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Sample records for microfluidics meet cell

  1. Microfluidic fuel cells

    NASA Astrophysics Data System (ADS)

    Kjeang, Erik

    Microfluidic fuel cell architectures are presented in this thesis. This work represents the mechanical and microfluidic portion of a microfluidic biofuel cell project. While the microfluidic fuel cells developed here are targeted to eventual integration with biocatalysts, the contributions of this thesis have more general applicability. The cell architectures are developed and evaluated based on conventional non-biological electrocatalysts. The fuel cells employ co-laminar flow of fuel and oxidant streams that do not require a membrane for physical separation, and comprise carbon or gold electrodes compatible with most enzyme immobilization schemes developed to date. The demonstrated microfluidic fuel cell architectures include the following: a single cell with planar gold electrodes and a grooved channel architecture that accommodates gaseous product evolution while preventing crossover effects; a single cell with planar carbon electrodes based on graphite rods; a three-dimensional hexagonal array cell based on multiple graphite rod electrodes with unique scale-up opportunities; a single cell with porous carbon electrodes that provides enhanced power output mainly attributed to the increased active area; a single cell with flow-through porous carbon electrodes that provides improved performance and overall energy conversion efficiency; and a single cell with flow-through porous gold electrodes with similar capabilities and reduced ohmic resistance. As compared to previous results, the microfluidic fuel cells developed in this work show improved fuel cell performance (both in terms of power density and efficiency). In addition, this dissertation includes the development of an integrated electrochemical velocimetry approach for microfluidic devices, and a computational modeling study of strategic enzyme patterning for microfluidic biofuel cells with consecutive reactions.

  2. Microfluidic Cell Culture Device

    NASA Technical Reports Server (NTRS)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  3. Digital Microfluidic Cell Culture.

    PubMed

    Ng, Alphonsus H C; Li, Bingyu Betty; Chamberlain, M Dean; Wheeler, Aaron R

    2015-01-01

    Digital microfluidics (DMF) is a droplet-based liquid-handling technology that has recently become popular for cell culture and analysis. In DMF, picoliter- to microliter-sized droplets are manipulated on a planar surface using electric fields, thus enabling software-reconfigurable operations on individual droplets, such as move, merge, split, and dispense from reservoirs. Using this technique, multistep cell-based processes can be carried out using simple and compact instrumentation, making DMF an attractive platform for eventual integration into routine biology workflows. In this review, we summarize the state-of-the-art in DMF cell culture, and describe design considerations, types of DMF cell culture, and cell-based applications of DMF. PMID:26643019

  4. Research highlights: microfluidics meets big data.

    PubMed

    Tseng, Peter; Weaver, Westbrook M; Masaeli, Mahdokht; Owsley, Keegan; Di Carlo, Dino

    2014-03-01

    In this issue we highlight a collection of recent work in which microfluidic parallelization and automation have been employed to address the increasing need for large amounts of quantitative data concerning cellular function--from correlating microRNA levels to protein expression, increasing the throughput and reducing the noise when studying protein dynamics in single-cells, and understanding how signal dynamics encodes information. The painstaking dissection of cellular pathways one protein at a time appears to be coming to an end, leading to more rapid discoveries which will inevitably translate to better cellular control--in producing useful gene products and treating disease at the individual cell level. From these studies it is also clear that development of large scale mutant or fusion libraries, automation of microscopy, image analysis, and data extraction will be key components as microfluidics contributes its strengths to aid systems biology moving forward. PMID:24473594

  5. Applications of Microfluidics in Stem Cell Biology.

    PubMed

    Zhang, Qiucen; Austin, Robert H

    2012-12-01

    Stem cell research can significantly benefit from recent advances of microfluidics technology. In a rationally designed microfluidics device, analyses of stem cells can be done in a much deeper and wider way than in a conventional tissue culture dish. Miniaturization makes analyses operated in a high-throughput fashion, while controls of fluids help to reconstruct the physiological environments. Through integration with present characterization tools like fluorescent microscope, microfluidics offers a systematic way to study the decision-making process of stem cells, which has attractive medical applications. In this paper, recent progress of microfluidics devices on stem cell research are discussed. The purpose of this review is to highlight some key features of microfluidics for stem cell biologists, as well as provide physicists/engineers an overview of how microfluidics has been and could be used for stem cell research. PMID:23336098

  6. Microfluidic tools for cell biological research

    PubMed Central

    Velve-Casquillas, Guilhem; Le Berre, Maël; Piel, Matthieu; Tran, Phong T.

    2010-01-01

    Summary Microfluidic technology is creating powerful tools for cell biologists to control the complete cellular microenvironment, leading to new questions and new discoveries. We review here the basic concepts and methodologies in designing microfluidic devices, and their diverse cell biological applications. PMID:21152269

  7. Microfluidic devices for cell cultivation and proliferation

    PubMed Central

    Tehranirokh, Masoomeh; Kouzani, Abbas Z.; Francis, Paul S.; Kanwar, Jagat R.

    2013-01-01

    Microfluidic technology provides precise, controlled-environment, cost-effective, compact, integrated, and high-throughput microsystems that are promising substitutes for conventional biological laboratory methods. In recent years, microfluidic cell culture devices have been used for applications such as tissue engineering, diagnostics, drug screening, immunology, cancer studies, stem cell proliferation and differentiation, and neurite guidance. Microfluidic technology allows dynamic cell culture in microperfusion systems to deliver continuous nutrient supplies for long term cell culture. It offers many opportunities to mimic the cell-cell and cell-extracellular matrix interactions of tissues by creating gradient concentrations of biochemical signals such as growth factors, chemokines, and hormones. Other applications of cell cultivation in microfluidic systems include high resolution cell patterning on a modified substrate with adhesive patterns and the reconstruction of complicated tissue architectures. In this review, recent advances in microfluidic platforms for cell culturing and proliferation, for both simple monolayer (2D) cell seeding processes and 3D configurations as accurate models of in vivo conditions, are examined. PMID:24273628

  8. A perspective on microfluidic biofuel cells

    PubMed Central

    Lee, Jin wook; Kjeang, Erik

    2010-01-01

    This review article presents how microfluidic technologies and biological materials are paired to assist in the development of low cost, green energy fuel cell systems. Miniaturized biological fuel cells, employing enzymes or microorganisms as biocatalysts in an environmentally benign configuration, can become an attractive candidate for small-scale power source applications such as biological sensors, implantable medical devices, and portable electronics. State-of-the-art biofuel cell technologies are reviewed with emphasis on microfabrication compatibility and microfluidic fuel cell designs. Integrated microfluidic biofuel cell prototypes are examined with comparisons of their performance achievements and fabrication methods. The technical challenges for further developments and the potential research opportunities for practical cell designs are discussed. PMID:21139699

  9. Microfluidic device for acoustic cell lysis

    DOEpatents

    Branch, Darren W.; Cooley, Erika Jane; Smith, Gennifer Tanabe; James, Conrad D.; McClain, Jaime L.

    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.

  10. Mechanically activated artificial cell by using microfluidics.

    PubMed

    Ho, Kenneth K Y; Lee, Lap Man; Liu, Allen P

    2016-01-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology. PMID:27610921

  11. Mechanically activated artificial cell by using microfluidics

    PubMed Central

    Ho, Kenneth K. Y.; Lee, Lap Man; Liu, Allen P.

    2016-01-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology. PMID:27610921

  12. Understanding cell passage through constricted microfluidic channels

    NASA Astrophysics Data System (ADS)

    Cartas-Ayala, Marco A.; Karnik, Rohit

    2012-11-01

    Recently, several microfluidic platforms have been proposed to characterize cells based on their behaviour during cell passage through constricted channels. Variables like transit time have been analyzed in disease states like sickle cell anemia, malaria and sepsis. Nevertheless, it is hard to make direct comparisons between different platforms and cell types. We present experimental results of the relationship between solid deformable particle properties, i.e. stiffness and relative particle size, and flow properties, i.e. particle's velocity. We measured the hydrodynamic variables during the flow of HL-60 cells, a white myeloid cell type, in narrow microfluidic square channels using a microfluidic differential manometer. We measured the flow force required to move cells of different sizes through microchannels and quantified friction forces opposing cell passage. We determined the non-dimensional parameters that influence the flow of cells and we used them to obtain a non dimensional expression that can be used to predict the forces needed to drive cells through microchannels. We found that the friction force needed to flow HL-60 through a microfluidic channel is the sum of two parts. The first part is a static friction force that is proportional to the force needed to keep the force compressed. The second part is a factor that is proportional to the cell velocity, hence a dynamic term, and slightly sensitive to the compressive force. We thank CONACYT (Mexican Science and Technology Council) for supporting this project, grant 205899.

  13. Uniform yeast cell assembly via microfluidics.

    PubMed

    Chang, Ya-Wen; He, Peng; Marquez, Samantha M; Cheng, Zhengdong

    2012-06-01

    This paper reports the use of microfluidic approaches for the fabrication of yeastosomes (yeast-celloidosomes) based on self-assembly of yeast cells onto liquid-solid or liquid-gas interfaces. Precise control over fluidic flows in droplet- and bubble-forming microfluidic devices allows production of monodispersed, size-selected templates. The general strategy to organize and assemble living cells is to tune electrostatic attractions between the template (gel or gas core) and the cells via surface charging. Layer-by-Layer (LbL) polyelectrolyte deposition was employed to invert or enhance charges of solid surfaces. We demonstrated the ability to produce high-quality, monolayer-shelled yeastosome structures under proper conditions when sufficient electrostatic driving forces are present. The combination of microfluidic fabrication with cell self-assembly enables a versatile platform for designing synthetic hierarchy bio-structures. PMID:22655026

  14. Rare cell isolation and analysis in microfluidics

    PubMed Central

    Chen, Yuchao; Li, Peng; Huang, Po-Hsun; Xie, Yuliang; Mai, John D.; Wang, Lin; Nguyen, Nam-Trung; Huang, Tony Jun

    2014-01-01

    Rare cells are low-abundance cells in a much larger population of background cells. Conventional benchtop techniques have limited capabilities to isolate and analyze rare cells because of their generally low selectivity and significant sample loss. Recent rapid advances in microfluidics have been providing robust solutions to the challenges in the isolation and analysis of rare cells. In addition to the apparent performance enhancements resulting in higher efficiencies and sensitivity levels, microfluidics provides other advanced features such as simpler handling of small sample volumes and multiplexing capabilities for high-throughput processing. All of these advantages make microfluidics an excellent platform to deal with the transport, isolation, and analysis of rare cells. Various cellular biomarkers, including physical properties, dielectric properties, as well as immunoaffinities, have been explored for isolating rare cells. In this Focus article, we discuss the design considerations of representative microfluidic devices for rare cell isolation and analysis. Examples from recently published works are discussed to highlight the advantages and limitations of the different techniques. Various applications of these techniques are then introduced. Finally, a perspective on the development trends and promising research directions in this field are proposed. PMID:24406985

  15. Microfluidic fuel cells for energy generation.

    PubMed

    Safdar, M; Jänis, J; Sánchez, S

    2016-08-01

    Sustainable energy generation is of recent interest due to a growing energy demand across the globe and increasing environmental issues caused by conventional non-renewable means of power generation. In the context of microsystems, portable electronics and lab-on-a-chip based (bio)chemical sensors would essentially require fully integrated, reliable means of power generation. Microfluidic-based fuel cells can offer unique advantages compared to conventional fuel cells such as high surface area-to-volume ratio, ease of integration, cost effectiveness and portability. Here, we summarize recent developments which utilize the potential of microfluidic devices for energy generation. PMID:27367869

  16. Differential white cell count by centrifugal microfluidics.

    SciTech Connect

    Sommer, Gregory Jon; Tentori, Augusto M.; Schaff, Ulrich Y.

    2010-07-01

    We present a method for counting white blood cells that is uniquely compatible with centrifugation based microfluidics. Blood is deposited on top of one or more layers of density media within a microfluidic disk. Spinning the disk causes the cell populations within whole blood to settle through the media, reaching an equilibrium based on the density of each cell type. Separation and fluorescence measurement of cell types stained with a DNA dye is demonstrated using this technique. The integrated signal from bands of fluorescent microspheres is shown to be proportional to their initial concentration in suspension. Among the current generation of medical diagnostics are devices based on the principle of centrifuging a CD sized disk functionalized with microfluidics. These portable 'lab on a disk' devices are capable of conducting multiple assays directly from a blood sample, embodied by platforms developed by Gyros, Samsung, and Abaxis. [1,2] However, no centrifugal platform to date includes a differential white blood cell count, which is an important metric complimentary to diagnostic assays. Measuring the differential white blood cell count (the relative fraction of granulocytes, lymphocytes, and monocytes) is a standard medical diagnostic technique useful for identifying sepsis, leukemia, AIDS, radiation exposure, and a host of other conditions that affect the immune system. Several methods exist for measuring the relative white blood cell count including flow cytometry, electrical impedance, and visual identification from a stained drop of blood under a microscope. However, none of these methods is easily incorporated into a centrifugal microfluidic diagnostic platform.

  17. 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. PMID:26773879

  18. Cell mechanics through analysis of cell trajectories in microfluidic channel

    NASA Astrophysics Data System (ADS)

    Bowie, Samuel; Alexeev, Alexander; Sulchek, Todd

    The understanding of dynamic cell behavior can aid in research ranging from the mechanistic causes of diseases to the development of microfluidic devices for cancer detection. Through analysis of trajectories captured from video of the cells moving in a specially designed microfluidic device, insight into the dynamic viscoelastic nature of cells can be found. The microfluidic device distinguishes cells viscoelastic properties through the use of angled ridges causing a series of compressions, resulting in differences in trajectories based on cell stiffness. Trajectories of cell passing through the device are collected using image processing methods and data mining techniques are used to relate the trajectories to cell properties obtained from experiments. Furthermore, numerical simulation of the cell and microfluidic device are used to match the experimental results from the trajectory analysis. Combination of the modeling and experimental data help to uncover how changes in cellular structures result in changes in mechanical properties.

  19. A 3-D microfluidic combinatorial cell array.

    PubMed

    Liu, Mike C; Tai, Yu-Chong

    2011-02-01

    We present the development of a three-dimensional (3-D) combinatorial cell culture array device featured with integrated three-input, eight-output combinatorial mixer and cell culture chambers. The device is designed for cell-based screening of multiple compounds simultaneously on a microfluidic platform. The final assembled device is composed of a porous membrane integrated in between a Parylene 3-D microfluidic chip and a PDMS microfluidic chip. The membrane turned the cell culture chambers into two-level configuration to facilitate cell loading and to maintain cells in a diffusion dominated space during device operation. Experimentally, we first characterized the combined compound concentration profile at each chamber using a fluorescence method. We then successfully demonstrated the functionality of the quantitative cell-based assay by culturing B35 rat neuronal cells on this device and screening the ability of three compounds (1,5-dihydroxyisoquinoline, deferoxamine, and 3-aminobenzoic acid) to attenuate cell death caused by cytotoxic hydrogen peroxide. In another experiment, we assayed for the combinatorial effects of three chemotherapeutic compound exposures (vinorelbine, paclitaxel, and γ-linolenic acid) on human breast cancer cells, MDA-MB-231. The same technology will enable the construction of inexpensive lab-on-a-chip devices with high-input combinatorial mixer for performing high-throughput cell-based assay and highly parallel and combinatorial chemical or biochemical reactions. PMID:21063783

  20. Advances in microfluidic cell separation and manipulation

    PubMed Central

    Jackson, Emily L; Lu, Hang

    2014-01-01

    Cellular separations are required in many contexts in biochemical and biomedical applications for the identification, isolation, and analysis of phenotypes or samples of interest. Microfluidics is uniquely suited for handling biological samples, and emerging technologies have become increasingly accessible tools for researchers and clinicians. Here, we review advances in the last few years in techniques for microfluidic cell separation and manipulation. Applications such as high-throughput cell and organism phenotypic screening, purification of heterogeneous stem cell populations, separation of blood components, and isolation of rare cells in patients highlight some of the areas in which these technologies show great potential. Continued advances in separation mechanisms and understanding of cellular systems will yield further improvements in the throughput, resolution, and robustness of techniques. PMID:24701393

  1. Cell-based bioassays in microfluidic systems

    NASA Astrophysics Data System (ADS)

    Itle, Laura J.; Zguris, Jeanna C.; Pishko, Michael V.

    2004-12-01

    The development of cell-based bioassays for high throughput drug screening or the sensing of biotoxins is contingent on the development of whole cell sensors for specific changes in intracellular conditions and the integration of those systems into sample delivery devices. Here we show the feasibility of using a 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate, a fluorescent dye capable of responding to changes in intracellular pH, as a detection method for the bacterial endotoxin, lipopolysaccharide. We used photolithography to entrap cells with this dye within poly(ethylene) glyocol diacrylate hydrogels in microfluidic channels. After 18 hours of exposure to lipopolysaccharide, we were able to see visible changes in the fluorescent pattern. This work shows the feasibility of using whole cell based biosensors within microfluidic networks to detect cellular changes in response to exogenous agents.

  2. Microfluidic Blood Cell Preparation: Now and Beyond

    PubMed Central

    Yu, Zeta Tak For; Yong, Koh Meng Aw; Fu, Jianping

    2014-01-01

    Blood plays an important role in homeostatic regulation with each of its cellular components having important therapeutic and diagnostic uses. Therefore, separation and sorting of blood cells has been of a great interest to clinicians and researchers. However, while conventional methods of processing blood have been successful in generating relatively pure fractions, they are time consuming, labor intensive, and are not optimal for processing small volume blood samples. In recent years, microfluidics has garnered great interest from clinicians and researchers as a powerful technology for separating blood into different cell fractions. As microfluidics involves fluid manipulation at the microscale level, it has the potential for achieving high-resolution separation and sorting of blood cells down to a single-cell level, with an added benefit of integrating physical and biological methods for blood cell separation and analysis on the same single chip platform. This paper will first review the conventional methods of processing and sorting blood cells, followed by a discussion on how microfluidics is emerging as an efficient tool to rapidly change the field of blood cell sorting for blood-based therapeutic and diagnostic applications. PMID:24515899

  3. Microfluidic tissue model for live cell screening.

    PubMed

    Lee, Philip J; Gaige, Terry A; Ghorashian, Navid; Hung, Paul J

    2007-01-01

    We have developed a microfluidic platform modeled after the physiologic microcirculation for multiplexed tissue-like culture and high-throughput analysis. Each microfabricated culture unit consisted of three functional components: a 50 microm wide cell culture pocket, an artificial endothelial barrier with 2 microm pores, and a nutrient transport channel. This configuration enabled a high density of cancer cells to be maintained for over 1 week in a solid tumor-like morphology when fed with continuous flow. The microfluidic chip contained 16 parallel units for "flow cell" based experiments where live cells were exposed to a soluble factor and analyzed via fluorescence microscopy or flow-through biochemistry. Each fluidically independent tissue unit contained approximately 500 cells fed with a continuous flow of 10 nL/min. As a demonstration, the toxicity profile of the anti-cancer drug paclitaxel was collected on HeLa cells cultured in the microfluidic format and compared with a 384-well dish for up to 5 days of continuous drug exposure. PMID:17585775

  4. Fundamentals of microfluidic cell culture in controlled microenvironments†

    PubMed Central

    Young, Edmond W. K.; Beebe, David J.

    2010-01-01

    Microfluidics has the potential to revolutionize the way we approach cell biology research. The dimensions of microfluidic channels are well suited to the physical scale of biological cells, and the many advantages of microfluidics make it an attractive platform for new techniques in biology. One of the key benefits of microfluidics for basic biology is the ability to control parameters of the cell microenvironment at relevant length and time scales. Considerable progress has been made in the design and use of novel microfluidic devices for culturing cells and for subsequent treatment and analysis. With the recent pace of scientific discovery, it is becoming increasingly important to evaluate existing tools and techniques, and to synthesize fundamental concepts that would further improve the efficiency of biological research at the microscale. This tutorial review integrates fundamental principles from cell biology and local microenvironments with cell culture techniques and concepts in microfluidics. Culturing cells in microscale environments requires knowledge of multiple disciplines including physics, biochemistry, and engineering. We discuss basic concepts related to the physical and biochemical microenvironments of the cell, physicochemical properties of that microenvironment, cell culture techniques, and practical knowledge of microfluidic device design and operation. We also discuss the most recent advances in microfluidic cell culture and their implications on the future of the field. The goal is to guide new and interested researchers to the important areas and challenges facing the scientific community as we strive toward full integration of microfluidics with biology. PMID:20179823

  5. Characterizing Cell Mechanics with AFM and Microfluidics

    NASA Astrophysics Data System (ADS)

    Walter, N.; Micoulet, A.; Suresh, S.; Spatz, J. P.

    2007-03-01

    Cell mechanical properties and functionality are mainly determined by the cytoskeleton, besides the cell membrane, the nucleus and the cytosol, and depend on various parameters e.g. surface chemistry and rigidity, surface area and time available for cell spreading, nutrients and drugs provided in the culture medium. Human epithelial pancreatic and mammary cancer cells and their keratin intermediate filaments are the main focus of our work. We use Atomic Force Microscopy (AFM) to study cells adhering to substrates and Microfluidic Channels to probe cells in suspension, respectively. Local and global properties are extracted by varying AFM probe tip size and the available adhesion area for cells. Depth-sensing, instrumented indentation tests with AFM show a clear difference in contact stiffness for cells that are spread of controlled substrates and those that are loosely attached. Microfluidic Channels are utilized in parallel to evaluate cell deformation and ``flow resistance'', which are dependent on channel cross section, flow rate, cell nucleus size and the mechanical properties of cytoskeleton and membrane. The results from the study are used to provide some broad and quantitative assessments of the connections between cellular/subcellular mechanics and biochemical origins of disease states.

  6. Microfluidic-chip platform for cell sorting

    NASA Astrophysics Data System (ADS)

    Malik, Sarul; Balyan, Prerna; Akhtar, J.; Agarwal, Ajay

    2016-04-01

    Cell sorting and separation are considered to be very crucial preparatory steps for numerous clinical diagnostics and therapeutics applications in cell biology research arena. Label free cell separation techniques acceptance rate has been increased to multifold by various research groups. Size based cell separation method focuses on the intrinsic properties of the cell which not only avoids clogging issues associated with mechanical and centrifugation filtration methods but also reduces the overall cost for the process. Consequentially flow based cell separation method for continuous flow has attracted the attention of millions. Due to the realization of structures close to particle size in micro dimensions, the microfluidic devices offer precise and rapid particle manipulation which ultimately leads to an extraordinary cell separation results. The proposed microfluidic device is fabricated to separate polystyrene beads of size 1 µm, 5 µm, 10 µm and 20 µm. The actual dimensions of blood corpuscles were kept in mind while deciding the particle size of polystyrene beads which are used as a model particles for study.

  7. Single cell microfluidics for systems oncology

    NASA Astrophysics Data System (ADS)

    Fan, Rong

    2012-02-01

    The singular term ``cancer'' is never one kind of disease, but deceivingly encompasses a large number of heterogeneous disease states, which makes it impossible to completely treat cancer using a generic approach. Rather systems approaches are urgently required to assess cancer heterogeneity, stratify patients and enable the most effective, individualized treatment. The heterogeneity of tumors at the single cell level is reflected by the hierarchical complexity of the tumor microenvironment. To identify all the cellular components, including both tumor and infiltrating immune cells, and to delineate the associated cell-to-cell signaling network that dictates tumor initiation, progression and metastasis, we developed a single cell microfluidics chip that can analyze a panel of proteins that are potentially associated inter-cellular signaling network in tumor microenvironment from hundreds of single cells in parallel. This platform integrates two advanced technologies -- microfluidic single cell handling and ultra-high density protein array. This device was first tested for highly multiplexed profiling of secreted proteins including tumor-immune signaling molecules from monocytic leukemia cells. We observed profound cellular heterogeneity with all functional phenotypes quantitatively identified. Correlation analysis further indicated the existence of an intercellular cytokine network in which TNFα-induced secondary signaling cascades further increased functional cellular diversity. It was also exploited to evaluate polyfunctionality of tumor antigen-specific T cells from melanoma patients being treated with adoptive T cell transfer immunotherapy. This platform could be further extended to analyze both solid tumor cells (e.g. human lung carcinoma cells) and infiltrating immune cells (e.g. macrophages) so as to enable systems analysis of the complex tumor microenvironment from small amounts of clinical specimens, e.g. skinny needle biopsies. Thus, it could potentially

  8. Digital microfluidic immunocytochemistry in single cells.

    PubMed

    Ng, Alphonsus H C; Dean Chamberlain, M; Situ, Haozhong; Lee, Victor; Wheeler, Aaron R

    2015-01-01

    We report a new technique called Digital microfluidic Immunocytochemistry in Single Cells (DISC). DISC automates protocols for cell culture, stimulation and immunocytochemistry, enabling the interrogation of protein phosphorylation on pulsing with stimulus for as little as 3 s. DISC was used to probe the phosphorylation states of platelet-derived growth factor receptor (PDGFR) and the downstream signalling protein, Akt, to evaluate concentration- and time-dependent effects of stimulation. The high time resolution of the technique allowed for surprising new observations-for example, a 10 s pulse stimulus of a low concentration of PDGF is sufficient to cause >30% of adherent fibroblasts to commit to Akt activation. With the ability to quantitatively probe signalling events with high time resolution at the single-cell level, we propose that DISC may be an important new technique for a wide range of applications, especially for screening signalling responses of a heterogeneous cell population. PMID:26104298

  9. Diffusion phenomena of cells and biomolecules in microfluidic devices

    PubMed Central

    Yildiz-Ozturk, Ece; Yesil-Celiktas, Ozlem

    2015-01-01

    Biomicrofluidics is an emerging field at the cross roads of microfluidics and life sciences which requires intensive research efforts in terms of introducing appropriate designs, production techniques, and analysis. The ultimate goal is to deliver innovative and cost-effective microfluidic devices to biotech, biomedical, and pharmaceutical industries. Therefore, creating an in-depth understanding of the transport phenomena of cells and biomolecules becomes vital and concurrently poses significant challenges. The present article outlines the recent advancements in diffusion phenomena of cells and biomolecules by highlighting transport principles from an engineering perspective, cell responses in microfluidic devices with emphases on diffusion- and flow-based microfluidic gradient platforms, macroscopic and microscopic approaches for investigating the diffusion phenomena of biomolecules, microfluidic platforms for the delivery of these molecules, as well as the state of the art in biological applications of mammalian cell responses and diffusion of biomolecules. PMID:26180576

  10. Recent Advances in Microfluidic Cell Separations

    PubMed Central

    Gao, Yan; Li, Wenjie; Pappas, Dimitri

    2013-01-01

    The isolation and sorting of cells has become an increasingly important step in chemical and biological analyses. As a unit operation in more complex analyses, isolating a phenotypically pure cell population from a heterogeneous sample presents unique challenges. Microfluidic systems are ideal platforms for performing cell separations, enabling integration with other techniques and enhancing traditional separation modalities. In recent years there have been several techniques that use surface antigen affinity, physical interactions, or a combination of the two to achieve high separation purity and efficiency. This review discusses methods including magnetophoretic, acoustophoretic, sedimentation, electric, and hydrodynamic methods for physical separations. We also discuss affinity methods, including magnetic sorting, flow sorting, and affinity capture. PMID:23778244

  11. Microfluidic Magnetic Bead Assay for Cell Detection.

    PubMed

    Liu, Fan; KC, Pawan; Zhang, Ge; Zhe, Jiang

    2016-01-01

    We present a novel cell detection device based on a magnetic bead cell assay and microfluidic Coulter counting technology. The device cannot only accurately measure cells size distribution and concentration but also detect specific target cells. The device consists of two identical micro Coulter counters separated by a fluid chamber where an external magnetic field is applied. Antibody-functionalized magnetic beads were bound to specific antigens expressed on the target cells. A high-gradient magnetic field was applied to the chamber closer to the second counter via an external cylindrical magnet. Because of the magnetic interaction between the magnetic beads and the magnetic field, target cells were retarded by the magnetic field; transit time of a target cell (bound with magnetic beads) passing through the second counter was longer than that through the first counter. In comparison, transit times of a nontarget cell remained nearly the same when it passed through both counters. Thus, from the transit time delay we can identify target cells and quantify their concentration in a cell suspension. The transit time and the size of each cell were accurately measured in terms of the width and amplitude of the resistive pulses generated from the two Coulter counters. Experiments demonstrated that for mixed cells with various target cell ratios, the transit time delay increased approximately linearly with the increasing target cell ratio. The limit of detection (LOD) of the assay was estimated to be 5.6% in terms of target cell ratio. Cell viability tests further demonstrated that most cells were viable after the detection. With the simple device configuration and easy sample preparation, this rapid and reliable method is expected to accurately detect target cells and could be applied to facilitate stem cell isolation and characterization. PMID:26636715

  12. Microfluidic approach of Sickled Cell Anemia

    NASA Astrophysics Data System (ADS)

    Abkarian, Manouk; Loiseau, Etienne; Massiera, Gladys

    2012-11-01

    Sickle Cell Anemia is a disorder of the microcirculation caused by a genetic point mutation that produces an altered hemoglobin protein called HbS. HbS self-assembles reversibly into long rope like fibers inside the red blood cells. The resulting distorded sickled red blood cells are believed to block the smallest capillaries of the tissues producing anemia. Despite the large amount of work that provided a thorough understanding of HbS polymerization in bulk as well as in intact red blood cells at rest, no consequent cellular scale approaches of the study of polymerization and its link to the capillary obstruction have been proposed in microflow, although the problem of obstruction is in essence a circulatory problem. Here, we use microfluidic channels, designed to mimic physiological conditions (flow velocity, oxygen concentration, hematocrit...) of the microcirculation to carry out a biomimetic study at the cellular scale of sickled cell vaso-occlusion. We show that flow geometry, oxygen concentration, white blood cells and free hemoglobin S are essential in the formation of original cell aggregates which could play a role in the vaso-occlusion events.

  13. Cell loss in integrated microfluidic device.

    PubMed

    Zhu, Liang; Peh, Xue Li; Ji, Hong Miao; Teo, Cheng Yong; Feng, Han Hua; Liu, Wen-Tso

    2007-10-01

    Cell loss during sample transporting from macro-components to micro-components in integrated microfluidic devices can considerably deteriorate cell detection sensitivity. This intrinsic cell loss was studied and effectively minimized through (a) increasing the tubing diameter connecting the sample storage and the micro-device, (b) applying a hydrodynamic focusing approach for sample delivering to reduce cells contacting and adhesion on the walls of micro-channel and chip inlet; (c) optimizing the filter design with a zigzag arrangement of pillars (13 microm in chamber depth and 0.8 microm in gap) to prolong the effective filter length, and iv) the use of diamond shaped pillar instead of normally used rectangular shape to reduce the gap length between any two given pillar (i.e. pressure drop) at the filter region. Cell trapping and immunofluorescent detection of 12 Giardia lamblia and 12 Cryptosporidium parvum cells in 150 microl solution and 50 MCF-7 breast cancer cells in 150 microl solution was completed within 15 min with trapping efficiencies improved from 79+/-11%, 50.8+/-5.5% and 41.3+/-3.6% without hydrodynamic focusing, respectively, to 90.8+/-5.8%, 89.8+/-16.6% and 77.0+/-9.2% with hydrodynamic focusing. PMID:17541747

  14. Microfluidic cell chips for high-throughput drug screening.

    PubMed

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

    2016-05-01

    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

  15. Virtual microfluidics for digital quantification and single-cell sequencing.

    PubMed

    Xu, Liyi; Brito, Ilana L; Alm, Eric J; Blainey, Paul C

    2016-09-01

    We have developed hydrogel-based virtual microfluidics as a simple and robust alternative to complex engineered microfluidic systems for the compartmentalization of nucleic acid amplification reactions. We applied in-gel digital multiple displacement amplification (dMDA) to purified DNA templates, cultured bacterial cells and human microbiome samples in the virtual microfluidics system, and demonstrated whole-genome sequencing of single-cell MDA products with excellent coverage uniformity and markedly reduced chimerism compared with products of liquid MDA reactions. PMID:27479330

  16. Microfluidics meets metabolomics to reveal the impact of Campylobacter jejuni infection on biochemical pathways.

    PubMed

    Mortensen, Ninell P; Mercier, Kelly A; McRitchie, Susan; Cavallo, Tammy B; Pathmasiri, Wimal; Stewart, Delisha; Sumner, Susan J

    2016-06-01

    Microfluidic devices that are currently being used in pharmaceutical research also have a significant potential for utilization in investigating exposure to infectious agents. We have established a microfluidic device cultured with Caco-2 cells, and utilized metabolomics to investigate the biochemical responses to the bacterial pathogen Campylobacter jejuni. In the microfluidic devices, Caco-2 cells polarize at day 5, are uniform, have defined brush borders and tight junctions, and form a mucus layer. Metabolomics analysis of cell culture media collected from both Caco-2 cell culture systems demonstrated a more metabolic homogenous biochemical profile in the media collected from microfluidic devices, compared with media collected from transwells. GeneGo pathway mapping indicated that aminoacyl-tRNA biosynthesis was perturbed by fluid flow, suggesting that fluid dynamics and shear stress impacts the cells translational quality control. Both microfluidic device and transwell culturing systems were used to investigate the impact of Campylobacter jejuni infection on biochemical processes. Caco-2 cells cultured in either system were infected at day 5 with C. jejuni 81-176 for 48 h. Metabolomics analysis clearly differentiated C. jejuni 81-176 infected and non-infected medias collected from the microfluidic devices, and demonstrated that C. jejuni 81-176 infection in microfluidic devices impacts branched-chain amino acid metabolism, glycolysis, and gluconeogenesis. In contrast, no distinction was seen in the biochemical profiles of infected versus non-infected media collected from cells cultured in transwells. Microfluidic culturing conditions demonstrated a more metabolically homogenous cell population, and present the opportunity for studying host-pathogen interactions for extended periods of time. PMID:27231016

  17. 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

  18. Digital Microfluidics for Manipulation and Analysis of a Single Cell.

    PubMed

    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

  19. Microfluidic systems for stem cell-based neural tissue engineering.

    PubMed

    Karimi, Mahdi; Bahrami, Sajad; Mirshekari, Hamed; Basri, Seyed Masoud Moosavi; Nik, Amirala Bakhshian; Aref, Amir R; Akbari, Mohsen; Hamblin, Michael R

    2016-07-01

    Neural tissue engineering aims at developing novel approaches for the treatment of diseases of the nervous system, by providing a permissive environment for the growth and differentiation of neural cells. Three-dimensional (3D) cell culture systems provide a closer biomimetic environment, and promote better cell differentiation and improved cell function, than could be achieved by conventional two-dimensional (2D) culture systems. With the recent advances in the discovery and introduction of different types of stem cells for tissue engineering, microfluidic platforms have provided an improved microenvironment for the 3D-culture of stem cells. Microfluidic systems can provide more precise control over the spatiotemporal distribution of chemical and physical cues at the cellular level compared to traditional systems. Various microsystems have been designed and fabricated for the purpose of neural tissue engineering. Enhanced neural migration and differentiation, and monitoring of these processes, as well as understanding the behavior of stem cells and their microenvironment have been obtained through application of different microfluidic-based stem cell culture and tissue engineering techniques. As the technology advances it may be possible to construct a "brain-on-a-chip". In this review, we describe the basics of stem cells and tissue engineering as well as microfluidics-based tissue engineering approaches. We review recent testing of various microfluidic approaches for stem cell-based neural tissue engineering. PMID:27296463

  20. Microfluidic System for Automated Cell-based Assays.

    PubMed

    Lee, Philip J; Ghorashian, Navid; Gaige, Terry A; Hung, Paul J

    2007-12-01

    Microfluidic cell culture is a promising technology for applications in the drug screening industry. Key benefits include improved biological function, higher quality cell-based data, reduced reagent consumption, and lower cost. In this work, we demonstrate how a microfluidic cell culture design was adapted to be compatible with the standard 96-well plate format. Key design features include the elimination of tubing and connectors, the ability to maintain long term continuous perfusion cell culture using a passive gravity driven pump, and direct analysis on the outlet wells of the microfluidic plate. A single microfluidic culture plate contained 8 independent flow units, each with 10(4) cells at a flow rate of 50 μl/day (6 minute residence time). The cytotoxicity of the anti-cancer drug etoposide was measured on HeLa cells cultured in this format, using a commercial lactate dehydrogenase (LDH) plate reader assay. The integration of microfluidic cell culture methods with commercial automation capabilities offers an exciting opportunity for improved cell-based screening. PMID:18172509

  1. Microfluidic integrated acoustic waving for manipulation of cells and molecules.

    PubMed

    Barani, Alireza; Paktinat, Hossein; Janmaleki, Mohsen; Mohammadi, Aminollah; Mosaddegh, Peiman; Fadaei-Tehrani, Alireza; Sanati-Nezhad, Amir

    2016-11-15

    Acoustophoresis with its simple and low-cost fabrication, rapid and localized fluid actuation, compatibility with microfluidic components, and biocompatibility for cellular studies, has been extensively integrated into microfluidics to provide on-chip microdevices for a variety of applications in biology, bioengineering and chemistry. Among different applications, noninvasive manipulation of cells and biomolecules are significantly important, which are addressed by acoustic-based microfluidics. Here in this paper, we briefly explain the principles and different configurations of acoustic wave and acoustic streaming for the manipulation of cells and molecules and overview its applications for single cell isolation, cell focusing and sorting, cell washing and patterning, cell-cell fusion and communication, and tissue engineering. We further discuss the application of acoustic-based microfluidic systems for the mixing and transport of liquids, manipulation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules, followed by explanation on the present challenges of acoustic-based microfluidics for the handling of cells and molecules, and highlighting the future directions. PMID:27262557

  2. Research on microfluidic chip and imaging system used to measure Ca2+ in cell

    NASA Astrophysics Data System (ADS)

    Zhou, Wei; Zhang, Sixiang; Ran, Dugang; Liu, Bao

    2009-07-01

    The microfluidic fluorescence detecting system which is used to measure the concentration of Ca2+ had been designed. On the microfluidic chip we designed, cell-dyeing, cell fostering, reagent injecting and other operations can be completed. The monochromatic light came from optical monochromator which can emit continuous spectrum was used to excitated the fluorescent probe in the cell, then the fluorescence signal and image were sampled by the PMT and CCD, at last the data was processed and the content of Ca2+ in the cell was figured out by using the fluorescence ratio method. Meanwhile, by using the system, the dynamic curve of [Ca2+]1 in cell was given after being stimulated by high K+. The precise result verifies that the system is stable and credible and it meets the requirement of detecting [Ca2+]i in live cells in the filed of physiology.

  3. Silicon based microfluidic cell for terahertz frequencies

    NASA Astrophysics Data System (ADS)

    Baragwanath, A. J.; Swift, G. P.; Dai, D.; Gallant, A. J.; Chamberlain, J. M.

    2010-07-01

    We present a detailed analysis of the design, fabrication and testing of a silicon based, microfluidic cell, for transmission terahertz time-domain spectroscopy. The sensitivity of the device is tested through a range of experiments involving primary alcohol/water mixtures. The dielectric properties of these solutions are subsequently extracted using a Nelder-Mead search algorithm, and are in good agreement with literature values obtained via alternative techniques. Quantities in the order of 2 μmol can be easily distinguished for primary alcohols in solution, even with the subwavelength optical path lengths used. A further display of the device sensitivity is shown through the analysis of commercial whiskeys, where there are clear, detectable differences between samples. Slight absorption variations were identified between samples of the same commercial brand, owing to a 2.5% difference in their alcoholic content. Results from data taken on subsequent days after system realignment are also presented, confirming the robustness of the technique, and the data extraction algorithm used. One final experiment, showing the possible use of this device to analyze aqueous biological samples is detailed; where biotin, a molecule known for its specific terahertz absorptions, is analyzed in solution. The device sensitivity is once again displayed, where quantities of 3 nmol can be clearly detected between samples.

