sPTFE). Using differences in adhesion strengths, a cell on a sPTFE substrate was picked up using a BSA-coated cup-chip, placed on a NH2 substrate, repeating this cell manipulation five times, and line patterning of cells was achieved. These results indicate that measurements of cell adhesion strength are fundamental to fabricate desired cell networks and the cup-chip is a useful tool for achieving easy cell manipulation.

Creating a Tiny Human Body on a Chip

ScienceCinema

Hunsberger, Maren; Soscia, Dave; Moya, Monica

2018-06-21

LLNL science communicator Maren Hunsberger takes us "Inside the Lab" to learn about the iChip (In-vitro Chip-based Human Investigational Platform) project at Lawrence Livermore National Laboratory. "One application of the iChip system would be to develop new pharmaceutical drugs," explains Dave Soscia, LLNL postdoc. "When you test in a mouse for example, it's not as close to the human system as you can get. If we can take human cells and put them on devices and actually mimic the structure and function of the organ systems in the human, we can actually replace animal testing and even make a better system for testing pharmaceutical drugs."

Thermometry in dielectrophoresis chips for contact-free cell handling

NASA Astrophysics Data System (ADS)

Jaeger, M. S.; Mueller, T.; Schnelle, T.

2007-01-01

Cell biology applications, protocols in immunology and stem cell research, require that individual cells are handled under strict control of their contacts to other cells or synthetic surfaces. Dielectrophoresis (DEP) in microfluidic chips is an established technique to investigate, group, wash, cultivate and sort cells contact-free under physiological conditions: microelectrode octode cages, versatile dielectrophoretic elements energized with radio frequency electric fields, stably trap single cells or cellular aggregates. For medical applications and cell cultivation, possible side effects of the dielectrophoretic manipulation, such as membrane polarization and Joule heating, have to be quantified. Therefore, we characterized the electric field-induced warming in dielectrophoretic cages using ohmic resistance measurements, fluorometry, liquid crystal beads, infra-red thermography and bubble size thermometry. We compare the results of these techniques with respect to the influences of voltage, electric conductivity of buffer, frequency, cage size and electrode surface. We conclude that in the culture medium thermal effects may be neglected if low voltages and an electric field-reducing phase pattern are used. Our experimental results provide explicit values for estimating the thermal effect on dielectrophoretically caged cells and show that Joule heating is best minimized by optimizing the cage geometry and reducing the buffer conductivity. The results may additionally serve to evaluate and improve theoretical predictions on field-induced effects. Based on present-day chip processing possibilities, DEP is well suited for the manipulation of cells.

Platforms for Single-Cell Collection and Analysis.

PubMed

Valihrach, Lukas; Androvic, Peter; Kubista, Mikael

2018-03-11

Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.

Platforms for Single-Cell Collection and Analysis

PubMed Central

Valihrach, Lukas; Androvic, Peter; Kubista, Mikael

2018-01-01

Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments. PMID:29534489

Clinical application of a microfluidic chip for immunocapture and quantification of circulating exosomes to assist breast cancer diagnosis and molecular classification.

PubMed

Fang, Shimeng; Tian, Hongzhu; Li, Xiancheng; Jin, Dong; Li, Xiaojie; Kong, Jing; Yang, Chun; Yang, Xuesong; Lu, Yao; Luo, Yong; Lin, Bingcheng; Niu, Weidong; Liu, Tingjiao

2017-01-01

Increasing attention has been attracted by exosomes in blood-based diagnosis because cancer cells release more exosomes in serum than normal cells and these exosomes overexpress a certain number of cancer-related biomarkers. However, capture and biomarker analysis of exosomes for clinical application are technically challenging. In this study, we developed a microfluidic chip for immunocapture and quantification of circulating exosomes from small sample volume and applied this device in clinical study. Circulating EpCAM-positive exosomes were measured in 6 cases breast cancer patients and 3 healthy controls to assist diagnosis. A significant increase in the EpCAM-positive exosome level in these patients was detected, compared to healthy controls. Furthermore, we quantified circulating HER2-positive exosomes in 19 cases of breast cancer patients for molecular classification. We demonstrated that the exosomal HER2 expression levels were almost consistent with that in tumor tissues assessed by immunohistochemical staining. The microfluidic chip might provide a new platform to assist breast cancer diagnosis and molecular classification.

Microfluidic, marker-free isolation of circulating tumor cells from blood samples

PubMed Central

Karabacak, Nezihi Murat; Spuhler, Philipp S; Fachin, Fabio; Lim, Eugene J; Pai, Vincent; Ozkumur, Emre; Martel, Joseph M; Kojic, Nikola; Smith, Kyle; Chen, Pin-i; Yang, Jennifer; Hwang, Henry; Morgan, Bailey; Trautwein, Julie; Barber, Thomas A; Stott, Shannon L; Maheswaran, Shyamala; Kapur, Ravi; Haber, Daniel A; Toner, Mehmet

2014-01-01

The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen–independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 107 cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2–5 d. PMID:24577360

Chip-based device for parallel sorting, amplification, detection, and identification of nucleic acid subsequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beer, Neil Reginald; Colston, Jr, Billy W.

An apparatus for chip-based sorting, amplification, detection, and identification of a sample having a planar substrate. The planar substrate is divided into cells. The cells are arranged on the planar substrate in rows and columns. Electrodes are located in the cells. A micro-reactor maker produces micro-reactors containing the sample. The micro-reactor maker is positioned to deliver the micro-reactors to the planar substrate. A microprocessor is connected to the electrodes for manipulating the micro-reactors on the planar substrate. A detector is positioned to interrogate the sample contained in the micro-reactors.

Towards toxicity detection using a lab-on-chip based on the integration of MOEMS and whole-cell sensors.

PubMed

Elman, Noel M; Ben-Yoav, Hadar; Sternheim, Marek; Rosen, Rachel; Krylov, Slava; Shacham-Diamand, Yosi

2008-06-15

A lab-on-chip consisting of a unique integration of whole-cell sensors, a MOEMS (Micro-Opto-Electro-Mechanical-System) modulator, and solid-state photo-detectors was implemented for the first time. Whole-cell sensors were genetically engineered to express a bioluminescent reporter (lux) as a function of the lac promoter. The MOEMS modulator was designed to overcome the inherent low frequency noise of solid-state photo-detectors by means of a previously reported modulation technique, named IHOS (Integrated Heterodyne Optical System). The bio-reporter signals were modulated prior to photo-detection, increasing the SNR of solid-state photo-detectors at least by three orders of magnitude. Experiments were performed using isopropyl-beta-d-thiogalactopyranoside (IPTG) as a preliminary step towards testing environmental toxicity. The inducer was used to trigger the expression response of the whole-cell sensors testing the sensitivity of the lab-on-chip. Low intensity bio-reporter optical signals were measured after the whole-cell sensors were exposed to IPTG concentrations of 0.1, 0.05, and 0.02mM. The experimental results reveal the potential of this technology for future implementation as an inexpensive massive method for rapid environmental toxicity detection.

A Novel Chip for Cyclic Stretch and Intermittent Hypoxia Cell Exposures Mimicking Obstructive Sleep Apnea.

PubMed

Campillo, Noelia; Jorba, Ignasi; Schaedel, Laura; Casals, Blai; Gozal, David; Farré, Ramon; Almendros, Isaac; Navajas, Daniel

2016-01-01

Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), plays a critical role in the pathogenesis of OSA-associated morbidities, especially in the cardiovascular and respiratory systems. Oxidative stress and inflammation induced by IH are suggested as main contributors of end-organ dysfunction in OSA patients and animal models. Since the molecular mechanisms underlying these in vivo pathological responses remain poorly understood, implementation of experimental in vitro cell-based systems capable of inducing high-frequency IH would be highly desirable. Here, we describe the design, fabrication, and validation of a versatile chip for subjecting cultured cells to fast changes in gas partial pressure and to cyclic stretch. The chip is fabricated with polydimethylsiloxane (PDMS) and consists of a cylindrical well-covered by a thin membrane. Cells cultured on top of the membrane can be subjected to fast changes in oxygen concentration (equilibrium time ~6 s). Moreover, cells can be subjected to cyclic stretch at cardiac or respiratory frequencies independently or simultaneously. Rat bone marrow-derived mesenchymal stem cells (MSCs) exposed to IH mimicking OSA and cyclic stretch at cardiac frequencies revealed that hypoxia-inducible factor 1α (HIF-1α) expression was increased in response to both stimuli. Thus, the chip provides a versatile tool for the study of cellular responses to cyclical hypoxia and stretch.