  4. Polar stimulation and constrained cell migration in microfluidic channels†

    PubMed Central

    Agrawal, Nitin; Mitchison, Timothy; Toner, Mehmet

    2010-01-01

    Asymmetrical delivery of stimuli to moving cells for perturbing spatially-heterogeneous intracellular signaling is an experimental challenge not adequately met by existing technologies. Here, we report a robust microfluidic platform allowing localized treatment of the front and/or back of moving cells which crawl through narrow channels that they completely occlude. The enabling technical element for this study is a novel design for precise, passive balancing of flow inside the microfluidic device by contacting two fluid streams before splitting them again. The microchannels constrain cell morphology and induce qualitative and quantitative changes in neutrophil chemotaxis that mimic cells crawling through tissues. PMID:18030401

  5. Can microfluidics address biomanufacturing challenges in drug/gene/cell therapies?

    PubMed

    Chan, Hon Fai; Ma, Siying; Leong, Kam W

    2016-06-01

    Translation of any inventions into products requires manufacturing. Development of drug/gene/cell delivery systems will eventually face manufacturing challenges, which require the establishment of standardized processes to produce biologically-relevant products of high quality without incurring prohibitive cost. Microfluidicu technologies present many advantages to improve the quality of drug/gene/cell delivery systems. They also offer the benefits of automation. What remains unclear is whether they can meet the scale-up requirement. In this perspective, we discuss the advantages of microfluidic-assisted synthesis of nanoscale drug/gene delivery systems, formation of microscale drug/cell-encapsulated particles, generation of genetically engineered cells and fabrication of macroscale drug/cell-loaded micro-/nano-fibers. We also highlight the scale-up challenges one would face in adopting microfluidic technologies for the manufacturing of these therapeutic delivery systems. PMID:27047674

  6. Can microfluidics address biomanufacturing challenges in drug/gene/cell therapies?

    PubMed Central

    Chan, Hon Fai; Ma, Siying; Leong, Kam W.

    2016-01-01

    Translation of any inventions into products requires manufacturing. Development of drug/gene/cell delivery systems will eventually face manufacturing challenges, which require the establishment of standardized processes to produce biologically-relevant products of high quality without incurring prohibitive cost. Microfluidicu technologies present many advantages to improve the quality of drug/gene/cell delivery systems. They also offer the benefits of automation. What remains unclear is whether they can meet the scale-up requirement. In this perspective, we discuss the advantages of microfluidic-assisted synthesis of nanoscale drug/gene delivery systems, formation of microscale drug/cell-encapsulated particles, generation of genetically engineered cells and fabrication of macroscale drug/cell-loaded micro-/nano-fibers. We also highlight the scale-up challenges one would face in adopting microfluidic technologies for the manufacturing of these therapeutic delivery systems. PMID:27047674

  7. Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

    PubMed Central

    Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

    2015-01-01

    This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications. PMID:25938973

  8. Microfluidics-Based Laser Guided Cell-Micropatterning System

    PubMed Central

    Erdman, Nick; Schmidt, Lucas; Qin, Wan; Yang, Xiaoqi; Lin, Yongliang; DeSilva, Mauris N; Gao, Bruce Z.

    2014-01-01

    The ability to place individual cells into an engineered microenvironment in a cell-culture model is critical for the study of in vivo-relevant cell-cell and cell-extracellular matrix interactions. Microfluidics provides a high-throughput modality to inject various cell types into a microenvironment. Laser guided systems provide the high spatial and temporal resolution necessary for single-cell micropatterning. Combining these two techniques, the authors designed, constructed, tested, and evaluated 1) a novel removable microfluidics-based cell-delivery biochip and 2) a combined system that uses the novel biochip coupled with a laser guided cell-micropatterning system to place individual cells into both 2D and 3D arrays. Cell-suspensions of chick forebrain neurons and glial cells were loaded into their respective inlet reservoirs and traversed the microfluidic channels until reaching the outlet ports. Individual cells were trapped and guided from the outlet of a microfluidic channel to a target site on the cell-culture substrate. At the target site, 2D and 3D pattern arrays were constructed with micron-level accuracy. Single-cell manipulation was accomplished at a rate of 150 μm/s in the radial plane and 50 μm/s in the axial direction of the laser beam. Results demonstrated that a single-cell can typically be patterned in 20-30 seconds, and that highly accurate and reproducible cellular arrays and systems can be achieved through coupling the microfluidics-based cell-delivery biochip with the laser guided system. PMID:25190714

  9. Microfluidics-based laser cell-micropatterning system.

    PubMed

    Erdman, Nick; Schmidt, Lucas; Qin, Wan; Yang, Xiaoqi; Lin, Yongliang; DeSilva, Mauris N; Gao, Bruce Z

    2014-09-01

    The ability to place individual cells into an engineered microenvironment in a cell-culture model is critical for the study of in vivo relevant cell-cell and cell-extracellular matrix interactions. Microfluidics provides a high-throughput modality to inject various cell types into a microenvironment. Laser guided systems provide the high spatial and temporal resolution necessary for single-cell micropatterning. Combining these two techniques, the authors designed, constructed, tested and evaluated (1) a novel removable microfluidics-based cell-delivery biochip and (2) a combined system that uses the novel biochip coupled with a laser guided cell-micropatterning system to place individual cells into both two-dimensional (2D) and three-dimensional (3D) arrays. Cell-suspensions of chick forebrain neurons and glial cells were loaded into their respective inlet reservoirs and traversed the microfluidic channels until reaching the outlet ports. Individual cells were trapped and guided from the outlet of a microfluidic channel to a target site on the cell-culture substrate. At the target site, 2D and 3D pattern arrays were constructed with micron-level accuracy. Single-cell manipulation was accomplished at a rate of 150 μm s(-1) in the radial plane and 50 μm s(-1) in the axial direction of the laser beam. Results demonstrated that a single-cell can typically be patterned in 20-30 s, and that highly accurate and reproducible cellular arrays and systems can be achieved through coupling the microfluidics-based cell-delivery biochip with the laser guided system. PMID:25190714

  10. Optical Oxygen Sensors for Applications in Microfluidic Cell Culture

    PubMed Central

    Grist, Samantha M.; Chrostowski, Lukas; Cheung, Karen C.

    2010-01-01

    The presence and concentration of oxygen in biological systems has a large impact on the behavior and viability of many types of cells, including the differentiation of stem cells or the growth of tumor cells. As a result, the integration of oxygen sensors within cell culture environments presents a powerful tool for quantifying the effects of oxygen concentrations on cell behavior, cell viability, and drug effectiveness. Because microfluidic cell culture environments are a promising alternative to traditional cell culture platforms, there is recent interest in integrating oxygen-sensing mechanisms with microfluidics for cell culture applications. Optical, luminescence-based oxygen sensors, in particular, show great promise in their ability to be integrated with microfluidics and cell culture systems. These sensors can be highly sensitive and do not consume oxygen or generate toxic byproducts in their sensing process. This paper presents a review of previously proposed optical oxygen sensor types, materials and formats most applicable to microfluidic cell culture, and analyzes their suitability for this and other in vitro applications. PMID:22163408

  11. Probing Embryonic Stem Cell Autocrine and Paracrine Signaling Using Microfluidics

    NASA Astrophysics Data System (ADS)

    Przybyla, Laralynne; Voldman, Joel

    2012-07-01

    Although stem cell fate is traditionally manipulated by exogenously altering the cells' extracellular signaling environment, the endogenous autocrine and paracrine signals produced by the cells also contribute to their two essential processes: self-renewal and differentiation. Autocrine and/or paracrine signals are fundamental to both embryonic stem cell self-renewal and early embryonic development, but the nature and contributions of these signals are often difficult to fully define using conventional methods. Microfluidic techniques have been used to explore the effects of cell-secreted signals by controlling cell organization or by providing precise control over the spatial and temporal cellular microenvironment. Here we review how such techniques have begun to be adapted for use with embryonic stem cells, and we illustrate how many remaining questions in embryonic stem cell biology could be addressed using microfluidic technologies.

  12. Microfluidic systems and methods of transport and lysis of cells and analysis of cell lysate

    DOEpatents

    Culbertson, Christopher T.; Jacobson, Stephen C.; McClain, Maxine A.; Ramsey, J. Michael

    2004-08-31

    Microfluidic systems and methods are disclosed which are adapted to transport and lyse cellular components of a test sample for analysis. The disclosed microfluidic systems and methods, which employ an electric field to rupture the cell membrane, cause unusually rapid lysis, thereby minimizing continued cellular activity and resulting in greater accuracy of analysis of cell processes.

  13. Microfluidic systems and methods for transport and lysis of cells and analysis of cell lysate

    DOEpatents

    Culbertson, Christopher T [Oak Ridge, TN; Jacobson, Stephen C [Knoxville, TN; McClain, Maxine A [Knoxville, TN; Ramsey, J Michael [Knoxville, TN

    2008-09-02

    Microfluidic systems and methods are disclosed which are adapted to transport and lyse cellular components of a test sample for analysis. The disclosed microfluidic systems and methods, which employ an electric field to rupture the cell membrane, cause unusually rapid lysis, thereby minimizing continued cellular activity and resulting in greater accuracy of analysis of cell processes.

  14. Development of novel microfluidic platforms for neural stem cell research

    NASA Astrophysics Data System (ADS)

    Chung, Bonggeun

    This dissertation describes the development and characterization of novel microfluidic platforms to study proliferation, differentiation, migration, and apoptosis of neural stem cells (NSCs). NSCs hold tremendous promise for fundamental biological studies and cell-based therapies in human disorders. NSCs are defined as cells that can self-renew yet maintain the ability to generate the three principal cell types of the central nervous system such as neurons, astrocytes, and oligodendrocytes. NSCs therefore have therapeutic possibilities in multiple neurodevelopmental and neurodegenerative diseases. Despite their promise, cell-based therapies are limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms can provide much greater control over cell microenvironments and optimize proliferation and differentiation conditions of cells exposed to combinatorial mixtures of growth factors. Human NSCs were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor mixture. NSCs proliferated and differentiated in a graded and proportional fashion that varied directly with growth factor concentration. In parallel to the study of growth and differentiation of NSCs, we are interested in proliferation and apoptosis of mouse NSCs exposed to morphogen gradients. Morphogen gradients are fundamental to animal brain development. Nonetheless, much controversy remains about the mechanisms by which morphogen gradients act on the developing brain. To overcome limitations of in-vitro models of gradients, we have developed a hybrid microfluidic platform that can mimic morphogen gradient profiles. Bone morphogenetic protein (BMP) activity in the developing cortex is graded and cortical NSC responses to BMPs are highly dependent on concentration and gradient slope of BMPs. To make novel microfluidic devices integrated with multiple functions, we have

  15. Digital microfluidics for automated hanging drop cell spheroid culture.

    PubMed

    Aijian, Andrew P; Garrell, Robin L

    2015-06-01

    Cell spheroids are multicellular aggregates, grown in vitro, that mimic the three-dimensional morphology of physiological tissues. Although there are numerous benefits to using spheroids in cell-based assays, the adoption of spheroids in routine biomedical research has been limited, in part, by the tedious workflow associated with spheroid formation and analysis. Here we describe a digital microfluidic platform that has been developed to automate liquid-handling protocols for the formation, maintenance, and analysis of multicellular spheroids in hanging drop culture. We show that droplets of liquid can be added to and extracted from through-holes, or "wells," and fabricated in the bottom plate of a digital microfluidic device, enabling the formation and assaying of hanging drops. Using this digital microfluidic platform, spheroids of mouse mesenchymal stem cells were formed and maintained in situ for 72 h, exhibiting good viability (>90%) and size uniformity (% coefficient of variation <10% intraexperiment, <20% interexperiment). A proof-of-principle drug screen was performed on human colorectal adenocarcinoma spheroids to demonstrate the ability to recapitulate physiologically relevant phenomena such as insulin-induced drug resistance. With automatable and flexible liquid handling, and a wide range of in situ sample preparation and analysis capabilities, the digital microfluidic platform provides a viable tool for automating cell spheroid culture and analysis. PMID:25510471

  16. Microfluidic jet injection for delivering macromolecules into cells

    PubMed Central

    Adamo, A.; Roushdy, O.; Dokov, R.; Sharei, A.; Jensen, K.F.

    2013-01-01

    We present a microfluidic based injection system designed to achieve intracellular delivery of macromolecules by directing a picoliter-jet of a solution towards individual cells. After discussing the concept, we present design specification and criteria, elucidate performance and discuss results. The method has the potential to be quantitative and high throughput, overcoming limitations of current intracellular delivery protocols. PMID:23956498

  17. Mosquitoes meet microfluidics: High-throughput microfluidic tools for insect-parasite ecology in field conditions

    NASA Astrophysics Data System (ADS)

    Prakash, Manu; Mukundarajan, Haripriya

    2013-11-01

    A simple bite from an insect is the transmission mechanism for many deadly diseases worldwide--including malaria, yellow fever, west nile and dengue. Very little is known about how populations of numerous insect species and disease-causing parasites interact in their natural habitats due to a lack of measurement techniques. At present, vector surveillance techniques involve manual capture by using humans as live bait, which is hard to justify on ethical grounds. Individual mosquitoes are manually dissected to isolate salivary glands to detect sporozites. With typical vector infection rates being very low even in endemic areas, it is almost impossible to get an accurate picture of disease distribution, in both space and time. Here we present novel high-throughput microfluidic tools for vector surveillance, specifically mosquitoes. A two-dimensional high density array with baits provide an integrated platform for multiplex PCR for detection of both vector and parasite species. Combining techniques from engineering and field ecology, methods and tools developed here will enable high-throughput measurement of infection rates for a number of diseases in mosquito populations in field conditions. Pew Foundation.

  18. Hydrogel microfluidics for the patterning of pluripotent stem cells

    NASA Astrophysics Data System (ADS)

    Cosson, S.; Lutolf, M. P.

    2014-03-01

    Biomolecular signaling is of utmost importance in governing many biological processes such as the patterning of the developing embryo where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro-scale is often far from ideal due to medium evaporation, limited space for cell growth or shear stress. To overcome these challenges, we here introduce a concept based on hydrogel microfluidics for decoupling conventional, macro-scale cell culture from precise biomolecule delivery through a gel layer. We demonstrate the spatiotemporally controlled neuronal commitment of mouse embryonic stem cells via delivery of retinoic acid gradients. This technique should be useful for testing the effect of dose and timing of biomolecules, singly or in combination, on stem cell fate.

  19. Microfluidic pretreatment of bacterial cells for analysis of intracellular contents

    NASA Astrophysics Data System (ADS)

    Wang, Hsiang-Yu; Lu, Chang; Banada, Padmapriya P.; Jagadeesan, Balamurugan; Bhunia, Arun K.

    2005-11-01

    Electrical lysis of biological cells on a microfluidic platform has been raising a lot of interests due to its applications in rapid recovering intracellular contents without introducing lytic agents. In this study, we demonstrated a simple microfluidic device which lysed green fluorescent protein (GFP) expressing E. coli cells under continuous DC voltage while cells flowed through. The cell lysis only happened in a defined section of a microfluidic channel due to the local field amplification by geometric modification. The geometric modification also effectively decreased the required voltage for lysis by several folds. We found that a local field strength of 1500V/cm was required for lysis of nearly 100% of E. coli cells. This lysis field strength was considerably lower than the value reported in the literature, possibly due to the longer duration of the field. The lysis was witnessed by plate count and fluorescence spectroscopy. The devices were fabricated using low-cost soft lithography with channel widths considerably larger than the cell size to avoid clogging and ensure stable performance. Our tool will be ideal for high throughput processing of a large number of cells. Furthermore, the application of continuous DC field makes it straightforward to couple our cell lysis device with on-chip electrophoresis to realize the integration of cell pretreatment and chemical analysis. In principle, the same approach can also be applied for the lysis of mammalian cells and for the electroporation and transfection.

  20. Microfluidic single-cell analysis of intracellular compounds

    PubMed Central

    Chao, Tzu-Chiao; Ros, Alexandra

    2008-01-01

    Biological analyses traditionally probe cell ensembles in the range of 103–106 cells, thereby completely averaging over relevant individual cell responses, such as differences in cell proliferation, responses to external stimuli or disease onset. In past years, this fact has been realized and increasing interest has evolved for single-cell analytical methods, which could give exciting new insights into genomics, proteomics, transcriptomics and systems biology. Microfluidic or lab-on-a-chip devices are the method of choice for single-cell analytical tools as they allow the integration of a variety of necessary process steps involved in single-cell analysis, such as selection, navigation, positioning or lysis of single cells as well as separation and detection of cellular analytes. Along with this advantageous integration, microfluidic devices confine single cells in compartments near their intrinsic volume, thus minimizing dilution effects and increasing detection sensitivity. This review overviews the developments and achievements of microfluidic single-cell analysis of intracellular compounds in the past few years, from proof-of-principle devices to applications demonstrating a high biological relevance. PMID:18682362

  1. Comparison of Inlet Geometry in Microfluidic Cell Affinity Chromatography

    PubMed Central

    Li, Peng; Tian, Yu; Pappas, Dimitri

    2011-01-01

    Cell separation based on microfluidic affinity chromatography is a widely used methodology in cell analysis research when rapid separations with high purity are needed. Several successful examples have been reported with high separation efficiency and purity; however, cell capture at the inlet area and inlet design has not been extensively described or studied. The most common inlets—used to connect the microfluidic chip to pumps, tubing, etc—are vertical (top-loading) inlets and parallel (in-line) inlets. In this work, we investigated the cell capture behavior near the affinity chip inlet area and compared the different performance of vertical inlet devices and parallel inlet devices. Vertical inlet devices showed significant cell capture capability near the inlet area, which led to the formation of cell blockages as the separation progressed. Cell density near the inlet area was much higher than the remaining channel, while for parallel inlet chips cell density at the inlet area was similar to the rest of the channel. In this paper, we discuss the effects of inlet type on chip fabrication, nonspecific binding, cell capture efficiency, and separation purity. We also discuss the possibility of using vertical inlets in negative selection separations. Our findings show that inlet design is critical and must be considered when fabricating cell affinity microfluidic devices. PMID:21207967

  2. Negative Enrichment of Target Cells by Microfluidic Affinity Chromatography

    PubMed Central

    Li, Peng; Gao, Yan; Pappas, Dimitri

    2011-01-01

    A three-dimensional microfluidic channel was developed for high purity cell separations. This system featured high capture affinity using multiple vertical inlets to an affinity surface. In cell separations, positive selection (capture of the target cell) is usually employed. Negative enrichment, the capture of non-target cells and elution of target cells, has distinct advantages over positive selection. In negative enrichment, target cells are not labeled, and are not subjected to strenuous elution conditions or dilution. As a result, negative enrichment systems are amenable to multi-step processes in microfluidic systems. In previous work, we reported cell capture enhancement effects at vertical inlets to the affinity surface. In this study, we designed a chip that has multiple vertical and horizontal channels, forming a three-dimensional separation system. Enrichment of target cells showed separation purities of 92-96%, compared with straight-channel systems (77% purity). A parallelized chip was also developed for increased sample throughput. A two-channel showed similar separation purity with twice the sample flow rate. This microfluidic system, featuring high separation purity, ease of fabrication and use, is suitable for cell separations when subsequent analysis of target cells is required. PMID:21939198

  3. A microfluidic system for dynamic yeast cell imaging.

    PubMed

    Lee, Philip J; Helman, Noah C; Lim, Wendell A; Hung, Paul J

    2008-01-01

    The investigation of cellular processes and gene regulatory networks within living cells requires the development of improved technology for dynamic, single cell imaging. Here, we demonstrate a microfluidic system capable of mechanical trapping of yeast cells with continuous flow and flow switching capability during time-lapse high magnification fluorescence imaging. The novel functionality of the system was validated by observing the response of pheromone-induced expression of GFP in Saccharomyces cerevisiae. PMID:18254385

  4. Microfluidic single-cell whole-transcriptome sequencing.

    PubMed

    Streets, Aaron M; Zhang, Xiannian; Cao, Chen; Pang, Yuhong; Wu, Xinglong; Xiong, Liang; Yang, Lu; Fu, Yusi; Zhao, Liang; Tang, Fuchou; Huang, Yanyi

    2014-05-13

    Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis. PMID:24782542

  5. Microfluidic approaches for epithelial cell layer culture and characterisation

    PubMed Central

    Thuenauer, Roland; Rodriguez-Boulan, Enrique; Römer, Winfried

    2014-01-01

    In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips, including methods to perform electrical impedance spectroscopy, methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry, techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress, and methods to carry out high-resolution imaging of vesicular trafficking with light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. PMID:24668405

  6. Microfluidic approaches for epithelial cell layer culture and characterisation.

    PubMed

    Thuenauer, Roland; Rodriguez-Boulan, Enrique; Römer, Winfried

    2014-07-01

    In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips; including methods to perform electrical impedance spectroscopy; methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry; techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress; and methods to carry out high-resolution imaging of vesicular trafficking using light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. PMID:24668405

  7. Separating Magnetically Labeled and Unlabeled Biological Cells within Microfluidic Channels

    NASA Astrophysics Data System (ADS)

    Byvank, Tom; Vieira, Greg; Miller, Brandon; Yu, Bo; Chalmers, Jeffrey; Lee, L. James; Sooryakumar, R.

    2011-03-01

    The transport of microscopic objects that rely on magnetic forces have numerous advantages including flexibility of controlling many design parameters and the long range magnetic interactions generally do not adversely affect biological or chemical interactions. We present results on the use of magnetic micro-arrays imprinted within polydimethylsiloxane (PDMS) microfluidic channels that benefit from these features and the ability to rapidly reprogram the magnetic energy landscape for cell manipulation and sorting applications. A central enabling feature is the very large, tunable, magnetic field gradients (> 10 4) that can be designed within the microfluidic architecture. Through use of antibody-conjugated magnetic microspheres to label biological cells, results on the transport and sorting of heterogeneous cell populations are presented. The effects of micro-array and fluid channel design parameters, competition between magnetic forces and hydrodynamic drag forces, and cell-labeling efficiency on cell separation are discussed.

  8. Microfluidic techniques for high throughput single cell analysis.

    PubMed

    Reece, Amy; Xia, Bingzhao; Jiang, Zhongliang; Noren, Benjamin; McBride, Ralph; Oakey, John

    2016-08-01

    The microfabrication of microfluidic control systems and the development of increasingly sensitive molecular amplification tools have enabled the miniaturization of single cells analytical platforms. Only recently has the throughput of these platforms increased to a level at which populations can be screened at the single cell level. Techniques based upon both active and passive manipulation are now capable of discriminating between single cell phenotypes for sorting, diagnostic or prognostic applications in a variety of clinical scenarios. The introduction of multiphase microfluidics enables the segmentation of single cells into biochemically discrete picoliter environments. The combination of these techniques are enabling a class of single cell analytical platforms within great potential for data driven biomedicine, genomics and transcriptomics. PMID:27032065

  9. Temporal Monitoring of Differentiated Human Airway Epithelial Cells Using Microfluidics

    PubMed Central

    Blume, Cornelia; Reale, Riccardo; Held, Marie; Millar, Timothy M.; Collins, Jane E.; Davies, Donna E.; Morgan, Hywel; Swindle, Emily J.

    2015-01-01

    The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL–8 release is detectable within the first 2h and peaks at 4–6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms. PMID:26436734

  10. Temporal Monitoring of Differentiated Human Airway Epithelial Cells Using Microfluidics.

    PubMed

    Blume, Cornelia; Reale, Riccardo; Held, Marie; Millar, Timothy M; Collins, Jane E; Davies, Donna E; Morgan, Hywel; Swindle, Emily J

    2015-01-01

    The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL-8 release is detectable within the first 2h and peaks at 4-6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms. PMID:26436734

  11. A microfluidic galvanic cell on a single layer of paper

    NASA Astrophysics Data System (ADS)

    Purohit, Krutarth H.; Emrani, Saina; Rodriguez, Sandra; Liaw, Shi-Shen; Pham, Linda; Galvan, Vicente; Domalaon, Kryls; Gomez, Frank A.; Haan, John L.

    2016-06-01

    Paper microfluidics is used to produce single layer galvanic and hybrid cells to produce energy that could power paper-based analytical sensors. When two aqueous streams are absorbed onto paper to establish co-laminar flow, the streams stay in contact with each other with limited mixing. The interface at which mixing occurs acts as a charge-transfer region, eliminating the need for a salt bridge. We designed a Cusbnd Zn galvanic cell that powers an LED when two are placed in series. We also used more powerful redox couples (formate and silver, formate and permanganate) to produce higher power density (18 and 3.1 mW mg-1 Pd). These power densities are greater than previously reported paper microfluidic fuel cells using formate or methanol. The single layer design is much more simplified than previous reports of multi-layer galvanic cells on paper.

  12. Isolating single cells in a neurosphere assay using inertial microfluidics

    PubMed Central

    Nathamgari, S. Shiva P.; Dong, Biqin; Zhou, Fan; Kang, Wonmo; Giraldo-Vela, Juan P.; McGuire, Tammy; McNaughton, Rebecca L.; Sun, Cheng; Kessler, John A.; Espinosa, Horacio D.

    2015-01-01

    Sphere forming assays are routinely used for in vitro propagation and differentiation of stem cells. Because the stem cell clusters can become heterogeneous and polyclonal, they must first be dissociated into a single cell suspension for further clonal analysis or differentiation studies. The dissociated population is marred by the presence of doublets, triplets and semi-cleaved/intact clusters which makes identification and further analysis of differentiation pathways difficult. In this work, we use inertial microfluidics to separate the single cells and clusters in a population of chemically dissociated neurospheres. In contrast to previous microfluidic sorting technologies which operated at high flow rates, we implement the spiral microfluidic channel in a novel focusing regime that occurs at lower flow rates. In this regime, the curvature-induced Dean’s force focuses the smaller, single cells towards the inner wall and the larger clusters towards the center. We further demonstrate that sorting in this low flow rate (and hence low shear stress) regime yields a high percentage (> 90%) of viable cells and preserves multipotency by differentiating the sorted neural stem cell population into neurons and astrocytes. The modularity of the device allows easy integration with other lab-on-a-chip devices for upstream mechanical dissociation and downstream high-throughput clonal analysis, localized electroporation and sampling. Although demonstrated in the case of the neurosphere assay, the method is equally applicable to other sphere forming assays. PMID:26511875

  13. Label-free cell separation and sorting in microfluidic systems

    PubMed Central

    Gossett, Daniel R.; Weaver, Westbrook M.; Mach, Albert J.; Hur, Soojung Claire; Tse, Henry Tat Kwong; Lee, Wonhee; Amini, Hamed

    2010-01-01

    Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible. Figure A wide range of microfluidic technologies have been developed to separate and sort cells by taking advantage of differences in their intrinsic biophysical properties PMID:20419490

  14. Perspective on Microfluidic Cell Separation: A Solved Problem?

    PubMed Central

    2015-01-01

    The purification and sorting of cells using microfluidic methodologies has been a remarkably active area of research over the past decade. Much of the scientific and technological work associated with microfluidic cell separation has been driven by needs in clinical diagnostics and therapeutic monitoring, most notably in the context of circulating tumor cells. The last several years have seen advances in a broad range of separation modalities ranging from miniaturized analogs of established techniques such as fluorescence- and magnetic-activated cell sorting (FACS and MACS, respectively), to more specialized approaches based on affinity, dielectrophoretic mobility, and inertial properties of cells. With several of these technologies nearing commercialization, there is a sense that the field of microfluidic cell separation has achieved a high level of maturity over an unusually short span of time. In this Perspective, we set the stage by describing major scientific and technological advances in this field and ask what the future holds. While many scientific questions remain unanswered and new compelling questions will undoubtedly arise, the relative maturity of this field poses some unique challenges. PMID:25350696

  15. Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

    PubMed

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Chen, Chia-Hung

    2016-07-15

    Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. PMID:26995287

  16. Dynamic cell culture: a microfluidic function generator for live cell microscopy.

    PubMed

    Lee, Philip J; Gaige, Terry A; Hung, Paul J

    2009-01-01

    We present a microfluidic system for time-lapsed, live cell microscopy with the ability to control solution exchange via a dynamic flow controller. The application specific microfluidic plates are designed to maintain adherent and non-adherent cell types for multiple days with continuous medium perfusion. Upstream channels with flow controlled via custom software allow the delivery of unique exposure profiles to the cultured cells, such as square waves, step functions, ramps, etc. PMID:19209350

  17. Microfluidic 3D cell culture: from tools to tissue models.

    PubMed

    van Duinen, Vincent; Trietsch, Sebastiaan J; Joore, Jos; Vulto, Paul; Hankemeier, Thomas

    2015-12-01

    The transition from 2D to 3D cell culture techniques is an important step in a trend towards better biomimetic tissue models. Microfluidics allows spatial control over fluids in micrometer-sized channels has become a valuable tool to further increase the physiological relevance of 3D cell culture by enabling spatially controlled co-cultures, perfusion flow and spatial control over of signaling gradients. This paper reviews most important developments in microfluidic 3D culture since 2012. Most efforts were exerted in the field of vasculature, both as a tissue on its own and as part of cancer models. We observe that the focus is shifting from tool building to implementation of specific tissue models. The next big challenge for the field is the full validation of these models and subsequently the implementation of these models in drug development pipelines of the pharmaceutical industry and ultimately in personalized medicine applications. PMID:26094109

  18. Microfluidic cytometric analysis of cancer cell transportability and invasiveness

    PubMed Central

    Liu, Zongbin; Lee, Yeonju; Jang, Joon hee; Li, Ying; Han, Xin; Yokoi, Kenji; Ferrari, Mauro; Zhou, Ledu; Qin, Lidong

    2015-01-01

    The extensive phenotypic and functional heterogeneity of cancer cells plays an important role in tumor progression and therapeutic resistance. Characterizing this heterogeneity and identifying invasive phenotype may provide possibility to improve chemotherapy treatment. By mimicking cancer cell perfusion through circulatory system in metastasis, we develop a unique microfluidic cytometry (MC) platform to separate cancer cells at high throughput, and further derive a physical parameter ‘transportability’ to characterize the ability to pass through micro-constrictions. The transportability is determined by cell stiffness and cell-surface frictional property, and can be used to probe tumor heterogeneity, discriminate more invasive phenotypes and correlate with biomarker expressions in breast cancer cells. Decreased cell stiffness and cell-surface frictional force leads to an increase in transportability and may be a feature of invasive cancer cells by promoting cell perfusion through narrow spaces in circulatory system. The MC-Chip provides a promising microfluidic platform for studying cell mechanics and transportability could be used as a novel marker for probing tumor heterogeneity and determining invasive phenotypes. PMID:26404901

  19. Inertial microfluidics for continuous separation of cells and particles

    NASA Astrophysics Data System (ADS)

    Chatterjee, Arpita; Kuntaegowdanahalli, Sathyakumar S.; Papautsky, Ian

    2011-02-01

    In this work we describe the use of inertial microfluidics for continuous multi-particle separation in a simple spiral microchannel. The inertial forces coupled with the rotational Dean drag force in the spiral microchannel geometry cause neutrally-buoyant particles and cells to occupy a single equilibrium position near the inner microchannel wall. This position is strongly dependent on the particle/cell diameter. Based on this concept, a 5-loop Archimedean spiral microchannel chip was used to demonstrate for the first time focusing and separation of four particles simultaneously. The polystyrene particles (7.32 μm, 10 μm, 15 μm, 20 μm in diameter) were selected for this work since they are compatible to the size of blood cells. The device exhibited an average 87% separation efficiency, which is comparable to that of other microfluidic separation systems. The simple planar structure and high sample throughput offered by this passive microfluidic approach makes it attractive for lab-on-a-chip integration in hematology applications.

  20. A microfluidic direct formate fuel cell on paper.

    PubMed

    Copenhaver, Thomas S; Purohit, Krutarth H; Domalaon, Kryls; Pham, Linda; Burgess, Brianna J; Manorothkul, Natalie; Galvan, Vicente; Sotez, Samantha; Gomez, Frank A; Haan, John L

    2015-08-01

    We describe the first direct formate fuel cell on a paper microfluidic platform. In traditional membrane-less microfluidic fuel cells (MFCs), external pumping consumes power produced by the fuel cell in order to maintain co-laminar flow of the anode stream and oxidant stream to prevent mixing. However, in paper microfluidics, capillary action drives flow while minimizing stream mixing. In this work, we demonstrate a paper MFC that uses formate and hydrogen peroxide as the anode fuel and cathode oxidant, respectively. Using these materials we achieve a maximum power density of nearly 2.5 mW/mg Pd. In a series configuration, our MFC achieves an open circuit voltage just over 1 V, and in a parallel configuration, short circuit of 20 mA absolute current. We also demonstrate that the MFC does not require continuous flow of fuel and oxidant to produce power. We found that we can pre-saturate the materials on the paper, stop the electrolyte flow, and still produce approximately 0.5 V for 15 min. This type of paper MFC has potential applications in point-of-care diagnostic devices and other electrochemical sensors. PMID:25546700

  1. AC Electrokinetic Cell Separation on a Microfluidic Device

    NASA Astrophysics Data System (ADS)

    Gagnon, Zachary; Chang, Hsueh-Chia

    2009-03-01

    Rapid cell separation and collection is demonstrated through the integration of electrokinetic pumps, dielectrophoretic (DEP) traps and field driven valves into a well designed microfluidic channel loop. We present the ground-up design and analysis of this fully functional microfluidic device for the rapid separation and collection of live and dead yeast cells and malaria red blood cells (RBCs) at low concentrations. DEP cell sorting and concentration schemes are based on the exploitation of cell specific DEP crossover frequencies (cof's). A rigorous DEP study of yeast and RBCs is presented and used to determine optimal conditions for cell separation. By utilizing a glutaraldehyde crosslinking cell fixation reaction that is sensitive to cell membrane protein concentration, we demonstrate the ability to further amplify these differences between healthy and unhealthy cells as well as stabilize their DEP cof's. Pumping is achieved with a new type of electrokinetic flow, AC electrothermal electro-osmosis (ETEO) and is shown to scale inversely with the field induced debye length and drive fluid velocities in excess of 6 mm/sec. The well characterized electrokinetic phenomena are integrated into a microchannel loop with a specifically designed electrode field penetration length for low concentration cell separation and concentration.

  2. Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

    PubMed Central

    Millet, Larry J.; Park, Kidong; Watkins, Nicholas N.; Hsia, K. Jimmy; Bashir, Rashid

    2011-01-01

    Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices. PMID:21339720

  3. A microfluidic device for epigenomic profiling using 100 cells.

    PubMed

    Cao, Zhenning; Chen, Changya; He, Bing; Tan, Kai; Lu, Chang

    2015-10-01

    The sensitivity of chromatin immunoprecipitation (ChIP) assays poses a major obstacle for epigenomic studies of low-abundance cells. Here we present a microfluidics-based ChIP-seq protocol using as few as 100 cells via drastically improved collection of high-quality ChIP-enriched DNA. Using this technology, we uncovered many new enhancers and super enhancers in hematopoietic stem and progenitor cells from mouse fetal liver, suggesting that enhancer activity is highly dynamic during early hematopoiesis. PMID:26214128

  4. Multiwell cell culture plate format with integrated microfluidic perfusion system

    NASA Astrophysics Data System (ADS)

    Domansky, Karel; Inman, Walker; Serdy, Jim; Griffith, Linda G.

    2006-01-01

    A new cell culture analog has been developed. It is based on the standard multiwell cell culture plate format but it provides perfused three-dimensional cell culture capability. The new capability is achieved by integrating microfluidic valves and pumps into the plate. The system provides a means to conduct high throughput assays for target validation and predictive toxicology in the drug discovery and development process. It can be also used for evaluation of long-term exposure to drugs or environmental agents or as a model to study viral hepatitis, cancer metastasis, and other diseases and pathological conditions.

  5. Microfluidic system for single cell sorting with optical tweezers

    NASA Astrophysics Data System (ADS)

    Bruns, Thomas; Becsi, Laszlo; Talkenberg, Marc; Wagner, Michael; Weber, Petra; Mescheder, Ulrich; Schneckenburger, Herbert

    2010-11-01

    A microfluidic system was developed and combined with optical tweezers for single cell sorting. This system consists of a glass chip of 300 μm thickness with an etched crosswise channel structure, a silicon layer for sealing and a PMMA substrate for tubular coupling. Selected cells are trapped and moved in perpendicular direction to the main flow for recovery in special reservoirs and further evaluation (e.g. by polymerase chain reaction, PCR). In addition, maximum light doses and exposure times for maintaining cell viability were determined.

  6. A microfluidic device for epigenomic profiling using 100 cells

    PubMed Central

    Cao, Zhenning; Chen, Changya; He, Bing; Tan, Kai; Lu, Chang

    2015-01-01

    The sensitivity of chromatin immunoprecipitation (ChIP) assays poses a major obstacle for epigenomic studies of low-abundance cells. Here we present a microfluidics-based ChIP-Seq protocol using as few as 100 cells via drastically improved collection of high-quality ChIP-enriched DNA. Using this technology, we uncovered many novel enhancers and super enhancers in hematopoietic stem and progenitor cells from mouse fetal liver, suggesting that enhancer activity is highly dynamic during early hematopoiesis. PMID:26214128

  7. Recent advances and future applications of microfluidic live-cell microarrays.

    PubMed

    Rothbauer, Mario; Wartmann, David; Charwat, Verena; Ertl, Peter

    2015-11-01

    Microfluidic live-cell microarrays show much promise as screening tools for biomedical research because they could shed light on key biological processes such as cell signaling and cell-to-cell and cell-to-substrate dynamic responses. While miniaturization reduces the need for expensive clinical grade reagents, the integration of functional components including micropumps, biosensors, actuators, mixers and gradient generators results in improved assay reliability, reproducibility and well-defined cell culture conditions. The present review addresses recent technological advances in microfluidic live-cell microarray technology with a special focus on the applications of microfluidic single-cell, multi-cell and 3D cell microarrays. PMID:26133396

  8. A microfluidic device enabling high-efficiency single cell trapping.

    PubMed

    Jin, D; Deng, B; Li, J X; Cai, W; Tu, L; Chen, J; Wu, Q; Wang, W H

    2015-01-01

    Single cell trapping increasingly serves as a key manipulation technique in single cell analysis for many cutting-edge cell studies. Due to their inherent advantages, microfluidic devices have been widely used to enable single cell immobilization. To further improve the single cell trapping efficiency, this paper reports on a passive hydrodynamic microfluidic device based on the "least flow resistance path" principle with geometry optimized in line with corresponding cell types. Different from serpentine structure, the core trapping structure of the micro-device consists of a series of concatenated T and inverse T junction pairs which function as bypassing channels and trapping constrictions. This new device enhances the single cell trapping efficiency from three aspects: (1) there is no need to deploy very long or complicated channels to adjust flow resistance, thus saving space for each trapping unit; (2) the trapping works in a "deterministic" manner, thus saving a great deal of cell samples; and (3) the compact configuration allows shorter flowing path of cells in multiple channels, thus increasing the speed and throughput of cell trapping. The mathematical model of the design was proposed and optimization of associated key geometric parameters was conducted based on computational fluid dynamics (CFD) simulation. As a proof demonstration, two types of PDMS microfluidic devices were fabricated to trap HeLa and HEK-293T cells with relatively significant differences in cell sizes. Experimental results showed 100% cell trapping and 90% single cell trapping over 4 × 100 trap sites for these two cell types, respectively. The space saving is estimated to be 2-fold and the cell trapping speed enhancement to be 3-fold compared to previously reported devices. This device can be used for trapping various types of cells and expanded to trap cells in the order of tens of thousands on 1-cm(2) scale area, as a promising tool to pattern large-scale single cells on specific

  9. Deformability-based microfluidic cell pairing and fusion.

    PubMed

    Dura, Burak; Liu, Yaoping; Voldman, Joel

    2014-08-01

    We present a microfluidic cell pairing device capable of sequential trapping and pairing of hundreds of cells using passive hydrodynamics and flow-induced deformation. We describe the design and operation principles of our device and show its applicability for cell fusion. Using our device, we achieved both homotypic and heterotypic cell pairing, demonstrating efficiencies up to 80%. The platform is compatible with fusion protocols based on biological, chemical and physical stimuli with fusion yields up to 95%. Our device further permits its disconnection from the fluidic hardware enabling its transportation for imaging and culture while maintaining cell registration on chip. Our design principles and cell trapping technique can readily be applied for different cell types and can be extended to trap and fuse multiple (>2) cell partners as demonstrated by our preliminary experiments. PMID:24898933

  10. Mammosphere culture of cancer stem cells in a microfluidic device

    NASA Astrophysics Data System (ADS)

    Saadin, Katayoon; White, Ian M.

    2012-03-01

    It is known that tumor-initiating cells with stem-like properties will form spherical colonies - termed mammospheres - when cultured in serum-free media on low-attachment substrates. Currently this assay is performed in commercially available 96-well trays with low-attachment surfaces. Here we report a novel microsystem that features on-chip mammosphere culture on low attachment surfaces. We have cultured mammospheres in this microsystem from well-studied human breast cancer cell lines. To enable the long-term culture of these unattached cells, we have integrated diffusion-based delivery columns that provide zero-convection delivery of reagents, such as fresh media, staining agents, or drugs. The multi-layer system consists of parallel cell-culture chambers on top of a low-attachment surface, connected vertically with a microfluidic reagent delivery layer. This design incorporates a reagent reservoir, which is necessary to reduce evaporation from the cell culture micro-chambers. The development of this microsystem will lead to the integration of mammosphere culture with other microfluidic functions, including circulating tumor cell recovery and high throughput drug screening. This will enable the cancer research community to achieve a much greater understanding of these tumor initiating cancer stem cells.

  11. A microfluidic platform for regulating signal transduction in single cells

    NASA Astrophysics Data System (ADS)

    Wong, Pak Kin; Yu, Fuqu; Sun, Ren; Ho, Chih-Ming

    2004-11-01

    Recent progress in micro cell culture systems has lead to new approaches in cell biology studies. Using micro devices for cell culturing possesses distinctive advantages over traditional methods. Length scale matching facilitates manipulation and detection at the single cell level. Previously, we have demonstrated generation of various stimulations such as spatial chemical gradient, electric field, and shear stress to study the dynamic responses of individual cells. Dynamic stimulations and continuous monitoring in a microfluidic system can be useful in studying different aspects of cellular process. In this work, we present a microfluidic platform for regulating nuclear factor kappa B (NF-kB) signal transduction in human embryonic kidney 293T cells. Time-varying bio-chemical stimulants, such as interleukin 1 and tumor necrosis factor, are introduced into the microchannel to activate the NF-kB signaling pathway. The dynamic responses of individual cells are monitored with the expression of reporter gene, green fluorescent protein. Regulation of the NF-kB activity is successfully demonstrated. This work is supported by CMISE through NASA URETI program.

  12. Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis

    PubMed Central

    Lo, Shih-Jie; Yao, Da-Jeng

    2015-01-01

    This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell. PMID:26213918

  13. Acoustophoretic sorting of viable mammalian cells in a microfluidic device.

    PubMed

    Yang, Allen H J; Soh, H Tom

    2012-12-18

    We report the first use of ultrasonic acoustophoresis for the label-free separation of viable and nonviable mammalian cells within a microfluidic device. Cells that have undergone apoptosis are physically smaller than viable cells, and our device exploits this fact to achieve efficient sorting based on the strong size dependence of acoustic radiation forces within a microchannel. As a model, we have selectively enriched viable MCF-7 breast tumor cells from heterogeneous mixtures of viable and nonviable cells. We found that this mode of separation is gentle and enables efficient, label-free isolation of viable cells from mixed samples containing 10(6) cells/mL at flow rates of up to 12 mL/h. We have extensively characterized the device, and we report the effects of piezoelectric voltage and sample flow rate on device performance and describe how these parameters can be tuned to optimize recovery, purity, or throughput. PMID:23157478

  14. Cell-free protein synthesis in microfluidic array devices.

    PubMed

    Mei, Qian; Fredrickson, Carl K; Simon, Andrew; Khnouf, Ruba; Fan, Z Hugh

    2007-01-01

    We report the development of a microfluidic array device for continuous-exchange, cell-free protein synthesis. The advantages of protein expression in the microfluidic array include (1) the potential to achieve high-throughput protein expression, matching the throughput of gene discovery; (2) more than 2 orders of magnitude reduction in reagent consumption, decreasing the cost of protein synthesis; and (3) the possibility to integrate with detection for rapid protein analysis, eliminating the need to harvest proteins. The device consists of an array of units, and each unit can be used for production of an individual protein. The unit comprises a tray chamber for in vitro protein expression and a well chamber as a nutrient reservoir. The tray is nested in the well, and they are separated by a dialysis membrane and connected through a microfluidic connection that provides a means to supply nutrients and remove the reaction byproducts. The device is demonstrated by synthesis of green fluorescent protein, chloramphenicol acetyl-transferase, and luciferase. Protein expression in the device lasts 5-10 times longer and the production yield is 13-22 times higher than in a microcentrifuge tube. In addition, we studied the effects of the operation temperature and hydrostatic flow on the protein production yield. PMID:17924644

  15. Hydrodynamic mechanisms of cell and particle trapping in microfluidics

    PubMed Central

    Karimi, A.; Yazdi, S.; Ardekani, A. M.

    2013-01-01

    Focusing and sorting cells and particles utilizing microfluidic phenomena have been flourishing areas of development in recent years. These processes are largely beneficial in biomedical applications and fundamental studies of cell biology as they provide cost-effective and point-of-care miniaturized diagnostic devices and rare cell enrichment techniques. Due to inherent problems of isolation methods based on the biomarkers and antigens, separation approaches exploiting physical characteristics of cells of interest, such as size, deformability, and electric and magnetic properties, have gained currency in many medical assays. Here, we present an overview of the cell/particle sorting techniques by harnessing intrinsic hydrodynamic effects in microchannels. Our emphasis is on the underlying fluid dynamical mechanisms causing cross stream migration of objects in shear and vortical flows. We also highlight the advantages and drawbacks of each method in terms of throughput, separation efficiency, and cell viability. Finally, we discuss the future research areas for extending the scope of hydrodynamic mechanisms and exploring new physical directions for microfluidic applications. PMID:24404005

  16. Microfluidic Transport in Microdevices for Rare Cell Capture

    PubMed Central

    Smith, James P.; Barbati, Alexander C.; Santana, Steven M.; Gleghorn, Jason P.; Kirby, Brian J.

    2013-01-01

    The isolation and capture of rare cells is a problem uniquely suited to microfluidic devices, in which geometries on the cellular length scale can be engineered and a wide range of chemical functionalizations can be implemented. The performance of such devices is primarily affected by the chemical interaction between the cell and the capture surface and the mechanics of cell– surface collision and adhesion. As rare cell capture technology has been summarized elsewhere [1], this article focuses on the fundamental adhesion and transport mechanisms in rare cell capture microdevices, and explores modern device design strategies in a transport context. The biorheology and engineering parameters of cell adhesion are defined; adhesion models and reaction kinetics briefly reviewed. Transport at the microscale, including diffusion and steric interactions that result in cell motion across streamlines, is discussed. The review concludes by discussing design strategies with a focus on leveraging the underlying transport phenomena to maximize device performance. PMID:23065634

  17. Computerized microfluidic cell culture using elastomeric channels and Braille displays

    PubMed Central

    Gu, Wei; Zhu, Xiaoyue; Futai, Nobuyuki; Cho, Brenda S.; Takayama, Shuichi

    2004-01-01

    Computer-controlled microfluidics would advance many types of cellular assays and microscale tissue engineering studies wherever spatiotemporal changes in fluidics need to be defined. However, this goal has been elusive because of the limited availability of integrated, programmable pumps and valves. This paper demonstrates how a refreshable Braille display, with its grid of 320 vertically moving pins, can power integrated pumps and valves through localized deformations of channel networks within elastic silicone rubber. The resulting computerized fluidic control is able to switch among: (i) rapid and efficient mixing between streams, (ii) multiple laminar flows with minimal mixing between streams, and (iii) segmented plug-flow of immiscible fluids within the same channel architecture. The same control method is used to precisely seed cells, compartmentalize them into distinct subpopulations through channel reconfiguration, and culture each cell subpopulation for up to 3 weeks under perfusion. These reliable microscale cell cultures showed gradients of cellular behavior from C2C12 myoblasts along channel lengths, as well as differences in cell density of undifferentiated myoblasts and differentiation patterns, both programmable through different flow rates of serum-containing media. This technology will allow future microscale tissue or cell studies to be more accessible, especially for high-throughput, complex, and long-term experiments. The microfluidic actuation method described is versatile and computer programmable, yet simple, well packaged, and portable enough for personal use. PMID:15514025

  18. Monitoring of chromosome dynamics of single yeast cells in a microfluidic platform with aperture cell traps.

    PubMed

    Jin, Si Hyung; Jang, Sung-Chan; Lee, Byungjin; Jeong, Heon-Ho; Jeong, Seong-Geun; Lee, Sung Sik; Kim, Keun Pil; Lee, Chang-Soo

    2016-04-12

    Chromosome movement plays important roles in DNA replication, repair, genetic recombination, and epigenetic phenomena during mitosis and meiosis. In particular, chromosome movement in the nuclear space is essential for the reorganization of the nucleus. However, conventional methods for analyzing the chromosome movements in vivo have been limited by technical constraints of cell trapping, cell cultivation, oxygenation, and in situ imaging. Here, we present a simple microfluidic platform with aperture-based cell trapping arrays to monitor the chromosome dynamics in single living cells for a desired period of time. Under the optimized conditions, our microfluidic platform shows a single-cell trapping efficiency of 57%. This microfluidic approach enables in situ imaging of intracellular dynamics in living cells responding to variable input stimuli under the well-controlled microenvironment. As a validation of this microfluidic platform, we investigate the fundamental features of the dynamic cellular response of the individual cells treated with different stimuli and drug. We prove the basis for dynamic chromosome movement in single yeast cells to be the telomere and nuclear envelope ensembles that attach to and move in concert with nuclear actin cables. Therefore, these results illustrate the monitoring of cellular functions and obtaining of dynamic information at a high spatiotemporal resolution through the integration of a simple microfluidic platform. PMID:26980179

  19. A microfluidic approach to parallelized transcriptional profiling of single cells

    PubMed Central

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

    2016-01-01

    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. PMID:27194954

  20. A simple and versatile microfluidic cell density gradient generator for quantum dot cytotoxicity assay.

    PubMed

    Wu, Jing; Chen, Qiushui; Liu, Wu; Lin, Jin-Ming

    2013-05-21

    In this work, a simple and versatile microfluidic cell density gradient generator was successfully developed for cytotoxicity of quantum dots (QDs) assay. The microfluidic cell density gradient generator is composed of eight parallel channels which are respectively surrounded by 1-8 microwells with optimized length and width. The cells fall into microwells by gravity and the cell densities are obviously dependent of microwell number. In a case study, HepG2 and MCF-7 cells were successfully utilized for generating cell density gradients on the microfluidic chip. The microfluidic cell density gradient generator was proved to be easily handled, cell-friendly and could be used to conduct the subsequent cell-based assay. As a proof-of-concept, QD cytotoxicity was evaluated and the results exhibited obvious cell density-dependence. For comparison, QD cytotoxicity was also investigated with a series of cell densities infused by pipette tips. Higher reproducibility was observed on the microfluidic cell density gradient generator and cell density was demonstrated to be a vital factor in cytotoxic study. With higher efficiency, controllability and reproducibility, the microfluidic cell density gradient generator could be integrated into microfluidic analysis systems to promote chip-based biological assay. PMID:23538998

  1. High-Throughput Microfluidic Device for Rare Cell Isolation

    PubMed Central

    Yang, Daniel; Leong, Serena; Lei, Andy; Sohn, Lydia L.

    2016-01-01

    Enumerating and analyzing circulating tumor cells (CTCs)—cells that have been shed from primary solid tumors—can potentially be used to determine patient prognosis and track the progression of disease. There is a great challenge to create an effective platform that can isolate these cells, as they are extremely rare: only 1-10 CTCs are present in a 7.5mL of a cancer patient's peripheral blood. We have developed a novel microfluidic system that can isolate CTC populations label free. Our system consists of a multistage separator that employs inertial migration to sort cells based on size. We demonstrate the feasibility of our device by sorting colloids that are comparable in size to red blood cells (RBCs) and CTCs. PMID:26937065

  2. Intracavity Microfluidic Laser Device for Single Cell Analysis

    NASA Astrophysics Data System (ADS)

    Gourley, Paul

    2015-03-01

    An intracavity microfluidic laser device has been developed to study bioparticles ranging in size from 50 nm to 20 μm (virons to organelles to whole cells). The versatile device can be operated used in several modes including static or flowing fluids, with or without molecular labels, and microscopic imaging and/or spectroscopy. It enables advantageous new ways to perform analyses of bioparticles for applications including cell biology, detection of disease and pathogens, environmental monitoring, pharmaceuticals, agriculture, and food processing. This talk will briefly summarize the physics of the device including its laser optics, fluid dynamics, and intracavity light interaction with cells. The talk will then focus on results of a study of mitochondria in normal and cancer liver cells. The study examines the transformation of intracellular and isolated mitochondria from the normal to disease state. The results highlight the unique utility of the device to rapidly assess biophysical changes arising from altered biomolecular states of cells and organelles.

  3. High-throughput microfluidic device for rare cell isolation

    NASA Astrophysics Data System (ADS)

    Yang, Daniel; Leong, Serena; Lei, Andy; Sohn, Lydia L.

    2015-06-01

    Enumerating and analyzing circulating tumor cells (CTCs)—cells that have been shed from primary solid tumors—can potentially be used to determine patient prognosis and track the progression of disease. There is a great challenge to create an effective platform that can isolate these cells, as they are extremely rare: only 1-10 CTCs are present in a 7.5mL of a cancer patient's peripheral blood. We have developed a novel microfluidic system that can isolate CTC populations label free. Our system consists of a multistage separator that employs inertial migration to sort cells based on size. We demonstrate the feasibility of our device by sorting colloids that are comparable in size to red blood cells (RBCs) and CTCs.

  4. Transport Mechanisms of Circulating Tumor Cells in Microfluidic Devices

    NASA Astrophysics Data System (ADS)

    Rangharajan, Kaushik; Conlisk, A. T.; Prakash, Shaurya

    2014-11-01

    Lab-on-a-chip (LoC) devices are becoming an essential tool for several emerging point-of-care healthcare needs and applications. Among the plethora of challenging problems in the personalized healthcare domain, early detection of cancer continues to be a challenge. For instance, identification of most tumors occurs by the time the tumor comprises approximately 1 billion cells, with poor prognosis for metastatic disease. The key obstacle in identifying and subsequent capture of circulating tumor cells (CTCs) is that the amount of CTCs in the blood stream is ~1 in 109 cells. The fundamental challenge in design and fabrication of microfluidic devices arises due to lack of information on suitable sorting needed for sample preparation before any labeling or capture scheme can be employed. Moreover, the ability to study these low concentration cells relies on knowledge of their physical and chemical properties, of which the physical properties are poorly understood. Also, nearly all existing microfluidic mixers were developed for aqueous electrolyte solutions to enhance mixing in traditional low Re flows. However, no systematic studies have developed design rules for particle mixing. Therefore, we present a numerical model to discuss design rules for microscale mixers and sorters for particle sorting for high efficiency antibody labeling of CTCs along with presenting a pathway for a device to capture CTCs without the need for labeling based on particle electrical properties. NSF Nanoscale Science and Engineering Center (NSEC) for the Affordable Nanoengineering of Polymeric Biomedical Devices EEC-0914790.

  5. Practical fabrication of microfluidic platforms for live-cell microscopy.

    PubMed

    Lorusso, Daniel; Nikolov, Hristo N; Milner, Jaques S; Ochotny, Noelle M; Sims, Stephen M; Dixon, S Jeffrey; Holdsworth, David W

    2016-10-01

    We describe a simple fabrication technique - targeted towards non-specialists - that allows for the production of leak-proof polydimethylsiloxane (PDMS) microfluidic devices that are compatible with live-cell microscopy. Thin PDMS base membranes were spin-coated onto a glass-bottom cell culture dish and then partially cured via microwave irradiation. PDMS chips were generated using a replica molding technique, and then sealed to the PDMS base membrane by microwave irradiation. Once a mold was generated, devices could be rapidly fabricated within hours. Fibronectin pre-treatment of the PDMS improved cell attachment. Coupling the device to programmable pumps allowed application of precise fluid flow rates through the channels. The transparency and minimal thickness of the device enabled compatibility with inverted light microscopy techniques (e.g. phase-contrast, fluorescence imaging, etc.). The key benefits of this technique are the use of standard laboratory equipment during fabrication and ease of implementation, helping to extend applications in live-cell microfluidics for scientists outside the engineering and core microdevice communities. PMID:27523472

  6. Wireless induction heating in a microfluidic device for cell lysis.

    PubMed

    Baek, Seung-ki; Min, Junghong; Park, Jung-Hwan

    2010-04-01

    A wireless induction heating system in a microfluidic device was devised for cell lysis to extract DNA and RNA from Escherichia coli. The thermal responses of nickel, iron and copper heating units were studied by applying an alternating magnetic field as a function of geometry of unit, strength of magnetic field, and kind of metal. Heating units were prepared by cutting metal film using a fiber laser, and the units were integrated into a microchannel system using a soft lithographic process. Variation and distribution of temperature on the surface of the heating units was observed using a thermographic camera and temperature labels. The amount of protein released from E. coli by thermal lysis was determined by protein concentration measurement. Hemoglobin released from red blood cells was observed using colorimetric intensity measurement. Extracted DNA was quantified by real-time polymerase chain reaction, and the profile was compared with that of a positive control of ultrasonically disrupted E. coli. The stability of RNA extracted by induction heating was quantified by the measurement of 23S/16S rRNA ratio and comparison with that by normal RNA extraction kit as a gold standard. A solid-shaped nickel structure was selected as the induction heating element in the microfluidic device because of the relatively small influence of geometries and faster thermal response.The amount of protein extracted from E. coli and hemoglobin released from red blood cells by induction heating of the nickel unit in the microfluidic device was proportional to the strength of the applied magnetic field. The lysis of E. coli by induction heating was as effective as lysis of DNA by the ultrasonication method because the threshold cycle values of the sample were compatible with those of the positive control as measured by ultrasonication. Thermal lysis of E. coli by induction heating represents a reasonable alternative to a commercial RNA extraction method as shown by the comparative

  7. Microfluidic technology enhances the potential of human pluripotent stem cells.

    PubMed

    Gagliano, Onelia; Elvassore, Nicola; Luni, Camilla

    2016-05-01

    Since the discovery of human somatic cell reprogramming, human induced pluripotent stem cells (hiPSC) have been increasingly recognized as the landmark for development of organs-on-chip. hiPSCs show a remarkable plasticity that is related to their ability to promptly respond to the surrounding environment. In vitro, the soluble culture microenvironment, with its critical balance between exogenous and cell-secreted factors, plays a great role in inducing hiPSC response, for both preserving pluripotency and controlling differentiation stages. Exploring the complexity of hiPSC microenvironment requires new experimental tools, as a tight control is limited within conventional culture dishes. Microfluidic technology is particularly attractive in hiPSC research because of its ability to mimic specific environmental cues by accurate control of soluble factors with high spatiotemporal resolution and in a high-throughput fashion. In this review, we highlight recent progress in hiPSC research enabled by microfluidic technology as well as new emerging scenarios. PMID:26772885

  8. High-throughput single-cell PCR using microfluidic emulsions

    NASA Astrophysics Data System (ADS)

    Guo, Mira; Mazutis, Linas; Agresti, Jeremy; Sommer, Morten; Dantas, Gautam; Church, George; Turnbaugh, Peter; Weitz, David

    2012-02-01

    The human gut and other environmental samples contain large populations of diverse bacteria that are poorly characterized and unculturable, yet have many functions relevant to human health. Our goal is to identify exactly which species carry some gene of interest, such as a carbohydrate metabolism gene. Conventional metagenomic assays sequence DNA extracted in bulk from populations of mixed cell types, and are therefore unable to associate a gene of interest with a species-identifying 16S gene, to determine that the two genes originated from the same cell. We solve this problem by microfluidically encapsulating single bacteria cells in drops, using PCR to amplify the two genes inside any drop whose encapsulated cell contains both genes, and sequencing the DNA from those drops that contain both amplification products.

  9. Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization

    PubMed Central

    Fan, Beiyuan; Li, Xiufeng; Chen, Deyong; Peng, Hongshang; Wang, Junbo; Chen, Jian

    2016-01-01

    This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on three key performance parameters (absolute quantification, sensitivity, and throughput). PMID:26891303

  10. Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization.

    PubMed

    Fan, Beiyuan; Li, Xiufeng; Chen, Deyong; Peng, Hongshang; Wang, Junbo; Chen, Jian

    2016-01-01

    This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on three key performance parameters (absolute quantification, sensitivity, and throughput). PMID:26891303

  11. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays.

    PubMed

    Hung, Paul J; Lee, Philip J; Sabounchi, Poorya; Lin, Robert; Lee, Luke P

    2005-01-01

    We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of approximately 2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology. PMID:15580587

  12. Research highlights: microfluidic-enabled single-cell epigenetics.

    PubMed

    Dhar, Manjima; Khojah, Reem; Tay, Andy; Di Carlo, Dino

    2015-11-01

    Individual cells are the fundamental unit of life with diverse functions from metabolism to motility. In multicellular organisms, a single genome can give rise to tremendous variability across tissues at the single-cell level due to epigenetic differences in the genes that are expressed. Signals from the local environment or a history of signals can drive these variations, and tissues have many cell types that play separate roles. This epigenetic heterogeneity is of biological importance in normal functions such as tissue morphogenesis and can contribute to development or resistance of cancer, or other disease states. Therefore, an improved understanding of variations at the single cell level are fundamental to understanding biology and developing new approaches to combating disease. Traditional approaches to characterize epigenetic modifications of chromatin or the transcriptome of cells have often focused on blended responses of many cells in a tissue; however, such bulk measures lose spatial and temporal differences that occur from cell to cell, and cannot uncover novel or rare populations of cells. Here we highlight a flurry of recent activity to identify the mRNA profiles from thousands of single-cells as well as chromatin accessibility and histone marks on single to few hundreds of cells. Microfluidics and microfabrication have played a central role in the range of new techniques, and will likely continue to impact their further development towards routine single-cell epigenetic analysis. PMID:26405849

  13. Suspended microfluidics

    PubMed Central

    Casavant, Benjamin P.; Berthier, Erwin; Theberge, Ashleigh B.; Berthier, Jean; Montanez-Sauri, Sara I.; Bischel, Lauren L.; Brakke, Kenneth; Hedman, Curtis J.; Bushman, Wade; Keller, Nancy P.; Beebe, David J.

    2013-01-01

    Although the field of microfluidics has made significant progress in bringing new tools to address biological questions, the accessibility and adoption of microfluidics within the life sciences are still limited. Open microfluidic systems have the potential to lower the barriers to adoption, but the absence of robust design rules has hindered their use. Here, we present an open microfluidic platform, suspended microfluidics, that uses surface tension to fill and maintain a fluid in microscale structures devoid of a ceiling and floor. We developed a simple and ubiquitous model predicting fluid flow in suspended microfluidic systems and show that it encompasses many known capillary phenomena. Suspended microfluidics was used to create arrays of collagen membranes, mico Dots (μDots), in a horizontal plane separating two fluidic chambers, demonstrating a transwell platform able to discern collective or individual cellular invasion. Further, we demonstrated that μDots can also be used as a simple multiplexed 3D cellular growth platform. Using the μDot array, we probed the combined effects of soluble factors and matrix components, finding that laminin mitigates the growth suppression properties of the matrix metalloproteinase inhibitor GM6001. Based on the same fluidic principles, we created a suspended microfluidic metabolite extraction platform using a multilayer biphasic system that leverages the accessibility of open microchannels to retrieve steroids and other metabolites readily from cell culture. Suspended microfluidics brings the high degree of fluidic control and unique functionality of closed microfluidics into the highly accessible and robust platform of open microfluidics. PMID:23729815

  14. Structural studies of enzyme-based microfluidic biofuel cells

    NASA Astrophysics Data System (ADS)

    Togo, Makoto; Takamura, Akimasa; Asai, Tatsuya; Kaji, Hirokazu; Nishizawa, Matsuhiko

    An enzyme-based glucose/O 2 biofuel cell was constructed within a microfluidic channel to study the influence of electrode configuration and fluidic channel height on cell performance. The cell was composed of a bilirubin oxidase (BOD)-adsorbed O 2 cathode and a glucose anode prepared by co-immobilization of glucose dehydrogenase (GDH), diaphorase (Dp) and VK 3-pendant poly- L-lysine. The consumption of O 2 at the upstream cathode protected the downstream anode from interfering O 2 molecules, and consequently improved the cell performance (maximum cell current) ca. 10% for the present cell. The cell performance was also affected by the channel height. The output current and power of a 0.1 mm-height cell was significantly less than those of a 1 mm-height cell because of the depletion of O 2, as determined by the shape of the E- I curve at the cathode. On the other hand, the volume density of current and power was several times higher for the narrower cell.

  15. Microfluidic-based single cell trapping using a combination of stagnation point flow and physical barrier

    NASA Astrophysics Data System (ADS)

    Yu, Miao; Chen, Zongzheng; Xiang, Cheng; Liu, Bo; Xie, Handi; Qin, Kairong

    2016-03-01

    Single cell trapping in vitro by microfluidic device is an emerging approach for the study of the relationship between single cells and their dynamic biochemical microenvironments. In this paper, a hydrodynamic-based microfluidic device for single cell trapping is designed using a combination of stagnation point flow and physical barrier. The microfluidic device overcomes the weakness of the traditional ones, which have been only based upon either stagnation point flows or physical barriers, and can conveniently load dynamic biochemical signals to the trapped cell. In addition, it can connect with a programmable syringe pump and a microscope to constitute an integrated experimental system. It is experimentally verified that the microfluidic system can trap single cells in vitro even under flow disturbance and conveniently load biochemical signals to the trapped cell. The designed micro-device would provide a simple yet effective experimental platform for further study of the interactions between single cells and their microenvironments.

  16. Microfluidic-based single cell trapping using a combination of stagnation point flow and physical barrier

    NASA Astrophysics Data System (ADS)

    Yu, Miao; Chen, Zongzheng; Xiang, Cheng; Liu, Bo; Xie, Handi; Qin, Kairong

    2016-06-01

    Single cell trapping in vitro by microfluidic device is an emerging approach for the study of the relationship between single cells and their dynamic biochemical microenvironments. In this paper, a hydrodynamic-based microfluidic device for single cell trapping is designed using a combination of stagnation point flow and physical barrier. The microfluidic device overcomes the weakness of the traditional ones, which have been only based upon either stagnation point flows or physical barriers, and can conveniently load dynamic biochemical signals to the trapped cell. In addition, it can connect with a programmable syringe pump and a microscope to constitute an integrated experimental system. It is experimentally verified that the microfluidic system can trap single cells in vitro even under flow disturbance and conveniently load biochemical signals to the trapped cell. The designed micro-device would provide a simple yet effective experimental platform for further study of the interactions between single cells and their microenvironments.

  17. An improved alkaline direct formate paper microfluidic fuel cell.

    PubMed

    Galvan, Vicente; Domalaon, Kryls; Tang, Catherine; Sotez, Samantha; Mendez, Alex; Jalali-Heravi, Mehdi; Purohit, Krutarth; Pham, Linda; Haan, John; Gomez, Frank A

    2016-02-01

    Paper-based microfluidic fuel cells (MFCs) are a potential replacement for traditional FCs and batteries due to their low cost, portability, and simplicity to operate. In MFCs, separate solutions of fuel and oxidant migrate through paper due to capillary action and laminar flow and, upon contact with each other and catalyst, produce electricity. In the present work, we describe an improved microfluidic paper-based direct formate FC (DFFC) employing formate and hydrogen peroxide as the anode fuel and cathode oxidant, respectively. The dimensions of the lateral column, current collectors, and cathode were optimized. A maximum power density of 2.53 mW/cm(2) was achieved with a DFFC of surface area 3.0 cm(2) , steel mesh as current collector, 5% carbon to paint mass ratio for cathode electrode and, 30% hydrogen peroxide. The longevity of the MFC's detailed herein is greater than eight hours with continuous flow of streams. In a series configuration, the MFCs generate sufficient energy to power light-emitting diodes and a handheld calculator. PMID:26572774

  18. Manufacturing and wetting low-cost microfluidic cell separation devices

    PubMed Central

    Pawell, Ryan S.; Inglis, David W.; Barber, Tracie J.; Taylor, Robert A.

    2013-01-01

    Deterministic lateral displacement (DLD) is a microfluidic size-based particle separation or filter technology with applications in cell separation and enrichment. Currently, there are no cost-effective manufacturing methods for this promising microfluidic technology. In this fabrication paper, however, we develop a simple, yet robust protocol for thermoplastic DLD devices using regulatory-approved materials and biocompatible methods. The final standalone device allowed for volumetric flow rates of 660 μl min−1 while reducing the manufacturing time to <1 h. Optical profilometry and image analysis were employed to assess manufacturing accuracy and precision; the average replicated post height was 0.48% less than the average post height on the master mold and the average replicated array pitch was 1.1% less than the original design with replicated posts heights of 62.1 ± 5.1 μm (mean ± 6 standard deviations) and replicated array pitches of 35.6 ± 0.31 μm. PMID:24404077

  19. Geometric effects in microfluidics on heterogeneous cell stress using an Eulerian-Lagrangian approach.

    PubMed

    Warren, K M; Mpagazehe, J N; LeDuc, P R; Higgs, C F

    2016-02-01

    The response of individual cells at the micro-scale in cell mechanics is important in understanding how they are affected by changing environments. To control cell stresses, microfluidics can be implemented since there is tremendous control over the geometry of the devices. Designing microfluidic devices to induce and manipulate stress levels on biological cells can be aided by computational modeling approaches. Such approaches serve as an efficient precursor to fabricating various microfluidic geometries that induce predictable levels of stress on biological cells, based on their mechanical properties. Here, a three-dimensional, multiphase computational fluid dynamics (CFD) modeling approach was implemented for soft biological materials. The computational model incorporates the physics of the particle dynamics, fluid dynamics and solid mechanics, which allows us to study how stresses affect the cells. By using an Eulerian-Lagrangian approach to treat the fluid domain as a continuum in the microfluidics, we are conducting studies of the cells' movement and the stresses applied to the cell. As a result of our studies, we were able to determine that a channel with periodically alternating columns of obstacles was capable of stressing cells at the highest rate, and that microfluidic systems can be engineered to impose heterogenous cell stresses through geometric configuring. We found that when using controlled geometries of the microfluidics channels with staggered obstructions, we could increase the maximum cell stress by nearly 200 times over cells flowing through microfluidic channels with no obstructions. Incorporating computational modeling in the design of microfluidic configurations for controllable cell stressing could help in the design of microfludic devices for stressing cells such as cell homogenizers. PMID:26753780

  20. Pumpless steady-flow microfluidic chip for cell culture.

    PubMed

    Marimuthu, Mohana; Kim, Sanghyo

    2013-06-15

    The current research engineered a pumpless energy-efficient microfluidic perfusion cell culture chip that works by modifying the basic gravity-driven siphon flow using an intravenous (IV) infusion set as a conventional, inexpensive, and sterile tool. The IV set was modified to control the constant hydrostatic head difference, thereby maintaining the steady flow rate medium perfusion. The micro-bioreactor chip demonstrated flexibility in controlling a wide range of flow rates from 0.1 to 10ml/min, among which 1- and 5-ml/min flow rates were examined as suitable shear flows for long-term dermal fibroblast cell culture, paving the way for artificial skin development. PMID:23453976

  1. Biological implications of polydimethylsiloxane-based microfluidic cell culture†

    PubMed Central

    Regehr, Keil J.; Domenech, Maribella; Koepsel, Justin T.; Carver, Kristopher C.; Ellison-Zelski, Stephanie J.; Murphy, William L.; Schuler, Linda A.; Alarid, Elaine T.; Beebe, David J.

    2009-01-01

    Polydimethylsiloxane (PDMS) has become a staple of the microfluidics community by virtue of its simple fabrication process and material attributes, such as gas permeability, optical transparency, and flexibility. As microfluidic systems are put toward biological problems and increasingly utilized as cell culture platforms, the material properties of PDMS must be considered in a biological context. Two properties of PDMS were addressed in this study: the leaching of uncured oligomers from the polymer network into microchannel media, and the absorption of small, hydrophobic molecules (i.e. estrogen) from serum-containing media into the polymer bulk. Uncured PDMS oligomers were detectable via MALDI-MS in microchannel media both before and after Soxhlet extraction of PDMS devices in ethanol. Additionally, PDMS oligomers were identified in the plasma membranes of NMuMG cells cultured in PDMS microchannels for 24 hours. Cells cultured in extracted microchannels also contained a detectable amount of uncured PDMS. It was shown that MCF-7 cells seeded directly on PDMS inserts were responsive to hydrophilic prolactin but not hydrophobic estrogen, reflecting its specificity for absorbing small, hydrophobic molecules; and the presence of PDMS floating in wells significantly reduced cellular response to estrogen in a serum-dependent manner. Quantification of estrogen via ELISA revealed that microchannel estrogen partitioned rapidly into the surrounding PDMS to a ratio of approximately 9:1. Pretreatments such as blocking with serum or pre-absorbing estrogen for 24 hours did not affect estrogen loss from PDMS-based microchannels. These findings highlight the importance of careful consideration of culture system properties when determining an appropriate environment for biological experiments. PMID:19606288

  2. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

    PubMed Central

    Yehezkel, Tuval Ben; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-01-01

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

  3. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.

    PubMed

    Ben Yehezkel, Tuval; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-02-29

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

  4. Direct integration of MEMS, dielectric pumping and cell manipulation with reversibly bonded gecko adhesive microfluidics

    NASA Astrophysics Data System (ADS)

    Warnat, S.; King, H.; Wasay, A.; Sameoto, D.; Hubbard, T.

    2016-09-01

    We present an approach to form a microfluidic environment on top of MEMS dies using reversibly bonded microfluidics. The reversible polymeric microfluidics moulds bond to the MEMS die using a gecko-inspired gasket architecture. In this study the formed microchannels are demonstrated in conjunction with a MEMS mechanical single cell testing environment for BioMEMS applications. A reversible microfluidics placement technique with an x-y and rotational accuracy of  ±2 µm and 1° respectively on a MEMS die was developed. No leaks were observed during pneumatic pumping of common cell media (PBS, sorbitol, water, seawater) through the fluidic channels. Thermal chevron actuators were successful operated inside this fluidic environment and a performance deviation of ~15% was measured compared to an open MEMS configuration. Latex micro-spheres were pumped using traveling wave di-electrophoresis and compared to an open (no-microfluidics) configuration with velocities of 24 µm s‑1 and 20 µm s‑1.

  5. A Rapidly Fabricated Microfluidic Chip for Cell Culture.

    PubMed

    Li, Rui; Lv, Xuefei; Hasan, Murtaza; Xu, Jiandong; Xu, Yuanqing; Zhang, Xingjian; Qin, Kuiwei; Wang, Jianshe; Zhou, Di; Deng, Yulin

    2016-04-01

    Microfluidic chips (μFC) are emerging as powerful tools in chemistry, biochemistry, nanotechnology and biotechnology. The microscale size, possibility of integration and high-throughput present huge technical potential to facilitate the research of cell behavior by creating in vivo-like microenvironments. Here, we have developed a new method for rapid fabrication of μFC with Norland Optical Adhesive 81 (NOA81) for multiple cell culture with high efficiency. The proposed method is more suitable for the early structure exploration stage of μFC than existing procedures since no templates are needed and fast fabrication methods are presented. Simple PDMS-NOA81-linked microvalves were embedded in the μFC to control or block the fluid flow effectively, which significantly broadened the applications of μFC. Various types of cells were integrated into the chip and normal viabilities were maintained up to 1 week. Besides, concentration gradient was generated to investigate the cells in the μFC responded to drug stimulation. The cells appeared different in terms of shape and proliferation that strongly demonstrated the potential application of our μFC in online drug delivery. The high biocompatibility of NOA81 and its facile fabrication (μFC) promise its use in various cell analyses, such as cell-cell interactions or tissue engineering. PMID:26657733

  6. Mechanical response of tumor cells flowing through a microfluidic capillary

    NASA Astrophysics Data System (ADS)

    Khan, Zeina S.; Kamyabi, Nabiollah; Hussain, Fazle; Vanapalli, Siva A.

    2014-03-01

    Circulating tumor cells, the primary cause of cancer metastasis, are transported throughout the body to distant organs by blood flow. Despite the importance of cell transport and deformability in the vasculature for cancer metastasis, quantitative understanding of the hydrodynamic interactions between the cells and the blood vessel walls is lacking. Using a model microfluidic capillary of rectangular cross-section with an on-chip manometer coupled with high speed video imaging, we quantitatively investigate the hydrodynamic behavior via the cell excess pressure drop. By characterizing our device with simple model systems including viscous drops and soft elastic particles, we find that the excess pressure drop shows no apparent dependence on elastic modulus or interfacial tension, but depends significantly on internal viscosity for moderate confinements and shear stresses within the physiological range of 1-10 Pa. This suggests that the metastatic potential of circulating cells can be characterized by the effective viscosity. We test this hypothesis with several tumor cell lines and find that the effective cell viscosity determined from excess pressure drop measurements can be used to differentiate highly from lowly invasive cells.

  7. Portable microfluidic cytometer for whole blood cell analysis

    NASA Astrophysics Data System (ADS)

    Grafton, Meggie M.; Zordan, Michael D.; Chuang, Han-Sheng; Rajdev, Pooja; Reece, Lisa M.; Irazoqui, Pedro P.; Wereley, Steven T.; Byrnes, Ron; Todd, Paul; Leary, James F.

    2010-02-01

    Lab-on-a-chip (LOC) systems allow complex laboratory assays to be carried out on a single chip using less time, reagents, and manpower than traditional methods. There are many chips addressing PCR and other DNA assays, but few that address blood cell analysis. Blood analysis, particularly of the cellular component, is highly important in both medical and scientific fields. Traditionally blood samples require a vial of blood, then several processing steps to separate and stain the various components, followed by the preparations for each specific assay to be performed. A LOC system for blood cell analysis and sorting would be ideal. The microfluidic-based system we have developed requires a mere drop of blood to be introduced onto the chip. Once on chip, the blood is mixed with both fluorescent and magnetic labels. The lab-on-a-chip device then uses a syringe drive to push the cells through the chip, while a permanent magnet is positioned to pull the magnetically labeled white blood cells to a separate channel. The white blood cells, labeled with different color fluorescent quantum dots (Qdots) conjugated to antibodies against WBC subpopulations, are analyzed and counted, while a sampling of red blood cells is also counted in a separate channel. This device will be capable of processing whole blood samples on location in a matter of minutes and displaying the cell count and should eventually find use in neonatology, AIDS and remote site applications.

  8. 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.