Ubiquitinylation of α-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

PubMed Central

Chau, Hien; Lozano, Andres M.; Hyman, Bradley T.; McLean, Pamela J.

2011-01-01

Parkinson's disease (PD) is a common neurodegenerative condition in which abnormalities in protein homeostasis, or proteostasis, may lead to accumulation of the protein α-synuclein (α-syn). Mutations within or multiplications of the gene encoding α-syn are known to cause genetic forms of PD and polymorphisms in the gene are recently established risk factors for idiopathic PD. α-syn is a major component of Lewy bodies, the intracellular proteinaceous inclusions which are pathological hallmarks of most forms of PD. Recent evidence demonstrates that α-syn can self associate into soluble oligomeric species and implicates these α-syn oligomers in cell death. We have previously shown that carboxyl terminus of Hsp70-interacting protein (CHIP), a co-chaperone molecule with E3 ubiquitin ligase activity, may reduce the levels of toxic α-syn oligomers. Here we demonstrate that α-syn is ubiquitinylated by CHIP both in vitro and in cells. We find that the products from ubiquitinylation by CHIP include both monoubiquitinylated and polyubiquitinylated forms of α-syn. We also demonstrate that CHIP and α-syn exist within a protein complex with the co-chaperone bcl-2-associated athanogene 5 (BAG5) in brain. The interaction of CHIP with BAG5 is mediated by Hsp70 which binds to the tetratricopeptide repeat domain of CHIP and the BAG domains of BAG5. The Hsp70-mediated association of BAG5 with CHIP results in inhibition of CHIP E3 ubiquitin ligase activity and subsequently reduces α-syn ubiquitinylation. Furthermore, we use a luciferase-based protein-fragment complementation assay of α-syn oligomerization to investigate regulation of α-syn oligomers by CHIP in living cells. We demonstrate that BAG5 mitigates the ability of CHIP to reduce α-syn oligomerization and that non-ubiquitinylated α-syn has an increased propensity for oligomerization. Thus, our results identify CHIP as an E3 ubiquitin ligase of α-syn and suggest a novel function for BAG5 as a modulator of CHIP E3 ubiquitin ligase activity with implications for CHIP-mediated regulation of α-syn oligomerization. PMID:21358815

Microfluidic chip for non-invasive analysis of tumor cells interaction with anti-cancer drug doxorubicin by AFM and Raman spectroscopy.

PubMed

Zhang, Han; Xiao, Lifu; Li, Qifei; Qi, Xiaojun; Zhou, Anhong

2018-03-01

Raman spectroscopy has been playing an increasingly significant role for cell classification. Here, we introduce a novel microfluidic chip for non-invasive Raman cell natural fingerprint collection. Traditional Raman spectroscopy measurement of the cells grown in a Polydimethylsiloxane (PDMS) based microfluidic device suffers from the background noise from the substrate materials of PDMS when intended to apply as an in vitro cell assay. To overcome this disadvantage, the current device is designed with a middle layer of PDMS layer sandwiched by two MgF 2 slides which minimize the PDMS background signal in Raman measurement. Three cancer cell lines, including a human lung cancer cell A549, and human breast cancer cell lines MDA-MB-231 and MDA-MB-231/BRMS1, were cultured in this microdevice separately for a period of three days to evaluate the biocompatibility of the microfluidic system. In addition, atomic force microscopy (AFM) was used to measure the Young's modulus and adhesion force of cancer cells at single cell level. The AFM results indicated that our microchannel environment did not seem to alter the cell biomechanical properties. The biochemical responses of cancer cells exposed to anti-cancer drug doxorubicin (DOX) up to 24 h were assessed by Raman spectroscopy. Principal component analysis over the Raman spectra indicated that cancer cells untreated and treated with DOX can be distinguished. This PDMS microfluidic device offers a non-invasive and reusable tool for in vitro Raman measurement of living cells, and can be potentially applied for anti-cancer drug screening.

Correlation between failure and local material property in chopped carbon fiber chip-reinforced sheet molding compound composites under tensile load

DOE PAGES

Tang, Haibin; Chen, Zhangxing; Zhou, Guowei; ...

2018-02-06

To develop further understanding towards the role of a heterogeneous microstructure on tensile crack initiation and failure behavior in chopped carbon fiber chip-reinforced composites, uni-axial tensile tests are performed on coupons cut from compression molded plaque with varying directions. Our experimental results indicate that failure initiation is relevant to the strain localization, and a new criterion with the nominal modulus to predict the failure location is proposed based on the strain analysis. Furthermore, optical microscopic images show that the nominal modulus is determined by the chip orientation distribution. At the area with low nominal modulus, it is found that chipsmore » are mostly aligning along directions transverse to loading direction and/or less concentrated, while at the area with high nominal modulus, more chips are aligning to tensile direction. On the basis of failure mechanism analysis, it is concluded that transversely-oriented chips or resin-rich regions are easier for damage initiation, while longitudinally-oriented chips postpone the fracture. Good agreement is found among failure mechanism, strain localization and chip orientation distribution.« less

Correlation between failure and local material property in chopped carbon fiber chip-reinforced sheet molding compound composites under tensile load

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Haibin; Chen, Zhangxing; Zhou, Guowei

To develop further understanding towards the role of a heterogeneous microstructure on tensile crack initiation and failure behavior in chopped carbon fiber chip-reinforced composites, uni-axial tensile tests are performed on coupons cut from compression molded plaque with varying directions. Our experimental results indicate that failure initiation is relevant to the strain localization, and a new criterion with the nominal modulus to predict the failure location is proposed based on the strain analysis. Furthermore, optical microscopic images show that the nominal modulus is determined by the chip orientation distribution. At the area with low nominal modulus, it is found that chipsmore » are mostly aligning along directions transverse to loading direction and/or less concentrated, while at the area with high nominal modulus, more chips are aligning to tensile direction. On the basis of failure mechanism analysis, it is concluded that transversely-oriented chips or resin-rich regions are easier for damage initiation, while longitudinally-oriented chips postpone the fracture. Good agreement is found among failure mechanism, strain localization and chip orientation distribution.« less

Ultrathin Polymer Membranes with Patterned, Micrometric Pores for Organs-on-Chips.

PubMed

Pensabene, Virginia; Costa, Lino; Terekhov, Alexander Y; Gnecco, Juan S; Wikswo, John P; Hofmeister, William H

2016-08-31

The basal lamina or basement membrane (BM) is a key physiological system that participates in physicochemical signaling between tissue types. Its formation and function are essential in tissue maintenance, growth, angiogenesis, disease progression, and immunology. In vitro models of the BM (e.g., Boyden and transwell chambers) are common in cell biology and lab-on-a-chip devices where cells require apical and basolateral polarization. Extravasation, intravasation, membrane transport of chemokines, cytokines, chemotaxis of cells, and other key functions are routinely studied in these models. The goal of the present study was to integrate a semipermeable ultrathin polymer membrane with precisely positioned pores of 2 μm diameter in a microfluidic device with apical and basolateral chambers. We selected poly(l-lactic acid) (PLLA), a transparent biocompatible polymer, to prepare the semipermeable ultrathin membranes. The pores were generated by pattern transfer using a three-step method coupling femtosecond laser machining, polymer replication, and spin coating. Each step of the fabrication process was characterized by scanning electron microscopy to investigate reliability of the process and fidelity of pattern transfer. In order to evaluate the compatibility of the fabrication method with organs-on-a-chip technology, porous PLLA membranes were embedded in polydimethylsiloxane (PDMS) microfluidic devices and used to grow human umbilical vein endothelial cells (HUVECS) on top of the membrane with perfusion through the basolateral chamber. Viability of cells, optical transparency of membranes and strong adhesion of PLLA to PDMS were observed, thus confirming the suitability of the prepared membranes for use in organs-on-a-chip devices.

Electrochemical Detection of Human Mesenchymal Stem Cell Differentiation on Fabricated Gold Nano-Dot Cell Chips.