  9. Microfluidics Technologies for Low Cell Number Chromatin Immunoprecipitation.

    PubMed

    Wu, Angela R; Quake, Stephen R

    2016-01-01

    Protein-DNA interactions are responsible for numerous critical cellular events: For example, gene expression and silencing are mediated by transcription factor protein binding and histone protein modifications, and DNA replication and repair rely on site-specific protein binding. Chromatin immunoprecipitation (ChIP) is the only molecular assay that directly determines, in a living cell, the binding association between a protein of interest and specific genomic loci. It is an indispensible tool in the biologist's toolbox, but the many limitations of this technique prevent broad adoption of ChIP in biological studies. The typical ChIP assay can take up to 1 wk to complete, and the process is technically tricky, yet tedious. The ChIP assay yields are also low, thus requiring on the order of millions to billions of cells as starting material, which makes the assay unfeasible for studies using rare or precious samples. For example, fluorescence-activated cell sorting (FACS) of cancer stem cells (CSCs) obtained from primary tumors, rarely yields more than ~100,000 CSCs per tumor. This protocol describes a microfluidics-based strategy for performing ChIP, which uses automation and scalability to reduce both total and hands-on assay time, and improve throughput. It allows whole fixed cells as input, and enables automated ChIP from as few as 2000 cells. PMID:26700100

  10. Microfluidic shear devices for quantitative analysis of cell adhesion.

    PubMed

    Lu, Hang; Koo, Lily Y; Wang, Wechung M; Lauffenburger, Douglas A; Griffith, Linda G; Jensen, Klavs F

    2004-09-15

    We describe the design, construction, and characterization of microfluidic devices for studying cell adhesion and cell mechanics. The method offers multiple advantages over previous approaches, including a wide range of distractive forces, high-throughput performance, simplicity in experimental setup and control, and potential for integration with other microanalytic modules. By manipulating the geometry and surface chemistry of the microdevices, we are able to vary the shear force and the biochemistry during an experiment. The dynamics of cell detachment under different conditions can be captured simultaneously using time-lapse videomicroscopy. We demonstrate assessment of cell adhesion to fibronectin-coated substrates as a function of the shear stress or fibronectin concentration in microchannels. Furthermore, a combined perfusion-shear device is designed to maintain cell viability for long-term culture as well as to introduce exogenous reagents for biochemical studies of cell adhesion regulation. In agreement with established literature, we show that fibroblasts cultured in the combined device reduced their adhesion strength to the substrate in response to epidermal growth factor stimulation. PMID:15362881

  11. Sequential flow membraneless microfluidic fuel cell with porous electrodes

    NASA Astrophysics Data System (ADS)

    Salloum, Kamil S.; Hayes, Joel R.; Friesen, Cody A.; Posner, Jonathan D.

    A novel convective flow membraneless microfluidic fuel cell with porous disk electrodes is described. In this fuel cell design, the fuel flows radially outward through a thin disk shaped anode and across a gap to a ring shaped cathode. An oxidant is introduced into the gap between anode and cathode and advects radially outward to the cathode. This fuel cell differs from previous membraneless designs in that the fuel and the oxidant flow in series, rather than in parallel, enabling independent control over the fuel and oxidant flow rate and the electrode areas. The cell uses formic acid as a fuel and potassium permanganate as the oxidant, both contained in a sulfuric acid electrolyte. The flow velocity field is examined using microscale particle image velocimetry and shown to be nearly axisymmetric and steady. The results show that increasing the electrolyte concentration reduces the cell Ohmic resistance, resulting in larger maximum currents and peak power densities. Increasing the flow rate delays the onset of mass transport and reduces Ohmic losses resulting in larger maximum currents and peak power densities. An average open circuit potential of 1.2 V is obtained with maximum current and power densities of 5.35 mA cm -2 and 2.8 mW cm -2, respectively (cell electrode area of 4.3 cm 2). At a flow rate of 100 μL min -1 a fuel utilization of 58% is obtained.

  12. Microfluidic microbial fuel cells: from membrane to membrane free

    NASA Astrophysics Data System (ADS)

    Yang, Yang; Ye, Dingding; Li, Jun; Zhu, Xun; Liao, Qiang; Zhang, Biao

    2016-08-01

    Microfluidic microbial fuel cells (MMFCs) are small carbon-neutral devices that use self-organized bacteria to degrade organic substrates and harness energy from the waste water. Conventional MMFCs have made great strides in the past decade and have overcome some limitations, such as high capital costs and low energy output. A co-laminar flow MFC has been first proposed in 2011 with the potential to be an attractively power source to niche applications. Co-laminar MFCs typically operate without any physical membranes separating the reactants, and bacterial ecosystems can be easily manipulated by regulating the inlet conditions. This paper highlights recent accomplishments in the development of co-laminar MFCs, emphasizing basic principles, mass transport and fluid dynamics including boundary layer theory, entrance conditions and mixing zone issues. Furthermore, the development of current techniques, major challenges and the potential research directions are discussed.

  13. A microfluidic chip with hydrodynamic traps for in vitro microscopic investigations of single cells

    NASA Astrophysics Data System (ADS)

    Kukhtevich, I. V.; Belousov, K. I.; Bukatin, A. S.; Dubina, M. V.; Evstrapov, A. A.

    2015-03-01

    The results on making a microfluidic chip for in vitro microscopic investigations of single cells are presented. Numerical simulation of the motion trajectories of microparticles makes it possible to determine the geometry of hydrodynamic traps, their number, and the trap arrangement in a reaction chamber. According to the developed design, microfluidic chips were fabricated from a SU-8 photoresist by photolithography. The microfluidic chips have been tested to prove their operating capacity for isolating and holding K562 human myeloid leukemia cells from a sample flow and their subsequent investigation by confocal laser scanning microscopy.

  14. 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. PMID:27108255

  15. Microfluidic Device for Studying Tumor Cell Extravasation in Cancer Metastasis

    SciTech Connect

    Lin, Henry K; Thundat, Thomas George; Evans III, Boyd Mccutchen; Datar, Ram H; Reese, Benjamin E; Zheng, Siyang

    2010-01-01

    Metastasis is the process by which cancer spreads to form secondary tumors at downstream locations throughout the body. This uncontrolled spreading is the leading cause of death in patients with epithelial cancers and is the main reason that suppressing and targeting cancer has proven to be so challenging. Tumor cell extravasation is one of the key steps in cancer s progression towards a metastatic state. This occurs when circulating tumor cells found within the blood stream are able to transmigrate through the endothelium lining and basement membrane of the vasculature to form metastatic tumors at secondary sites within the body. Predicting the likelihood of this occurrence in patients, or being able to determine specific markers involved in this process could lead to preventative measures targeting these types of cancer; moreover, this may lead to the discovery of novel anti-metastatic drugs. We have developed a microfluidic device that has shown the extravasation of fluorescently labeled tumor cells across an endothelial cell lined membrane coated with matrigel followed by the formation of colonies. This device provides the advantages of combining a controlled environment, mimicking that found within the body, with real-time monitoring capabilities allowing for the study of these biomarkers and cellular interactions along with other potential mechanisms involved in the process of extravasation.

  16. Magnetic microfluidic system for isolation of single cells

    NASA Astrophysics Data System (ADS)

    Mitterboeck, Richard; Kokkinis, Georgios; Berris, Theocharis; Keplinger, Franz; Giouroudi, Ioanna

    2015-06-01

    This paper presents the design and realization of a compact, portable and cost effective microfluidic system for isolation and detection of rare circulating tumor cells (CTCs) in suspension. The innovative aspect of the proposed isolation method is that it utilizes superparamagnetic particles (SMPs) to label CTCs and then isolate those using microtraps with integrated current carrying microconductors. The magnetically labeled and trapped CTCs can then be detected by integrated magnetic microsensors e.g. giant magnetoresistive (GMR) or giant magnetoimpedance (GMI) sensors. The channel and trap dimensions are optimized to protect the cells from shear stress and achieve high trapping efficiency. These intact single CTCs can then be used for additional analysis, testing and patient specific drug screening. Being able to analyze the CTCs metastasis-driving capabilities on the single cell level is considered of great importance for developing patient specific therapies. Experiments showed that it is possible to capture single labeled cells in multiple microtraps and hold them there without permanent electric current and magnetic field.

  17. Microeddies as microfluidic elements: Reactors and cell traps

    NASA Astrophysics Data System (ADS)

    Lutz, Barry R.

    2003-07-01

    Microfluidic applications generally seek to control fluids, reagents, and objects at the microscale, and the development of individual components to either mimic traditional processes or to realize novel processes remains important to development in the field. This work focuses on microscopic acoustic streaming eddies as hydrodynamic microreactors and traps for microscopic objects including motile cells. Four microeddies were created around a stationary cylinder (radius 406 mum) by oscillating the surrounding fluid (audible frequency). Concentration images measured using Raman spectroscopy show that eddies act as hydrodynamic "vessels" for reagents dosed from the cylinder (an electrode), and the oscillation amplitude and reagent dosing rate quantitatively controlled the eddy composition. These "vessels" were used to quantify the antioxidant properties of vitamin C against an electrogenerated oxidant. Material balances over the eddy yield a reactor model identical to a two-input CSTR (i.e., perfect backmixing model); and the mean reactor residence time, Damkohler number, and reagent feed ratio are quantitatively related to eddy properties. The CSTR model fit to data for a range of reactor conversions gives the homogeneous rate constant for vitamin C oxidation, showing that the composition of microeddy reactors can be controlled quantitatively. The cylinder and oscillating fluid were incorporated into microscale channels to provide a route to integration with more conventional microfluidic applications. Detailed flow measurements describe the three-dimensional acoustic streaming flow structure, and theory relates measured flow features to frequency and geometry through simple scaling. These channel-based microeddies show an impressive ability to trap microscopic objects at fixed positions in three-dimensions. Microeddies formed in a microchannel (425 mum depth) collect and trap motile phytoplankton (P. micans) and microspheres (˜20--0 mum diameter). The trap

  18. Cell Separation by Non-Inertial Force Fields in Microfluidic Systems

    PubMed Central

    Tsutsui, Hideaki; Ho, Chih-Ming

    2009-01-01

    Cell and microparticle separation in microfluidic systems has recently gained significant attention in sample preparations for biological and chemical studies. Microfluidic separation is typically achieved by applying differential forces on the target particles to guide them into different paths. This paper reviews basic concepts and novel designs of such microfluidic separators with emphasis on the use of non-inertial force fields, including dielectrophoretic force, optical gradient force, magnetic force, and acoustic primary radiation force. Comparisons of separation performances with discussions on physiological effects and instrumentation issues toward point-of-care devices are provided as references for choosing appropriate separation methods for various applications. PMID:20046897

  19. Biofilm responses to smooth flow fields and chemical gradients in novel microfluidic flow cells.

    PubMed

    Song, Jisun L; Au, Kelly H; Huynh, Kimberly T; Packman, Aaron I

    2014-03-01

    We present two novel microfluidic flow cells developed to provide reliable control of flow distributions and chemical gradients in biofilm studies. We developed a single-inlet microfluidic flow cell to support biofilm growth under a uniform velocity field, and a double-inlet flow cell to provide a very smooth transverse concentration gradient. Both flow cells consist of a layer of polydimethylsiloxane (PDMS) bonded to glass cover slips and were fabricated using the replica molding technique. We demonstrate the capabilities of the flow cells by quantifying flow patterns before and after growth of Pseudomonas aeruginosa biofilms through particle imaging velocimetry, and by evaluating concentration gradients within the double-inlet microfluidic flow cell. Biofilm growth substantially increased flow complexity by diverting flow around biomass, creating high- and low-velocity regions and surface friction. Under a glucose gradient in the double-inlet flow cell, P. aeruginosa biofilms grew in proportion to the local glucose concentration, producing distinct spatial patterns in biofilm biomass relative to the imposed glucose gradient. When biofilms were subjected to a ciprofloxacin gradient, spatial patterns of fractions of dead cells were also in proportion to the local antibiotic concentration. These results demonstrate that the microfluidic flow cells are suitable for quantifying flow complexities resulting from flow-biofilm interactions and investigating spatial patterns of biofilm growth under chemical gradients. These novel microfluidic flow cells will facilitate biofilm research that requires flow control and in situ imaging, particularly investigations of biofilm-environment interactions. PMID:24038055

  20. Biofilm responses to smooth flow fields and chemical gradients in novel microfluidic flow cells

    PubMed Central

    Song, Jisun L.; Au, Kelly H.; Huynh, Kimberly T.

    2013-01-01

    We present two novel microfluidic flow cells developed to provide reliable control of flow distributions and chemical gradients in biofilm studies. We developed a single-inlet microfluidic flow cell to support biofilm growth under a uniform velocity field, and a double-inlet flow cell to provide a very smooth transverse concentration gradient. Both flow cells consist of a layer of polydimethylsiloxane (PDMS) bonded to glass cover slips and were fabricated using the replica molding technique. We demonstrate the capabilities of the flow cells by quantifying flow patterns before and after growth of Pseudomonas aeruginosa biofilms through particle imaging velocimetry, and by evaluating concentration gradients within the double-inlet microfluidic flow cell. Biofilm growth substantially increased flow complexity by diverting flow around biomass, creating high- and low-velocity regions and surface friction. Under a glucose gradient in the double-inlet flow cell, P. aeruginosa biofilms grew in proportion to the local glucose concentration, producing distinct spatial patterns in biofilm biomass relative to the imposed glucose gradient. When biofilms were subjected to a ciprofloxacin gradient, spatial patterns of fractions of dead cells were also in proportion to the local antibiotic concentration. These results demonstrate that the microfluidic flow cells are suitable for quantifying flow complexities resulting from flow-biofilm interactions and investigating spatial patterns of biofilm growth under chemical gradients. These novel microfluidic flow cells will facilitate biofilm research that requires flow control and in situ imaging, particularly investigations of biofilm-environment interactions. PMID:24038055

  1. Light scattering characterization of single biological cells in a microfluidic cytometer

    NASA Astrophysics Data System (ADS)

    Su, Xuantao; Kirkwood, Sean E.; Gul, Hilal; Singh, Kirat; Islam, Md. Z.; Janowska-Wieczorek, Anna; Rozmus, Wojciech; Tsui, Ying Y.

    2009-06-01

    The characterization of single biological cells in a microfluidic flow by using a 2D light scattering microfluidic cytometric technique is described. Laser light is coupled into a microfluidic cytometer via an optical fiber to illuminate a single scatterer in a fluidic flow. The 2D light scattering patterns are obtained by using a charge-coupled device (CCD) detector. The system is tested by using standard polystyrene beads of 4 μm and 9.6 μm in diameter, and the bead experimental results agree well with 1D Mie theory simulation results. Experiments on yeast cells are performed using the microfluidic cytometer. Cell results are studied by finite-difference time-domain (FDTD) method, which can simulate light scattering from non-homogeneous cells. For example, a complex biological cell model with inner mitochondrial distribution is studied by FDTD in this paper. Considering the yeast cell size variations, the yeast cell 2D scatter patterns agree well with the FDTD 2D simulation patterns. The system is capable of obtaining 2D side scatter patterns from a single biological cell which may contain rich information on the biological cell inner structures. The integration of light scattering, microfluidics and fiber optics described here may ultimately allow the development of a lab-on-chip cytometer for label-free detection of diseases at a single cell level.

  2. Particle dynamics and particle-cell interaction in microfluidic systems

    NASA Astrophysics Data System (ADS)

    Stamm, Matthew T.

    Particle-laden flow in a microchannel resulting in aggregation of microparticles was investigated to determine the dependence of the cluster growth rate on the following parameters: suspension void fraction, shear strain rate, and channel-height to particle-diameter ratio. The growth rate of an average cluster was found to increase linearly with suspension void fraction, and to obey a power-law relationships with shear strain rate as S 0.9 and channel-height to particle-diameter ratio as (h/d )--3.5. Ceramic liposomal nanoparticles and silica microparticles were functionalized with antibodies that act as targeting ligands. The bio-functionality and physical integrity of the cerasomes were characterized. Surface functionalization allows cerasomes to deliver drugs with selectivity and specificity that is not possible using standard liposomes. The functionalized particle-target cell binding process was characterized using BT-20 breast cancer cells. Two microfluidic systems were used; one with both species in suspension, the other with cells immobilized inside a microchannel and particle suspension as the mobile phase. Effects of incubation time, particle concentration, and shear strain rate on particle-cell binding were investigated. With both species in suspension, the particle-cell binding process was found to be reasonably well-described by a first-order model. Particle desorption and cellular loss of binding affinity in time were found to be negligible; cell-particle-cell interaction was identified as the limiting mechanism in particle-cell binding. Findings suggest that separation of a bound particle from a cell may be detrimental to cellular binding affinity. Cell-particle-cell interactions were prevented by immobilizing cells inside a microchannel. The initial stage of particle-cell binding was investigated and was again found to be reasonably well-described by a first-order model. For both systems, the time constant was found to be inversely proportional to

  3. Direct formic acid microfluidic fuel cell design and performance evolution

    NASA Astrophysics Data System (ADS)

    Moreno-Zuria, A.; Dector, A.; Cuevas-Muñiz, F. M.; Esquivel, J. P.; Sabaté, N.; Ledesma-García, J.; Arriaga, L. G.; Chávez-Ramírez, A. U.

    2014-12-01

    This work reports the evolution of design, fabrication and testing of direct formic acid microfluidic fuel cells (DFAμFFC), the architecture and channel dimensions are miniaturized from a thousand to few cents of micrometers. Three generations of DFAμFFCs are presented, from the initial Y-shape configuration made by a hot pressing technique; evolving into a novel miniaturized fuel cell based on microfabrication technology using SU-8 photoresist as core material; to the last air-breathing μFFC with enhanced performance and built with low cost materials and processes. The three devices were evaluated in acidic media in the presence of formic acid as fuel and oxygen/air as oxidant. Commercial Pt/C (30 wt. % E-TEK) and Pd/C XC-72 (20 wt. %, E-TEK) were used as cathode and anode electrodes respectively. The air-breathing μFFC generation, delivered up to 27.3 mW cm-2 for at least 30 min, which is a competitive power density value at the lowest fuel flow of 200 μL min-1 reported to date.

  4. Response of microfluidic fuel cells to secondary flows

    NASA Astrophysics Data System (ADS)

    Rossi, Massimiliano; Kähler, Christian J.

    2013-11-01

    Microfluidic or membraneless fuel cells (MFCs) are a recent class of miniaturized fuel cells (Ferrigno et al. 2002, Choban et al. 2004) composed by a microchannel in which a parallel laminar stream of two fluids, a fuel and an oxidant, is established. The fuel and oxidant remain in contact but do not mix due to the absence of turbulence. The simple architecture and the fact that no expensive proton exchange membranes are needed make this configuration technologically very attractive, however the efficiency especially in terms of fuel utilization is still too low to be competitive for practical applications. One limitation is given by the formation of depletion boundary layers at the electrodes that worsen the red-ox reactions. A way to reduce this problem is to use transversal secondary flows to stir the fluid streams and replenish the depletion layers. In this study, we intend to characterize the performance of MFC with curved channels in which the transversal secondary flows are present in the form of two counter-rotating vortices known as Dean vortices. The characterization will be completed by simultaneous measurements of the current intensity and of the flow velocity performed with 3D Astigmatic Particle Tracking Velocimetry.

  5. Characterization of a microfluidic microbial fuel cell as a power generator based on a nickel electrode.

    PubMed

    Mardanpour, Mohammad Mahdi; Yaghmaei, Soheila

    2016-05-15

    This study reports the fabrication of a microfluidic microbial fuel cell (MFC) using nickel as a novel alternative for conventional electrodes and a non-phatogenic strain of Escherichia coli as the biocatalyst. The feasibility of a microfluidic MFC as an efficient power generator for production of bioelectricity from glucose and urea as organic substrates in human blood and urine for implantable medical devices (IMDs) was investigated. A maximum open circuit potential of 459 mV was achieved for the batch-fed microfluidic MFC. During continuous mode operation, a maximum power density of 104 Wm(-3) was obtained with nutrient broth. For the glucose-fed microfluidic MFC, the maximum power density of 5.2 μW cm(-2) obtained in this study is significantly greater than the power densities reported previously for microsized MFCs and glucose fuel cells. The maximum power density of 14 Wm(-3) obtained using urea indicates the successful performance of a microfluidic MFC using human excreta. It features high power density, self-regeneration, waste management and a low production cost (<$1), which suggest it as a promising alternative to conventional power supplies for IMDs. The performance of the microfluidic MFC as a power supply was characterized based on polarization behavior and cell potential in different substrates, operational modes, and concentrations. PMID:26720922

  6. 3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs.

    PubMed

    Knowlton, Stephanie; Yu, Chu Hsiang; Ersoy, Fulya; Emadi, Sharareh; Khademhosseini, Ali; Tasoglu, Savas

    2016-06-01

    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. PMID:27321481

  7. Automated cell viability assessment using a microfluidics based portable imaging flow analyzer.

    PubMed

    Jagannadh, Veerendra Kalyan; Adhikari, Jayesh Vasudeva; Gorthi, Sai Siva

    2015-03-01

    In this work, we report a system-level integration of portable microscopy and microfluidics for the realization of optofluidic imaging flow analyzer with a throughput of 450 cells/s. With the use of a cellphone augmented with off-the-shelf optical components and custom designed microfluidics, we demonstrate a portable optofluidic imaging flow analyzer. A multiple microfluidic channel geometry was employed to demonstrate the enhancement of throughput in the context of low frame-rate imaging systems. Using the cell-phone based digital imaging flow analyzer, we have imaged yeast cells present in a suspension. By digitally processing the recorded videos of the flow stream on the cellphone, we demonstrated an automated cell viability assessment of the yeast cell population. In addition, we also demonstrate the suitability of the system for blood cell counting. PMID:26015835

  8. Automated cell viability assessment using a microfluidics based portable imaging flow analyzer

    PubMed Central

    Jagannadh, Veerendra Kalyan; Adhikari, Jayesh Vasudeva; Gorthi, Sai Siva

    2015-01-01

    In this work, we report a system-level integration of portable microscopy and microfluidics for the realization of optofluidic imaging flow analyzer with a throughput of 450 cells/s. With the use of a cellphone augmented with off-the-shelf optical components and custom designed microfluidics, we demonstrate a portable optofluidic imaging flow analyzer. A multiple microfluidic channel geometry was employed to demonstrate the enhancement of throughput in the context of low frame-rate imaging systems. Using the cell-phone based digital imaging flow analyzer, we have imaged yeast cells present in a suspension. By digitally processing the recorded videos of the flow stream on the cellphone, we demonstrated an automated cell viability assessment of the yeast cell population. In addition, we also demonstrate the suitability of the system for blood cell counting. PMID:26015835

  9. In vitro microfluidic model of sickle cell disease

    NASA Astrophysics Data System (ADS)

    Wood, D. K.; Higgins, J. M.; Mahadevan, L.; Bhatia, S. N.

    2010-03-01

    The pathophysiology of sickle cell disease is complicated by the multiscale processes that link the molecular genotype to the organismal phenotype: hemoglobin polymerization occurring in milliseconds, microscopic cellular sickling in a few seconds or less, and macroscopic vessel occlusion over a time scale of minutes. The rheology of sickle blood, which captures many of these processes, can be studied in vitro using physical tools and insights. We present a minimal microfluidic device in which blood flow dynamics can be directly manipulated by modulating physical factors such as oxygen concentration, capillary size, and fluid shear. We have used this system to map out the phase space of blood flow with respect to a combination of geometric, physical, chemical, and biological parameters. We show that morphological changes in erythrocytes due to sickle hemoglobin polymerization and melting are alone sufficient to change blood rheology. We characterize whole blood from many patients in this device and correlate in vitro performance to clinical outcomes, suggesting the potential utility of such a device for patient monitoring. Our experimental study integrates the dynamics of many of the processes associated with vasoocclusion and provides a potential tool for optimizing and individualizing treatment, and identifying new therapies.

  10. Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device.

    PubMed

    Valero, A; Post, J N; van Nieuwkasteele, J W; Ter Braak, P M; Kruijer, W; van den Berg, A

    2008-01-01

    There is great interest in genetic modification of bone marrow-derived mesenchymal stem cells (MSC), not only for research purposes but also for use in (autologous) patient-derived-patient-used transplantations. A major drawback of bulk methods for genetic modifications of (stem) cells, like bulk-electroporation, is its limited yield of DNA transfection (typically then 10%). This is even more limited when cells are present at very low numbers, as is the case for stem cells. Here we present an alternative technology to transfect cells with high efficiency (>75%), based on single cell electroporation in a microfluidic device. In a first experiment we show that we can successfully transport propidium iodide (PI) into single mouse myoblastic C2C12 cells. Subsequently, we show the use of this microfluidic device to perform successful electroporation of single mouse myoblastic C2C12 cells and single human MSC with vector DNA encoding a green fluorescent-erk1 fusion protein (EGFP-ERK1 (MAPK3)). Finally, we performed electroporation in combination with live imaging of protein expression and dynamics in response to extracellular stimuli, by fibroblast growth factor (FGF-2). We observed nuclear translocation of EGFP-ERK1 in both cell types within 15 min after FGF-2 stimulation. Due to the successful and promising results, we predict that microfluidic devices can be used for highly efficient small-scale 'genetic modification' of cells, and biological experimentation, offering possibilities to study cellular processes at the single cell level. Future applications might be small-scale production of cells for therapeutic application under controlled conditions. PMID:18094762

  11. Microfluidic device for mechanical dissociation of cancer cell aggregates into single cells

    PubMed Central

    Pennell, Marissa; Troiani, Marco; Haun, Jered B.

    2014-01-01

    Tumors tissues house a diverse array of cell types, requiring powerful cell-based analysis methods to characterize different cell subtypes. Tumor tissue is dissociated into single cells by treatment with proteolytic enzymes, followed by mechanical disruption using vortexing or pipetting. These procedures can be incomplete and require significant time, and the latter mechanical treatments are poorly defined and controlled. Here, we present a novel microfluidic device to improve mechanical dissociation of digested tissue and cell aggregates into single cells. The device design includes a network of branching channels that range in size from millimeters down to hundreds of microns. The channels also contain flow constrictions that generate well-defined regions of high shear force, which we refer to as “hydrodynamic micro-scalpels,” to progressively disaggregate tissue fragments and clusters into single cells. We show using in vitro cancer cell models that the microfluidic device significantly enhances cell recovery in comparison to mechanical disruption by pipetting and vortexing digestion with trypsin or incubation with EDTA. Notably, the device enabled superior results to be obtained after shorter proteolytic digestion times, resulting in fully viable cells in less than ten minutes. The device could also be operated under enzyme-free conditions that could better maintain expression of certain surface markers. The microfluidic format is advantageous because it enables application of well-defined mechanical forces and rapid processing times. Furthermore, it may be possible to directly integrate downstream processing and detection operations to create integrated cell-based analysis platforms. The enhanced capabilities enabled by our novel device may help promote applications of single cell detection and purification techniques to tumor tissue specimens, advancing the current understanding of cancer biology and enabling molecular diagnostics in clinical settings

  12. Microfluidic 3D cell culture: potential application for tissue-based bioassays

    PubMed Central

    Li, XiuJun (James); Valadez, Alejandra V.; Zuo, Peng; Nie, Zhihong

    2014-01-01

    Current fundamental investigations of human biology and the development of therapeutic drugs, commonly rely on two-dimensional (2D) monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function, physiology of living tissues, as well as highly complex and dynamic three-dimensional (3D) environments in vivo. The microfluidic technology can provide micro-scale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in microfluidic technology for 3D cell culture and their biological applications. PMID:22793034

  13. Microfluidic chips for the study of cell migration under the effect of chemicals

    NASA Astrophysics Data System (ADS)

    Kukhtevich, I. V.; Belousov, K. I.; Bukatin, A. S.; Chubinskiy-Nadezhdin, V. I.; Vasileva, V. Yu.; Negulyaev, Yu. A.; Evstrapov, A. A.

    2016-05-01

    Numerical simulation of the formation of a chemoattractant gradient in reaction chambers of a chip having different geometries enabled the determination of a structure suitable for the study of cell migration, in accordance with which hybrid polymer-glass microfluidic devices were manufactured. Verification of the procedures of alignment of cells in the reaction chamber of the chip by centrifugal force and subsequent culturing of the cells showed that microfluidic chips can be used to study cell migration under the effect of the chemoattractant gradient in vitro.

  14. Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture.

    PubMed

    Utech, Stefanie; Prodanovic, Radivoje; Mao, Angelo S; Ostafe, Raluca; Mooney, David J; Weitz, David A

    2015-08-01

    Monodisperse alginate microgels (10-50 μm) are created via droplet-based microfluidics by a novel crosslinking procedure. Ionic crosslinking of alginate is induced by release of chelated calcium ions. The process separates droplet formation and gelation reaction enabling excellent control over size and homogeneity under mild reaction conditions. Living mesenchymal stem cells are encapsulated and cultured in the generated 3D microenvironments. PMID:26039892

  15. Microfluidic platform for the study of intercellular communication via soluble factor-cell and cell-cell paracrine signaling.

    PubMed

    Byrne, Matthew B; Trump, Lisa; Desai, Amit V; Schook, Lawrence B; Gaskins, H Rex; Kenis, Paul J A

    2014-07-01

    Diffusion of autocrine and paracrine signaling molecules allows cells to communicate in the absence of physical contact. This chemical-based, long-range communication serves crucial roles in tissue function, activation of the immune system, and other physiological functions. Despite its importance, few in vitro methods to study cell-cell signaling through paracrine factors are available today. Here, we report the design and validation of a microfluidic platform that enables (i) soluble molecule-cell and/or (ii) cell-cell paracrine signaling. In the microfluidic platform, multiple cell populations can be introduced into parallel channels. The channels are separated by arrays of posts allowing diffusion of paracrine molecules between cell populations. A computational analysis was performed to aid design of the microfluidic platform. Specifically, it revealed that channel spacing affects both spatial and temporal distribution of signaling molecules, while the initial concentration of the signaling molecule mainly affects the concentration of the signaling molecules excreted by the cells. To validate the microfluidic platform, a model system composed of the signaling molecule lipopolysaccharide, mouse macrophages, and engineered human embryonic kidney cells was introduced into the platform. Upon diffusion from the first channel to the second channel, lipopolysaccharide activates the macrophages which begin to produce TNF-α. The TNF-α diffuses from the second channel to the third channel to stimulate the kidney cells, which express green fluorescent protein (GFP) in response. By increasing the initial lipopolysaccharide concentration an increase in fluorescent response was recorded, demonstrating the ability to quantify intercellular communication between 3D cellular constructs using the microfluidic platform reported here. Overall, these studies provide a detailed analysis on how concentration of the initial signaling molecules, spatiotemporal dynamics, and inter

  16. Droplet-based microfluidics for artificial cell generation: a brief review.

    PubMed

    Martino, Chiara; deMello, Andrew J

    2016-08-01

    Artificial cells are best defined as micrometre-sized structures able to mimic many of the morphological and functional characteristics of a living cell. In this mini-review, we describe progress in the application of droplet-based microfluidics for the generation of artificial cells and protocells. PMID:27499841

  17. A microfluidic device for uniform-sized cell spheroids formation, culture, harvesting and flow cytometry analysis.

    PubMed

    Patra, Bishnubrata; Chen, Ying-Hua; Peng, Chien-Chung; Lin, Shiang-Chi; Lee, Chau-Hwang; Tung, Yi-Chung

    2013-01-01

    Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell-cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers. PMID:24396525

  18. Handling and analysis of cells and bioparticles on centrifugal microfluidic platforms.

    PubMed

    Burger, Robert; Ducrée, Jens

    2012-05-01

    Microfluidic systems for cell separation and analysis have attracted increasing research activity over the past decades. In particular, the prospect of integrating all steps from sample preparation to assay readout in a single microfluidic cartridge, which is inserted into a compact, portable and potentially low-cost instrument, bears great promise to leverage next-generation diagnostic products and to advance life-science research with novel cell and particle manipulation, and analysis tools. Within the range of microfluidic actuation principles available, the centrifugal force is exceptionally well suited for cell handling due to its rotationally induced 'artificial gravity field', which can be varied over several orders of magnitude and which can manipulate bioparticles even in the absence of flow. We will survey how the base centrifugal force has been combined with the hydrodynamic Stokes drag, magnetic, dielectrophoretic and other forces to enable multidimensional separation and manipulation. The same centrifugal microfluidic toolbox has also been applied to investigate particles such as biofunctionalized beads, bacteria and multicellular microorganisms. This review summarizes the significant progress in modular unit operations such as cell removal, filtering, lysis, separation, sorting, encapsulation, trapping, assaying, sensing, cytometry and detection, even derived from low-cost conventional optical disc drive technology (e.g., CD and DVD), towards integrated and automated centrifugal microfluidic platforms for the handling and analysis of cells and bioparticles. PMID:22616705

  19. Fuel cell-powered microfluidic platform for lab-on-a-chip applications.

    PubMed

    Esquivel, Juan Pablo; Castellarnau, Marc; Senn, Tobias; Löchel, Bernd; Samitier, Josep; Sabaté, Neus

    2012-01-01

    The achievement of a higher degree of integration of components--especially micropumps and power sources--is a challenge currently being pursued to obtain portable and totally autonomous microfluidic devices. This paper presents the integration of a micro direct methanol fuel cell (μDMFC) in a microfluidic platform as a smart solution to provide both electrical and pumping power to a Lab-on-a-Chip system. In this system the electric power produced by the fuel cell is available to enable most of the functionalites required by the microfluidic chip, while the generated CO(2) from the electrochemical reaction produces a pressure capable of pumping a liquid volume through a microchannel. The control of the fuel cell operating conditions allows regulation of the flow rate of a liquid sample through a microfluidic network. The relation between sample flow rate and the current generated by the fuel cell is practically linear, achieving values in the range of 4-18 μL min(-1) while having an available power between 1-4 mW. This permits adjusting the desired flow rate for a given application by controlling the fuel cell output conditions and foresees a fully autonomous analytical Lab-on-a-Chip in which the same device would provide the electrical power to a detection module and at the same time use the CO(2) pumping action to flow the required analytes through a particular microfluidic design. PMID:22072241

  20. A Microfluidic Platform for High-throughput Single-cell Isolation and Culture.

    PubMed

    Lin, Ching-Hui; Chang, Hao-Chen; Hsu, Chia-Hsien

    2016-01-01

    Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency. PMID:27341146

  1. Characterization of a single cell of Chlorella in a microfluidic channel using amperometric electrode arrays.

    PubMed

    Song, Young Seok; Bai, Seoung Jai

    2014-11-01

    Electrochemical characteristics of O2 and/or mediators secreted by a single cell of Chlorella fusea were analyzed by using amperometric measurements on microelectrodes embedded in a microfluidic device. A single cell was trapped in a microfluidic channel, which simplifies the mass transfer phenomenon, i.e., one-dimensional distribution of solutes in the channel. Such amperometric measurements allowed us to obtain more refined data in a localized space and to understand photosynthetic behavior of algae at the single cell level. In addition, the concentration of a photosynthetic mediator, p-benzoquinone, was numerically calculated by using the finite element method. PMID:24966046

  2. Single-cell analysis and sorting using droplet-based microfluidics

    PubMed Central

    Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

    2014-01-01

    We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. as an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. the beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ~200 Hz as well as cell enrichment. the microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ~1 million cells, the microfluidic operations require 2–6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5–7 d. PMID:23558786

  3. Simulation of malaria-infected red blood cells in microfluidic channels: Passage and blockage

    PubMed Central

    Wu, Tenghu; Feng, James J.

    2013-01-01

    Malaria-infected red blood cells (iRBCs) become less deformable with the progression of infection and tend to occlude microcapillaries. This process has been investigated in vitro using microfluidic channels. The objective of this paper is to provide a quantitative basis for interpreting the experimental observations of iRBC occlusion of microfluidic channels. Using a particle-based model for the iRBC, we simulate the traverse of iRBCs through a converging microfluidic channel and explore the progressive loss of cell deformability due to three factors: the stiffening of the membrane, the reduction of the cell's surface-volume ratio, and the growing solid parasites inside the cell. When examined individually, each factor tends to hinder the passage of the iRBC and lengthen the transit time. Moreover, at sufficient magnitude, each may lead to obstruction of narrow microfluidic channels. We then integrate the three factors into a series of simulations that mimic the development of malaria infection through the ring, trophozoite, and schizont stages. These simulations successfully reproduce the experimental observation that with progression of infection, the iRBC transitions from passage to blockage in larger and larger channels. The numerical results suggest a scheme for quantifying iRBC rigidification through microfluidic measurements of the critical pressure required for passage. PMID:24404048

  4. Microfluidic assay-based optical measurement techniques for cell analysis: A review of recent progress.

    PubMed

    Choi, Jong-Ryul; Song, Hyerin; Sung, Jong Hwan; Kim, Donghyun; Kim, Kyujung

    2016-03-15

    Since the early 2000s, microfluidic cell culture systems have attracted significant attention as a promising alternative to conventional cell culture methods and the importance of designing an efficient detection system to analyze cell behavior on a chip in real time is raised. For this reason, various measurement techniques for microfluidic devices have been developed with the development of microfluidic assays for high-throughput screening and mimicking of in vivo conditions. In this review, we discuss optical measurement techniques for microfluidic assays. First of all, the recent development of fluorescence- and absorbance-based optical measurement systems is described. Next, advanced optical detection systems are introduced with respect to three emphases: 1) optimization for long-term, real-time, and in situ measurements; 2) performance improvements; and 3) multimodal analysis conjugations. Moreover, we explore presents future prospects for the establishment of optical detection systems following the development of complex, multi-dimensional microfluidic cell culture assays to mimic in vivo tissue, organ, and human systems. PMID:26409023

  5. Microfluidic enrichment for the single cell analysis of circulating tumor cells.

    PubMed

    Yeo, Trifanny; Tan, Swee Jin; Lim, Chew Leng; Lau, Dawn Ping Xi; Chua, Yong Wei; Krisna, Sai Sakktee; Iyer, Gopal; Tan, Gek San; Lim, Tony Kiat Hon; Tan, Daniel S W; Lim, Wan-Teck; Lim, Chwee Teck

    2016-01-01

    Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy. PMID:26924553

  6. Microfluidic enrichment for the single cell analysis of circulating tumor cells

    PubMed Central

    Yeo, Trifanny; Tan, Swee Jin; Lim, Chew Leng; Lau, Dawn Ping Xi; Chua, Yong Wei; Krisna, Sai Sakktee; Iyer, Gopal; Tan, Gek San; Lim, Tony Kiat Hon; Tan, Daniel S.W.; Lim, Wan-Teck; Lim, Chwee Teck

    2016-01-01

    Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy. PMID:26924553

  7. Microfluidic electrochemical reactors

    DOEpatents

    Nuzzo, Ralph G.; Mitrovski, Svetlana M.

    2011-03-22

    A microfluidic electrochemical reactor includes an electrode and one or more microfluidic channels on the electrode, where the microfluidic channels are covered with a membrane containing a gas permeable polymer. The distance between the electrode and the membrane is less than 500 micrometers. The microfluidic electrochemical reactor can provide for increased reaction rates in electrochemical reactions using a gaseous reactant, as compared to conventional electrochemical cells. Microfluidic electrochemical reactors can be incorporated into devices for applications such as fuel cells, electrochemical analysis, microfluidic actuation, pH gradient formation.

  8. Enhanced biofilm distribution and cell performance of microfluidic microbial fuel cells with multiple anolyte inlets.

    PubMed

    Yang, Yang; Ye, Dingding; Liao, Qiang; Zhang, Pengqing; Zhu, Xun; Li, Jun; Fu, Qian

    2016-05-15

    A laminar-flow controlled microfluidic microbial fuel cell (MMFC) is considered as a promising approach to be a bio-electrochemical system (BES). But poor bacterial colonization and low power generation are two severe bottlenecks to restrict its development. In this study, we reported a MMFC with multiple anolyte inlets (MMFC-MI) to enhance the biofilm formation and promote the power density of MMFCs. Voltage profiles during the inoculation process demonstrated MMFC-MI had a faster start-up process than the conventional microfluidic microbial fuel cell with one inlet (MMFC-OI). Meanwhile, benefited from the periodical replenishment of boundary layer near the electrode, a more densely-packed bacterial aggregation was observed along the flow direction and also the substantially low internal resistance for MMFC-MI. Most importantly, the output power density of MMFC-MI was the highest value among the reported µl-scale MFCs to our best knowledge. The presented MMFC-MI appears promising for bio-chip technology and extends the scope of microfluidic energy. PMID:26735875

  9. A digital microfluidic method for multiplexed cell-based apoptosis assays.

    PubMed

    Bogojevic, Dario; Chamberlain, M Dean; Barbulovic-Nad, Irena; Wheeler, Aaron R

    2012-02-01

    Digital microfluidics (DMF), a fluid-handling technique in which picolitre-microlitre droplets are manipulated electrostatically on an array of electrodes, has recently become popular for applications in chemistry and biology. DMF devices are reconfigurable, have no moving parts, and are compatible with conventional high-throughput screening infrastructure (e.g., multiwell plate readers). For these and other reasons, digital microfluidics has been touted as being a potentially useful new tool for applications in multiplexed screening. Here, we introduce the first digital microfluidic platform used to implement parallel-scale cell-based assays. A fluorogenic apoptosis assay for caspase-3 activity was chosen as a model system because of the popularity of apoptosis as a target for anti-cancer drug discovery research. Dose-response profiles of caspase-3 activity as a function of staurosporine concentration were generated using both the digital microfluidic method and conventional techniques (i.e., pipetting, aspiration, and 96-well plates.) As expected, the digital microfluidic method had a 33-fold reduction in reagent consumption relative to the conventional technique. Although both types of methods used the same detector (a benchtop multiwell plate reader), the data generated by the digital microfluidic method had lower detection limits and greater dynamic range because apoptotic cells were much less likely to de-laminate when exposed to droplet manipulation by DMF relative to pipetting/aspiration in multiwell plates. We propose that the techniques described here represent an important milestone in the development of digital microfluidics as a useful tool for parallel cell-based screening and other applications. PMID:22159547

  10. Method of measuring nitric oxide release by vascular endothelial cells grown in microfluidic channels

    NASA Astrophysics Data System (ADS)

    Hosseinpour, S.; Liu, A. C.; Barakat, A. I.; Choy, J. C.; Gray, B. L.