PubMed

An, Jeung Hee; Kim, Seung U; Park, Mi-Kyung; Choi, Jeong Woo

2015-10-01

Human mesenchymal stem cells (MSCs) have the capacity for self-renewal and maintain pluripotency, which is defined by their ability to differentiate into cells such as osteoblasts, neurons, and glial cells. In this study, we report a method for defining the status of human MSCs based on electrochemical detection systems. Gold nano-dot structures were fabricated using a nanoporous alumina mask, and the structural formations were confirmed by scanning electron microscopy (SEM). Human MSCs were allowed to attach to RGD (Arg-Gly-Asp) peptide nanopatterned surfaces, and electrochemical tools were applied to the MSCs attached on the chip surface. The cultured MSCs were shown to differentiate into neural cell types, as indicated by immunocytochemical staining for tyrosine hydroxylase and beta tubulin III. Following treatment with basic fibroblast growth factor (bFGF) for 14 days, most of the B10 cells exhibited bipolar or multipolar morphology with branched processes, and the proportion of B10 cells expressing neuronal cell markers considerably increased. Electrophysiological recordings from MSCs treated with bFGF for 5-14 days were examined with cyclic voltammetry, and the electrochemical signals were shown to increase during differentiation from MSCs to neuronal cells. This human MSC cell line is a useful tool for studying organogenesis, specifically neurogenesis, and in addition, the cell line provides a valuable source of cells for cell therapy. The electrochemical measurement system proposed here could be utilized in electrical cell chips for numerous applications, including cell differentiation, disease diagnosis, drug detection, and on-site monitoring.

Recent advances in particle and droplet manipulation for lab-on-a-chip devices based on surface acoustic waves.

PubMed

Wang, Zhuochen; Zhe, Jiang

2011-04-07

Manipulation of microscale particles and fluid liquid droplets is an important task for lab-on-a-chip devices for numerous biological researches and applications, such as cell detection and tissue engineering. Particle manipulation techniques based on surface acoustic waves (SAWs) appear effective for lab-on-a-chip devices because they are non-invasive, compatible with soft lithography micromachining, have high energy density, and work for nearly any type of microscale particles. Here we review the most recent research and development of the past two years in SAW based particle and liquid droplet manipulation for lab-on-a-chip devices including particle focusing and separation, particle alignment and patterning, particle directing, and liquid droplet delivery.

Revisiting lab-on-a-chip technology for drug discovery.

PubMed

Neuži, Pavel; Giselbrecht, Stefan; Länge, Kerstin; Huang, Tony Jun; Manz, Andreas

2012-08-01

The field of microfluidics or lab-on-a-chip technology aims to improve and extend the possibilities of bioassays, cell biology and biomedical research based on the idea of miniaturization. Microfluidic systems allow more accurate modelling of physiological situations for both fundamental research and drug development, and enable systematic high-volume testing for various aspects of drug discovery. Microfluidic systems are in development that not only model biological environments but also physically mimic biological tissues and organs; such 'organs on a chip' could have an important role in expediting early stages of drug discovery and help reduce reliance on animal testing. This Review highlights the latest lab-on-a-chip technologies for drug discovery and discusses the potential for future developments in this field.

Real-time bacterial microcolony counting using on-chip microscopy

NASA Astrophysics Data System (ADS)

Jung, Jae Hee; Lee, Jung Eun

2016-02-01

Observing microbial colonies is the standard method for determining the microbe titer and investigating the behaviors of microbes. Here, we report an automated, real-time bacterial microcolony-counting system implemented on a wide field-of-view (FOV), on-chip microscopy platform, termed ePetri. Using sub-pixel sweeping microscopy (SPSM) with a super-resolution algorithm, this system offers the ability to dynamically track individual bacterial microcolonies over a wide FOV of 5.7 mm × 4.3 mm without requiring a moving stage or lens. As a demonstration, we obtained high-resolution time-series images of S. epidermidis at 20-min intervals. We implemented an image-processing algorithm to analyze the spatiotemporal distribution of microcolonies, the development of which could be observed from a single bacterial cell. Test bacterial colonies with a minimum diameter of 20 μm could be enumerated within 6 h. We showed that our approach not only provides results that are comparable to conventional colony-counting assays but also can be used to monitor the dynamics of colony formation and growth. This microcolony-counting system using on-chip microscopy represents a new platform that substantially reduces the detection time for bacterial colony counting. It uses chip-scale image acquisition and is a simple and compact solution for the automation of colony-counting assays and microbe behavior analysis with applications in antibacterial drug discovery.

Real-time bacterial microcolony counting using on-chip microscopy

PubMed Central

Jung, Jae Hee; Lee, Jung Eun

2016-01-01

Observing microbial colonies is the standard method for determining the microbe titer and investigating the behaviors of microbes. Here, we report an automated, real-time bacterial microcolony-counting system implemented on a wide field-of-view (FOV), on-chip microscopy platform, termed ePetri. Using sub-pixel sweeping microscopy (SPSM) with a super-resolution algorithm, this system offers the ability to dynamically track individual bacterial microcolonies over a wide FOV of 5.7 mm × 4.3 mm without requiring a moving stage or lens. As a demonstration, we obtained high-resolution time-series images of S. epidermidis at 20-min intervals. We implemented an image-processing algorithm to analyze the spatiotemporal distribution of microcolonies, the development of which could be observed from a single bacterial cell. Test bacterial colonies with a minimum diameter of 20 μm could be enumerated within 6 h. We showed that our approach not only provides results that are comparable to conventional colony-counting assays but also can be used to monitor the dynamics of colony formation and growth. This microcolony-counting system using on-chip microscopy represents a new platform that substantially reduces the detection time for bacterial colony counting. It uses chip-scale image acquisition and is a simple and compact solution for the automation of colony-counting assays and microbe behavior analysis with applications in antibacterial drug discovery. PMID:26902822

Microfluidic Platform for Parallel Single Cell Analysis for Diagnostic Applications.

PubMed

Le Gac, Séverine

2017-01-01

Cell populations are heterogeneous: they can comprise different cell types or even cells at different stages of the cell cycle and/or of biological processes. Furthermore, molecular processes taking place in cells are stochastic in nature. Therefore, cellular analysis must be brought down to the single cell level to get useful insight into biological processes, and to access essential molecular information that would be lost when using a cell population analysis approach. Furthermore, to fully characterize a cell population, ideally, information both at the single cell level and on the whole cell population is required, which calls for analyzing each individual cell in a population in a parallel manner. This single cell level analysis approach is particularly important for diagnostic applications to unravel molecular perturbations at the onset of a disease, to identify biomarkers, and for personalized medicine, not only because of the heterogeneity of the cell sample, but also due to the availability of a reduced amount of cells, or even unique cells. This chapter presents a versatile platform meant for the parallel analysis of individual cells, with a particular focus on diagnostic applications and the analysis of cancer cells. We first describe one essential step of this parallel single cell analysis protocol, which is the trapping of individual cells in dedicated structures. Following this, we report different steps of a whole analytical process, including on-chip cell staining and imaging, cell membrane permeabilization and/or lysis using either chemical or physical means, and retrieval of the cell molecular content in dedicated channels for further analysis. This series of experiments illustrates the versatility of the herein-presented platform and its suitability for various analysis schemes and different analytical purposes.

Two-Dimensional Algal Collection and Assembly by Combining AC-Dielectrophoresis with Fluorescence Detection for Contaminant-Induced Oxidative Stress Sensing.

PubMed

Siebman, Coralie; Velev, Orlin D; Slaveykova, Vera I

2015-06-15

An alternative current (AC) dielectrophoretic lab-on-chip setup was evaluated as a rapid tool of capture and assembly of microalga Chlamydomonas reinhardtii in two-dimensional (2D) close-packed arrays. An electric field of 100 V·cm⁻¹, 100 Hz applied for 30 min was found optimal to collect and assemble the algae into single-layer structures of closely packed cells without inducing cellular oxidative stress. Combined with oxidative stress specific staining and fluorescence microscopy detection, the capability of using the 2D whole-cell assembly on-chip to follow the reactive oxygen species (ROS) production and oxidative stress during short-term exposure to several environmental contaminants, including mercury, methylmercury, copper, copper oxide nanoparticles (CuO-NPs), and diuron was explored. The results showed significant increase of the cellular ROS when C. reinhardtii was exposed to high concentrations of methylmercury, CuO-NPs, and 10⁻⁵ M Cu. Overall, this study demonstrates the potential of combining AC-dielectrophoretically assembled two-dimensional algal structures with cell metabolic analysis using fluorescence staining, as a rapid analytical tool for probing the effect of contaminants in highly impacted environment.

Integration of systems biology with organs-on-chips to humanize therapeutic development

NASA Astrophysics Data System (ADS)

Edington, Collin D.; Cirit, Murat; Chen, Wen Li Kelly; Clark, Amanda M.; Wells, Alan; Trumper, David L.; Griffith, Linda G.

2017-02-01

"Mice are not little people" - a refrain becoming louder as the gaps between animal models and human disease become more apparent. At the same time, three emerging approaches are headed toward integration: powerful systems biology analysis of cell-cell and intracellular signaling networks in patient-derived samples; 3D tissue engineered models of human organ systems, often made from stem cells; and micro-fluidic and meso-fluidic devices that enable living systems to be sustained, perturbed and analyzed for weeks in culture. Integration of these rapidly moving fields has the potential to revolutionize development of therapeutics for complex, chronic diseases, including those that have weak genetic bases and substantial contributions from gene-environment interactions. Technical challenges in modeling complex diseases with "organs on chips" approaches include the need for relatively large tissue masses and organ-organ cross talk to capture systemic effects, such that current microfluidic formats often fail to capture the required scale and complexity for interconnected systems. These constraints drive development of new strategies for designing in vitro models, including perfusing organ models, as well as "mesofluidic" pumping and circulation in platforms connecting several organ systems, to achieve the appropriate physiological relevance.

Automated microfluidic platform of bead-based electrochemical immunosensor integrated with bioreactor for continual monitoring of cell secreted biomarkers

NASA Astrophysics Data System (ADS)

Riahi, Reza; Shaegh, Seyed Ali Mousavi; Ghaderi, Masoumeh; Zhang, Yu Shrike; Shin, Su Ryon; Aleman, Julio; Massa, Solange; Kim, Duckjin; Dokmeci, Mehmet Remzi; Khademhosseini, Ali

2016-04-01

There is an increasing interest in developing microfluidic bioreactors and organs-on-a-chip platforms combined with sensing capabilities for continual monitoring of cell-secreted biomarkers. Conventional approaches such as ELISA and mass spectroscopy cannot satisfy the needs of continual monitoring as they are labor-intensive and not easily integrable with low-volume bioreactors. This paper reports on the development of an automated microfluidic bead-based electrochemical immunosensor for in-line measurement of cell-secreted biomarkers. For the operation of the multi-use immunosensor, disposable magnetic microbeads were used to immobilize biomarker-recognition molecules. Microvalves were further integrated in the microfluidic immunosensor chip to achieve programmable operations of the immunoassay including bead loading and unloading, binding, washing, and electrochemical sensing. The platform allowed convenient integration of the immunosensor with liver-on-chips to carry out continual quantification of biomarkers secreted from hepatocytes. Transferrin and albumin productions were monitored during a 5-day hepatotoxicity assessment in which human primary hepatocytes cultured in the bioreactor were treated with acetaminophen. Taken together, our unique microfluidic immunosensor provides a new platform for in-line detection of biomarkers in low volumes and long-term in vitro assessments of cellular functions in microfluidic bioreactors and organs-on-chips.

Automated microfluidic platform of bead-based electrochemical immunosensor integrated with bioreactor for continual monitoring of cell secreted biomarkers

PubMed Central

Riahi, Reza; Shaegh, Seyed Ali Mousavi; Ghaderi, Masoumeh; Zhang, Yu Shrike; Shin, Su Ryon; Aleman, Julio; Massa, Solange; Kim, Duckjin; Dokmeci, Mehmet Remzi; Khademhosseini, Ali

2016-01-01

There is an increasing interest in developing microfluidic bioreactors and organs-on-a-chip platforms combined with sensing capabilities for continual monitoring of cell-secreted biomarkers. Conventional approaches such as ELISA and mass spectroscopy cannot satisfy the needs of continual monitoring as they are labor-intensive and not easily integrable with low-volume bioreactors. This paper reports on the development of an automated microfluidic bead-based electrochemical immunosensor for in-line measurement of cell-secreted biomarkers. For the operation of the multi-use immunosensor, disposable magnetic microbeads were used to immobilize biomarker-recognition molecules. Microvalves were further integrated in the microfluidic immunosensor chip to achieve programmable operations of the immunoassay including bead loading and unloading, binding, washing, and electrochemical sensing. The platform allowed convenient integration of the immunosensor with liver-on-chips to carry out continual quantification of biomarkers secreted from hepatocytes. Transferrin and albumin productions were monitored during a 5-day hepatotoxicity assessment in which human primary hepatocytes cultured in the bioreactor were treated with acetaminophen. Taken together, our unique microfluidic immunosensor provides a new platform for in-line detection of biomarkers in low volumes and long-term in vitro assessments of cellular functions in microfluidic bioreactors and organs-on-chips. PMID:27098564

C-terminus of HSC70-Interacting Protein (CHIP) Inhibits Adipocyte Differentiation via Ubiquitin- and Proteasome-Mediated Degradation of PPARγ

PubMed Central

Kim, Jung-Hoon; Shin, Soyeon; Seo, Jinho; Lee, Eun-Woo; Jeong, Manhyung; Lee, Min-sik; Han, Hyun-Ji; Song, Jaewhan

2017-01-01

PPARγ (Peroxisome proliferator-activated receptor γ) is a nuclear receptor involved in lipid homeostasis and related metabolic diseases. Acting as a transcription factor, PPARγ is a master regulator for adipocyte differentiation. Here, we reveal that CHIP (C-terminus of HSC70-interacting protein) suppresses adipocyte differentiation by functioning as an E3 ligase of PPARγ. CHIP directly binds to and induces ubiquitylation of the PPARγ protein, leading to proteasome-dependent degradation. Stable overexpression or knockdown of CHIP inhibited or promoted adipogenesis, respectively, in 3T3-L1 cells. On the other hand, a CHIP mutant defective in E3 ligase could neither regulate PPARγ protein levels nor suppress adipogenesis, indicating the importance of CHIP-mediated ubiquitylation of PPARγ in adipocyte differentiation. Lastly, a CHIP null embryo fibroblast exhibited augmented adipocyte differentiation with increases in PPARγ and its target protein levels. In conclusion, CHIP acts as an E3 ligase of PPARγ, suppressing PPARγ-mediated adipogenesis. PMID:28059128

Genome-wide methylation analysis identifies genes silenced in non-seminoma cell lines

PubMed Central

Noor, Dzul Azri Mohamed; Jeyapalan, Jennie N; Alhazmi, Safiah; Carr, Matthew; Squibb, Benjamin; Wallace, Claire; Tan, Christopher; Cusack, Martin; Hughes, Jaime; Reader, Tom; Shipley, Janet; Sheer, Denise; Scotting, Paul J

2016-01-01

Silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the genome-wide effect of DNA methylation on gene expression has been analysed in relatively few cancers. Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. Previous analyses have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. The genomic targets that are methylated, the extent to which this results in gene silencing and the identity of the silenced genes most likely to play a role in the tumours’ biology have not yet been established. In this study, genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data from primary tumours to address this question. Genome methylation was analysed using the Illumina infinium HumanMethylome450 bead chip system and gene expression was analysed using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Regulation by methylation was confirmed by demethylation using 5-aza-2-deoxycytidine and reverse transcription–quantitative PCR. Large differences in the level of methylation of the CpG islands of individual genes between tumour cell lines correlated well with differential gene expression. Treatment of non-seminoma cells with 5-aza-2-deoxycytidine verified that methylation of all genes tested played a role in their silencing in yolk sac tumour cells and many of these genes were also differentially expressed in primary tumours. Genes silenced by methylation in the various GCT cell lines were identified. Several pluripotency-associated genes were identified as a major functional group of silenced genes. PMID:29263807

Genome-wide methylation analysis identifies genes silenced in non-seminoma cell lines.

PubMed

Noor, Dzul Azri Mohamed; Jeyapalan, Jennie N; Alhazmi, Safiah; Carr, Matthew; Squibb, Benjamin; Wallace, Claire; Tan, Christopher; Cusack, Martin; Hughes, Jaime; Reader, Tom; Shipley, Janet; Sheer, Denise; Scotting, Paul J

2016-01-01

Silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the genome-wide effect of DNA methylation on gene expression has been analysed in relatively few cancers. Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. Previous analyses have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. The genomic targets that are methylated, the extent to which this results in gene silencing and the identity of the silenced genes most likely to play a role in the tumours' biology have not yet been established. In this study, genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data from primary tumours to address this question. Genome methylation was analysed using the Illumina infinium HumanMethylome450 bead chip system and gene expression was analysed using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Regulation by methylation was confirmed by demethylation using 5-aza-2-deoxycytidine and reverse transcription-quantitative PCR. Large differences in the level of methylation of the CpG islands of individual genes between tumour cell lines correlated well with differential gene expression. Treatment of non-seminoma cells with 5-aza-2-deoxycytidine verified that methylation of all genes tested played a role in their silencing in yolk sac tumour cells and many of these genes were also differentially expressed in primary tumours. Genes silenced by methylation in the various GCT cell lines were identified. Several pluripotency-associated genes were identified as a major functional group of silenced genes.