    2014-03-01

    In this paper, a simple and versatile method is presented which enables detection of nitric oxide (NO) released from vascular endothelial cells (ECs) cultured in microfluidic structures. The culturing system and NO measurement method allow cell shape to be controlled in a non-invasive manner using microfluidic structures while NO release is monitored for cell shape versus function studies. The culturing system consists of arrays of polydimethylsiloxane (PDMS) fluidic channels 120 micrometers in depth and ranging from 100 micrometers to 3 mm in width. The number of channels in each array is varied to yield a constant cell culture surface area (75 mm2) independent of channel width. The channel surfaces are collagen-coated and ECs are cultured to confluence within the channels. A cell scraper is then used to scrape extraneous cells cultured between channels, and NO measurements are made 18 to 24 hours later. A chemiluminescence-based sensor system (NOA 280i, Sievers NO Analyzer) is utilized to measure sample NO. Initial results indicate that NO concentrations can be measured from different microfluidic channel-containing samples using this method. It is shown that there is no significant difference in NO concentration derived from channels of different widths even though the degree of cell elongation varies due to physical constraint by microfluidic channel walls. However, cells treated with TNFα release more NO than untreated cells in fluidic channels, which is comparable to the function of ECs cultured in conventional culturing systems such as culturing dishes.

  11. High throughput and multiplex localization of proteins and cells for in situ micropatterning using pneumatic microfluidics.

    PubMed

    Wang, Jian-Chun; Liu, Wenming; Tu, Qin; Ma, Chao; Zhao, Lei; Wang, Yaolei; Ouyang, Jia; Pang, Long; Wang, Jinyi

    2015-02-01

    Micropatterning technologies are emerging as an enabling tool for various microfluidic-based applications in life sciences. However, the high throughput and multiplex localization of multiple bio-components in a microfluidic device has not yet been well established. In this paper, we describe a simple and in situ micropatterning method using an integrated microfluidic device with pneumatic microstructures (PμSs) for highly controllable immobilization of both proteins and cells in a high throughput, geometry-dynamic, and multi-patterning way. The precise Pluronic F127 passivation of a microchamber surface except the PμS-blocked regions was performed and characterized, and the spatial dynamics and consistency of both the PμSs and protein/cell micropatterning were optically evaluated and quantitatively demonstrated too. Furthermore, a systematic investigation of PμS-assisted micropatterning in microfluidics was carried out. The feature of high throughput and spatial control of micropatterning can be simply realized by using the well-designed PμS arrays. Meanwhile, the co-micropatterning of different proteins (bovine serum albumin and chicken egg albumin) and cells (human umbilical vein endothelial cells and human hepatocellular carcinoma cells) in a microfluidic device was successfully accomplished with the orderly serial manipulation of PμS groups. We demonstrate that PμS-assisted micropatterning can be applied as a convenient microfluidic component for large-scale and diversified protein/cell patterning and manipulation, which could be useful for cell-based tissue organization, high-throughput imaging, protein-related interactions and immunoassays. PMID:25453039

  12. Image-guided precision manipulation of cells and nanoparticles in microfluidics

    NASA Astrophysics Data System (ADS)

    Cummins, Zachary

    Manipulation of single cells and particles is important to biology and nanotechnology. Our electrokinetic (EK) tweezers manipulate objects in simple microfluidic devices using gentle fluid and electric forces under vision-based feedback control. In this dissertation, I detail a user-friendly implementation of EK tweezers that allows users to select, position, and assemble cells and nanoparticles. This EK system was used to measure attachment forces between living breast cancer cells, trap single quantum dots with 45 nm accuracy, build nanophotonic circuits, and scan optical properties of nanowires. With a novel multi-layer microfluidic device, EK was also used to guide single microspheres along complex 3D trajectories. The schemes, software, and methods developed here can be used in many settings to precisely manipulate most visible objects, assemble objects into useful structures, and improve the function of lab-on-a-chip microfluidic systems.

  13. Study of Chemotaxis and Cell-Cell Interactions in Cancer with Microfluidic Devices.

    PubMed

    Sai, Jiqing; Rogers, Matthew; Hockemeyer, Kathryn; Wikswo, John P; Richmond, Ann

    2016-01-01

    Microfluidic devices have very broad applications in biological assays from simple chemotaxis assays to much more complicated 3D bioreactors. In this chapter, we describe the design and methods for performing chemotaxis assays using simple microfluidic chemotaxis chambers. With these devices, using real-time video microscopy we can examine the chemotactic responses of neutrophil-like cells under conditions of varying gradient steepness or flow rate and then utilize software programs to calculate the speed and angles of cell migration as gradient steepness and flow are varied. Considering the shearing force generated on the cells by the constant flow that is required to produce and maintain a stable gradient, the trajectories of the cell migration will reflect the net result of both shear force generated by flow and the chemotactic force resulting from the chemokine gradient. Moreover, the effects of mutations in chemokine receptors or the presence of inhibitors of intracellular signals required for gradient sensing can be evaluated in real time. We also describe a method to monitor intracellular signals required for cells to alter cell polarity in response to an abrupt switch in gradient direction. Lastly, we demonstrate an in vitro method for studying the interactions of human cancer cells with human endothelial cells, fibroblasts, and leukocytes, as well as environmental chemokines and cytokines, using 3D microbioreactors that mimic the in vivo microenvironment. PMID:26921940

  14. Separation of biological cells in a microfluidic device using surface acoustic waves (SAWs)

    NASA Astrophysics Data System (ADS)

    Ai, Ye; Marrone, Babetta L.

    2014-03-01

    In this study, a surface acoustic wave (SAW)-based microfluidic device has been developed to separate heterogeneous particle or cell mixtures in a continuous flow using acoustophoresis. The microfluidic device is comprised of two components, a SAW transducer and a microfluidic channel made of polydimethylsiloxane (PDMS). The SAW transducer was fabricated by patterning two pairs of interdigital electrodes on a lithium niobate (LiNbO3) piezoelectric substrate. When exciting the SAW transducer by AC signals, a standing SAW is generated along the cross-section of the channel. Solid particles immersed in the standing SAW field are accordingly pushed to the pressure node arising from the acoustic radiation force acting on the particles, referring to the acoustic particle-focusing phenomenon. Acoustic radiation force highly depends on the particle properties, resulting in different acoustic responses for different types of cells. A numerical model, coupling the piezoelectric effect in the solid substrate and acoustic pressure in the fluid, was developed to provide a better understanding of SAW-based particle manipulation. Separation of two types of fluorescent particles has been demonstrated using the developed SAW-based microfluidic device. An efficient separation of E. coli bacteria from peripheral blood mononuclear cell (PBMC) samples has also been successfully achieved. The purity of separated E. coli bacteria and separated PBMCs were over 95% and 91%, respectively, obtained by a flow cytometric analysis. The developed microfluidic device can efficiently separate E. coli bacteria from biological samples, which has potential applications in biomedical analysis and clinical diagnosis.

  15. Oxygen-Purged Microfluidic Device to Enhance Cell Viability in Photopolymerized PEG Hydrogel Microparticles.

    PubMed

    Xia, Bingzhao; Krutkramelis, Kaspars; Oakey, John

    2016-07-11

    Encapsulating cells within biocompatible materials is a widely used strategy for cell delivery and tissue engineering. While cells are commonly suspended within bulk hydrogel-forming solutions during gelation, substantial interest in the microfluidic fabrication of miniaturized cell encapsulation vehicles has more recently emerged. Here, we utilize multiphase microfluidics to encapsulate cells within photopolymerized picoliter-volume water-in-oil droplets at high production rates. The photoinitiated polymerization of polyethylene glycol diacrylate (PEGDA) is used to continuously produce solid particles from aqueous liquid drops containing cells and hydrogel forming solution. It is well understood that this photoinitiated addition reaction is inhibited by oxygen. In contrast to bulk polymerization in which ambient oxygen is rapidly and harmlessly consumed, allowing the polymerization reaction to proceed, photopolymerization within air permeable polydimethylsiloxane (PDMS) microfluidic devices allows oxygen to be replenished by diffusion as it is depleted. This sustained presence of oxygen and the consequential accumulation of peroxy radicals produce a dramatic effect upon both droplet polymerization and post-encapsulation cell viability. In this work we employ a nitrogen microjacketed microfluidic device to purge oxygen from flowing fluids during photopolymerization. By increasing the purging nitrogen pressure, oxygen concentration was attenuated, and increased post-encapsulation cell viability was achieved. A reaction-diffusion model was used to predict the cumulative intradroplet concentration of peroxy radicals, which corresponded directly to post-encapsulation cell viability. The nitrogen-jacketed microfluidic device presented here allows the droplet oxygen concentration to be finely tuned during cell encapsulation, leading to high post-encapsulation cell viability. PMID:27285343

  16. Tunable Microfluidic Devices for Hydrodynamic Fractionation of Cells and Beads: A Review

    PubMed Central

    Alvankarian, Jafar; Majlis, Burhanuddin Yeop

    2015-01-01

    The adjustable microfluidic devices that have been developed for hydrodynamic-based fractionation of beads and cells are important for fast performance tunability through interaction of mechanical properties of particles in fluid flow and mechanically flexible microstructures. In this review, the research works reported on fabrication and testing of the tunable elastomeric microfluidic devices for applications such as separation, filtration, isolation, and trapping of single or bulk of microbeads or cells are discussed. Such microfluidic systems for rapid performance alteration are classified in two groups of bulk deformation of microdevices using external mechanical forces, and local deformation of microstructures using flexible membrane by pneumatic pressure. The main advantage of membrane-based tunable systems has been addressed to be the high capability of integration with other microdevice components. The stretchable devices based on bulk deformation of microstructures have in common advantage of simplicity in design and fabrication process. PMID:26610519

  17. A laminar flow microfluidic fuel cell for detection of hexavalent chromium concentration.

    PubMed

    Ye, Dingding; Yang, Yang; Li, Jun; Zhu, Xun; Liao, Qiang; Zhang, Biao

    2015-11-01

    An electrochemical hexavalent chromium concentration sensor based on a microfluidic fuel cell is presented. The correlation between current density and chromium concentration is established in this report. Three related operation parameters are investigated, including pH values, temperature, and external resistance on the sensor performance. The results show that the current density increases with increasing temperature and the sensor produces a maximum regression coefficient at the catholyte pH value of 1.0. Moreover, it is found that the external resistance has a great influence on the linearity and current densities of the microfluidic sensor. Owing to the membraneless structure and the steady co-laminar flow inside the microchannel, the microfluidic sensor exhibits short response time to hexavalent chromium concentration. The laminar flow fuel cell sensor provides a new and simple method for detecting hexavalent chromium concentration in the industrial wastewater. PMID:26649130

  18. 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. PMID:26487900

  19. Rapid prototyping of arrayed microfluidic systems in polystyrene for cell-based assays

    PubMed Central

    Young, Edmond W.K.; Berthier, Erwin; Guckenberger, David J.; Sackmann, Eric; Lamers, Casey; Meyvantsson, Ivar; Huttenlocher, Anna; Beebe, David J.

    2011-01-01

    Microfluidic cell-based systems have enabled the study of cellular phenomena with improved spatiotemporal control of the microenvironment and at increased throughput. While PDMS has emerged as the most popular material in microfluidics research, it has specific limitations that prevent microfluidic platforms from achieving their full potential. We present here a complete process, ranging from mold design to embossing and bonding, that describes the fabrication of polystyrene (PS) microfluidic devices with similar cost and time expenditures as PDMS-based devices. Emphasis was placed on creating methods that can compete with PDMS fabrication methods in terms of robustness, complexity and time requirements. To achieve this goal several improvements were made to remove critical bottlenecks in existing PS embossing methods. First, traditional lithography techniques were adapted to fabricate bulk epoxy molds capable of resisting high temperatures and pressures. Second, a method was developed to emboss through-holes in a PS layer, enabling creation of large arrays of independent microfluidic systems on a single device without need to manually create access ports. Third, thermal bonding of PS layers was optimized in order to achieve quality bonding over large arrays of microsystems. The choice of materials and methods were validated for biological function using two different cell-based applications to demonstrate the versatility of our streamlined fabrication process. PMID:21261280

  20. Real-time optical pH measurement in a standard microfluidic cell culture system.

    PubMed

    Magnusson, Einar B; Halldorsson, Skarphedinn; Fleming, Ronan M T; Leosson, Kristjan

    2013-01-01

    The rapid growth of microfluidic cell culturing in biological and biomedical research and industry calls for fast, non-invasive and reliable methods of evaluating conditions such as pH inside a microfluidic system. We show that by careful calibration it is possible to measure pH within microfluidic chambers with high accuracy and precision, using a direct single-pass measurement of light absorption in a commercially available phenol-red-containing cell culture medium. The measurement is carried out using a standard laboratory microscope and, contrary to previously reported methods, requires no modification of the microfluidic device design. We demonstrate the validity of this method by measuring absorption of light transmitted through 30-micrometer thick microfluidic chambers, using an inverted microscope fitted with a scientific-grade digital camera and two bandpass filters. In the pH range of 7-8, our measurements have a standard deviation and absolute error below 0.05 for a measurement volume smaller than 4 nL. PMID:24049695

  1. Computational cell analysis for label-free detection of cell properties in a microfluidic laminar flow.

    PubMed

    Zhang, Alex Ce; Gu, Yi; Han, Yuanyuan; Mei, Zhe; Chiu, Yu-Jui; Geng, Lina; Cho, Sung Hwan; Lo, Yu-Hwa

    2016-06-20

    Although a flow cytometer, being one of the most popular research and clinical tools for biomedicine, can analyze cells based on the cell size, internal structures such as granularity, and molecular markers, it provides little information about the physical properties of cells such as cell stiffness and physical interactions between the cell membrane and fluid. In this paper, we propose a computational cell analysis technique using cells' different equilibrium positions in a laminar flow. This method utilizes a spatial coding technique to acquire the spatial position of the cell in a microfluidic channel and then uses mathematical algorithms to calculate the ratio of cell mixtures. Most uniquely, the invented computational cell analysis technique can unequivocally detect the subpopulation of each cell type without labeling even when the cell type shows a substantial overlap in the distribution plot with other cell types, a scenario limiting the use of conventional flow cytometers and machine learning techniques. To prove this concept, we have applied the computation method to distinguish live and fixed cancer cells without labeling, count neutrophils from human blood, and distinguish drug treated cells from untreated cells. Our work paves the way for using computation algorithms and fluidic dynamic properties for cell classification, a label-free method that can potentially classify over 200 types of human cells. Being a highly cost-effective cell analysis method complementary to flow cytometers, our method can offer orthogonal tests in companion with flow cytometers to provide crucial information for biomedical samples. PMID:27163941

  2. A simple microfluidic device for the deformability assessment of blood cells in a continuous flow.

    PubMed

    Rodrigues, Raquel O; Pinho, Diana; Faustino, Vera; Lima, Rui

    2015-12-01

    Blood flow presents several interesting phenomena in microcirculation that can be used to develop microfluidic devices capable to promote blood cells separation and analysis in continuous flow. In the last decade there have been numerous microfluidic studies focused on the deformation of red blood cells (RBCs) flowing through geometries mimicking microvessels. In contrast, studies focusing on the deformation of white blood cells (WBCs) are scarce despite this phenomenon often happens in the microcirculation. In this work, we present a novel integrative microfluidic device able to perform continuous separation of a desired amount of blood cells, without clogging or jamming, and at the same time, capable to assess the deformation index (DI) of both WBCs and RBCs. To determine the DI of both WBCs and RBCs, a hyperbolic converging microchannel was used, as well as a suitable image analysis technique to measure the DIs of these blood cells along the regions of interest. The results show that the WBCs have a much lower deformability than RBCs when subjected to the same in vitro flow conditions, which is directly related to their cytoskeleton and nucleus contents. The proposed strategy can be easily transformed into a simple and inexpensive diagnostic microfluidic system to simultaneously separate and assess blood cells deformability. PMID:26482154

  3. MEMS-based flow cytometry: microfluidics-based cell identification system by fluorescent imaging.

    PubMed

    Wu, W K; Liang, C K; Huang, J Z

    2004-01-01

    This study utilizes MEMS technology to realize a novel low-cost microfluidics-based biochip system for flow-type cell handling. Powered by vacuum pump, the microfluidic driving system enables cells to move in order one by one in the biochip by an effect of sheath flow prefocus. Then, cells are guided to a fluorescent inspection region where two detection tasks such as cell image identification and cell counting are conducted. Currently, the glass-based biochip has been manufactured and all the related devices have been well set up in our laboratory. With this proposed prototype system, typical results about cell separation of yeast cell and PC-3 cell are available and their separated images are also presented, respectively. PMID:17270801

  4. Microfluidics Meets Dilute Solution Viscometry: An Undergraduate Laboratory to Determine Polymer Molecular Weight Using a Microviscometer

    ERIC Educational Resources Information Center

    Pety, Stephen J.; Lu, Hang; Thio, Yonathan S.

    2011-01-01

    This paper describes a student laboratory experiment to determine the molecular weight of a polymer sample by measuring the viscosity of dilute polymer solutions in a PDMS microfluidic viscometer. Sample data are given for aqueous solutions of poly(ethylene oxide) (PEO). A demonstration of shear thinning behavior using the microviscometer is…

  5. Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture.

    PubMed

    Dura, Burak; Servos, Mariah M; Barry, Rachel M; Ploegh, Hidde L; Dougan, Stephanie K; Voldman, Joel

    2016-06-28

    Resolving how the early signaling events initiated by cell-cell interactions are transduced into diverse functional outcomes necessitates correlated measurements at various stages. Typical approaches that rely on bulk cocultures and population-wide correlations, however, only reveal these relationships broadly at the population level, not within each individual cell. Here, we present a microfluidics-based cell-cell interaction assay that enables longitudinal investigation of lymphocyte interactions at the single-cell level through microfluidic cell pairing, on-chip culture, and multiparameter assays, and allows recovery of desired cell pairs by micromanipulation for off-chip culture and analyses. Well-defined initiation of interactions enables probing cellular responses from the very onset, permitting single-cell correlation analyses between early signaling dynamics and later-stage functional outcomes within same cells. We demonstrate the utility of this microfluidic assay with natural killer cells interacting with tumor cells, and our findings suggest a possible role for the strength of early calcium signaling in selective coordination of subsequent cytotoxicity and IFN-gamma production. Collectively, our experiments demonstrate that this new approach is well-suited for resolving the relationships between complex immune responses within each individual cell. PMID:27303033

  6. The Effects of Nanotexturing Microfluidic Platforms to Isolate Brain Tumor Cells

    NASA Astrophysics Data System (ADS)

    Islam, Muhymin; Sajid, Adeel; Kim, Young-Tae; Iqbal, Samir M.

    2015-03-01

    Detection of tumor cells in the early stages of disease requires sensitive and selective approaches. Nanotextured polydimethylsiloxane (PDMS) substrates were implemented to detect metastatic human glioblastoma (hGBM) cells. RNA aptamers that were specific to epidermal growth factor receptors (EGFR) were used to functionalize the substrates. EGFR is known to be overexpressed on many cancer cells including hGBM. Nanotextured PDMS was prepared by micro reactive ion etching. PDMS surfaces became hydrophilic uponnanotexturing. Nanotextured substrates were incubated in tumor cell solution and density of captured cells was determined. Nanotextured PDMS provided >300% cell capture compared to plain PDMS due to increased effective surface area of roughened substrates at nanoscale as well as mire focal points for cell adhesion. Next, aptamer functionalized nanotextured PDMS was incorporated in microfluidic device to detect tumor cells at different flow velocities. The shear stress introduced by the flow pressure and heterogeneity of the EGFR overexpression on cell membranes of the tumor cells had significant impact on the cell capture efficiency of aptamer anchored nanotextured microfluidic devices. Eventually tumor cells were detected from the mixture of white blood cells at an efficiency of 73% using the microfluidic device. The interplay of binding energies and surface energies was major factor in this system. Support Acknowledged from NSF through ECCS-1407990.

  7. 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.

  8. Separation of two phenotypically similar cell types via a single common marker in microfluidic channels.

    PubMed

    Vickers, Dwayne A L; Chory, Emma J; Murthy, Shashi K

    2012-09-21

    To isolate clinically and biologically relevant cell types from a heterogeneous population, fluorescent or magnetic tagging together with knowledge of surface biomarker profiles represents the state of the art. To date, it remains exceedingly difficult to separate phenotypically and physically similar cell types from a mixed population. We report a microfluidic platform engineered to separate two highly similar cell types using a single antibody by taking advantage of subtle variations in surface receptor density and cell size. This platform utilizes antibody-conjugated surfaces in microfluidic channels together with precise modulation of fluid shear stresses to accomplish selective fractionation in a continuous flow process. Antibody conjugation density variation on the adhesive surfaces is achieved by covalently immobilizing an antibody in the presence of poly(ethylene glycol). This platform is used to demonstrate separation of two CD31 positive cell types, human umbilical vein endothelial cells and human micro vascular endothelial cells. PMID:22782544

  9. A Microfluidic Bioreactor for Toxicity Testing of Stem Cell Derived 3D Cardiac Bodies.

    PubMed

    Christoffersson, Jonas; Bergström, Gunnar; Schwanke, Kristin; Kempf, Henning; Zweigerdt, Robert; Mandenius, Carl-Fredrik

    2016-01-01

    Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here, we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging. PMID:27052611

  10. A Microfluidic Approach for Inducing Cell Rotation by Means of Hydrodynamic Forces.

    PubMed

    Torino, Stefania; Iodice, Mario; Rendina, Ivo; Coppola, Giuseppe; Schonbrun, Ethan

    2016-01-01

    Microfluidic technology allows to realize devices in which cells can be imaged in their three-dimensional shape. However, there are still some limitations in the method, due to the fact that cells follow a straight path while they are flowing in a channel. This can result in a loss in information, since only one side of the cell will be visible. Our work has started from the consideration that if a cell rotates, it is possible to overcome this problem. Several approaches have been proposed for cell manipulation in microfluidics. In our approach, cells are controlled by only taking advantages of hydrodynamic forces. Two different devices have been designed, realized, and tested. The first device induces cell rotation in a plane that is parallel (in-plane) to the observation plane, while the second one induce rotation in a plane perpendicular (out-of-plane) to the observation plane. PMID:27548187

  11. Microfluidic device for high-yield pairing and fusion of stem cells with somatic cells

    NASA Astrophysics Data System (ADS)

    Gel, Murat; Hirano, Kunio; Oana, Hidehiro; Kotera, Hidetoshi; Tada, Takashi; Washizu, Masao

    2011-12-01

    Electro cell fusion has significant potential as a biotechnology tool with applications ranging from antibody production to cellular reprogramming. However due to low fusion efficiency of the conventional electro fusion methodology the true potential of the technique has not been reached. In this paper, we report a new method which takes cell fusion efficiency two orders magnitude higher than the conventional electro fusion method. The new method, based on one-toone pairing, fusion and selection of fused cells was developed using a microfabricated device. The device was composed of two microfluidic channels, a micro slit array and a petri dish integrated with electrodes. The electrodes positioned in each channel were used to generate electric field lines concentrating in the micro slits. Cells were introduced into channels and brought in to contact through the micro slit array using dielectrophoresis. The cells in contact were fused by applying a DC pulse to electrodes. As the electric field lines were concentrated at the micro slits the membrane potential was induced only at the vicinity of the micro slits, namely only at the cell-cell contact point. This mechanism assured the minimum damage to cells in the fusion as well as the ability to control the strength and location of induced membrane potential. We introduced mouse embryonic stem cells and mouse embryonic fibroblasts to the microfluidic channels and demonstrated high-yield fusion (> 80%). Post-fusion study showed the method can generate viable hybrids of stem cells and embryonic fibroblasts. Multinucleated hybrid cells adhering on the chip surface were routinely obtained by using this method and on-chip culturing.

  12. Hydrodynamic guiding for addressing subsets of immobilized cells and molecules in microfluidic systems

    PubMed Central

    Brevig, Thomas; Krühne, Ulrich; Kahn, Rachel A; Ahl, Thomas; Beyer, Michael; Pedersen, Lars H

    2003-01-01

    Background The interest in microfluidics and surface patterning is increasing as the use of these technologies in diverse biomedical applications is substantiated. Controlled molecular and cellular surface patterning is a costly and time-consuming process. Methods for keeping multiple separate experimental conditions on a patterned area are, therefore, needed to amplify the amount of biological information that can be retrieved from a patterned surface area. We describe, in three examples of biomedical applications, how this can be achieved in an open microfluidic system, by hydrodynamically guiding sample fluid over biological molecules and living cells immobilized on a surface. Results A microfluidic format of a standard assay for cell-membrane integrity showed a fast and dose-dependent toxicity of saponin on mammalian cells. A model of the interactions of human mononuclear leukocytes and endothelial cells was established. By contrast to static adhesion assays, cell-cell adhesion in this dynamic model depended on cytokine-mediated activation of both endothelial and blood cells. The microfluidic system allowed the use of unprocessed blood as sample material, and a specific and fast immunoassay for measuring the concentration of C-reactive protein in whole blood was demonstrated. Conclusion The use of hydrodynamic guiding made multiple and dynamic experimental conditions on a small surface area possible. The ability to change the direction of flow and produce two-dimensional grids can increase the number of reactions per surface area even further. The described microfluidic system is widely applicable, and can take advantage of surfaces produced by current and future techniques for patterning in the micro- and nanometer scale. PMID:12875662

  13. On-chip gradient generation in 256 microfluidic cell cultures: simulation and experimental validation.

    PubMed

    Somaweera, Himali; Haputhanthri, Shehan O; Ibraguimov, Akif; Pappas, Dimitri

    2015-08-01

    A microfluidic diffusion diluter was used to create a stable concentration gradient for dose response studies. The microfluidic diffusion diluter used in this study consisted of 128 culture chambers on each side of the main fluidic channel. A calibration method was used to find unknown concentrations with 12% error. Flow rate dependent studies showed that changing the flow rates generated different gradient patterns. Mathematical simulations using COMSOL Multi-physics were performed to validate the experimental data. The experimental data obtained for the flow rate studies agreed with the simulation results. Cells could be loaded into culture chambers using vacuum actuation and cultured for long times under low shear stress. Decreasing the size of the culture chambers resulted in faster gradient formation (20 min). Mass transport into the side channels of the microfluidic diffusion diluter used in this study is an important factor in creating the gradient using diffusional mixing as a function of the distance. To demonstrate the device's utility, an H2O2 gradient was generated while culturing Ramos cells. Cell viability was assayed in the 256 culture chambers, each at a discrete H2O2 concentration. As expected, the cell viability for the high concentration side channels increased (by injecting H2O2) whereas the cell viability in the low concentration side channels decreased along the chip due to diffusional mixing as a function of distance. COMSOL simulations were used to identify the effective concentration of H2O2 for cell viability in each side chamber at 45 min. The gradient effects were confirmed using traditional H2O2 culture experiments. Viability of cells in the microfluidic device under gradient conditions showed a linear relationship with the viability of the traditional culture experiment. Development of the microfluidic device used in this study could be used to study hundreds of concentrations of a compound in a single experiment. PMID:26050759

  14. Modeling the role of nuclear mechanics in determining cell shape and motility through microfluidic channels

    NASA Astrophysics Data System (ADS)

    Shechter, Jake; Maki, Kara; Das, Moumita

    2014-03-01

    Cell mechanics and migration through tight spaces are critical to life processes such as immune response and fertilization, in several diseases, and in diagnostics and drug delivery. For example, breast cancer cells have been shown to deform more easily and transit more rapidly through microfluidic channels than healthy breast cells. In this computational biophysics project, we simulate a cell moving through a microfluidic channel. We calculate the deformation energy of a model cell, which includes contributions from the cell cytoskeleton and the cell nucleus. We study how the model cell deforms in response to external forces, focusing on the deformability of the cell as it squeezes into and through a microfluidic channel and how the nucleus plays a part in this. Recent experiments suggest that the nucleus can be up to an order of magnitude stiffer than the rest of the cell and our results may provide insights into how the nucleus influences cell mechanics and migration. This work was supported by a FEAD grant from the College of Science at Rochester Institute of Technology.

  15. High-throughput microfluidic device for single cell analysis using multiple integrated soft lithographic pumps.

    PubMed

    Patabadige, Damith E W; Mickleburgh, Tom; Ferris, Lorin; Brummer, Gage; Culbertson, Anne H; Culbertson, Christopher T

    2016-05-01

    The ability to accurately control fluid transport in microfluidic devices is key for developing high-throughput methods for single cell analysis. Making small, reproducible changes to flow rates, however, to optimize lysis and injection using pumps external to the microfluidic device are challenging and time-consuming. To improve the throughput and increase the number of cells analyzed, we have integrated previously reported micropumps into a microfluidic device that can increase the cell analysis rate to ∼1000 cells/h and operate for over an hour continuously. In order to increase the flow rates sufficiently to handle cells at a higher throughput, three sets of pumps were multiplexed. These pumps are simple, low-cost, durable, easy to fabricate, and biocompatible. They provide precise control of the flow rate up to 9.2 nL/s. These devices were used to automatically transport, lyse, and electrophoretically separate T-Lymphocyte cells loaded with Oregon green and 6-carboxyfluorescein. Peak overlap statistics predicted the number of fully resolved single-cell electropherograms seen. In addition, there was no change in the average fluorescent dye peak areas indicating that the cells remained intact and the dyes did not leak out of the cells over the 1 h analysis time. The cell lysate peak area distribution followed that expected of an asynchronous steady-state population of immortalized cells. PMID:26887846

  16. Development of microfluidic system and optical tweezers for electrophysiological investigations of an individual cell

    NASA Astrophysics Data System (ADS)

    Alrifaiy, A.; Bitaraf, N.; Lindahl, O.; Ramser, K.

    2010-08-01

    We present a new approach of combining Lab-on-a-chip technologies with optical manipulation technique for accurate investigations in the field of cell biology. A general concept was to develop and combine different methods to perform advanced electrophysiological investigations of an individual living cell under optimal control of the surrounding environment. The conventional patch clamp technique was customized by modifying the open system with a gas-tight multifunctional microfluidics system and optical trapping technique (optical tweezers). The system offers possibilities to measure the electrical signaling and activity of the neuron under optimum conditions of hypoxia and anoxia while the oxygenation state is controlled optically by means of a spectroscopic technique. A cellbased microfluidics system with an integrated patch clamp pipette was developed successfully. Selectively, an individual neuron is manipulated within the microchannels of the microfluidic system under a sufficient control of the environment. Experiments were performed to manipulate single yeast cell and red blood cell (RBC) optically through the microfluidics system toward an integrated patch clamp pipette. An absorption spectrum of a single RCB was recorded which showed that laser light did not impinge on the spectroscopic spectrum of light. This is promising for further development of a complete lab-on-a-chip system for patch clamp measurements.

  17. When cells and microbes meet in Krakow.

    PubMed

    Rajalingam, Krishnaraj; van der Goot, F Gisou

    2011-03-01

    The sixth edition of the biannual 'At the Joint Edge of Cellular Microbiology & Cell Biology' meeting took place in October 2010 in Krakow with the support of EMBO. The meeting highlighted that research at the interface between cell biology and microbiology continues to bloom, and that cell biologists still have a lot to learn from bugs, as do microbiologists from cell biology. PMID:21350503

  18. Development of a microfluidic platform with integrated power splitting waveguides for optogenetic neural cell stimulation.

    PubMed

    Feng, Hongtao; Shu, Weiliang; Chen, Xi; Zhang, Yuanyuan; Lu, Yi; Wang, Liping; Chen, Yan

    2015-10-01

    We present a microfluidic platform with integrated power splitting waveguides for optogenetic neural cell stimulation. A liquid-core/PDMS-cladding waveguide with a power splitter design was integrated with a neural cell culture chamber to provide a simple way of precise localized optical stimulation. The parallel on-chip excitation of individual neural cells using a single optical fiber input is demonstrated for optogenetic neural cell studies, and the excitation of each individual waveguide can be independently controlled by pneumatic valves. Light delivery and loss mechanisms through the waveguides were studied and characterized. The waveguide power splitter platform is capable of providing sufficient irradiance to evoke spikes in ChR2-expressing neural cells. The system enables high-resolution stimulation of neural cells in a controllable manner. The microfluidic platform described here represents a novel methodology for studying optogenetics in a compact integrated system with high spatial resolutions. PMID:26371060

  19. Microfluidic sieve valves

    SciTech Connect

    Quake, Stephen R; Marcus, Joshua S; Hansen, Carl L

    2015-01-13

    Sieve valves for use in microfluidic device are provided. The valves are useful for impeding the flow of particles, such as chromatography beads or cells, in a microfluidic channel while allowing liquid solution to pass through the valve. The valves find particular use in making microfluidic chromatography modules.

  20. High-Throughput Microfluidic Device for Circulating Tumor Cell Isolation from Whole Blood

    PubMed Central

    Yang, Daniel K.; Leong, Serena; Sohn, Lydia L.

    2016-01-01

    Circulating tumor cells (CTCs) are promising markers to determine cancer patient prognosis and track disease response to therapy. We present a multi-stage microfluidic device we have developed that utilizes inertial and Dean drag forces for isolating CTCs from whole blood. We demonstrate a 94.2% ± 2.1% recovery of cancer cells with our device when screening whole blood spiked with MCF-7 GFP cells.

  1. Preparation of Nucleic Acid Libraries for Personalized Sequencing Systems Using an Integrated Microfluidic Hub Technology (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

    ScienceCinema

    Patel, Kamlesh D [Ken]; SNL,

    2013-01-25

    Kamlesh (Ken) Patel from Sandia National Laboratories (Livermore, California) presents "Preparation of Nucleic Acid Libraries for Personalized Sequencing Systems Using an Integrated Microfluidic Hub Technology " at the 7th Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting held in June, 2012 in Santa Fe, NM.

  2. An integrated microfluidic device for rapid cell lysis and DNA purification of epithelial cell samples.

    PubMed

    Ha, Seung-Mo; Cho, Woong; Ahn, Yoomin; Hwang, Seung Yong

    2011-05-01

    In this paper, we describe the design and fabrication of a microfluidic device for cell lysis and DNA purification, and the results of device tests using a real sample of buccal cells. Cell lysis was thermally executed for two minutes at 80 degrees C in a serpentine type microreactor (20 microL) using an Au microheater with a microsensor. The DNA was then mixed with other residual products and purified by a new filtration process involving micropillars and 50-80 microm microbeads. The entire process of sample loading, cell lysis, DNA purification, and sample extraction was successfully completed in the microchip within five minutes. Sample preparation within the microchip was verified by performing a SY158 gene PCR analysis and gel electrophoresis on the products obtained from the chip. The new purification method enhanced DNA purity from 0.93 to 1.62 after purification. PMID:21780436

  3. Maximizing Fibroblast Adhesion on Protein-Coated Surfaces Using Microfluidic Cell Printing

    PubMed Central

    Davidoff, S.N.; Au, D.; Gale, B.K.; Brooks, B.D.; Brooks, A.E.

    2015-01-01

    translation of in vitro cell based assays to in vivo cellular response is imprecise at best. The advent of three-dimensional cell cultures in addition to bioreactor type microfluidics has improved the situation. However, these technical advances cannot be easily combined due to practical limitations. Development of a vertical microfluidic cell printer overcomes this obstacle, providing the ability to more closely recapitulate complex cellular environments and responses. As a proof of concept, we investigated the adhesion of fibroblasts under flow on protein-coated surfaces using a novel vertical microfluidic print head to isolate and manipulate both mechanical and biological factors as a model of fibroblast behavior during the foreign body response following implant insertion. A low flow rate with larger microfluidic channels onto a serum-coated surface has been determined to allow the highest density of viable fibroblasts to attach to the surface. While these insights into fibroblast surface attachment may lead to better material designs, the methods developed herein will certainly be useful as a biomaterials testing platform. PMID:26989480

  4. Entropy-based separation of yeast cells using a microfluidic system of conjoined spheres

    SciTech Connect

    Huang, Kai-Jian; Qin, S.-J. Bai, Zhong-Chen; Zhang, Xin; Mai, John D.

    2013-11-21

    A physical model is derived to create a biological cell separator that is based on controlling the entropy in a microfluidic system having conjoined spherical structures. A one-dimensional simplified model of this three-dimensional problem in terms of the corresponding effects of entropy on the Brownian motion of particles is presented. This dynamic mechanism is based on the Langevin equation from statistical thermodynamics and takes advantage of the characteristics of the Fokker-Planck equation. This mechanism can be applied to manipulate biological particles inside a microfluidic system with identical, conjoined, spherical compartments. This theoretical analysis is verified by performing a rapid and a simple technique for separating yeast cells in these conjoined, spherical microfluidic structures. The experimental results basically match with our theoretical model and we further analyze the parameters which can be used to control this separation mechanism. Both numerical simulations and experimental results show that the motion of the particles depends on the geometrical boundary conditions of the microfluidic system and the initial concentration of the diffusing material. This theoretical model can be implemented in future biophysics devices for the optimized design of passive cell sorters.

  5. Biodegradable microsphere-mediated cell perforation in microfluidic channel using femtosecond laser

    NASA Astrophysics Data System (ADS)

    Ishii, Atsuhiro; Ariyasu, Kazumasa; Mitsuhashi, Tatsuki; Heinemann, Dag; Heisterkamp, Alexander; Terakawa, Mitsuhiro

    2016-05-01

    The use of small particles has expanded the capability of ultrashort pulsed laser optoinjection technology toward simultaneous treatment of multiple cells. The microfluidic platform is one of the attractive systems that has obtained synergy with laser-based technology for cell manipulation, including optoinjection. We have demonstrated the delivery of molecules into suspended-flowing cells in a microfluidic channel by using biodegradable polymer microspheres and a near-infrared femtosecond laser pulse. The use of polylactic-co-glycolic acid microspheres realized not only a higher optoinjection ratio compared to that with polylactic acid microspheres but also avoids optical damage to the microfluidic chip, which is attributable to its higher optical intensity enhancement at the localized spot under a microsphere. Interestingly, optoinjection ratios to nucleus showed a difference for adhered cells and suspended cells. The use of biodegradable polymer microspheres provides high throughput optoinjection; i.e., multiple cells can be treated in a short time, which is promising for various applications in cell analysis, drug delivery, and ex vivo gene transfection to bone marrow cells and stem cells without concerns about residual microspheres.

  6. Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.

    PubMed

    Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

    2010-08-17

    We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost. PMID:20679245

  7. Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

    PubMed Central

    Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

    2010-01-01

    We propose a unique method for cell sorting, “Ephesia,” using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples—blood, pleural effusion, and fine needle aspirates— issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost. PMID:20679245

  8. Spatial control over cell attachment by partial solvent entrapment of poly lysine in microfluidic channels

    PubMed Central

    Baman, Nicki K; Schneider, Galen B; Terry, Treniece L; Zaharias, Rebecca; Salem, Aliasger K

    2006-01-01

    We demonstrate spatial control over cell attachment on biodegradable surfaces by flowing cell adhesive poly (D-lysine) (PDL) in a trifluoroethanol (TFE)–water mixture through microfluidic channels placed on a biodegradable poly (lactic acid)–poly (ethylene glycol) (PLA–PEG) substrate. The partial solvent mixture swells the PLA–PEG within the confines of the microfluidic channels allowing PDL to diffuse on to the surface gel layer. When excess water is flowed through the channels substituting the TFE–water mixture, the swollen PLA surface collapses, entrapping PDL polymer. Results using preosteoblast human palatal mesenchymal cells (HEPM) indicate that this new procedure can be used for facile attachment of cells in localized regions. The PEG component of the PLA–PEG copolymer prevents cells from binding to the nonpatterned regions. PMID:17722538

  9. 3D pulsed laser-triggered high-speed microfluidic fluorescence-activated cell sorter.

    PubMed

    Chen, Yue; Wu, Ting-Hsiang; Kung, Yu-Chun; Teitell, Michael A; Chiou, Pei-Yu

    2013-11-12

    We report a 3D microfluidic pulsed laser-triggered fluorescence-activated cell sorter capable of sorting at a throughput of 23 000 cells per s with 90% purity in high-purity mode and at a throughput of 45 000 cells per s with 45% purity in enrichment mode in one stage and in a single channel. This performance is realized by exciting laser-induced cavitation bubbles in a 3D PDMS microfluidic channel to generate high-speed liquid jets that deflect detected fluorescent cells and particles focused by 3D sheath flows. The ultrafast switching mechanism (20 μs complete on-off cycle), small liquid jet perturbation volume, and three-dimensional sheath flow focusing for accurate timing control of fast (1.5 m s(-1)) passing cells and particles are three critical factors enabling high-purity sorting at high-throughput in this sorter. PMID:23844418

  10. Microfluidic isolation of cancer-cell-derived microvesicles from hetergeneous extracellular shed vesicle populations

    PubMed Central

    Santana, Steven M.; Antonyak, Marc A.; Cerione, Richard A.

    2015-01-01

    Extracellular shed vesicles, including exosomes and microvesicles, are disseminated throughout the body and represent an important conduit of cell communication. Cancer-cell-derived microvesicles have potential as a cancer biomarker as they help shape the tumor microenvironment to promote the growth of the primary tumor and prime the metastatic niche. It is likely that, in cancer cell cultures, the two constituent extracellular shed vesicle subpopulations, observed in dynamic light scattering, represent an exosome population and a cancer-cell-specific microvesicle population and that extracellular shed vesicle size provides information about provenance and cargo. We have designed and implemented a novel microfluidic technology that separates microvesicles, as a function of diameter, from heterogeneous populations of cancer-cell-derived extracellular shed vesicles. We measured cargo carried by the microvesicle subpopulation processed through this microfluidic platform. Such analyses could enable future investigations to more accurately and reliably determine provenance, functional activity, and mechanisms of transformation in cancer. PMID:25342569

  11. Microfluidic isolation of cancer-cell-derived microvesicles from hetergeneous extracellular shed vesicle populations.

    PubMed

    Santana, Steven M; Antonyak, Marc A; Cerione, Richard A; Kirby, Brian J

    2014-12-01

    Extracellular shed vesicles, including exosomes and microvesicles, are disseminated throughout the body and represent an important conduit of cell communication. Cancer-cell-derived microvesicles have potential as a cancer biomarker as they help shape the tumor microenvironment to promote the growth of the primary tumor and prime the metastatic niche. It is likely that, in cancer cell cultures, the two constituent extracellular shed vesicle subpopulations, observed in dynamic light scattering, represent an exosome population and a cancer-cell-specific microvesicle population and that extracellular shed vesicle size provides information about provenance and cargo. We have designed and implemented a novel microfluidic technology that separates microvesicles, as a function of diameter, from heterogeneous populations of cancer-cell-derived extracellular shed vesicles. We measured cargo carried by the microvesicle subpopulation processed through this microfluidic platform. Such analyses could enable future investigations to more accurately and reliably determine provenance, functional activity, and mechanisms of transformation in cancer. PMID:25342569

  12. Probing the mechanical properties of brain cancer cells using a microfluidic cell squeezer device

    PubMed Central

    Khan, Z. S.; Vanapalli, S. A.

    2013-01-01

    Despite being invasive within surrounding brain tissues and the central nervous system, little is known about the mechanical properties of brain tumor cells in comparison with benign cells. Here, we present the first measurements of the peak pressure drop due to the passage of benign and cancerous brain cells through confined microchannels in a “microfluidic cell squeezer” device, as well as the elongation, speed, and entry time of the cells in confined channels. We find that cancerous and benign brain cells cannot be differentiated based on speeds or elongation. We have found that the entry time into a narrow constriction is a more sensitive indicator of the differences between malignant and healthy glial cells than pressure drops. Importantly, we also find that brain tumor cells take a longer time to squeeze through a constriction and migrate more slowly than benign cells in two dimensional wound healing assays. Based on these observations, we arrive at the surprising conclusion that the prevailing notion of extraneural cancer cells being more mechanically compliant than benign cells may not apply to brain cancer cells. PMID:24403988

  13. Comparison of Chip Inlet Geometry in Microfluidic Devices for Cell Studies.

    PubMed

    Sun, Yung-Shin

    2016-01-01

    Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies best mimicking the in vivo micro-environment. These devices are capable of creating precise and controllable surroundings of pH value, temperature, salt concentration, and other physical or chemical stimuli. Various cell studies such as chemotaxis and electrotaxis can be performed by using such devices. Moreover, microfluidic chips are designed and fabricated for applications in cell separations such as circulating tumor cell (CTC) chips. Usually, there are two most commonly used inlets in connecting the microfluidic chip to sample/reagent loading tubes: the vertical (top-loading) inlet and the parallel (in-line) inlet. Designing this macro-to-micro interface is believed to play an important role in device performance. In this study, by using the commercial COMSOL Multiphysics software, we compared the cell capture behavior in microfluidic devices with different inlet types and sample flow velocities. Three different inlets were constructed: the vertical inlet, the parallel inlet, and the vertically parallel inlet. We investigated the velocity field, the flow streamline, the cell capture rate, and the laminar shear stress in these inlets. It was concluded that the inlet should be designed depending on the experimental purpose, i.e., one wants to maximize or minimize cell capture. Also, although increasing the flow velocity could reduce cell sedimentation, too high shear stresses are thought harmful to cells. Our findings indicate that the inlet design and flow velocity are crucial and should be well considered in fabricating microfluidic devices for cell studies. PMID:27314318

  14. Uncovering stem-cell heterogeneity in the microniche with label-free microfluidics

    NASA Astrophysics Data System (ADS)

    Sohn, Lydia L.

    2013-03-01

    Better suited for large number of cells from bulk tissue, traditional cell-screening techniques, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), cannot easily screen stem or progenitor cells from minute populations found in their physiological niches. Furthermore, they rely upon irreversible antibody binding, potentially altering cell properties, including gene expression and regenerative capacity. We have developed a label-free, single-cell analysis microfluidic platform capable of quantifying cell-surface marker expression of functional organ stem cells directly isolated from their micro-anatomical niche. With this platform, we have screened single quiescent muscle stem (satellite) cells derived from single myofibers, and we have uncovered an important heterogeneity in the surface-marker expression of these cells. By sorting the screened cells with our microfluidic device, we have determined what this heterogeneity means in terms of muscle stem-cell functionality. For instance, we show that the levels of beta1-integrin can predict the differentiation capacity of quiescent satellite cells, and in contrast to recent literature, that some CXCR4 + cells are not myogenic. Our results provide the first direct demonstration of a microniche-specific variation in gene expression in stem cells of the same lineage. Overall, our label-free, single-cell analysis and cell-sorting platform could be extended to other systems involving rare-cell subsets. This work was funded by the W. M. Keck Foundation, NIH, and California Institute of Regenerative Medicine

  15. Microfluidic cell sorter for use in developing red fluorescent proteins with improved photostability

    PubMed Central

    Davis, Lloyd M.; Lubbeck, Jennifer L.; Dean, Kevin M.; Palmer, Amy E.; Jimenez, Ralph

    2014-01-01

    This paper presents a novel microfluidic cytometer for mammalian cells that rapidly measures the irreversible photobleaching of red fluorescent proteins expressed within each cell and achieves high purity (>99%) selection of individual cells based on these measurements. The selection is achieved by using sub-millisecond timed control of a piezo-tilt mirror to steer a focused 1064-nm laser spot for optical gradient force switching following analysis of the fluorescence signals from passage of the cell through a series of 532-nm laser beams. In transit through each beam, the fluorescent proteins within the cell undergo conversion to dark states, but the microfluidic chip enables the cell to pass sufficiently slowly that recovery from reversible dark states occurs between beams, thereby enabling irreversible photobleaching to be quantified separately from the reversible dark-state conversion. The microfluidic platform achieves sorting of samples down to sub-millilitre volumes with minimal loss, wherein collected cells remain alive and can subsequently proliferate. The instrument provides a unique first tool for rapid selection of individual mammalian cells on the merits of photostability and is likely to form the basis of subsequent lab-on-a-chip platforms that combine photobleaching with other spectroscopic measurements for on-going research to develop advanced red fluorescent proteins by screening of genetic libraries. PMID:23636097

  16. Surface Design for Efficient Capturing of Rare Cells in Microfluidic Device

    NASA Astrophysics Data System (ADS)

    Liu, Yaling; Depietro, Dan; Thomas, Antony; Chen, Chi-Mon; Yang, Shu

    2011-11-01

    This work aims to design, fabricate, and characterize a micro-patterned surface that will be integrated into microfluidic devices to enhance particle and rare cell capture efficiency. Capture of ultralow concentration of circulating tumor cells in a blood sample is of vital importance for early diagnostics of cancer diseases. Despite the significant progress achieved in development of cell capture techniques, the enhancement in capture efficiency is still limited and often accompanied with drawbacks such as low throughput, low selectivity, pre-diluting requirement, and cell viability issues. The goal of this work is to design a biomimetic surface that could significantly enhance particle/cell capture efficacy through computational modeling, surface patterning, and microfluidic integration and testing. A PDMS surface with microscale ripples is functionalized with epithelial cell adhesion molecule (EpCAM) to capture prostate cancer PC3 cells. Our microfluid chip with micropatterns has shown significantly higher cell capture efficiency and selectivity compared to the chips with plane surface or classical herringbone-grooves.

  17. Surface Design for Efficient Capturing of Rare Cells in Microfluidic Device

    NASA Astrophysics Data System (ADS)

    Liu, Yaling; Thomas, Antony; Chen, Chi-Mon; Yang, Shu

    2012-02-01

    This work aims to design, fabricate, and characterize a micro-patterned surface that will be integrated into microfluidic devices to enhance particle and rare cell capture efficiency. Capture of ultralow concentration of circulating tumor cells in a blood sample is of vital importance for early diagnostics of cancer diseases. Despite the significant progress achieved in development of cell capture techniques, the enhancement in capture efficiency is still limited and often accompanied with drawbacks such as low throughput, low selectivity, pre-diluting requirement, and cell viability issues. The goal of this work is to design a biomimetic surface that could significantly enhance particle/cell capture efficacy through computational modeling, surface patterning, and microfluidic integration and testing. A PDMS surface with microscale ripples is functionalized with epithelial cell adhesion molecule (EpCAM) to capture prostate cancer PC3 cells. Our microfluid chip with micropatterns has shown significantly higher cell capture efficiency and selectivity compared to the chips with plane surface or classical herringbone-grooves.

  18. Designing and modeling a centrifugal microfluidic device to separate target blood cells

    NASA Astrophysics Data System (ADS)

    Shamloo, Amir; Selahi, AmirAli; Madadelahi, Masoud

    2016-03-01

    The objective of this study is to design a novel and efficient portable lab-on-a-CD (LOCD) microfluidic device for separation of specific cells (target cells) using magnetic beads. In this study the results are shown for neutrophils as target cells. However, other kinds of target cells can be separated in a similar approach. The designed microfluidics can be utilized as a point of care system for neutrophil detection. This microfluidic system employs centrifugal and magnetic forces for separation. After model validation by the experimental data in the literature (that may be used as a design tool for developing centrifugo-magnetophoretic devices), two models are presented for separation of target cells using magnetic beads. The first model consists of one container in the inlet section and two containers in the outlets. Initially, the inlet container is filled with diluted blood sample which is a mixture of red blood cells (RBCs) plus neutrophils which are attached to Magnetic beads. It is shown that by using centrifugal and magnetic forces, this model can separate all neutrophils with recovery factor of ~100%. In the second model, due to excess of magnetic beads in usual experimental analysis (to ensure that all target cells are attached to them) the geometry is improved by adding a third outlet for these free magnetic beads. It is shown that at angular velocity of 45 rad s-1, recovery factor of 100% is achievable for RBCs, free magnetic beads and neutrophils as target cells.

  19. 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.

  20. Rapid and cheap prototyping of a microfluidic cell sorter.

    PubMed

    Islam, M Z; McMullin, J N; Tsui, Y Y

    2011-05-01

    Development of a microfluidic device is generally based on fabrication-design-fabrication loop, as, unlike the microelectronics design, there is no rigorous simulation-based verification of the chip before fabrication. This usually results in extremely long, and hence expensive, product development cycle if micro/nano fabrication facilities are used from the beginning of the cycle. Here, we illustrate a novel approach of device prototyping that is fast, cheap, reliable, and most importantly, this technique can be adopted even if no state-of-the-art microfabrication facility is available. A water-jet machine is used to cut the desired microfluidic channels into a thin steel plate which is then used as a template to cut the channels into a thin sheet of a transparent and cheap polymer material named Surlyn® by using a Hot Knife™. The feature-inscribed Surlyn sheet is bonded in between two microscope glass slides by utilizing the techniques which has been being used in curing polymer film between dual layer automotive glasses for years. Optical fibers are inserted from the sides of chip and are bonded by UV epoxy. To study the applicability of this prototyping approach, we made a basic microfluidic sorter and tested its functionalities. Sample containing microparticles is injected into the chip. Light from a 532-nm diode laser is coupled into the optical fiber that delivers light to the interrogation region in the channel. The emitted light from the particle is collected by a photodiode (PD) placed over the detection window. The device sorts the particles into the sorted or waste outlets depending on the level of the PD signal. We used fluorescent latex beads to test the detection and sorting functionalities of the device. We found that the system could detect all the beads that passed through its geometric observation region and could sort almost all the beads it detected. PMID:21491584

  1. Engineered three-dimensional microfluidic device for interrogating cell-cell interactions in the tumor microenvironment

    PubMed Central

    Hockemeyer, K.; Janetopoulos, C.; Terekhov, A.; Hofmeister, W.; Vilgelm, A.; Costa, Lino; Wikswo, J. P.; Richmond, A.

    2014-01-01

    Stromal cells in the tumor microenvironment play a key role in the metastatic properties of a tumor. It is recognized that cancer-associated fibroblasts (CAFs) and endothelial cells secrete factors capable of influencing tumor cell migration into the blood or lymphatic vessels. We developed a microfluidic device that can be used to image the interactions between stromal cells and tumor cell spheroids in a three dimensional (3D) microenvironment while enabling external control of interstitial flow at an interface, which supports endothelial cells. The apparatus couples a 200-μm channel with a semicircular well to mimic the interface of a blood vessel with the stroma, and the design allows for visualization of the interactions of interstitial flow, endothelial cells, leukocytes, and fibroblasts with the tumor cells. We observed that normal tissue-associated fibroblasts (NAFs) contribute to the “single file” pattern of migration of tumor cells from the spheroid in the 3D microenvironment. In contrast, CAFs induce a rapid dispersion of tumor cells out of the spheroid with migration into the 3D matrix. Moreover, treatment of tumor spheroid cultures with the chemokine CXCL12 mimics the effect of the CAFs, resulting in similar patterns of dispersal of the tumor cells from the spheroid. Conversely, addition of CXCL12 to co-cultures of NAFs with tumor spheroids did not mimic the effects observed with CAF co-cultures, suggesting that NAFs produce factors that stabilize the tumor spheroids to reduce their migration in response to CXCL12. PMID:25379090

  2. Engineered three-dimensional microfluidic device for interrogating cell-cell interactions in the tumor microenvironment.

    PubMed

    Hockemeyer, K; Janetopoulos, C; Terekhov, A; Hofmeister, W; Vilgelm, A; Costa, Lino; Wikswo, J P; Richmond, A

    2014-07-01

    Stromal cells in the tumor microenvironment play a key role in the metastatic properties of a tumor. It is recognized that cancer-associated fibroblasts (CAFs) and endothelial cells secrete factors capable of influencing tumor cell migration into the blood or lymphatic vessels. We developed a microfluidic device that can be used to image the interactions between stromal cells and tumor cell spheroids in a three dimensional (3D) microenvironment while enabling external control of interstitial flow at an interface, which supports endothelial cells. The apparatus couples a 200-μm channel with a semicircular well to mimic the interface of a blood vessel with the stroma, and the design allows for visualization of the interactions of interstitial flow, endothelial cells, leukocytes, and fibroblasts with the tumor cells. We observed that normal tissue-associated fibroblasts (NAFs) contribute to the "single file" pattern of migration of tumor cells from the spheroid in the 3D microenvironment. In contrast, CAFs induce a rapid dispersion of tumor cells out of the spheroid with migration into the 3D matrix. Moreover, treatment of tumor spheroid cultures with the chemokine CXCL12 mimics the effect of the CAFs, resulting in similar patterns of dispersal of the tumor cells from the spheroid. Conversely, addition of CXCL12 to co-cultures of NAFs with tumor spheroids did not mimic the effects observed with CAF co-cultures, suggesting that NAFs produce factors that stabilize the tumor spheroids to reduce their migration in response to CXCL12. PMID:25379090

  3. Enhancement of Renal Epithelial Cell Functions through Microfluidic-Based Coculture with Adipose-Derived Stem Cells

    PubMed Central

    Huang, Hui-Chun; Chang, Ya-Ju; Chen, Wan-Chun; Harn, Hans I-Chen; Tang, Ming-Jer

    2013-01-01

    Current hemodialysis has functional limitations and is insufficient for renal transplantation. The bioartificial tubule device has been developed to contribute to metabolic functions by implanting renal epithelial cells into hollow tubes and showed a higher survival rate in acute kidney injury patients. In healthy kidney, epithelial cells are surrounded by various types of cells that interact with extracellular matrices, which are primarily composed of laminin and collagen. The current study developed a microfluidic coculture platform to enhance epithelial cell function in bioartificial microenvironments with multiple microfluidic channels that are microfabricated by polydimethylsiloxane. Collagen gel (CG) encapsulated with adipose-derived stem cells (CG-ASC) was injected into a central microfluidic channel for three-dimensional (3D) culture. The resuspended Madin-Darby canine kidney (MDCK) cells were injected into nascent channels and formed an epithelial monolayer. In comparison to coculture different cells using the commercial transwell system, the current coculture device allowed living cell monitoring of both the MDCK epithelial monolayer and CG-ASC in a 3D microenvironment. By coculture with CG-ASC, the cell height was increased with columnar shapes in MDCK. Promotion of cilia formation and functional expression of the ion transport protein in MDCK were also observed in the cocultured microfluidic device. When applying fluid flow, the intracellular protein dynamics can be monitored in the current platform by using the time-lapse confocal microscopy and transfection of GFP-tubulin plasmid in MDCK. Thus, this microfluidic coculture device provides the renal epithelial cells with both morphological and functional improvements that may avail to develop bioartificial renal chips. PMID:23557379

  4. Portable sub-terahertz resonance spectrometer combined with microfluidic sample cell

    NASA Astrophysics Data System (ADS)

    Ferrance, Jerome P.; Khromov, Alexander; Moyer, Aaron; Khromova, Tatiana; Gelmont, Boris; Sizov, Igor; Globus, Tatiana

    2013-05-01

    Radiation in the Terahertz frequency range interacts with vibrations in the weakest molecular couplings such as hydrogen bonding, van der Waals forces, and hydrophobic interactions. The work presented demonstrates our efforts towards the development of a microfluidic device as the sample cell for presenting liquid samples within the detection region of a novel sub-THz spectrometer. The continuous-wave, frequency-domain spectrometer, operating at room temperature between 315 and 480 GHz with spectral resolution of 0.3 GHz, already demonstrated highly intense and specific signatures from nanogram samples of dry biological molecules and whole bacterial cells. The very low absorption by water in this sample cell will allow for the use of liquid samples to present cells and molecules in their natural environment. The microfluidic device design utilizes a set of channels formed with metal sidewalls to enhance the interaction between the THz radiation and the sample, increasing the sensitivity of the system. Combined with near field effects, through use of a detection probe close to the surface of the sample cell, spatial resolution less than the diffraction limit can be achieved, further reducing the amount of sample required for analysis. This work focuses on the design, and fabrication methods, which will allow implementation of the microfluidic sample cell device within the THz spectrometer. The device will be utilized for characterization of different cell types, showing that THz interrogation of liquid samples is possible.

  5. Particle collision dynamics in periodic asymmetric microfluidic obstacle arrays for rare cell capture

    NASA Astrophysics Data System (ADS)

    Smith, James; Gleghorn, Jason; Kirby, Brian

    2012-11-01

    Particle-obstacle collision dynamics in periodic microfluidic obstacle arrays are presented in the context of microfluidic devices for the capture of rare cells, such as circulating tumor cells (CTCs). A coupled CFD-particle advection simulation was used to calculate particle trajectories for low Reynolds number, low Stokes number flows. A rich range of deterministic transport modes was identified as a function of array geometry, and the resulting particle size-dependent collision rate highlights the usefulness of these arrays for high-efficiency, high-purity rare cell capture. A reduced-order model, assuming unidirectional flow and infinitesimal obstacles, captures most of the details of transport in these systems with an O (104) computational saving; this model is a useful tool for rapidly exploring a large design space and optimizing geometries for a specific rare cell capture application. Results of the CFD simulations, reduced-order ballistic models, and experiments with polystyrene particles and cancer cells indicate that array geometry is central to rare cell capture and that simple models can be used to inform the design of these microfluidic devices. Present affiliation: Department of Chemical and Biological Engineering, Princeton University.

  6. Time-resolved NMR metabolomics of plant cells based on a microfluidic chip.

    PubMed

    Maisch, Jan; Kreppenhofer, Kristina; Büchler, Silke; Merle, Christian; Sobich, Shukhrat; Görling, Benjamin; Luy, Burkhard; Ahrens, Ralf; Guber, Andreas E; Nick, Peter

    2016-08-01

    The plant secondary metabolism generates numerous compounds harbouring pharmaceutical activity. In plants, these compounds are typically formed by different and specialised cell types that have to interact constituting a metabolic process chain. This interactivity impedes biotechnological production of secondary compounds, because cell differentiation is suppressed under the conditions of a batch bio-fermenter. We present a novel strategy to address this limitation using a biomimetic approach, where we simulate the situation in a real tissue by a microfluidic chamber system, where plant cells can be integrated into a process flow. We show that walled cells of the plant model tobacco BY-2 can be successfully cultivated in this system and that physiological parameters (such as cell viability, mitotic index and division synchrony) can be preserved over several days. The microfluidic design allows to resolve dynamic changes of specific metabolites over different stages of culture development. These results serve as proof-of-principle that a microfluidic organisation of cultivated plant cells can mimic the metabolic flows in a real plant tissue. PMID:27318870

  7. Sequencing Single Cell Microbial Genomes with Microfluidic Amplifications Tools (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

    SciTech Connect

    Quake, Steve

    2011-10-12

    Stanford University's Steve Quake on "Sequencing Single Cell Microbial Genomes with Microfluidic Amplification Tools" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

  8. Sequencing Single Cell Microbial Genomes with Microfluidic Amplifications Tools (MICW - Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

    ScienceCinema

    Quake, Steve [University of Stanford

    2013-01-22

    Stanford University's Steve Quake on "Sequencing Single Cell Microbial Genomes with Microfluidic Amplification Tools" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

  9. Rapid localized cell trapping on biodegradable polymers using cell surface derivatization and microfluidic networking.

    PubMed

    Sinclair, Jason; Salem, Aliasger K

    2006-03-01

    Spatial control over cell attachment is essential for controlling cell behavior and engineering cell-based sensor arrays. Here we report on a patterning procedure that can be utilized on a wide range of adherent and non-adherent cell types without the need to identify the exact peptide sequence or extracellular matrix (ECM) necessary for optimal cell attachment. This is achieved by converting native sialic residues present on the surface of most cells into non-native aldehydes using a mild sodium periodate treatment. The aldehyde groups are then reacted with biotin hydrazide to produce biotinylated cells. Avidin is patterned onto the surface of a biotinylated biodegradable block copolymer, polylactide-poly(ethylene glycol)-biotin (PLA-PEG-biotin) by microfluidic networking using a PDMS stamp. The biotinylated cells then bind specifically to the patterned avidin regions. The PEG that is presented from the PLA-PEG-biotin copolymer in the regions without avidin immobilization minimizes cell binding in the non-patterned regions. PMID:16307795

  10. A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry

    NASA Astrophysics Data System (ADS)

    Patibandla, Phani K.; Estrada, Rosendo; Kannan, Manasaa; Sethu, Palaniappan

    2014-03-01

    Flow cytometry is a powerful technique capable of simultaneous multi-parametric analysis of heterogeneous cell populations for research and clinical applications. In recent years, the flow cytometer has been miniaturized and made portable for application in clinical- and resource-limited settings. The sample preparation procedure, i.e. labeling of cells with antibodies conjugated to fluorescent labels, is a time consuming (˜45 min) and labor-intensive procedure. Microfluidics provides enabling technologies to accomplish rapid and automated sample preparation. Using an integrated microfluidic device consisting of a labeling and washing module, we demonstrate a new protocol that can eliminate sample handling and accomplish sample and reagent metering, high-efficiency mixing, labeling and washing in rapid automated fashion. The labeling module consists of a long microfluidic channel with an integrated chaotic mixer. Samples and reagents are precisely metered into this device to accomplish rapid and high-efficiency mixing. The mixed sample and reagents are collected in a holding syringe and held for up to 8 min following which the mixture is introduced into an inertial washing module to obtain ‘analysis-ready’ samples. The washing module consists of a high aspect ratio channel capable of focusing cells to equilibrium positions close to the channel walls. By introducing the cells and labeling reagents in a narrow stream at the center of the channel flanked on both sides by a wash buffer, the elution of cells into the wash buffer away from the free unbound antibodies is accomplished. After initial calibration experiments to determine appropriate ‘holding time’ to allow antibody binding, both modules were used in conjunction to label MOLT-3 cells (T lymphoblast cell line) with three different antibodies simultaneously. Results confirm no significant difference in mean fluorescence intensity values for all three antibodies labels (p < 0.01) between the

  11. Diagnostics of tumor cells by combination of Raman spectroscopy and microfluidics

    NASA Astrophysics Data System (ADS)

    Neugebauer, U.; Dochow, S.; Krafft, C.; Bocklitz, T.; Clement, J. H.; Popp, J.

    2011-07-01

    Circulating epithelial tumor cells are of increasing importance for tumor diagnosis and therapy monitoring of cancer patients. The definite identification of the rare tumor cells within numerous blood cells is challenging. Therefore, within the research initiative "Jenaer Zell-Identifizierungs-Gruppe" (JenZIG) we develop new methods for cell identification, micromanipulation and sorting based on spectroscopic methods and microfluidic systems. In this contribution we show, that classification models based on Raman spectroscopic analysis allow a precise discrimination of tumor cells from non-tumor cells with high prediction accuracies, up to more than 99% for dried cells. That holds true for unknown cell mixtures (tumor cells and leukocytes/erythrocytes) under dried conditions as well as in solution using the Raman laser as an optical tweezers to keep the cells in focus. We extended our studies by using a capillary system consisting of a quartz capillary, fiber optics and an adjustable fitting to trap cells. This system allows a prediction accuracy of 92.2% on the single cell level, and is a prerequisite for the development of a cell sorting and identification device based on a microfluidic chip. Initial experiments show that tumor cell lines can be differentiated from healthy leukocyte cells with an accuracy of more than 98%.

  12. Single Cell Mass Measurement Using Drag Force Inside Lab-on-Chip Microfluidics System.

    PubMed

    Rahman, Md Habibur; Ahmad, Mohd Ridzuan; Takeuchi, Masaru; Nakajima, Masahiro; Hasegawa, Yasuhisa; Fukuda, Toshio

    2015-12-01

    Single cell mass (SCM) is an intrinsic property of single cell, it arouses a great interest among scientists as cell mass depends on the synthesis of proteins, DNA replication, cell wall stiffness, cell cytoplasm density, cell growth, ribosome, and other analogous of organisms. To date, several great strides have been taken to the advancements of SCM measurement techniques. Nevertheless, more works are required to enable the technology to push frontier in deep analysis of SCM measurement, hence to elucidate intracellular properties. In this paper, we present a lab-on-chip microfluidics system for SCM measurement, related with the force required to drag a single cell and Newton's law of motion inside microfluidics channel. Drag force on the cell was generated by a pressure driven syringe micropump and the motion of the cell was measured using optical observation under an inverted microscope. This approach of measuring SCM was calibrated using known mass (77.3 pg) of a polystyrene particle of 5.2 μm diameter. Furthermore, we used Saccharomyces cerevisiae baker's yeast cells of different sizes ([Formula: see text] diameter) for SCM measurement. Mass of 4.4 μm diameter of single yeast cell was measured as 2.12 pg which is in the range of previously reported single yeast cell mass (2-3 pg). In addition, we also studied the relation between SCM and single cell size. Results showed that single yeast cell mass increases exponentially with the increasing of single cell size. PMID:26761952

  13. Rapid characterization of the biomechanical properties of drug-treated cells in a microfluidic device

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaofei; Chu, Henry K.; Zhang, Yang; Bai, Guohua; Wang, Kaiqun; Tan, Qiulin; Sun, Dong

    2015-10-01

    Cell mechanics is closely related to many cell functions. Recent studies have suggested that the deformability of cells can be an effective biomarker to indicate the onset and progression of diseases. In this paper, a microfluidic chip is designed for rapid characterization of the mechanics of drug-treated cells through stretching with dielectrophoresis (DEP) force. This chip was fabricated using PDMS and micro-electrodes were integrated and patterned on the ITO layer of the chip. Leukemia NB4 cells were considered and the effect of all-trans retinoic acid (ATRA) drug on NB4 cells were examined via the microfluidic chip. To induce a DEP force onto the cell, a relatively weak ac voltage was utilized to immobilize a cell at one side of the electrodes. The applied voltage was then increased to 3.5 V pp and the cell started to be stretched along the applied electric field lines. The elongation of the cell was observed using an optical microscope and the results showed that both types of cells were deformed by the induced DEP force. The strain of the NB4 cell without the drug treatment was recorded to be about 0.08 (time t = 180 s) and the drug-treated NB4 cell was about 0.21 (time t = 180 s), indicating a decrease in the stiffness after drug treatment. The elastic modulus of the cell was also evaluated and the modulus changed from 140 Pa to 41 Pa after drug treatment. This microfluidic chip can provide a simple and rapid platform for measuring the change in the biomechanical properties of cells and can potentially be used as the tool to determine the biomechanical effects of different drug treatments for drug discovery and development applications.

  14. Microfluidic Capture of Endothelial Colony-Forming Cells from Human Adult Peripheral Blood: Phenotypic and Functional Validation In Vivo

    PubMed Central

    Lin, Ruei-Zeng; Hatch, Adam; Antontsev, Victor G.; Murthy, Shashi K.

    2015-01-01

    Introduction: Endothelial colony-forming cells (ECFCs) are endothelial progenitors that circulate in peripheral blood and are currently the subject of intensive investigation due to their therapeutic potential. However, in adults, ECFCs comprise a very small subset among circulating cells, which makes their isolation a challenge. Materials and Methods: Currently, the standard method for ECFC isolation relies on the separation of mononuclear cells and erythrocyte lysis, steps that are time consuming and known to increase cell loss. Alternatively, we previously developed a novel disposable microfluidic platform containing antibody-functionalized degradable hydrogel coatings that is ideally suited for capturing low-abundance circulating cells from unprocessed blood. In this study, we reasoned that this microfluidic approach could effectively isolate rare ECFCs by virtue of their CD34 expression. Results: We conducted preclinical experiments with peripheral blood from four adult volunteers and demonstrated that the actual microfluidic capture of circulating CD34+ cells from unprocessed blood was compatible with the subsequent differentiation of these cells into ECFCs. Moreover, the ECFC yield obtained with the microfluidic system was comparable to that of the standard method. Importantly, we unequivocally validated the phenotypical and functional properties of the captured ECFCs, including the ability to form microvascular networks following transplantation into immunodeficient mice. Discussion: We showed that the simplicity and versatility of our microfluidic system could be very instrumental for ECFC isolation while preserving their therapeutic potential. We anticipate our results will facilitate additional development of clinically suitable microfluidic devices by the vascular therapeutic and diagnostic industry. PMID:25091645

  15. Volumetric measurement of human red blood cells by MOSFET-based microfluidic gate.

    PubMed

    Guo, Jinhong; Ai, Ye; Cheng, Yuanbing; Li, Chang Ming; Kang, Yuejun; Wang, Zhiming

    2015-08-01

    In this paper, we present a MOSFET-based (metal oxide semiconductor field-effect transistor) microfluidic gate to characterize the translocation of red blood cells (RBCs) through a gate. In the microfluidic system, the bias voltage modulated by the particles or biological cells is connected to the gate of MOSFET. The particles or cells can be detected by monitoring the MOSFET drain current instead of DC/AC-gating method across the electronic gate. Polystyrene particles with various standard sizes are utilized to calibrate the proposed device. Furthermore, RBCs from both adults and newborn blood sample are used to characterize the performance of the device in distinguishing the two types of RBCs. As compared to conventional DC/AC current modulation method, the proposed device demonstrates a higher sensitivity and is capable of being a promising platform for bioassay analysis. PMID:25349117

  16. A microfluidic live cell assay to study anthrax toxin induced cell lethality assisted by conditioned medium

    PubMed Central

    Shen, Jie; Cai, Changzu; Yu, Zhilong; Pang, Yuhong; Zhou, Ying; Qian, Lili; Wei, Wensheng; Huang, Yanyi

    2015-01-01

    It is technically challenging to investigate the function of secreted protein in real time by supply of conditioned medium that contains secreted protein of interest. The internalization of anthrax toxin is facilitated by a secreted protein Dickkopf-1 (DKK1) and its receptor, and eventually leads to cell lethality. To monitor the dynamic interplay between these components in live cells, we use an integrated microfluidic device to perform the cell viability assays with real-time controlled culture microenvironment in parallel. Conditioned medium, which contains the secreted proteins from specific cell lines, can be continuously pumped towards the cells that exposed to toxin. The exogenous DKK1 secreted from distant cells is able to rescue the sensitivity to toxin for those DKK1-knocked-down cells. This high-throughput assay allows us to precisely quantify the dynamic interaction between key components that cause cell death, and provide independent evidence of the function of DKK1 in the complex process of anthrax toxin internalization. PMID:25731605

  17. Magnetophoresis 'meets' viscoelasticity: deterministic separation of magnetic particles in a modular microfluidic device.

    PubMed

    Del Giudice, Francesco; Madadi, Hojjat; Villone, Massimiliano M; D'Avino, Gaetano; Cusano, Angela M; Vecchione, Raffaele; Ventre, Maurizio; Maffettone, Pier Luca; Netti, Paolo A

    2015-04-21

    The deflection of magnetic beads in a microfluidic channel through magnetophoresis can be improved if the particles are somehow focused along the same streamline in the device. We design and fabricate a microfluidic device made of two modules, each one performing a unit operation. A suspension of magnetic beads in a viscoelastic medium is fed to the first module, which is a straight rectangular-shaped channel. Here, the magnetic particles are focused by exploiting fluid viscoelasticity. Such a channel is one inlet of the second module, which is a H-shaped channel, where a buffer stream is injected in the second inlet. A permanent magnet is used to displace the magnetic beads from the original to the buffer stream. Experiments with a Newtonian suspending fluid, where no focusing occurs, are carried out for comparison. When viscoelastic focusing and magnetophoresis are combined, magnetic particles can be deterministically separated from the original streamflow to the buffer, thus leading to a high deflection efficiency (up to ~96%) in a wide range of flow rates. The effect of the focusing length on the deflection of particles is also investigated. Finally, the proposed modular device is tested to separate magnetic and non-magnetic beads. PMID:25732596

  18. Cardiac Meets Skeletal: What's New in Microfluidic Models for Muscle Tissue Engineering.

    PubMed

    Visone, Roberta; Gilardi, Mara; Marsano, Anna; Rasponi, Marco; Bersini, Simone; Moretti, Matteo

    2016-01-01

    In the last few years microfluidics and microfabrication technique principles have been extensively exploited for biomedical applications. In this framework, organs-on-a-chip represent promising tools to reproduce key features of functional tissue units within microscale culture chambers. These systems offer the possibility to investigate the effects of biochemical, mechanical, and electrical stimulations, which are usually applied to enhance the functionality of the engineered tissues. Since the functionality of muscle tissues relies on the 3D organization and on the perfect coupling between electrochemical stimulation and mechanical contraction, great efforts have been devoted to generate biomimetic skeletal and cardiac systems to allow high-throughput pathophysiological studies and drug screening. This review critically analyzes microfluidic platforms that were designed for skeletal and cardiac muscle tissue engineering. Our aim is to highlight which specific features of the engineered systems promoted a typical reorganization of the engineered construct and to discuss how promising design solutions exploited for skeletal muscle models could be applied to improve cardiac tissue models and vice versa. PMID:27571058

  19. Microfluidic devices with permeable polymer barriers for capture and transport of biomolecules and cells

    PubMed Central

    Lee, Ho Suk; Chu, Wai Keung; Zhang, Kun

    2013-01-01

    We report a method for fabricating permeable polymer microstructure barriers in polydimethylsiloxane (PDMS) microfluidic devices and the use of the devices to capture and transport DNA and cells. The polymer microstructure in a desired location in a fluidic channel is formed in situ by the polymerization of acrylamide and polyethylene diacrylate cross-linker (PEG-DA) monomer in a solution which is trapped in the location using a pair of PDMS valves. The porous polymer microstructure provides a mechanical barrier to convective fluid flow in the channel or between two microfluidic chambers while it still conducts ions or small charged species under an electric field, allowing for the rapid capture and transport of biomolecules and cells by electrophoresis. We have demonstrated the application of the devices for the rapid capture and efficient release of bacteriophage λ genomic DNA, solution exchange and for the transport and capture of HeLa cells. Our devices will enable the multi-step processing of biomolecules and cells or individual cells within a single microfluidic chamber. PMID:23828542

  20. Single Cell Response to Time-dependent Stimuli using a Microfluidic Bioreactor

    NASA Astrophysics Data System (ADS)

    Johnson-Chavarria, Eric M.; Agrawal, Utsav; Tanyeri, Melikhan; Kuhlman, Thomas E.; Schroeder, Charles M.

    2014-03-01

    Cellular adaptation is critical for survival under uncertain or dynamic environmental conditions. Recent studies have reported the ability of biological systems to implement low-pass filters to distinguish high frequency noise in environmental stimuli from lower frequency input signals, yet we still lack a complete understanding of this phenomenon. In this work, we report a microfluidic-based platform for single cell analysis that provides dynamic control over periodic, time-dependent culture media. Single cells are confined in free solution by the sole action of gentle fluid flow, thereby enabling non-perturbative trapping of cells for long time scales. In this way, our microfluidic-based technique provides the ability to control external stimuli with precise methods while observing non-adherent cells over long timescales. Using this approach, we observed intranucleoid diffusion of genetic repressor proteins released from a chromosomal binding array. Overall, this microfluidic approach provides a direct method for sustaining periodic environmental conditions, measuring growth rates, and detecting gene expression of single cells in free solution. Funded by NIH Pathway to Independence (PI) Award, 4R00HG004183-03. This work was supported by the National Science Foundation through a Graduate Research Fellowship to Eric M. Johnson-Chavarria.

  1. Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging.