A dual-docking microfluidic cell migration assay (D2-Chip) for testing neutrophil chemotaxis and the memory effect.

PubMed

Yang, Ke; Wu, Jiandong; Xu, Guoqing; Xie, Dongxue; Peretz-Soroka, Hagit; Santos, Susy; Alexander, Murray; Zhu, Ling; Zhang, Michael; Liu, Yong; Lin, Francis

2017-04-18

Chemotaxis is a classic mechanism for guiding cell migration and an important topic in both fundamental cell biology and health sciences. Neutrophils are a widely used model to study eukaryotic cell migration and neutrophil chemotaxis itself can lead to protective or harmful immune actions to the body. While much has been learnt from past research about how neutrophils effectively navigate through a chemoattractant gradient, many interesting questions remain unclear. For example, while it is tempting to model neutrophil chemotaxis using the well-established biased random walk theory, the experimental proof was challenged by the cell's highly persistent migrating nature. A special experimental design is required to test the key predictions from the random walk model. Another question that has interested the cell migration community for decades concerns the existence of chemotactic memory and its underlying mechanism. Although chemotactic memory has been suggested in various studies, a clear quantitative experimental demonstration will improve our understanding of the migratory memory effect. Motivated by these questions, we developed a microfluidic cell migration assay (so-called dual-docking chip or D 2 -Chip) that can test both the biased random walk model and the memory effect for neutrophil chemotaxis on a single chip enabled by multi-region gradient generation and dual-region cell alignment. Our results provide experimental support for the biased random walk model and chemotactic memory for neutrophil chemotaxis. Quantitative data analyses provide new insights into neutrophil chemotaxis and memory by making connections to entropic disorder, cell morphology and oscillating migratory response.

Automatic extraction and processing of small RNAs on a multi-well/multi-channel (M&M) chip.

PubMed

Zhong, Runtao; Flack, Kenneth; Zhong, Wenwan

2012-12-07

The study of the regulatory roles in small RNAs can be accelerated by techniques that permit simple, low-cost, and rapid extraction of small RNAs from a small number of cells. In order to ensure highly specific and sensitive detection, the extracted RNAs should be free of the background nucleic acids and present stably in a small volume. To meet these criteria, we designed a multi-well/multi-channel (M&M) chip to carry out automatic and selective isolation of small RNAs via solid-phase extraction (SPE), followed by reverse-transcription (RT) to convert them to the more stable cDNAs in a final volume of 2 μL. Droplets containing buffers for RNA binding, washing, and elution were trapped in microwells, which were connected by one channel, and suspended in mineral oil. The silica magnetic particles (SMPs) for SPE were moved along the channel from well to well, i.e. in between droplets, by a fixed magnet and a translation stage, allowing the nucleic acid fragments to bind to the SMPs, be washed, and then be eluted for RT reaction within 15 minutes. RNAs shorter than 63 nt were selectively enriched from cell lysates, with recovery comparable to that of a commercial kit. Physical separation of the droplets on our M&M chip allowed the usage of multiple channels for parallel processing of multiple samples. It also permitted smooth integration with on-chip RT-PCR, which simultaneously detected the target microRNA, mir-191, expressed in fewer than 10 cancer cells. Our results have demonstrated that the M&M chip device is a valuable and cost-saving platform for studying small RNA expression patterns in a limited number of cells with reasonable sample throughput.

Neural Stem Cell Differentiation Using Microfluidic Device-Generated Growth Factor Gradient.

PubMed

Kim, Ji Hyeon; Sim, Jiyeon; Kim, Hyun-Jung

2018-04-11

Neural stem cells (NSCs) have the ability to self-renew and differentiate into multiple nervous system cell types. During embryonic development, the concentrations of soluble biological molecules have a critical role in controlling cell proliferation, migration, differentiation and apoptosis. In an effort to find optimal culture conditions for the generation of desired cell types in vitro , we used a microfluidic chip-generated growth factor gradient system. In the current study, NSCs in the microfluidic device remained healthy during the entire period of cell culture, and proliferated and differentiated in response to the concentration gradient of growth factors (epithermal growth factor and basic fibroblast growth factor). We also showed that overexpression of ASCL1 in NSCs increased neuronal differentiation depending on the concentration gradient of growth factors generated in the microfluidic gradient chip. The microfluidic system allowed us to study concentration-dependent effects of growth factors within a single device, while a traditional system requires multiple independent cultures using fixed growth factor concentrations. Our study suggests that the microfluidic gradient-generating chip is a powerful tool for determining the optimal culture conditions.

High-performance genetic analysis on microfabricated capillary array electrophoresis plastic chips fabricated by injection molding.

PubMed

Dang, Fuquan; Tabata, Osamu; Kurokawa, Masaya; Ewis, Ashraf A; Zhang, Lihua; Yamaoka, Yoshihisa; Shinohara, Shouji; Shinohara, Yasuo; Ishikawa, Mitsuru; Baba, Yoshinobu

2005-04-01

We have developed a novel technique for mass production of microfabricated capillary array electrophoresis (mu-CAE) plastic chips for high-speed, high-throughput genetic analysis. The mu-CAE chips, containing 10 individual separation channels of 50-microm width, 50-microm depth, and a 100-microm lane-to-lane spacing at the detection region and a sacrificial channel network, were fabricated on a poly(methyl methacrylate) substrate by injection molding and then bonded manually using a pressure-sensitive sealing tape within several seconds at room temperature. The conditions for injection molding and bonding were carefully characterized to yield mu-CAE chips with well-defined channel and injection structures. A CCD camera equipped with an image intensifier was used to monitor simultaneously the separation in a 10-channel array with laser-induced fluorescence detection. High-performance electrophoretic separations of phiX174 HaeIII DNA restriction fragments and PCR products related to the human beta-globin gene and SP-B gene (the surfactant protein B) have been demonstrated on mu-CAE plastic chips using a methylcellulose sieving matrix in individual channels. The current work demonstrated greatly simplified the fabrication process as well as a detection scheme for mu-CAE chips and will bring the low-cost mass production and application of mu-CAE plastic chips for genetic analysis.

Course 8: Biological Physics in Silico

NASA Astrophysics Data System (ADS)

Austin, R. H.

1 Why micro/nanofabrication? Lecture 1a: Hydrodynamic Transport 1 Introduction: The need to control flows in 2 1/2 D 2 Somewhat simple hydrodynamics in 2 1/2 D 3 The N-port injector idea 4 Conclusion Lecture 1b: Dielectrophoresis and Microfabrication 1 Introduction 2 Methods 3 Results 4 Data and analysis 5 Origin of the low frequency dielectrophoretic force in DNA 6 Conclusion Lecture 2a: Hex Arrays 1 Introduction 2 Experimental approach 3 Conclusions Lecture 2b: The DNA Prism 1 Introduction 2 Design 3 Results 4 Conclusions Lecture 2c: Bigger is Better in Rachets 1 The problems with insulators in rachets 2 An experimental test 3 Conclusions Lecture 3: Going After Epigenetics 1 Introduction 2 The nearfield scanner 3 The chip 4 Experiments with molecules 5 Conclusions Lecture 4: Fractionating Cells 1 Introduction 2 Blood specifics 3 Magnetic separation 4 Microfabrication 5 Magnetic field gradients 6 Device interface 7 A preliminary blood cell run 8 Conclusions Lecture 5: Protein Folding on a Chip 1 Introduction 2 Technology 3 Experiments 4 Conclusions

A microfluidic co-culture system to monitor tumor-stromal interactions on a chip

PubMed Central

Menon, Nishanth V.; Cao, Bin; Lim, Mayasari; Kang, Yuejun

2014-01-01

The living cells are arranged in a complex natural environment wherein they interact with extracellular matrix and other neighboring cells. Cell-cell interactions, especially those between distinct phenotypes, have attracted particular interest due to the significant physiological relevance they can reveal for both fundamental and applied biomedical research. To study cell-cell interactions, it is necessary to develop co-culture systems, where different cell types can be cultured within the same confined space. Although the current advancement in lab-on-a-chip technology has allowed the creation of in vitro models to mimic the complexity of in vivo environment, it is still rather challenging to create such co-culture systems for easy control of different colonies of cells. In this paper, we have demonstrated a straightforward method for the development of an on-chip co-culture system. It involves a series of steps to selectively change the surface property for discriminative cell seeding and to induce cellular interaction in a co-culture region. Bone marrow stromal cells (HS5) and a liver tumor cell line (HuH7) have been used to demonstrate this co-culture model. The cell migration and cellular interaction have been analyzed using microscopy and biochemical assays. This co-culture system could be used as a disease model to obtain biological insight of pathological progression, as well as a tool to evaluate the efficacy of different drugs for pharmaceutical studies. PMID:25553194

Characterization of size-dependent mechanical properties of tip-growing cells using a lab-on-chip device.

PubMed

Hu, Chengzhi; Munglani, Gautam; Vogler, Hannes; Ndinyanka Fabrice, Tohnyui; Shamsudhin, Naveen; Wittel, Falk K; Ringli, Christoph; Grossniklaus, Ueli; Herrmann, Hans J; Nelson, Bradley J

2016-12-20

Quantification of mechanical properties of tissues, living cells, and cellular components is crucial for the modeling of plant developmental processes such as mechanotransduction. Pollen tubes are tip-growing cells that provide an ideal system to study the mechanical properties at the single cell level. In this article, a lab-on-a-chip (LOC) device is developed to quantitatively measure the biomechanical properties of lily (Lilium longiflorum) pollen tubes. A single pollen tube is fixed inside the microfluidic chip at a specific orientation and subjected to compression by a soft membrane. By comparing the deformation of the pollen tube at a given external load (compressibility) and the effect of turgor pressure on the tube diameter (stretch ratio) with finite element modeling, its mechanical properties are determined. The turgor pressure and wall stiffness of the pollen tubes are found to decrease considerably with increasing initial diameter of the pollen tubes. This observation supports the hypothesis that tip-growth is regulated by a delicate balance between turgor pressure and wall stiffness. The LOC device is modular and adaptable to a variety of cells that exhibit tip-growth, allowing for the straightforward measurement of mechanical properties.

Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses.

PubMed

Teste, Bruno; Champ, Jerome; Londono-Vallejo, Arturo; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis; Draskovic, Irena; Mottet, Guillaume

2017-01-31

Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

Vacuum-assisted cell loading enables shear-free mammalian microfluidic culture

PubMed Central

Kolnik, Martin; Tsimring, Lev S; Hasty, Je

2012-01-01

Microfluidic perfusion cultures for mammalian cells provide a novel means for probing single-cell behavior but require the management of culture parameters such as flow-induced shear stress. Methods to eliminate shear stress generally focus on capturing cells in regions with high resistance to fluid flow. Here, we present a novel trapping design to easily and reliably load a high density of cells into culture chambers that are extremely isolated from potentially damaging flow effects. We utilize a transient on-chip vacuum to remove air from the culture chambers and rapidly replace the volume with a liquid cell suspension. We demonstrate the ability of this simple and robust method to load and culture three commonly used cell lines. We show how the incorporation of an on-chip function generator can be used for dynamic stimulation of cells during long-term continuous perfusion culture. PMID:22961584

Kidney-on-a-Chip: a New Technology for Predicting Drug Efficacy, Interactions, and Drug-induced Nephrotoxicity.

PubMed

Lee, Jeonghwan; Kim, Sejoong

2018-03-08

The kidneys play a pivotal role in most drug-removal processes and are important when evaluating drug safety. Kidney dysfunction resulting from various drugs is an important issue in clinical practice and during the drug development process. Traditional in vivo animal experiments are limited with respect to evaluating drug efficacy and nephrotoxicity due to discrepancies in drug pharmacokinetics and pharmacodynamics between humans and animals, and static cell culture experiments cannot fully reflect the actual microphysiological environment in humans. A kidney-on-a-chip is a microfluidic device that allows the culture of living renal cells in 3-dimensional channels and mimics the human microphysiological environment, thus simulating the actual drug filtering, absorption, and secretion process.. In this review, we discuss recent developments in microfluidic culturing technique and describe current and future kidney-on-a-chip applications. We focus on pharmacological interactions and drug-induced nephrotoxicity, and additionally discuss the development of multi-organ chips and their possible applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

On-chip magnetically actuated robot with ultrasonic vibration for single cell manipulations.

PubMed

Hagiwara, Masaya; Kawahara, Tomohiro; Yamanishi, Yoko; Masuda, Taisuke; Feng, Lin; Arai, Fumihito

2011-06-21

This paper presents an innovative driving method for an on-chip robot actuated by permanent magnets in a microfluidic chip. A piezoelectric ceramic is applied to induce ultrasonic vibration to the microfluidic chip and the high-frequency vibration reduces the effective friction on the MMT significantly. As a result, we achieved 1.1 micrometre positioning accuracy of the microrobot, which is 100 times higher accuracy than without vibration. The response speed is also improved and the microrobot can be actuated with a speed of 5.5 mm s(-1) in 3 degrees of freedom. The novelty of the ultrasonic vibration appears in the output force as well. Contrary to the reduction of friction on the microrobot, the output force increased twice as much by the ultrasonic vibration. Using this high accuracy, high speed, and high power microrobot, swine oocyte manipulations are presented in a microfluidic chip.

Organs-on-chips at the frontiers of drug discovery

PubMed Central

Esch, Eric W.; Bahinski, Anthony; Huh, Dongeun

2016-01-01

Improving the effectiveness of preclinical predictions of human drug responses is critical to reducing costly failures in clinical trials. Recent advances in cell biology, microfabrication and microfluidics have enabled the development of microengineered models of the functional units of human organs — known as organs-on-chips — that could provide the basis for preclinical assays with greater predictive power. Here, we examine the new opportunities for the application of organ-on-chip technologies in a range of areas in preclinical drug discovery, such as target identification and validation, target-based screening, and phenotypic screening. We also discuss emerging drug discovery opportunities enabled by organs-on-chips, as well as important challenges in realizing the full potential of this technology. PMID:25792263

Design of a dual-mode electrochemical measurement and analysis system.

PubMed

Yang, Jr-Fu; Wei, Chia-Ling; Wu, Jian-Fu; Liu, Bin-Da

2013-01-01

A dual-mode electrochemical measurement and analysis system is proposed. This system includes a dual-mode chip, which was designed and fabricated by using TSMC 0.35 µm 3.3 V/5 V 2P4M mixed-signal CMOS process. Two electrochemical measurement and analysis methods, chronopotentiometry and voltammetry, can be performed by using the proposed chip and system. The proposed chip and system are verified successfully by performing voltammetry and chronopotentiometry on solutions.

A multidisciplinary study using in vivo tumor models and microfluidic cell-on-chip approach to explore the cross-talk between cancer and immune cells.

PubMed

Mattei, Fabrizio; Schiavoni, Giovanna; De Ninno, Adele; Lucarini, Valeria; Sestili, Paola; Sistigu, Antonella; Fragale, Alessandra; Sanchez, Massimo; Spada, Massimo; Gerardino, Annamaria; Belardelli, Filippo; Businaro, Luca; Gabriele, Lucia

2014-10-01

A full elucidation of events occurring inside the cancer microenvironment is fundamental for the optimization of more effective therapies. In the present study, the cross-talk between cancer and immune cells was examined by employing mice deficient (KO) in interferon regulatory factor (IRF)-8, a transcription factor essential for induction of competent immune responses. The in vivo results showed that IRF-8 KO mice were highly permissive to B16.F10 melanoma growth and metastasis due to failure of their immune cells to exert proper immunosurveillance. These events were found to be dependent on soluble factors released by cells of the immune system capable of shaping the malignant phenotype of melanoma cells. An on-chip model was then generated to further explore the reciprocal interactions between the B16.F10 and immune cells. B16.F10 and immune cells were co-cultured in a microfluidic device composed of three culturing chambers suitably inter-connected by an array of microchannels; mutual interactions were then followed using time-lapse microscopy. It was observed that WT immune cells migrated through the microchannels towards the B16.F10 cells, establishing tight interactions that in turn limited tumor spread. In contrast, IRF-8 KO immune cells poorly interacted with the melanoma cells, resulting in a more invasive behavior of the B16.F10 cells. These results suggest that IRF-8 expression plays a key role in the cross-talk between melanoma and immune cells, and under-score the value of cell-on-chip approaches as useful in vitro tools to reconstruct complex in vivo microenvironments on a microscale level to explore cell interactions such as those occurring within a cancer immunoenvironment.

Creating a Tiny Human Body on a Chip

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hunsberger, Maren; Soscia, Dave; Moya, Monica

LLNL science communicator Maren Hunsberger takes us "Inside the Lab" to learn about the iChip (In-vitro Chip-based Human Investigational Platform) project at Lawrence Livermore National Laboratory. "One application of the iChip system would be to develop new pharmaceutical drugs," explains Dave Soscia, LLNL postdoc. "When you test in a mouse for example, it's not as close to the human system as you can get. If we can take human cells and put them on devices and actually mimic the structure and function of the organ systems in the human, we can actually replace animal testing and even make a bettermore » system for testing pharmaceutical drugs."« less

Mobile Phone Chips Reduce Increases in EEG Brain Activity Induced by Mobile Phone-Emitted Electromagnetic Fields.