    PubMed

    Quinto-Su, Pedro A; Lai, Hsuan-Hong; Yoon, Helen H; Sims, Christopher E; Allbritton, Nancy L; Venugopalan, Vasan

    2008-03-01

    We use time-resolved imaging to examine the lysis dynamics of non-adherent BAF-3 cells within a microfluidic channel produced by the delivery of single highly-focused 540 ps duration laser pulses at lambda = 532 nm. Time-resolved bright-field images reveal that the delivery of the pulsed laser microbeam results in the formation of a laser-induced plasma followed by shock wave emission and cavitation bubble formation. The confinement offered by the microfluidic channel constrains substantially the cavitation bubble expansion and results in significant deformation of the PDMS channel walls. To examine the cell lysis and dispersal of the cellular contents, we acquire time-resolved fluorescence images of the process in which the cells were loaded with a fluorescent dye. These fluorescence images reveal cell lysis to occur on the nanosecond to microsecond time scale by the plasma formation and cavitation bubble dynamics. Moreover, the time-resolved fluorescence images show that while the cellular contents are dispersed by the expansion of the laser-induced cavitation bubble, the flow associated with the bubble collapse subsequently re-localizes the cellular contents to a small region. This capacity of pulsed laser microbeam irradiation to achieve rapid cell lysis in microfluidic channels with minimal dilution of the cellular contents has important implications for their use in lab-on-a-chip applications. PMID:18305858

  2. Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging

    PubMed Central

    Quinto-Su, Pedro A.; Lai, Hsuan-Hong; Yoon, Helen H.; Sims, Christopher E.; Allbritton, Nancy L.; Venugopalan, Vasan

    2008-01-01

    We use time-resolved imaging to examine the lysis dynamics of non-adherent BAF-3 cells within a microfluidic channel produced by the delivery of single highly-focused 540 ps duration laser pulses at λ = 532 nm. Time-resolved bright-field images reveal that the delivery of the pulsed laser microbeam results in the formation of a laser-induced plasma followed by shock wave emission and cavitation bubble formation. The confinement offered by the microfluidic channel constrains substantially the cavitation bubble expansion and results in significant deformation of the PDMS channel walls. To examine the cell lysis and dispersal of the cellular contents, we acquire time-resolved fluorescence images of the process in which the cells were loaded with a fluorescent dye. These fluorescence images reveal cell lysis to occur on the nanosecond to microsecond time scale by the plasma formation and cavitation bubble dynamics. Moreover, the time-resolved fluorescence images show that while the cellular contents are dispersed by the expansion of the laser-induced cavitation bubble, the flow associated with the bubble collapse subsequently re-localizes the cellular contents to a small region. This capacity of pulsed laser microbeam irradiation to achieve rapid cell lysis in microfluidic channels with minimal dilution of the cellular contents has important implications for their use in lab-on-a-chip applications. PMID:18305858

  3. Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies.

    PubMed

    Wang, Xiaolin; Chen, Shuxun; Kong, Marco; Wang, Zuankai; Costa, Kevin D; Li, Ronald A; Sun, Dong

    2011-11-01

    Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper, we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells, we develop an image processing methodology with a recognition capability of multiple features, e.g., cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern, in which single as well as multiple laser traps are employed for cell transportation, and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach. PMID:21918752

  4. Induction of sustained glycolytic oscillations in single yeast cells using microfluidics and optical tweezers

    NASA Astrophysics Data System (ADS)

    Gustavsson, Anna-Karin; Adiels, Caroline B.; Goksör, Mattias

    2012-10-01

    Yeast glycolytic oscillations have been studied since the 1950s in cell free extracts and in intact cells. Until recently, sustained oscillations have only been observed in intact cells at the population level. The aim of this study was to investigate sustained glycolytic oscillations in single cells. Optical tweezers were used to position yeast cells in arrays with variable cell density in the junction of a microfluidic flow chamber. The microfluidic flow chambers were fabricated using soft lithography and the flow rates in the different inlet channels were individually controlled by syringe pumps. Due to the low Reynolds number, the solutions mixed by diffusion only. The environment in the junction of the chamber could thus be controlled by changing the flow rates in the inlet channels, with a complete change of environment within 2 s. The optimum position of the cell array was determined by simulations, to ensure complete coverage of the intended solution without any concentration gradients over the cell array. Using a DAPI filter set, the NADH auto fluorescence could be monitored in up to 100 cells simultaneously. Sustained oscillations were successfully induced in individual, isolated cells within specific flow rates and concentrations of glucose and cyanide. By changing the flow rates without changing the surrounding solution, it was found that the cell behavior was dependent on the concentration of chemicals in the medium rather than the flow rates in the range tested. Furthermore, by packing cells tightly, cell-to-cell interaction and synchronization could be studied.

  5. Specific capture and temperature-mediated release of cells in an aptamer-based microfluidic device†

    PubMed Central

    Zhu, Jing; Nguyen, ThaiHuu; Pei, Renjun; Stojanovic, Milan; Lin, Qiao

    2014-01-01

    Isolation of cells from heterogeneous mixtures is critically important in both basic cell biology studies and clinical diagnostics. Cell isolation can be realized based on physical properties such as size, density and electrical properties. Alternatively, affinity binding of target cells by surface-immobilized ligands, such as antibodies, can be used to achieve specific cell isolation. Microfluidics technology has recently been used in conjunction with antibody-based affinity isolation methods to capture, purify and isolate cells with higher yield rates, better efficiencies and lower costs. However, a method that allows easy release and collection of live cells from affinity surfaces for subsequent analysis and detection has yet to be developed. This paper presents a microfluidic device that not only achieves specific affinity capture and enrichment, but also enables non-destructive, temperature-mediated release and retrieval of cells. Specific cell capture is achieved using surface-immobilized aptamers in a microchamber. Release of the captured cells is realized by a moderate temperature change, effected via integrated heaters and a temperature sensor, to reversibly disrupt the cell-aptamer interaction. Experimental results with CCRF-CEM cells have demonstrated that the device is capable of specific capture and temperature-mediated release of cells, that the released cells remain viable and that the aptamer-functionalized surface is regenerable. PMID:22854859

  6. Poly(dimethylsiloxane) thin films as biocompatible coatings for microfluidic devices : cell culture and flow studies with glial cells.

    SciTech Connect

    Peterson, Sophie Louise; Sasaki, Darryl Yoshio; Gourley, Paul Lee; McDonald, Anthony Eugene

    2004-06-01

    Oxygen plasma treatment of poly(dimethylsiloxane) (PDMS) thin films produced a hydrophilic surface that was biocompatible and resistant to biofouling in microfluidic studies. Thin film coatings of PDMS were previously developed to provide protection for semiconductor-based microoptical devices from rapid degradation by biofluids. However, the hydrophobic surface of native PDMS induced rapid clogging of microfluidic channels with glial cells. To evaluate the various issues of surface hydrophobicity and chemistry on material biocompatibility, we tested both native and oxidized PDMS (ox-PDMS) coatings as well as bare silicon and hydrophobic alkane and hydrophilic oligoethylene glycol silane monolayer coated under both cell culture and microfluidic studies. For the culture studies, the observed trend was that the hydrophilic surfaces supported cell adhesion and growth, whereas the hydrophobic ones were inhibitive. However, for the fluidic studies, a glass-silicon microfluidic device coated with the hydrophilic ox-PDMS had an unperturbed flow rate over 14 min of operation, whereas the uncoated device suffered a loss in rate of 12%, and the native PDMS coating showed a loss of nearly 40%. Possible protein modification of the surfaces from the culture medium also were examined with adsorbed films of albumin, collagen, and fibrinogen to evaluate their effect on cell adhesion.

  7. Single-Cell Genetic Analysis Using Automated Microfluidics to Resolve Somatic Mosaicism.

    PubMed

    Szulwach, Keith E; Chen, Peilin; Wang, Xiaohui; Wang, Jing; Weaver, Lesley S; Gonzales, Michael L; Sun, Gang; Unger, Marc A; Ramakrishnan, Ramesh

    2015-01-01

    Somatic mosaicism occurs throughout normal development and contributes to numerous disease etiologies, including tumorigenesis and neurological disorders. Intratumor genetic heterogeneity is inherent to many cancers, creating challenges for effective treatments. Unfortunately, analysis of bulk DNA masks subclonal phylogenetic architectures created by the acquisition and distribution of somatic mutations amongst cells. As a result, single-cell genetic analysis is becoming recognized as vital for accurately characterizing cancers. Despite this, methods for single-cell genetics are lacking. Here we present an automated microfluidic workflow enabling efficient cell capture, lysis, and whole genome amplification (WGA). We find that ~90% of the genome is accessible in single cells with improved uniformity relative to current single-cell WGA methods. Allelic dropout (ADO) rates were limited to 13.75% and variant false discovery rates (SNV FDR) were 4.11x10(-6), on average. Application to ER-/PR-/HER2+ breast cancer cells and matched normal controls identified novel mutations that arose in a subpopulation of cells and effectively resolved the segregation of known cancer-related mutations with single-cell resolution. Finally, we demonstrate effective cell classification using mutation profiles with 10X average exome coverage depth per cell. Our data demonstrate an efficient automated microfluidic platform for single-cell WGA that enables the resolution of somatic mutation patterns in single cells. PMID:26302375

  8. Microfluidic device with chemical gradient for single-cell cytotoxicity assays.

    PubMed

    Hosokawa, Masahito; Hayashi, Takuma; Mori, Tetsushi; Yoshino, Tomoko; Nakasono, Satoshi; Matsunaga, Tadashi

    2011-05-15

    Here, we report the fabrication of a chemical gradient microfluidic device for single-cell cytotoxicity assays. This device consists of a microfluidic chemical gradient generator and a microcavity array that enables entrapment of cells with high efficiency at 88 ± 6% of the loaded cells. A 2-fold logarithmic chemical gradient generator that is capable of generating a serial 2-fold gradient was designed and then integrated with the microcavity array. High density single-cell entrapment was demonstrated in the device without cell damage, which was performed in 30 s. Finally, we validated the feasibility of this device to perform cytotoxicity assays by exposing cells to potassium cyanide (0-100 μM KCN). The device captured images of 4000 single cells affected by 6 concentrations of KCN and determined cell viability by counting the effected cells. Image scanning of the microcavity array was completed within 10 min using a 10× objective lens and a motorized stage. Aligning cells on the microcavity array eases cell counting, observation, imaging, and evaluation of singular cells. Thus, this platform was able to determine the cytotoxicity of chemicals at a single-cell level, as well as trace the cytotoxicity over time. This device and method will be useful for cytotoxicity analysis and basic biomedical research. PMID:21526753

  9. Continuous nucleus extraction by optically-induced cell lysis on a batch-type microfluidic platform.

    PubMed

    Huang, Shih-Hsuan; Hung, Lien-Yu; Lee, Gwo-Bin

    2016-04-12

    The extraction of a cell's nucleus is an essential technique required for a number of procedures, such as disease diagnosis, genetic replication, and animal cloning. However, existing nucleus extraction techniques are relatively inefficient and labor-intensive. Therefore, this study presents an innovative, microfluidics-based approach featuring optically-induced cell lysis (OICL) for nucleus extraction and collection in an automatic format. In comparison to previous micro-devices designed for nucleus extraction, the new OICL device designed herein is superior in terms of flexibility, selectivity, and efficiency. To facilitate this OICL module for continuous nucleus extraction, we further integrated an optically-induced dielectrophoresis (ODEP) module with the OICL device within the microfluidic chip. This on-chip integration circumvents the need for highly trained personnel and expensive, cumbersome equipment. Specifically, this microfluidic system automates four steps by 1) automatically focusing and transporting cells, 2) releasing the nuclei on the OICL module, 3) isolating the nuclei on the ODEP module, and 4) collecting the nuclei in the outlet chamber. The efficiency of cell membrane lysis and the ODEP nucleus separation was measured to be 78.04 ± 5.70% and 80.90 ± 5.98%, respectively, leading to an overall nucleus extraction efficiency of 58.21 ± 2.21%. These results demonstrate that this microfluidics-based system can successfully perform nucleus extraction, and the integrated platform is therefore promising in cell fusion technology with the goal of achieving genetic replication, or even animal cloning, in the near future. PMID:26987542

  10. Paramagnetic Structures within a Microfluidic Channel for Enhanced Immunomagnetic Isolation and Surface Patterning of Cells

    NASA Astrophysics Data System (ADS)

    Sun, Chen; Hassanisaber, Hamid; Yu, Richard; Ma, Sai; Verbridge, Scott S.; Lu, Chang

    2016-07-01

    In this report, we demonstrate a unique method for embedding magnetic structures inside a microfluidic channel for cell isolation. We used a molding process to fabricate these structures out of a ferrofluid of cobalt ferrite nanoparticles. We show that the embedded magnetic structures significantly increased the magnetic field in the channel, resulting in up to 4-fold enhancement in immunomagnetic capture as compared with a channel without these embedded magnetic structures. We also studied the spatial distribution of trapped cells both experimentally and computationally. We determined that the surface pattern of these trapped cells was determined by both location of the magnet and layout of the in-channel magnetic structures. Our magnetic structure embedded microfluidic device achieved over 90% capture efficiency at a flow velocity of 4 mm/s, a speed that was roughly two orders of magnitude faster than previous microfluidic systems used for a similar purpose. We envision that our technology will provide a powerful tool for detection and enrichment of rare cells from biological samples.

  11. Paramagnetic Structures within a Microfluidic Channel for Enhanced Immunomagnetic Isolation and Surface Patterning of Cells

    PubMed Central

    Sun, Chen; Hassanisaber, Hamid; Yu, Richard; Ma, Sai; Verbridge, Scott S.; Lu, Chang

    2016-01-01

    In this report, we demonstrate a unique method for embedding magnetic structures inside a microfluidic channel for cell isolation. We used a molding process to fabricate these structures out of a ferrofluid of cobalt ferrite nanoparticles. We show that the embedded magnetic structures significantly increased the magnetic field in the channel, resulting in up to 4-fold enhancement in immunomagnetic capture as compared with a channel without these embedded magnetic structures. We also studied the spatial distribution of trapped cells both experimentally and computationally. We determined that the surface pattern of these trapped cells was determined by both location of the magnet and layout of the in-channel magnetic structures. Our magnetic structure embedded microfluidic device achieved over 90% capture efficiency at a flow velocity of 4 mm/s, a speed that was roughly two orders of magnitude faster than previous microfluidic systems used for a similar purpose. We envision that our technology will provide a powerful tool for detection and enrichment of rare cells from biological samples. PMID:27388549

  12. A Two-Stage Microfluidic Device for the Isolation and Capture of Circulating Tumor Cells

    NASA Astrophysics Data System (ADS)

    Cook, Andrew; Belsare, Sayali; Giorgio, Todd; Mu, Richard

    2014-11-01

    Analysis of circulating tumor cells (CTCs) can be critical for studying how tumors grow and metastasize, in addition to personalizing treatment for cancer patients. CTCs are rare events in blood, making it difficult to remove CTCs from the blood stream. Two microfluidic devices have been developed to separate CTCs from blood. The first is a double spiral device that focuses cells into streams, the positions of which are determined by cell diameter. The second device uses ligand-coated magnetic nanoparticles that selectively attach to CTCs. The nanoparticles then pull CTCs out of solution using a magnetic field. These two devices will be combined into a single 2-stage microfluidic device that will capture CTCs more efficiently than either device on its own. The first stage depletes the number of blood cells in the sample by size-based separation. The second stage will magnetically remove CTCs from solution for study and culturing. Thus far, size-based separation has been achieved. Research will also focus on understanding the equations that govern fluid dynamics and magnetic fields in order to determine how the manipulation of microfluidic parameters, such as dimensions and flow rate, will affect integration and optimization of the 2-stage device. NSF-CREST: Center for Physics and Chemistry of Materials. HRD-0420516; Department of Defense, Peer Reviewed Medical Research Program Award W81XWH-13-1-0397.

  13. Hybrid microfluidic fuel cell based on Laccase/C and AuAg/C electrodes.

    PubMed

    López-González, B; Dector, A; Cuevas-Muñiz, F M; Arjona, N; Cruz-Madrid, C; Arana-Cuenca, A; Guerra-Balcázar, M; Arriaga, L G; Ledesma-García, J

    2014-12-15

    A hybrid glucose microfluidic fuel cell composed of an enzymatic cathode (Laccase/ABTS/C) and an inorganic anode (AuAg/C) was developed and tested. The enzymatic cathode was prepared by adsorption of 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and Laccase on Vulcan XC-72, which act as a redox mediator, enzymatic catalyst and support, respectively. The Laccase/ABTS/C composite was characterised by Fourier Transform Infrared (FTIR) Spectroscopy, streaming current measurements (Zeta potential) and cyclic voltammetry. The AuAg/C anode catalyst was characterised by Transmission electron microscopy (TEM) and cyclic voltammetry. The hybrid microfluidic fuel cell exhibited excellent performance with a maximum power density value (i.e., 0.45 mW cm(-2)) that is the highest reported to date. The cell also exhibited acceptable stability over the course of several days. In addition, a Mexican endemic Laccase was used as the biocathode electrode and evaluated in the hybrid microfluidic fuel cell generating 0.5 mW cm(-2) of maximum power density. PMID:25016252

  14. Paramagnetic Structures within a Microfluidic Channel for Enhanced Immunomagnetic Isolation and Surface Patterning of Cells.

    PubMed

    Sun, Chen; Hassanisaber, Hamid; Yu, Richard; Ma, Sai; Verbridge, Scott S; Lu, Chang

    2016-01-01

    In this report, we demonstrate a unique method for embedding magnetic structures inside a microfluidic channel for cell isolation. We used a molding process to fabricate these structures out of a ferrofluid of cobalt ferrite nanoparticles. We show that the embedded magnetic structures significantly increased the magnetic field in the channel, resulting in up to 4-fold enhancement in immunomagnetic capture as compared with a channel without these embedded magnetic structures. We also studied the spatial distribution of trapped cells both experimentally and computationally. We determined that the surface pattern of these trapped cells was determined by both location of the magnet and layout of the in-channel magnetic structures. Our magnetic structure embedded microfluidic device achieved over 90% capture efficiency at a flow velocity of 4 mm/s, a speed that was roughly two orders of magnitude faster than previous microfluidic systems used for a similar purpose. We envision that our technology will provide a powerful tool for detection and enrichment of rare cells from biological samples. PMID:27388549

  15. Hot embossing for fabrication of a microfluidic 3D cell culture platform

    PubMed Central

    Jeon, Jessie S.; Chung, Seok; Kamm, Roger D.; Charest, Joseph L.

    2011-01-01

    Clinically relevant studies of cell function in vitro require a physiologically-representative microenvironment possessing aspects such as a 3D extracellular matrix (ECM) and controlled biochemical and biophysical parameters. A polydimethylsiloxane (PDMS) microfluidic system with a 3D collagen gel has previously served for analysis of factors inducing different responses of cells in a 3D microenvironment under controlled biochemical and biophysical parameters. In the present study, applying the known commercially-viable manufacturing methods to a cyclic olefin copolymer (COC) material resulted in a microfluidic device with enhanced 3D gel capabilities, controlled surface properties, and improved potential to serve high-volume applications. Hot embossing and roller lamination molded and sealed the microfluidic device. A combination of oxygen plasma and thermal treatments enhanced the sealing, ensured proper placement of the 3D gel, and created controlled and stable surface properties within the device. Culture of cells in the new device indicated no adverse effects of the COC material or processing as compared to previous PDMS devices. The results demonstrate a methodology to transition microfludic devices for 3D cell culture from scientific research to high-volume applications with broad clinical impact. PMID:21113663

  16. Phenotype classification of single cells using SRS microscopy, RNA sequencing, and microfluidics (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Streets, Aaron M.; Cao, Chen; Zhang, Xiannian; Huang, Yanyi

    2016-03-01

    Phenotype classification of single cells reveals biological variation that is masked in ensemble measurement. This heterogeneity is found in gene and protein expression as well as in cell morphology. Many techniques are available to probe phenotypic heterogeneity at the single cell level, for example quantitative imaging and single-cell RNA sequencing, but it is difficult to perform multiple assays on the same single cell. In order to directly track correlation between morphology and gene expression at the single cell level, we developed a microfluidic platform for quantitative coherent Raman imaging and immediate RNA sequencing (RNA-Seq) of single cells. With this device we actively sort and trap cells for analysis with stimulated Raman scattering microscopy (SRS). The cells are then processed in parallel pipelines for lysis, and preparation of cDNA for high-throughput transcriptome sequencing. SRS microscopy offers three-dimensional imaging with chemical specificity for quantitative analysis of protein and lipid distribution in single cells. Meanwhile, the microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved RNA-Seq detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. By combining coherent Raman microscopy with RNA sequencing, we can better understand the relationship between cellular morphology and gene expression at the single-cell level.

  17. Analysis of pairwise cell interactions using an integrated dielectrophoretic-microfluidic system

    PubMed Central

    Yin, Zhizhong; Noren, David; Wang, C Joanne; Hang, Rob; Levchenko, Andre

    2008-01-01

    Blood vessel formation, during either normal vascular reconstruction or pathogenic tumour formation, relies upon highly organized cell–cell interactions. Isolating the function of any particular component of this cell–cell communication is often difficult, given the vast complexity of communication networks in multicellular systems. One way to address this problem is to analyse cell–cell communication on the most elementary scale—cell pairs. Here, we describe an integrated dielectrophoretic (DEP)-microfluidic device allowing for such analysis. Single cancer and endothelial cells (ECs) and cell pairs were patterned using DEP force and cultured within a minimally stressful microfluidic channel network. Controlling both the initial cell positions and extracellular environment, we investigated cell motility in homo- and heterotypic cell pairs under diverse conditions. We found that secreted collagen IV and soluble vascular endothelial growth factor have considerable guidance effect on ECs at the level of two interacting cells. Cell interaction rules extracted from the experiments of cell pairs were used to mathematically predict branching patterns characteristic of developing multicellular blood vessels. This integrative analysis method can be extended to other systems involving complex multicellular interactions. PMID:19096359

  18. A microfluidic array with cellular valving for single cell co-culture.

    PubMed

    Frimat, Jean-Philippe; Becker, Marco; Chiang, Ya-Yu; Marggraf, Ulrich; Janasek, Dirk; Hengstler, Jan G; Franzke, Joachim; West, Jonathan

    2011-01-21

    We present a highly parallel microfluidic approach for contacting single cell pairs. The approach combines a differential fluidic resistance trapping method with a novel cellular valving principle for homotypic and heterotypic single cell co-culturing. Differential fluidic resistance was used for sequential single cell arraying, with the adhesion and flattening of viable cells within the microstructured environment acting to produce valves in the open state. Reversal of the flow was used for the sequential single cell arraying of the second cell type. Plasma stencilling, along the linear path of least resistance, was required to confine the cells within the trap regions. Prime flow conditions with minimal shear stress were identified for highly efficient cell arraying (∼99%) and long term cell culture. Larger trap dimensions enabled the highest levels of cell pairing (∼70%). The single cell co-cultures were in close proximity for the formation of connexon structures and the study of contact modes of communication. The research further highlights the possibility of using the natural behaviour of cells as the working principle behind responsive microfluidic elements. PMID:20978708

  19. Probing Hypoxia-Induced Staurosporine Resistance in Prostate Cancer Cells with a Microfluidic Culture System

    PubMed Central

    Khanal, Grishma; Hiemstra, Scott

    2014-01-01

    A microfluidic system for cell culture and drug response studies was developed to elucidate the effects of hypoxia on drug susceptibility. Drug response studies were performed in prostate cancer cells and Ramos B cells under normoxic and hypoxic conditions. A vacuum actuated microfluidic culture device was used for cell culture and PC3 cells were cultured in the chip up to 16 hours. Cells were treated with several concentrations of staurosporine and apoptosis was assayed using the fluorescent probes MitoTracker Red and Annexin-V. For hypoxic samples, the chip was placed in a hypoxia chamber and pre-conditioned at <1% oxygen before inducing the cells with staurosporine. Cells exposed to 2 μM staurosporine were 32% ± 10% apoptotic under normoxic conditions but only 1.5% ± 12% apoptotic under hypoxic conditions. As little as 1 hour of hypoxic preconditioning increased drug resistance. Cell apoptosis correlated with drug dose, although in each case hypoxia reduced the apoptotic fraction significantly. Given the rapid nature of cell adaptation to hypoxia, this chip and analysis approach can be used to identify compounds that can induce cell death in hypoxic tumor cells rapidly. PMID:24479128

  20. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing

    PubMed Central

    Woodruff, Kristina; Maerkl, Sebastian J.

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  1. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing.

    PubMed

    Woodruff, Kristina; Maerkl, Sebastian J

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  2. Microfluidic Cell Sorting: A Review of the Advances in the Separation of Cells from Debulking to Rare Cell Isolation

    PubMed Central

    Shields, C. Wyatt; Reyes, Catherine D.; López, Gabriel P.

    2015-01-01

    Accurate and high throughput cell sorting is a critical enabling technology in molecular and cellular biology, biotechnology, and medicine. While conventional methods can provide high efficiency sorting in short timescales, advances in microfluidics have enabled the realization of miniaturized devices offering similar capabilities that exploit a variety of physical principles. We classify these technologies as either active or passive. Active systems generally use external fields (e.g., acoustic, electric, magnetic, and optical) to impose forces to displace cells for sorting, whereas passive systems use inertial forces, filters, and adhesion mechanisms to purify cell populations. Cell sorting on microchips provides numerous advantages over conventional methods by reducing the size of necessary equipment, eliminating potentially biohazardous aerosols, and simplifying the complex protocols commonly associated with cell sorting. Additionally, microchip devices are well suited for parallelization, enabling complete lab-on-a-chip devices for cellular isolation, analysis, and experimental processing. In this review, we examine the breadth of microfluidic cell sorting technologies, while focusing on those that offer the greatest potential for translation into clinical and industrial practice and that offer multiple, useful functions. We organize these sorting technologies by the type of cell preparation required (i.e., fluorescent label-based sorting, bead-based sorting, and label-free sorting) as well as by the physical principles underlying each sorting mechanism. PMID:25598308

  3. Pulsed laser triggered high speed microfluidic fluorescence activated cell sorter†‡

    PubMed Central

    Wu, Ting-Hsiang; Chen, Yue; Park, Sung-Yong; Hong, Jason; Teslaa, Tara; Zhong, Jiang F.; Di Carlo, Dino; Teitell, Michael A.

    2014-01-01

    We report a high speed and high purity pulsed laser triggered fluorescence activated cell sorter (PLACS) with a sorting throughput up to 20 000 mammalian cells s−1 with 37% sorting purity, 90% cell viability in enrichment mode, and >90% purity in high purity mode at 1500 cells s−1 or 3000 beads s−1. Fast switching (30 μs) and a small perturbation volume (~90 pL) is achieved by a unique sorting mechanism in which explosive vapor bubbles are generated using focused laser pulses in a single layer microfluidic PDMS channel. PMID:22361780

  4. A microfluidic cell-trapping device for single-cell tracking of host-microbe interactions.

    PubMed

    Delincé, Matthieu J; Bureau, Jean-Baptiste; López-Jiménez, Ana Teresa; Cosson, Pierre; Soldati, Thierry; McKinney, John D

    2016-08-16

    The impact of cellular individuality on host-microbe interactions is increasingly appreciated but studying the temporal dynamics of single-cell behavior in this context remains technically challenging. Here we present a microfluidic platform, InfectChip, to trap motile infected cells for high-resolution time-lapse microscopy. This approach allows the direct visualization of all stages of infection, from bacterial uptake to death of the bacterium or host cell, over extended periods of time. We demonstrate the utility of this approach by co-culturing an established host-cell model, Dictyostelium discoideum, with the extracellular pathogen Klebsiella pneumoniae or the intracellular pathogen Mycobacterium marinum. We show that the outcome of such infections is surprisingly heterogeneous, ranging from abortive infection to death of the bacterium or host cell. InfectChip thus provides a simple method to dissect the time-course of host-microbe interactions at the single-cell level, yielding new insights that could not be gleaned from conventional population-based measurements. PMID:27425421

  5. A simple microfluidic device to study cell-scale endothelial mechanotransduction.

    PubMed

    Lafaurie-Janvore, Julie; Antoine, Elizabeth E; Perkins, Sidney J; Babataheri, Avin; Barakat, Abdul I

    2016-08-01

    Atherosclerosis is triggered by chronic inflammation of arterial endothelial cells (ECs). Because atherosclerosis develops preferentially in regions where blood flow is disturbed and where ECs have a cuboidal morphology, the interplay between EC shape and mechanotransduction events is of primary interest. In this work we present a simple microfluidic device to study relationships between cell shape and EC response to fluid shear stress. Adhesive micropatterns are used to non-invasively control EC elongation and orientation at both the monolayer and single cell levels. The micropatterned substrate is coupled to a microfluidic chamber that allows precise control of the flow field, high-resolution live-cell imaging during flow experiments, and in situ immunostaining. Using micro particle image velocimetry, we show that cells within the chamber alter the local flow field so that the shear stress on the cell surface is significantly higher than the wall shear stress in regions containing no cells. In response to flow, we observe the formation of lamellipodia in the downstream portion of the EC and cell retraction in the upstream portion. We quantify flow-induced calcium mobilization at the single cell level for cells cultured on unpatterned surfaces or on adhesive lines oriented either parallel or orthogonal to the flow. Finally, we demonstrate flow-induced intracellular calcium waves and show that the direction of propagation of these waves is determined by cell polarization rather than by the flow direction. The combined versatility and simplicity of this microfluidic device renders it very useful for studying relationships between EC shape and mechanosensitivity. PMID:27402497

  6. Single cell functional analysis of multiple myeloma cell populations correlates with diffusion profiles in static microfluidic coculture systems.

    PubMed

    Moore, Thomas A; Young, Edmond W K

    2016-07-01

    Microfluidic cell culture systems are becoming increasingly useful for studying biology questions, particularly those involving small cell populations that are cultured within microscale geometries mimicking the complex cellular microenvironment. Depending on the geometry and spatial organization of these cell populations, however, paracrine signaling between cell types can depend critically on spatial concentration profiles of soluble factors generated by diffusive transport. In scenarios where single cell data are acquired to study cell population heterogeneities in functional response, uncertainty associated with concentration profiles can lead to interpretation bias. To address this issue and provide important evidence on how diffusion develops within typical microfluidic cell culture systems, a combination of experimental and computational approaches were applied to measure and predict concentration patterns within microfluidic geometries, and characterize the functional response of culture cells based on single-cell resolution transcription factor activation. Using a model coculture system consisting of multiple myeloma cells (MMCs) and neighboring bone marrow stromal cells (BMSCs), we measured concentrations of three cytokines (IL-6, VEGF, and TNF-α) in conditioned media collected from separate culture compartments using a multiplex ELISA system. A 3D numerical model was developed to predict biomolecular diffusion and resulting concentration profiles within the tested microsystems and compared with experimental diffusion of 20 kDa FITC-Dextran. Finally, diffusion was further characterized by controlling exogenous IL-6 diffusion and the coculture spatial configuration of BMSCs to stimulate STAT3 nuclear translocation in MMCs. Results showed agreement between numerical and experimental results, provided evidence of a shallow concentration gradient across the center well of the microsystem that did not lead to a bias in results, and demonstrated that

  7. Quantitative Study of Cell Invasion Process under Extracellular Stimulation of Cytokine in a Microfluidic Device

    PubMed Central

    Lei, Kin Fong; Tseng, Hsueh-Peng; Lee, Chia-Yi; Tsang, Ngan-Ming

    2016-01-01

    Cell invasion is the first step of cancer metastasis that is the primary cause of death for cancer patients and defined as cell movement through extracellular matrix (ECM). Investigation of the correlation between cell invasive and extracellular stimulation is critical for the inhabitation of metastatic dissemination. Conventional cell invasion assay is based on Boyden chamber assay, which has a number of limitations. In this work, a microfluidic device incorporating with impedance measurement technique was developed for quantitative investigation of cell invasion process. The device consisted of 2 reservoirs connecting with a microchannel filled with hydrogel. Malignant cells invaded along the microchannel and impedance measurement was concurrently conducted by measuring across electrodes located at the bottom of the microchannel. Therefore, cell invasion process could be monitored in real-time and non-invasive manner. Also, cell invasion rate was then calculated to study the correlation between cell invasion and extracellular stimulation, i.e., IL-6 cytokine. Results showed that cell invasion rate was directly proportional to the IL-6 concentration. The microfluidic device provides a reliable and convenient platform for cell-based assays to facilitate more quantitative assessments in cancer research. PMID:27150137

  8. Quantitative Study of Cell Invasion Process under Extracellular Stimulation of Cytokine in a Microfluidic Device.

    PubMed

    Lei, Kin Fong; Tseng, Hsueh-Peng; Lee, Chia-Yi; Tsang, Ngan-Ming

    2016-01-01

    Cell invasion is the first step of cancer metastasis that is the primary cause of death for cancer patients and defined as cell movement through extracellular matrix (ECM). Investigation of the correlation between cell invasive and extracellular stimulation is critical for the inhabitation of metastatic dissemination. Conventional cell invasion assay is based on Boyden chamber assay, which has a number of limitations. In this work, a microfluidic device incorporating with impedance measurement technique was developed for quantitative investigation of cell invasion process. The device consisted of 2 reservoirs connecting with a microchannel filled with hydrogel. Malignant cells invaded along the microchannel and impedance measurement was concurrently conducted by measuring across electrodes located at the bottom of the microchannel. Therefore, cell invasion process could be monitored in real-time and non-invasive manner. Also, cell invasion rate was then calculated to study the correlation between cell invasion and extracellular stimulation, i.e., IL-6 cytokine. Results showed that cell invasion rate was directly proportional to the IL-6 concentration. The microfluidic device provides a reliable and convenient platform for cell-based assays to facilitate more quantitative assessments in cancer research. PMID:27150137

  9. Quantitative Study of Cell Invasion Process under Extracellular Stimulation of Cytokine in a Microfluidic Device

    NASA Astrophysics Data System (ADS)

    Lei, Kin Fong; Tseng, Hsueh-Peng; Lee, Chia-Yi; Tsang, Ngan-Ming

    2016-05-01

    Cell invasion is the first step of cancer metastasis that is the primary cause of death for cancer patients and defined as cell movement through extracellular matrix (ECM). Investigation of the correlation between cell invasive and extracellular stimulation is critical for the inhabitation of metastatic dissemination. Conventional cell invasion assay is based on Boyden chamber assay, which has a number of limitations. In this work, a microfluidic device incorporating with impedance measurement technique was developed for quantitative investigation of cell invasion process. The device consisted of 2 reservoirs connecting with a microchannel filled with hydrogel. Malignant cells invaded along the microchannel and impedance measurement was concurrently conducted by measuring across electrodes located at the bottom of the microchannel. Therefore, cell invasion process could be monitored in real-time and non-invasive manner. Also, cell invasion rate was then calculated to study the correlation between cell invasion and extracellular stimulation, i.e., IL-6 cytokine. Results showed that cell invasion rate was directly proportional to the IL-6 concentration. The microfluidic device provides a reliable and convenient platform for cell-based assays to facilitate more quantitative assessments in cancer research.

  10. Microfluidic co-culture platform to quantify chemotaxis of primary stem cells.

    PubMed

    Tatárová, Z; Abbuehl, J P; Maerkl, S; Huelsken, J

    2016-05-21

    Functional analysis of primary tissue-specific stem cells is hampered by their rarity. Here we describe a greatly miniaturized microfluidic device for the multiplexed, quantitative analysis of the chemotactic properties of primary, bone marrow-derived mesenchymal stem cells (MSC). The device was integrated within a fully customized platform that both increased the viability of stem cells ex vivo and simplified manipulation during multidimensional acquisition. Since primary stem cells can be isolated only in limited number, we optimized the design for efficient cell trapping from low volume and low concentration cell suspensions. Using nanoliter volumes and automated microfluidic controls for pulsed medium supply, our platform is able to create stable gradients of chemoattractant secreted from mammalian producer cells within the device, as was visualized by a secreted NeonGreen fluorescent reporter. The design was functionally validated by a CXCL/CXCR ligand/receptor combination resulting in preferential migration of primary, non-passaged MSC. Stable gradient formation prolonged assay duration and resulted in enhanced response rates for slowly migrating stem cells. Time-lapse video microscopy facilitated determining a number of migratory properties based on single cell analysis. Jackknife-resampling revealed that our assay requires only 120 cells to obtain statistically significant results, enabling new approaches in the research on rare primary stem cells. Compartmentalization of the device not only facilitated such quantitative measurements but will also permit future, high-throughput functional screens. PMID:27137768

  11. Microfluidic Device

    NASA Technical Reports Server (NTRS)

    Tai, Yu-Chong (Inventor); Zheng, Siyang (Inventor); Lin, Jeffrey Chun-Hui (Inventor); Kasdan, Harvey (Inventor)

    2015-01-01

    Described herein are particular embodiments relating to a microfluidic device that may be utilized for cell sensing, counting, and/or sorting. Particular aspects relate to a microfabricated device that is capable of differentiating single cell types from dense cell populations. One particular embodiment relates a device and methods of using the same for sensing, counting, and/or sorting leukocytes from whole, undiluted blood samples.

  12. Microfluidic Device

    NASA Technical Reports Server (NTRS)

    Tai, Yu-Chong (Inventor); Zheng, Siyang (Inventor); Lin, Jeffrey Chun-Hui (Inventor); Kasdan, Harvey L. (Inventor)

    2016-01-01

    Described herein are particular embodiments relating to a microfluidic device that may be utilized for cell sensing, counting, and/or sorting. Particular aspects relate to a microfabricated device that is capable of differentiating single cell types from dense cell populations. One particular embodiment relates a device and methods of using the same for sensing, counting, and/or sorting leukocytes from whole, undiluted blood samples.

  13. A cell-based sensor of fluid shear stress for microfluidics.

    PubMed

    Varma, Sarvesh; Voldman, Joel

    2015-03-21

    Microsystems designed for cell-based studies or applications inherently require fluid handling. Flows within such systems inevitably generate fluid shear stress (FSS) that may adversely affect cell health. Simple assays of cell viability, morphology or growth are typically reported to indicate any gross disturbances to cell physiology. However, no straightforward metric exists to specifically evaluate physiological implications of FSS within microfluidic devices, or among competing microfluidic technologies. This paper presents the first genetically encoded cell sensors that fluoresce in a quantitative fashion upon FSS pathway activation. We picked a widely used cell line (NIH3T3s) and created a transcriptional cell-sensor where fluorescence turns on when transcription of a relevant FSS-induced protein is initiated. Specifically, we chose Early Growth Factor-1 (a mechanosensitive protein) upregulation as the node for FSS detection. We verified our sensor pathway specificity and functionality by noting induced fluorescence in response to chemical induction of the FSS pathway, seen both through microscopy and flow cytometry. Importantly, we found our cell sensors to be inducible by a range of FSS intensities and durations, with a limit of detection of 2 dynes cm(-2) when applied for 30 minutes. Additionally, our cell-sensors proved their versatility by showing induction sensitivity when made to flow through an inertial microfluidic device environment with typical flow conditions. We anticipate these cell sensors to have wide application in the microsystems community, allowing the device designer to engineer systems with acceptable FSS, and enabling the end-user to evaluate the impact of FSS upon their assay of interest. PMID:25648195

  14. Microfluidics for T- Lymphocyte Cell Separation and Inflammation Monitoring in Burn Patients

    PubMed Central

    Rosenbach, Alan E.; Koria, Piyush; Goverman, Jeremy; Kotz, Kenneth T.; Gupta, Amit; Yu, Ming; Fagan, Shawn P.; Irimia, Daniel; Tompkins, Ronald G.