PubMed

Henz, Diana; Schöllhorn, Wolfgang I; Poeggeler, Burkhard

2018-01-01

Recent neurophysiological studies indicate that exposure to electromagnetic fields (EMFs) generated by mobile phone radiation can exert effects on brain activity. One technical solution to reduce effects of EMFs in mobile phone use is provided in mobile phone chips that are applied to mobile phones or attached to their surfaces. To date, there are no systematical studies on the effects of mobile phone chip application on brain activity and the underlying neural mechanisms. The present study investigated whether mobile phone chips that are applied to mobile phones reduce effects of EMFs emitted by mobile phone radiation on electroencephalographic (EEG) brain activity in a laboratory study. Thirty participants volunteered in the present study. Experimental conditions (mobile phone chip, placebo chip, no chip) were set up in a randomized within-subjects design. Spontaneous EEG was recorded before and after mobile phone exposure for two 2-min sequences at resting conditions. During mobile phone exposure, spontaneous EEG was recorded for 30 min during resting conditions, and 5 min during performance of an attention test (d2-R). Results showed increased activity in the theta, alpha, beta and gamma bands during EMF exposure in the placebo and no chip conditions. Application of the mobile phone chip reduced effects of EMFs on EEG brain activity and attentional performance significantly. Attentional performance level was maintained regarding number of edited characters. Further, a dipole analysis revealed different underlying activation patterns in the chip condition compared to the placebo chip and no chip conditions. Finally, a correlational analysis for the EEG frequency bands and electromagnetic high-frequency (HF) emission showed significant correlations in the placebo chip and no chip condition for the theta, alpha, beta, and gamma bands. In the chip condition, a significant correlation of HF with the theta and alpha bands, but not with the beta and gamma bands was shown. We hypothesize that a reduction of EEG beta and gamma activation constitutes the key neural mechanism in mobile phone chip use that supports the brain to a degree in maintaining its natural activity and performance level during mobile phone use.

Mobile Phone Chips Reduce Increases in EEG Brain Activity Induced by Mobile Phone-Emitted Electromagnetic Fields

PubMed Central

Henz, Diana; Schöllhorn, Wolfgang I.; Poeggeler, Burkhard

2018-01-01

Recent neurophysiological studies indicate that exposure to electromagnetic fields (EMFs) generated by mobile phone radiation can exert effects on brain activity. One technical solution to reduce effects of EMFs in mobile phone use is provided in mobile phone chips that are applied to mobile phones or attached to their surfaces. To date, there are no systematical studies on the effects of mobile phone chip application on brain activity and the underlying neural mechanisms. The present study investigated whether mobile phone chips that are applied to mobile phones reduce effects of EMFs emitted by mobile phone radiation on electroencephalographic (EEG) brain activity in a laboratory study. Thirty participants volunteered in the present study. Experimental conditions (mobile phone chip, placebo chip, no chip) were set up in a randomized within-subjects design. Spontaneous EEG was recorded before and after mobile phone exposure for two 2-min sequences at resting conditions. During mobile phone exposure, spontaneous EEG was recorded for 30 min during resting conditions, and 5 min during performance of an attention test (d2-R). Results showed increased activity in the theta, alpha, beta and gamma bands during EMF exposure in the placebo and no chip conditions. Application of the mobile phone chip reduced effects of EMFs on EEG brain activity and attentional performance significantly. Attentional performance level was maintained regarding number of edited characters. Further, a dipole analysis revealed different underlying activation patterns in the chip condition compared to the placebo chip and no chip conditions. Finally, a correlational analysis for the EEG frequency bands and electromagnetic high-frequency (HF) emission showed significant correlations in the placebo chip and no chip condition for the theta, alpha, beta, and gamma bands. In the chip condition, a significant correlation of HF with the theta and alpha bands, but not with the beta and gamma bands was shown. We hypothesize that a reduction of EEG beta and gamma activation constitutes the key neural mechanism in mobile phone chip use that supports the brain to a degree in maintaining its natural activity and performance level during mobile phone use. PMID:29670503

Comparison of the osteogenic potential of bone dust and iliac bone chip.

PubMed

Ye, Shuai; Seo, Kyu-Bum; Park, Byung-Hyun; Song, Kyung-Jin; Kim, Jung-Ryul; Jang, Kyu-Yun; Chae, Young Ju; Lee, Kwang-Bok

2013-11-01

There is no comparative study of the in vitro and in vivo osteogenic potential of iliac bone chips (autogenous iliac cancellous bone chips) compared with bone dusts generated during the decortication process with a high-speed burr in spine fracture or fusion surgery. To compare the osteogenic potential of three sizes of bone dusts with iliac bone chips and to determine whether bone dusts can be used as a bone graft substitute. In vitro and in vivo study. Bone chips were harvested from the posterior superior iliac spine and bone dusts from the vertebrae of 15 patients who underwent spinal fracture surgery. Bone dust was divided into three groups: small (3 mm), middle (4 mm), and large (5 mm) according to the size of the burr tip. A comparison was made using a cell proliferation assay, alkaline phosphatase (ALP) activity, the degree of mineralization in an in vitro model, and radiographic and histologic studies (the change of absorbable area and tissue density) after implantation of the various materials into back muscles of nude mice. Although all three bone dust groups were less active with regard to cell proliferation, ALP activity, and the degree of mineralization, than were bone chips, they still exhibited osteogenic potential. Furthermore, there was no significant difference among the three bone dust groups. The three bone dust groups did show greater absorbable area and change of the tissue density than did the iliac bone chip group. Again, there was no significant difference among the three bone dust groups in this regard. Histologically, specimens from the bone dust groups had a higher osteoclast cell number than specimens from the iliac bone chip group. The osteogenic potential of bone dusts is lower than that of iliac bone chips, and the absorption speed of bone dusts in vivo is faster than that of iliac bone chips. The increased resorption speed appeared to result from an increase in osteoclast cell number. Therefore, caution needs to be used when surgeons employ bone dust as a bone graft substitute. Copyright © 2013 Elsevier Inc. All rights reserved.

An Experimental Study of Dependence of Optimum TBM Cutter Spacing on Pre-set Penetration Depth in Sandstone Fragmentation

NASA Astrophysics Data System (ADS)

Han, D. Y.; Cao, P.; Liu, J.; Zhu, J. B.

2017-12-01

Cutter spacing is an essential parameter in the TBM design. However, few efforts have been made to study the optimum cutter spacing incorporating penetration depth. To investigate the influence of pre-set penetration depth and cutter spacing on sandstone breakage and TBM performance, a series of sequential laboratory indentation tests were performed in a biaxial compression state. Effects of parameters including penetration force, penetration depth, chip mass, chip size distribution, groove volume, specific energy and maximum angle of lateral crack were investigated. Results show that the total mass of chips, the groove volume and the observed optimum cutter spacing increase with increasing pre-set penetration depth. It is also found that the total mass of chips could be an alternative means to determine optimum cutter spacing. In addition, analysis of chip size distribution suggests that the mass of large chips is dominated by both cutter spacing and pre-set penetration depth. After fractal dimension analysis, we found that cutter spacing and pre-set penetration depth have negligible influence on the formation of small chips and that small chips are formed due to squeezing of cutters and surface abrasion caused by shear failure. Analysis on specific energy indicates that the observed optimum spacing/penetration ratio is 10 for the sandstone, at which, the specific energy and the maximum angle of lateral cracks are smallest. The findings in this paper contribute to better understanding of the coupled effect of cutter spacing and pre-set penetration depth on TBM performance and rock breakage, and provide some guidelines for cutter arrangement.

Lab-on-a-chip and SDS-PAGE analysis of hemolymph protein profile from Rhipicephalus microplus (Acari: Ixodidae) infected with entomopathogenic nematode and fungus.

PubMed

Golo, Patrícia Silva; Dos Santos, Alessa Siqueira de Oliveira; Monteiro, Caio Marcio Oliveira; Perinotto, Wendell Marcelo de Souza; Quinelato, Simone; Camargo, Mariana Guedes; de Sá, Fillipe Araujo; Angelo, Isabele da Costa; Martins, Marta Fonseca; Prata, Marcia Cristina de Azevedo; Bittencourt, Vânia Rita Elias Pinheiro

2016-09-01

In the present study, lab-on-a-chip electrophoresis (LoaC) was suggested as an alternative method to the conventional polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) to analyze raw cell-free tick hemolymph. Rhipicephalus microplus females were exposed to the entomopathogenic fungus Metarhizium anisopliae senso latu IBCB 116 strain and/or to the entomopathogenic nematode Heterorhabditis indica LPP1 strain. Hemolymph from not exposed or exposed ticks was collected 16 and 24 h after exposure and analyze by SDS-PAGE or LoaC. SDS-PAGE yielded 15 bands and LoaC electrophoresis 17 bands. Despite the differences in the number of bands, when the hemolymph protein profiles of exposed or unexposed ticks were compared in the same method, no suppressing or additional bands were detected among the treatments regardless the method (i.e., SDS-PAGE or chip electrophoresis using the Protein 230 Kit®). The potential of LoaC electrophoresis to detect protein bands from tick hemolymph was considered more efficient in comparison to the detection obtained using the traditional SDS-PAGE method, especially when it comes to protein subunits heavier than 100 KDa. LoaC electrophoresis provided a very good reproducibility, and is much faster than the conventional SDS-PAGE method, which requires several hours for one analysis. Despite both methods can be used to analyze tick hemolymph composition, LoaC was considered more suitable for cell-free hemolymph protein separation and detection. LoaC hemolymph band percent data reported changes in key proteins (i.e., HeLp and vitellogenin) exceptionally important for tick embryogenesis. This study reported, for the first time, tick hemolymph protein profile using LoaC.

Effects of electrolyzed oxidizing water on reducing Listeria monocytogenes contamination on seafood processing surfaces.

PubMed

Liu, Chengchu; Duan, Jingyun; Su, Yi-Cheng

2006-02-15

The effects of electrolyzed oxidizing (EO) water on reducing Listeria monocytogenes contamination on seafood processing surfaces were studied. Chips (5 x 5 cm(2)) of stainless steel sheet (SS), ceramic tile (CT), and floor tile (FT) with and without crabmeat residue on the surface were inoculated with L. monocytogenes and soaked in tap or EO water for 5 min. Viable cells of L. monocytogenes were detected on all chip surfaces with or without crabmeat residue after being held at room temperature for 1 h. Soaking contaminated chips in tap water resulted in small-degree reductions of the organism (0.40-0.66 log cfu/chip on clean surfaces and 0.78-1.33 log cfu/chip on dirty surfaces). Treatments of EO water significantly (p<0.05) reduced L. monocytogenes on clean surfaces (3.73 log on SS, 4.24 log on CT, and 5.12 log on FT). Presence of crabmeat residue on chip surfaces reduced the effectiveness of EO water on inactivating Listeria cells. However, treatments of EO water also resulted in significant reductions of L. monocytogenes on dirty surfaces (2.33 log on SS and CT and 1.52 log on FT) when compared with tap water treatments. The antimicrobial activity of EO water was positively correlated with its chlorine content. High oxidation-reduction potential (ORP) of EO water also contributed significantly to its antimicrobial activity against L. monocytogenes. EO water was more effective than chlorine water on inactivating L. monocytogenes on surfaces and could be used as a chlorine alternative for sanitation purpose. Application of EO water following a thorough cleaning process could greatly reduce L. monocytogenes contamination in seafood processing environments.

ITO/gold nanoparticle/RGD peptide composites to enhance electrochemical signals and proliferation of human neural stem cells.

PubMed

Kim, Tae-Hyung; El-Said, Waleed Ahmed; An, Jeung Hee; Choi, Jeong-Woo

2013-04-01

A cell chip composed of ITO, gold nanoparticles (GNP) and RGD-MAP-C peptide composites was fabricated to enhance the electrochemical signals and proliferation of undifferentiated human neural stem cells (HB1.F3). The structural characteristics of the fabricated surfaces were confirmed by both scanning electron microscopy and surface-enhanced Raman spectroscopy. HB1.F3 cells were allowed to attach to various composites electrodes in the cell chip and the material-dependent effects on electrochemical signals and cell proliferation were analyzed. The ITO/60 nm GNP/RGD-MAP-C composite electrode was found to be the best material in regards to enhancing the voltammetric signals of HB1.F3 cells when exposed to cyclic voltammetry, as well as for increasing cell proliferation. Differential pulse voltammetry was performed to evaluate the adverse effects of doxorubicin on HB1.F3 cells. In these experiments, negative correlations between cell viability and chemical concentrations were obseved, which were more sensitive than MTT viability assay especially at low concentrations (<0.1 μg/mL). In this basic science study, a cell chip composed of ITO, gold nanoparticles and RGD-MAP-C peptide composites was fabricated to enhance electrochemical signals and proliferation of undifferentiated human neural stem cells (HB1.F3). The ITO/60 nm GNP/RGD-MAP-C composite electrode was found to best enhance the voltammetric signals of the studied cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Cryopreservation of adhered mammalian cells on a microfluidic device: Toward ready-to-use cell-based experimental platforms.

PubMed

Kondo, Eitaro; Wada, Ken-ichi; Hosokawa, Kazuo; Maeda, Mizuo

2016-01-01

In this paper, we describe cryopreservation of mammalian cells in the adhered state on a microfluidic device (microdevice) for the first time. HeLa, NIH3T3, MCF-7, and PC12 cells were cultured on a microdevice in which a commercial polystyrene dish surface was used as the cell adhesion surface. Without cell-detaching treatment, the microdevice was stored in a freezer at -80°C. After thawing, we observed a greater number of live cells on the microdevice than those on a control culture dish. Although the effectiveness of the microdevice varied depending on the cell type and surface coating, the trend was consistent. We confirmed that the phenotype of the PC12 cells to differentiate into neuron-like cells was kept after the on-chip cryopreservation, and that the results of cytotoxicity test of cisplatin against the HeLa cells were essentially unchanged by the on-chip cryopreservation. These findings will open up a new possibility of ready-to-use cell-based experimental platforms. © 2015 Wiley Periodicals, Inc.

Microfluidic LC Device with Orthogonal Sample Extraction for On-Chip MALDI-MS Detection

PubMed Central

Lazar, Iulia M.; Kabulski, Jarod L.

2013-01-01

A microfluidic device that enables on-chip matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) detection for liquid chromatography (LC) separations is described. The device comprises an array of functional elements to carry out LC separations, integrates a novel microchip-MS interface to facilitate the orthogonal transposition of the microfluidic LC channel into an array of reservoirs, and enables sensitive MALDI-MS detection directly from the chip. Essentially, the device provides a snapshot MALDI-MS map of the content of the separation channel present on the chip. The detection of proteins with biomarker potential from MCF10A breast epithelial cell extracts, and detection limits in the low fmol range, are demonstrated. In addition, the design of the novel LC-MALDI-MS chip entices the promotion of a new concept for performing sample separations within the limited time-frame that accompanies the dead-volume of a separation channel. PMID:23592150

Multi-angle lensless digital holography for depth resolved imaging on a chip.

PubMed

Su, Ting-Wei; Isikman, Serhan O; Bishara, Waheb; Tseng, Derek; Erlinger, Anthony; Ozcan, Aydogan

2010-04-26

A multi-angle lensfree holographic imaging platform that can accurately characterize both the axial and lateral positions of cells located within multi-layered micro-channels is introduced. In this platform, lensfree digital holograms of the micro-objects on the chip are recorded at different illumination angles using partially coherent illumination. These digital holograms start to shift laterally on the sensor plane as the illumination angle of the source is tilted. Since the exact amount of this lateral shift of each object hologram can be calculated with an accuracy that beats the diffraction limit of light, the height of each cell from the substrate can be determined over a large field of view without the use of any lenses. We demonstrate the proof of concept of this multi-angle lensless imaging platform by using light emitting diodes to characterize various sized microparticles located on a chip with sub-micron axial and lateral localization over approximately 60 mm(2) field of view. Furthermore, we successfully apply this lensless imaging approach to simultaneously characterize blood samples located at multi-layered micro-channels in terms of the counts, individual thicknesses and the volumes of the cells at each layer. Because this platform does not require any lenses, lasers or other bulky optical/mechanical components, it provides a compact and high-throughput alternative to conventional approaches for cytometry and diagnostics applications involving lab on a chip systems.

Carboxyl Terminus of HSC70-interacting Protein (CHIP) Down-regulates NF-κB-inducing Kinase (NIK) and Suppresses NIK-induced Liver Injury*

PubMed Central

Jiang, Bijie; Shen, Hong; Chen, Zheng; Yin, Lei; Zan, Linsen; Rui, Liangyou

2015-01-01

Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease. PMID:25792747