    2011-01-01

    Severe burns result in T-lymphocyte specific immunologic changes. In addition to decreased levels of circulating lymphocytes, changes in cytokine secretion and receptor expression also take place. Our finer understanding of the inflammatory response has led to the development of immune-targeted therapeutics, requiring specialized gene-expression monitoring. The emerging field of bio-micro-electromechanical systems can be used to isolate highly pure T-lymphocytes in a clinically relevant and timely manner for downstream genomic analysis. Blood samples from healthy volunteers and burn-injured patients were introduced into microfluidic devices developed in our laboratory. Utilizing cell-affinity-chromatography for positive selection of T-lymphocytes, the devices served as a platform for RNA extraction and downstream cytokine analysis via quantitative real time PCR. From a 0.5-mL whole blood sample, the microfluidic devices captured highly pure T-lymphocytes from healthy volunteers and burn-injured patients. Cell capture was of sufficient quantity, and extracted RNA was of sufficient quality, for evaluating the gene expression of cytokines: interferon-gamma, interleukin-2, interleukin-4, and interleukin-10. Microfluidics is a useful tool in processing blood from burn-injured patients. Though in its very early stages of development, cell-specific information obtained by this platform/technology will likely be an important component of near-patient molecular diagnostics and personalized medicine. PMID:21348958

  15. Modeling of Shear-Induced Red Blood Cell Migration for Guiding Microfluidic Device Design

    NASA Astrophysics Data System (ADS)

    Durant, Eden; Higgins, Adam; Sharp, Kendra

    2014-11-01

    Through refinement and extension of a two-phase flow model previously reported for modeling blood in cylindrical flows (Gidaspow, 2009), we have developed a computational model for blood flow in complex microfluidic. Treating plasma as a Newtonian fluid and the Red Blood Cells (RBCs) as a granular phase, whose local concentrations are determined statistically, we have captured the migration of RBCs and concomitant formation of a cell free plasma layer at the channel walls. This model provides us with a three-dimensional distribution of RBCs and the development of the stead-state flow profile, and enables us to study the influence of complex microfluidic geometries, including flow obstacles and varying channel dimensions, on the rate and extent of RBC margination. Simulations on 50 and 100 micron square channels match observed trends including decreasing RBC margination rate in larger channels, increasing RBC margination rate with higher hematocrit, and decreasing cell free layer width with increasing hematocrit. This predictive capability will allow microfluidic devices to be tailored and optimized for specific biomedical applications such as separation of blood constituents.

  16. A Microfluidic Device to Sort Cells Based on Dynamic Response to a Stimulus

    PubMed Central

    Mathuru, Ajay Sriram; Burkholder, William F.; Jesuthasan, Suresh J.

    2013-01-01

    Single cell techniques permit the analysis of cellular properties that are obscured by studying the average behavior of cell populations. One way to determine how gene expression contributes to phenotypic differences among cells is to combine functional analysis with transcriptional profiling of single cells. Here we describe a microfluidic device for monitoring the responses of single cells to a ligand and then collecting cells of interest for transcriptional profiling or other assays. As a test, cells from the olfactory epithelium of zebrafish were screened by calcium imaging to identify sensory neurons that were responsive to the odorant L-lysine. Single cells were subsequently recovered for transcriptional profiling by qRT-PCR. Responsive cells all expressed TRPC2 but not OMP, consistent with known properties of amino-acid sensitive olfactory neurons. The device can be adapted for other areas in biology where there is a need to sort and analyze cells based on their signaling responses. PMID:24250795

  17. Rare cell chemiluminescence detection based on aptamer-specific capture in microfluidic channels.

    PubMed

    Liu, Wu; Wei, Huibin; Lin, Zhen; Mao, Sifeng; Lin, Jin-Ming

    2011-10-15

    An aptamer-based "sandwich" approach combined with the chemiluminescence (CL) analysis was developed for the capture and detection of rare cells on a microfluidic chip. Aptamers were immobilized on microfluidic channels to achieve capture and isolation of the specific cells from a cell mixture. The capture efficiency for target cells was more than 70% with the purity greater than 97%, when the content of the target cells was between 0.5% and 10% in the initial cell mixture. Gold nanoparticles (Au NPs) modified with aptamers were then added in to bind on the cells and trigger a CL reaction. A satisfactory linearity of the log/log calibration curve between the CL intensity and the number of target cells was observed with a low detection limit of 30 target cells in a 3 μL cell mixture. Spiked whole blood samples were also used to verify the practicality of the present method. This work demonstrated the potential application of the cheap and rapid CL detection into the early diagnosis of cancers. PMID:21856143

  18. Microfluidic single-cell analysis links boundary environments and individual microbial phenotypes.

    PubMed

    Dusny, Christian; Schmid, Andreas

    2015-06-01

    Life is based on the cell as the elementary replicative and self-sustaining biological unit. Each single cell constitutes an independent and highly dynamic system with a remarkable individuality in a multitude of physiological traits and responses to environmental fluctuations. However, with traditional population-based cultivation set-ups, it is not possible to decouple inherent stochastic processes and extracellular contributions to phenotypic individuality for two central reasons: the lack of environmental control and the occlusion of single-cell dynamics by the population average. With microfluidic single-cell analysis as a new cell assay format, these issues can now be addressed, enabling cultivation and time-resolved analysis of single cells in precisely manipulable extracellular environments beyond the bulk. In this article, we explore the interplay of cellular physiology and environment at a single-cell level. We review biological basics that govern the functional state of the cell and put them in context with physical fundamentals that shape the extracellular environment. Furthermore, the significance of single-cell growth rates as pivotal descriptors for global cellular physiology is discussed and highlighted by selected studies. These examples illustrate the unique opportunities of microfluidic single-cell cultivation in combination with growth rate analysis, addressing questions of fundamental bio(techno)logical interest. PMID:25330456

  19. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels

    PubMed Central

    Shin, Yoojin; Han, Sewoon; Jeon, Jessie S.; Yamamoto, Kyoko; Zervantonakis, Ioannis K.; Sudo, Ryo; Kamm, Roger D.; Chung, Seok

    2014-01-01

    This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel incorporating chambers between surface-accessible microchannels. Using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of <1mm, thereby replicating many aspects of the in vivo microenvironment. Capabilities exist for time-dependent manipulation of flows and concentration gradients as well as high-resolution real-time imaging for observing spatial-temporal single cell behavior, cell-cell communication, cell-matrix interactions and cell population dynamics. These heterotypic cell type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 days to fabricate the system and experiments can run for up to several weeks. PMID:22678430

  20. Design of a microfluidic strategy for trapping and screening single cells.

    PubMed

    Occhetta, Paola; Licini, Mara; Redaelli, Alberto; Rasponi, Marco

    2016-01-01

    Traditionally, in vitro investigations on biology and physiology of cells rely on averaging the responses eliciting from heterogeneous cell populations, thus being unsuitable for assessing individual cell behaviors in response to external stimulations. In the last years, great interest has thus been focused on single cell analysis and screening, which represents a promising tool aiming at pursuing the direct and deterministic control over cause-effect relationships guiding cell behavior. In this regard, a high-throughput microfluidic platform for trapping and culturing adherent single cells was presented. A single cell trapping mechanism was implemented based on dynamic variation of fluidic resistances. A round-shaped culture chamber (Φ = 250 µm, h = 25 µm) was conceived presenting two connections with a main fluidic path: (i) an upper wide opening, and (ii) a bottom trapping junction which modulates the hydraulic resistance. Starting from eight different layouts, the chamber geometry was computationally optimized for maximizing the single cell trapping efficacy and then integrated in a polydimethylsiloxane (PDMS) microfluidic device. The final platform consists in (i) 288 chambers for trapping single cells organized in six culture units, independently addressable through the lines of (ii) a chaotic-mixer based serial dilution generator (SDG), designed for creating spatio-temporally controlled patterns of both soluble factors and non-diffusive particles. The device was experimentally validated by trapping polystyrene microspheres, featuring diameters comparable to cell size (Φ = 10 µm). PMID:26651214

  1. Microfluidic Device for Stem Cell Differentiation and Localized Electroporation of Postmitotic Neurons

    PubMed Central

    Kang, Wonmo; Giraldo-Vela, Juan P.; Nathamgari, S. Shiva P.; McGuire, Tammy; McNaughton, Rebecca L.; Kessler, John A.; Espinosa, Horacio D.

    2014-01-01

    New techniques to deliver of nucleic acids and other molecules for gene editing and gene expression profiling, which can be performed with minimal perturbation to cell growth or differentiation, are essential for advancing biological research. Studying cells in their natural state, with temporal control, is particularly important for primary cells that are derived by differentiation from stem cells and are adherent, e.g., neurons. Existing high-throughput transfection methods either require cells to be in suspension or are highly toxic and limited to a single transfection per experiment. Here we present a microfluidic device that couples on-chip culture of adherent cells and transfection by localized electroporation. Integrated microchannels allow long-term cell culture on the device and repeated temporal transfection. The microfluidic device was validated by first performing electroporation of HeLa and HT1080 cells, with transfection efficiencies of ~95% for propidium iodide and up to 50% for plasmids. Application to primary cells was demonstrated by on-chip differentiation of neural stem cells and transfection of postmitotic neurons with a green fluorescent protein plasmid. PMID:25205561

  2. 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. PMID:25060749

  3. Interaction between microfluidic droplets in a Hele-Shaw cell

    NASA Astrophysics Data System (ADS)

    Sarig, Itai; Starosvetsky, Yuli; Gat, Amir

    2015-11-01

    Various fluidic systems, such as chemical and biological lab-on-a-chip devices, involve motion of multiple droplets within an immersing fluid in narrow micro-channels. Modeling the dynamics of such systems requires calculation of the forces of interaction between the moving droplets. These forces are commonly approximated by superposition of dipoles solutions, which requires an assumption of sufficiently large distance between the droplets. In this work we obtain exact solutions for two droplets, and a droplet within a droplet, located within a moving immersing fluid and without limitation on the distance between the droplets. This is achieved by solution of the Laplace equation for the pressure in a bi-polar coordinate system, Fourier method and transformation and calculation of the force in a Cartesian coordinate system. Our results are validated with numerical computations, experimental data and with the existing dipole-based models. We utilize the results to calculate the dynamics of a droplet within a droplet, and of two close droplets, located within an immersing fluid with oscillating speed. The obtained results may be used to study the dynamics of dense droplet lattices, common to many current micro-fluidic systems.

  4. A microfluidic device for physical trapping and electrical lysis of bacterial cells

    NASA Astrophysics Data System (ADS)

    Bao, Ning; Lu, Chang

    2008-05-01

    In this letter, we report a simple microfluidic device that integrates the capture of bacterial cells using a microscale bead array and the rapid electrical lysis for release of intracellular materials. We study the retention of Escherichia coli cells with different concentrations in this type of bead array and the optimal electrical parameters for the electroporative release of intracellular proteins. Our design provides a simple solution to the extraction of intracellular materials from a bacterial cell population based entirely on physical methods without applying chemical or biological reagents.

  5. Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics

    PubMed Central

    Sarkar, Aniruddh; Hou, Han Wei; Mahan, Alison. E.; Han, Jongyoon; Alter, Galit

    2016-01-01

    Isolation of low abundance proteins or rare cells from complex mixtures, such as blood, is required for many diagnostic, therapeutic and research applications. Current affinity-based protein or cell separation methods use binary ‘bind-elute’ separations and are inefficient when applied to the isolation of multiple low-abundance proteins or cell types. We present a method for rapid and multiplexed, yet inexpensive, affinity-based isolation of both proteins and cells, using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets–cells or proteins–bind to beads of different sizes, which are then sorted by flowing them through a spiral microfluidic channel. This technique performs continuous-flow, high throughput affinity-separation of milligram-scale protein samples or millions of cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from peripheral blood mononuclear cells or whole blood. We use the technique to isolate low abundance antibodies specific to different HIV antigens and rare HIV-specific cells from blood obtained from HIV+ patients. PMID:27026280

  6. Microfluidic devices for cell culture and handling in organ-on-a-chip applications

    NASA Astrophysics Data System (ADS)

    Becker, Holger; Schulz, Ingo; Mosig, Alexander; Jahn, Tobias; Gärtner, Claudia

    2014-03-01

    For many problems in system biology or pharmacology, in-vivo-like models of cell-cell interactions or organ functions are highly sought after. Conventional stationary cell culture in 2D plates quickly reaches its limitations with respect to an in-vivo like expression and function of individual cell types. Microfabrication technologies and microfluidics offer an attractive solution to these problems. The ability to generate flow as well as geometrical conditions for cell culture and manipulation close to the in-vivo situation allows for an improved design of experiments and the modeling of organ-like functionalities. Furthermore, reduced internal volumes lead to a reduction in reagent volumes necessary as well as an increased assay sensitivity. In this paper we present a range of microfluidic devices designed for the co-culturing of a variety of cells. The influence of substrate materials and surface chemistry on the cell morphology and viability for long-term cell culture has been investigated as well as strategies and medium supply for on-chip cell cultivation.

  7. Single-Cell Chemical Lysis on Microfluidic Chips with Arrays of Microwells

    PubMed Central

    Jen, Chun-Ping; Hsiao, Ju-Hsiu; Maslov, Nikolay A.

    2012-01-01

    Many conventional biochemical assays are performed using populations of cells to determine their quantitative biomolecular profiles. However, population averages do not reflect actual physiological processes in individual cells, which occur either on short time scales or nonsynchronously. Therefore, accurate analysis at the single-cell level has become a highly attractive tool for investigating cellular content. Microfluidic chips with arrays of microwells were developed for single-cell chemical lysis in the present study. The cellular occupancy in 30-μm-diameter microwells (91.45%) was higher than that in 20-μm-diameter microwells (83.19%) at an injection flow rate of 2.8 μL/min. However, most of the occupied 20-μm-diameter microwells contained individual cells. The results of chemical lysis experiments at the single-cell level indicate that cell membranes were gradually lysed as the lysis buffer was injected; they were fully lysed after 12 s. Single-cell chemical lysis was demonstrated in the proposed microfluidic chip, which is suitable for high-throughput cell lysis. PMID:22368473

  8. Cell stimulus and lysis in a microfluidic device with segmented gas-liquid flow.

    PubMed

    El-Ali, Jamil; Gaudet, Suzanne; Günther, Axel; Sorger, Peter K; Jensen, Klavs F

    2005-06-01

    We describe a microfluidic device with rapid stimulus and lysis of mammalian cells for resolving fast transient responses in cell signaling networks. The device uses segmented gas-liquid flow to enhance mixing and has integrated thermoelectric heaters and coolers to control the temperature during cell stimulus and lysis. Potential negative effects of segmented flow on cell responses are investigated in three different cell types, with no morphological changes and no activation of the cell stress-sensitive mitogen activated protein kinases observed. Jurkat E6-1 cells are stimulated in the device using alpha-CD3, and the resulting activations of ERK and JNK are presented for different time points. Stimulation of cells performed on chip results in pathway activation identical to that of conventionally treated cells under the same conditions. PMID:15924398

  9. Soft inertial microfluidics for high throughput separation of bacteria from human blood cells

    SciTech Connect

    Wu, Zhigang; Willing, Ben; Bjerketorp, Joakim; Jansson, Janet K.; Hjort, Klas

    2009-01-05

    We developed a new approach to separate bacteria from human blood cells based on soft inertial force induced migration with flow defined curved and focused sample flow inside a microfluidic device. This approach relies on a combination of an asymmetrical sheath flow and proper channel geometry to generate a soft inertial force on the sample fluid in the curved and focused sample flow segment to deflect larger particles away while the smaller ones are kept on or near the original flow streamline. The curved and focused sample flow and inertial effect were visualized and verified using a fluorescent dye primed in the device. First the particle behavior was studied in detail using 9.9 and 1.0 {micro}m particles with a polymer-based prototype. The prototype device is compact with an active size of 3 mm{sup 2}. The soft inertial effect and deflection distance were proportional to the fluid Reynolds number (Re) and particle Reynolds number (Re{sub p}), respectively. We successfully demonstrated separation of bacteria (Escherichia coli) from human red blood cells at high cell concentrations (above 10{sup 8}/mL), using a sample flow rate of up to 18 {micro}L/min. This resulted in at least a 300-fold enrichment of bacteria at a wide range of flow rates with a controlled flow spreading. The separated cells were proven to be viable. Proteins from fractions before and after cell separation were analyzed by gel electrophoresis and staining to verify the removal of red blood cell proteins from the bacterial cell fraction. This novel microfluidic process is robust, reproducible, simple to perform, and has a high throughput compared to other cell sorting systems. Microfluidic systems based on these principles could easily be manufactured for clinical laboratory and biomedical applications.

  10. Cell culture monitoring for drug screening and cancer research: a transparent, microfluidic, multi-sensor microsystem.

    PubMed

    Weltin, Andreas; Slotwinski, Kinga; Kieninger, Jochen; Moser, Isabella; Jobst, Gerhard; Wego, Marcus; Ehret, Ralf; Urban, Gerald A

    2014-01-01

    We present a novel, multiparametric microphysiometry system for the dynamic online monitoring of human cancer cell metabolism. The optically transparent, modular, hybrid microsystem is based on a glass chip and combines a cell cultivation chamber, microfluidics and metabolic monitoring with fully integrated chemo- and biosensors. pH and oxygen are measured in the cell culture area, and biosensors for lactate and glucose are connected downstream by microfluidics. The wafer-level fabrication features thin-film platinum and iridium oxide microelectrodes on a glass chip, microfluidics in an epoxy resist, a hybrid assembly and an on-chip reference electrode. The reliable analytical performance of the sensors in cell culture medium was demonstrated. The pH sensors exhibit a long-term stable, linear response. The oxygen sensors show a linear behaviour, which is also observed for low oxygen concentrations. Glucose and lactate measurements show a linear, long-term stable, selective and reversible behaviour in the desired range. T98G human brain cancer cells were cultivated and cell culture metabolism was measured on-chip. Stop/flow cycles were applied and extracellular acidification, respiration, glucose consumption and lactate production were quantified. Long-term metabolic rates were determined and all parameters could be measured in the outlet channel. A placement downstream of the cell cultivation area for biosensors was realised. A highly effective medium exchange and undiluted sampling from the cell culture chamber with low flow rates (2 μl min(-1)) and low volumes (15 μl per cycle) were achieved. The drug screening application was demonstrated by detecting alteration and recovery effects of cellular metabolism induced by the addition of substances to the medium. PMID:24217869

  11. Single cell rheometry with a microfluidic constriction: Quantitative control of friction and fluid leaks between cell and channel walls

    PubMed Central

    Preira, Pascal; Valignat, Marie-Pierre; Bico, José; Théodoly, Olivier

    2013-01-01

    We report how cell rheology measurements can be performed by monitoring the deformation of a cell in a microfluidic constriction, provided that friction and fluid leaks effects between the cell and the walls of the microchannels are correctly taken into account. Indeed, the mismatch between the rounded shapes of cells and the angular cross-section of standard microfluidic channels hampers efficient obstruction of the channel by an incoming cell. Moreover, friction forces between a cell and channels walls have never been characterized. Both effects impede a quantitative determination of forces experienced by cells in a constriction. Our study is based on a new microfluidic device composed of two successive constrictions, combined with optical interference microscopy measurements to characterize the contact zone between the cell and the walls of the channel. A cell squeezed in a first constriction obstructs most of the channel cross-section, which strongly limits leaks around cells. The rheological properties of the cell are subsequently probed during its entry in a second narrower constriction. The pressure force is determined from the pressure drop across the device, the cell velocity, and the width of the gutters formed between the cell and the corners of the channel. The additional friction force, which has never been analyzed for moving and constrained cells before, is found to involve both hydrodynamic lubrication and surface forces. This friction results in the existence of a threshold for moving the cells and leads to a non-linear behavior at low velocity. The friction force can nevertheless be assessed in the linear regime. Finally, an apparent viscosity of single cells can be estimated from a numerical prediction of the viscous dissipation induced by a small step in the channel. A preliminary application of our method yields an apparent loss modulus on the order of 100 Pa s for leukocytes THP-1 cells, in agreement with the literature data. PMID:24404016

  12. Continuous-flow microfluidic blood cell sorting for unprocessed whole blood using surface-micromachined microfiltration membranes.

    PubMed

    Li, Xiang; Chen, Weiqiang; Liu, Guangyu; Lu, Wei; Fu, Jianping

    2014-07-21

    White blood cells (WBCs) constitute about 0.1% of the blood cells, yet they play a critical role in innate and adaptive immune responses against pathogenic infections, allergic conditions, and malignancies and thus contain rich information about the immune status of the body. Rapid isolation of WBCs directly from whole blood is a prerequisite for any integrated immunoassay platform designed for examining WBC phenotypes and functions; however, such functionality is still challenging for blood-on-a-chip systems, as existing microfluidic cell sorting techniques are inadequate for efficiently processing unprocessed whole blood on chip with concurrent high throughput and cell purity. Herein we report a microfluidic chip for continuous-flow isolation and sorting of WBCs from whole blood with high throughput and separation efficiency. The microfluidic cell sorting chip leveraged the crossflow filtration scheme in conjunction with a surface-micromachined poly(dimethylsiloxane) (PDMS) microfiltration membrane (PMM) with high porosity. With a sample throughput of 1 mL h(-1), the microfluidic cell sorting chip could recover 27.4 ± 4.9% WBCs with a purity of 93.5 ± 0.5%. By virtue of its separation efficiency, ease of sample recovery, and high throughput enabled by its continuous-flow operation, the microfluidic cell sorting chip holds promise as an upstream component for blood sample preparation and analysis in integrated blood-on-a-chip systems. PMID:24895109

  13. Continuous-flow microfluidic blood cell sorting for unprocessed whole blood using surface-micromachined microfiltration membranes

    PubMed Central

    Li, Xiang; Chen, Weiqiang; Liu, Guangyu; Lu, Wei; Fu, Jianping

    2014-01-01

    White blood cells (WBCs) constitute about 0.1% of the blood cells, yet they play a critical role in innate and adaptive immune responses against pathogenic infections, allergic conditions, and malignancies and thus contain rich information about the immune status of the body. Rapid isolation of WBCs directly from whole blood is a prerequisite for any integrated immunoassay platform designed for examining WBC phenotypes and functions; however, such functionality is still challenging for blood-on-a-chip systems, as existing microfluidic cell sorting techniques are inadequate for efficiently processing unprocessed whole blood on chip with concurrent high throughput and cell purity. Herein we report a microfluidic chip for continuous-flow isolation and sorting of WBCs from whole blood with high throughput and separation efficiency. The microfluidic cell sorting chip leveraged the crossflow filtration scheme in conjunction with a surface-micromachined poly(dimethylsiloxane) (PDMS) microfiltration membrane (PMM) with high porosity. With a sample throughput of 1 mL hr−1, the microfluidic cell sorting chip could recover 27.4 ± 4.9% WBCs with a purity of 93.5 ± 0.5%. By virtue of its separation efficiency, ease of sample recovery, and high throughput enabled by its continuous-flow operation, the microfluidic cell sorting chip holds promise as an upstream component for blood sample preparation and analysis in integrated blood-on-a-chip systems. PMID:24895109

  14. Spatiotemporal microbial single-cell analysis using a high-throughput microfluidics cultivation platform.

    PubMed

    Grünberger, Alexander; Probst, Christopher; Helfrich, Stefan; Nanda, Arun; Stute, Birgit; Wiechert, Wolfgang; von Lieres, Eric; Nöh, Katharina; Frunzke, Julia; Kohlheyer, Dietrich

    2015-12-01

    Cell-to-cell heterogeneity typically evolves due to a manifold of biological and environmental factors and special phenotypes are often relevant for the fate of the whole population but challenging to detect during conventional analysis. We demonstrate a microfluidic single-cell cultivation platform that incorporates several hundred growth chambers, in which isogenic bacteria microcolonies growing in cell monolayers are tracked by automated time-lapse microscopy with spatiotemporal resolution. The device was not explicitly developed for a specific organism, but has a very generic configuration suitable for various different microbial organisms. In the present study, we analyzed Corynebacterium glutamicum microcolonies, thereby generating complete lineage trees and detailed single-cell data on division behavior and morphology in order to demonstrate the platform's overall capabilities. Furthermore, the occurrence of spontaneously induced stress in individual C. glutamicum cells was investigated by analyzing strains with genetically encoded reporter systems and optically visualizing SOS response. The experiments revealed spontaneous SOS induction in the absence of any external trigger comparable to results obtained by flow cytometry (FC) analyzing cell samples from conventional shake flask cultivation. Our microfluidic setup delivers detailed single-cell data with spatial and temporal resolution; complementary information to conventional FC results. PMID:26348020

  15. Microfluidics-Based Single-Cell Functional Proteomics for Fundamental and Applied Biomedical Applications

    NASA Astrophysics Data System (ADS)

    Yu, Jing; Zhou, Jing; Sutherland, Alex; Wei, Wei; Shin, Young Shik; Xue, Min; Heath, James R.

    2014-06-01

    We review an emerging microfluidics-based toolkit for single-cell functional proteomics. Functional proteins include, but are not limited to, the secreted signaling proteins that can reflect the biological behaviors of immune cells or the intracellular phosphoproteins associated with growth factor-stimulated signaling networks. Advantages of the microfluidics platforms are multiple. First, 20 or more functional proteins may be assayed simultaneously from statistical numbers of single cells. Second, cell behaviors (e.g., motility) may be correlated with protein assays. Third, extensions to quantized cell populations can permit measurements of cell-cell interactions. Fourth, rare cells can be functionally identified and then separated for further analysis or culturing. Finally, certain assay types can provide a conduit between biology and the physicochemical laws. We discuss the history and challenges of the field then review design concepts and uses of the microchip platforms that have been reported, with an eye toward biomedical applications. We then look to the future of the field.

  16. Role of Microfluidics in Blood-Brain Barrier Permeability Cell Culture Modeling: Relevance to CNS Disorders.

    PubMed

    Rusanov, Alexander L; Luzgina, Natalia G; Barreto, George E; Aliev, Gjumrakch

    2016-01-01

    In vitro modeling of the human blood-brain barrier (BBB) is critical for pre-clinical evaluation and predicting the permeability of newly developed potentially neurotoxic and neurotrophic drugs. Here we summarize the specific structural and functional features of endothelial cells as a key component of the BBB and compare analysis of different cell culture models in reflecting these features. Particular attention is paid to cellular models of the BBB in microfluidic devices capable of circulating nutrient media to simulate the blood flow of the brain. In these conditions, it is possible to reproduce a number of factors affecting endothelial cells under physiological conditions, including shear stress. In comparison with static cell models, concentration gradients, which determine the velocity of transport of substances, reproduce more accurately conditions of nutrient medium flow, since they eliminate the accumulation of substances near the basal membrane of cells, not typical for the situation in vivo. Co-cultivation of different types of cells forming the BBB, in separate cell chambers connected by microchannels, allows to evaluate the mutual influences of cells under normal conditions and when exposed to the test substance. New experimental possibilities that can be achieved through modeling of BBB in microfluidic devices determine the feasibility of their use in the practice for pre-clinical studies of novel drugs against neurodegenerative diseases. PMID:26831260

  17. Microfluidic Assay To Study the Combinatorial Impact of Substrate Properties on Mesenchymal Stem Cell Migration.

    PubMed

    Menon, Nishanth V; Chuah, Yon Jin; Phey, Samantha; Zhang, Ying; Wu, Yingnan; Chan, Vincent; Kang, Yuejun

    2015-08-12

    As an alternative to complex and costly in vivo models, microfluidic in vitro models are being widely used to study various physiological phenomena. It is of particular interest to study cell migration in a controlled microenvironment because of its vital role in a large number of physiological processes, such as wound healing, disease progression, and tissue regeneration. Cell migration has been shown to be affected by variations in the biochemical and physical properties of the extracellular matrix (ECM). To study the combinatorial impact of the ECM physical properties on cell migration, we have developed a microfluidic assay to induce migration of human bone marrow derived mesenchymal stem cells (hBMSCs) on polydimethylsiloxane (PDMS) substrates with varying combinatorial properties (hydrophobicity, stiffness, and roughness). The results show that although the initial cell adhesion and viability appear similar on all PDMS samples, the cell spreading and migration are enhanced on PDMS samples exhibiting intermediate levels of hydrophobicity, stiffness, and roughness. This study suggests that there is a particular range of substrate properties for optimal cell spreading and migration. The influence of substrate properties on hBMSC migration can help understand the physical cues that affect cell migration, which may facilitate the development of optimized engineered scaffolds with desired properties for tissue regeneration applications. PMID:26186177

  18. Microfabrication of a two-stage BioMEMS microfluidic cell sorter

    NASA Astrophysics Data System (ADS)

    Grafton, Meggie M.; Geheb, Benjamin; Jang, Jae Hyuk; Chuang, Han-Sheng; Rajdev, Pooja; Reece, Lisa M.; Irazoqui, Pedro P.; Wereley, Steven T.; Jung, Byunghoo; Leary, James F.

    2009-02-01

    Point-of-care devices represent the future for medical technologies. Current diagnostic tools are cumbersome, expensive, complicated, and often at risk for contamination. There is a need for cost effective, portable, closed-system, high-speed cell screening and cell isolating device. A microfabricated, exponentially-staging, BioMEMS microfluidic cytometer/cell sorting device offers these advantages over current technologies. A two-stage branched architecture allows the study of inter-particle spacing, flow relations, pressure measurements, and cell behavior in an environment where fluorescence detection is used to identify and analyze certain cellular characteristics. This device was microfabricated using the polymer PDMS to transmit light effectively, to be inexpensive and disposable, and to be easy to manipulate. For initial prototyping, an inverted fluorescent Nikon microscope provided the necessary excitation to view the particles and cells. For the portable device, avalanche photo diodes (APDs) and light emitting diodes (LEDs) are being incorporated into the device for the detection and excitation respectively. For low light level applications, sigma-delta modulation methods are being applied to reduce noise susceptibility and to detect the APD signal more efficiently. In addition, a data acquisition system (DAQ) has been designed that can effectively track signals from a cell sorter using a digital signal processing (DSP) board and a laptop computer. Currently elastomeric valves for diverting flow have been incorporated into the microfluidic chip. Measurements are being made of the effects of the microfluidics valve structures, or the simple opening and closing of selected channels to divert flow and cells down specific channels depending on their measured properties.

  19. Single cell membrane poration by bubble-induced microjets in a microfluidic chip.

    PubMed

    Li, Z G; Liu, A Q; Klaseboer, E; Zhang, J B; Ohl, C D

    2013-03-21

    This paper demonstrates membrane poration of a single suspension cell due to a fast liquid microjet. The jet is formed during the collapse of a laser induced bubble created at a variable stand-off distance from the target cell. The cell is trapped by a converging structure within a microfluidic chip. The asymmetrical growth and collapse of the cavitation bubble next to the cell lead to the microjetting, which deforms and porates the cell membrane. In the experiments, the membrane porations of myeloma cells are probed with the uptake of trypan blue. Time-resolved studies of the diffusion of trypan blue show a marked dependency on the bubble dynamics, i.e. the stand-off distance. The penetration length of the dye increases with shorter distances. Numerical simulations of the diffusion process agree with larger pores formed on the cell membrane. This method allows for a fast, repeatable, and localized rupture of membranes of individual cells in suspension. PMID:23364762

  20. Streamline based design guideline for deterministic microfluidic hydrodynamic single cell traps

    PubMed Central

    Shenoy, Aditi; Smith, Richard

    2015-01-01

    A prerequisite for single cell study is the capture and isolation of individual cells. In microfluidic devices, cell capture is often achieved by means of trapping. While many microfluidic trapping techniques exist, hydrodynamic methods are particularly attractive due to their simplicity and scalability. However, current design guidelines for single cell hydrodynamic traps predominantly rely on flow resistance manipulation or qualitative streamline analysis without considering the target particle size. This lack of quantitative design criteria from first principles often leads to non-optimal probabilistic trapping. In this work, we describe an analytical design guideline for deterministic single cell hydrodynamic trapping through the optimization of streamline distributions under laminar flow with cell size as a key parameter. Using this guideline, we demonstrate an example design which can achieve 100% capture efficiency for a given particle size. Finite element modelling was used to determine the design parameters necessary for optimal trapping. The simulation results were subsequently confirmed with on-chip microbead and white blood cell trapping experiments. PMID:25825618

  1. Characterization of subcellular morphology of single yeast cells using high frequency microfluidic impedance cytometer.

    PubMed

    Haandbæk, Niels; Bürgel, Sebastian C; Heer, Flavio; Hierlemann, Andreas

    2014-01-21

    Single-cell impedance cytometry is an electrical analysis method, which has been used to count and discriminate cells on the basis of their dielectric properties. The method has several advantages, such as being label free and requiring minimal sample preparation. So far, however, it has been limited to measuring cell properties that are visible at low frequencies, such as size and membrane capacitance. We demonstrate a microfluidic single cell impedance cytometer capable of dielectric characterization of single cells at frequencies up to 500 MHz. This device features a more than ten-fold increased frequency range compared to other devices and enables the study of both low and high frequency dielectric properties in parallel. The increased frequency range potentially allows for characterization of subcellular features in addition to the properties that are visible at lower frequencies. The capabilities of the cytometer are demonstrated by discriminating wild-type yeast from a mutant, which differs in size and distribution of vacuoles in the intracellular fluid. This discrimination is based on the differences in dielectric properties at frequencies around 250 MHz. The results are compared to a 3D finite-element model of the microfluidic channel accommodating either a wild-type or a mutant yeast cell. The model is used to derive quantitative values to characterize the dielectric properties of the cells. PMID:24264643

  2. A microfluidic manifold with a single pump system to generate highly mono-disperse alginate beads for cell encapsulation

    PubMed Central

    Kim, Choong; Park, Juyoung

    2014-01-01

    Cell encapsulation technology is a promising strategy applicable to tissue engineering and cell therapy. Many advanced microencapsulation chips that function via multiple syringe pumps have been developed to generate mono-disperse hydrogel beads encapsulating cells. However, their operation is difficult and only trained microfluidic engineers can use them with dexterity. Hence, we propose a microfluidic manifold system, driven by a single syringe pump, which can enable the setup of automated flow sequences and generate highly mono-disperse alginate beads by minimizing disturbances to the pump pressure. The encapsulation of P19 mouse embryonic carcinoma cells and embryonic body formation are demonstrated to prove the efficiency of the proposed system. PMID:25587376

  3. Microfluidic Probe for Single-Cell Lysis and Analysis in Adherent Tissue Culture

    PubMed Central

    Lauffenburger, Douglas A.; Han, Jongyoon

    2014-01-01

    Single-cell analysis provides information critical to understanding key disease processes that are characterized by significant cellular heterogeneity. Few current methods allow single-cell measurement without removing cells from the context of interest, which not only destroys contextual information but also may perturb the process under study. Here we present a microfluidic probe that lyses single adherent cells from standard tissue culture and captures the contents to perform single-cell biochemical assays. We use this probe to measure kinase and housekeeping protein activities, separately or simultaneously, from single human hepatocellular carcinoma cells in adherent culture. This tool has the valuable ability to perform measurements that clarify connections between extracellular context, signals and responses, especially in cases where only a few cells exhibit a characteristic of interest. PMID:24594667

  4. Direct 3D-printing of cell-laden constructs in microfluidic architectures.

    PubMed

    Liu, Justin; Hwang, Henry H; Wang, Pengrui; Whang, Grace; Chen, Shaochen

    2016-04-21

    Microfluidic platforms have greatly benefited the biological and medical fields, however standard practices require a high cost of entry in terms of time and energy. The utilization of three-dimensional (3D) printing technologies has greatly enhanced the ability to iterate and build functional devices with unique functions. However, their inability to fabricate within microfluidic devices greatly increases the cost of producing several different devices to examine different scientific questions. In this work, a variable height micromixer (VHM) is fabricated using projection 3D-printing combined with soft lithography. Theoretical and flow experiments demonstrate that altering the local z-heights of VHM improved mixing at lower flow rates than simple geometries. Mixing of two fluids occurs as low as 320 μL min(-1) in VHM whereas the planar zigzag region requires a flow rate of 2.4 mL min(-1) before full mixing occurred. Following device printing, to further demonstrate the ability of this projection-based method, complex, user-defined cell-laden scaffolds are directly printed inside the VHM. The utilization of this unique ability to produce 3D tissue models within a microfluidic system could offer a unique platform for medical diagnostics and disease modeling. PMID:26980159

  5. Engineering anastomosis between living capillary networks and endothelial cell-lined microfluidic channels.

    PubMed

    Wang, Xiaolin; Phan, Duc T T; Sobrino, Agua; George, Steven C; Hughes, Christopher C W; Lee, Abraham P

    2016-01-21

    This paper reports a method for generating an intact and perfusable microvascular network that connects to microfluidic channels without appreciable leakage. This platform incorporates different stages of vascular development including vasculogenesis, endothelial cell (EC) lining, sprouting angiogenesis, and anastomosis in sequential order. After formation of a capillary network inside the tissue chamber via vasculogenesis, the adjacent microfluidic channels are lined with a monolayer of ECs, which then serve as the high-pressure input ("artery") and low pressure output ("vein") conduits. To promote a tight interconnection between the artery/vein and the capillary network, sprouting angiogenesis is induced, which promotes anastomosis of the vasculature inside the tissue chamber with the EC lining along the microfluidic channels. Flow of fluorescent microparticles confirms the perfusability of the lumenized microvascular network, and minimal leakage of 70 kDa FITC-dextran confirms physiologic tightness of the EC junctions and completeness of the interconnections between artery/vein and the capillary network. This versatile device design and its robust construction methodology establish a physiological transport model of interconnected perfused vessels from artery to vascularized tissue to vein. The system has utility in a wide range of organ-on-a-chip applications as it enables the physiological vascular interconnection of multiple on-chip tissue constructs that can serve as disease models for drug screening. PMID:26616908

  6. Red Blood Cell Dispersion in Morphologically-Inspired Microfluidic Models of Alveolar Capillary Networks

    NASA Astrophysics Data System (ADS)

    Stauber, Hagit; Fishler, Rami; Waisman, Dan; Sznitman, Josue

    2014-11-01

    Microfluidics is frequently used to study blood flow characteristics in microcapillary networks and investigate transport properties of red blood cells (RBC). To date, most of microfluidic studies have not focused on the specific morphology of alveolar capillary networks (ACN), with characteristic length scales of ~ 5 μm, known to give rise to organ-specific blood flow characteristics. To better understand flow characteristics and dispersion of RBCs in ACNs, we have designed morphologically-inspired microfluidic models of alveolar capillary beds at a real scale. We fabricate lab-on-chip devices featuring confined staggered pillar arrays with diameters of ~ 10 μm, representative of the dense ACN capillary meshes. Devices are supplied by an external reservoir containing whole blood at various hematocrit levels, to mimic RBC perfusion (Re < 0.01) within alveolar capillaries. Whole-field velocity patterns are imaged (PIV) and RBC motion is tracked using particle tracking velocimetry (PTV) from which dispersion coefficients are extracted. Our efforts are aimed at delivering a real-scale quantitative description of the pulmonary ACN microcirculation.

  7. A Mechanically Tunable Microfluidic Cell-Trapping Device

    PubMed Central

    Zhu, Jing; Shang, Junyi; Olsen, Timothy; Liu, Kun; Brenner, David; Lin, Qiao

    2015-01-01

    Controlled manipulation, such as isolation, positioning and trapping of cells, is important in basic biological research and clinical diagnostics. Micro/nanotechnologies have been enabling more effective and efficient cell trapping than possible with conventional platforms. Currently available micro/nanoscale methods for cell trapping, however, still lack flexibility in precisely controlling the number of trapped cells. We exploited the large compliance of elastomers to create an array of cell-trapping microstructures, whose dimensions can be mechanically modulated by inducing uniformly distributed strain via application of external force on the chip. The device consists of two elastomer polydimethylsiloxane (PDMS) sheets, one of which bears dam-like, cup-shaped geometries to physically capture cells. The mechanical modulation is used to tune the characteristics of cell trapping to capture a predetermined number of cells, from single cells to multiple cells. Thus, enhanced utility and flexibility for practical applications can be attained, as demonstrated by tunable trapping of MCF-7 cells, a human breast cancer cell line. PMID:25821347

  8. Droplet microfluidic technology for single-cell high-throughput screening.

    PubMed

    Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J Brian; Rothberg, Jonathan M; Link, Darren R; Perrimon, Norbert; Samuels, Michael L

    2009-08-25

    We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses. PMID:19617544

  9. Microfluidic direct writer with integrated declogging mechanism for fabricating cell-laden hydrogel constructs.

    PubMed

    Ghorbanian, Setareh; Qasaimeh, Mohammad A; Akbari, Mohsen; Tamayol, Ali; Juncker, David

    2014-06-01

    Cell distribution and nutrient supply in 3D cell-laden hydrogel scaffolds are critical and should mimic the in vivo cellular environment, but been difficult to control with conventional fabrication methods. Here, we present a microfluidic direct writer (MFDW) to construct 3D cell-laden hydrogel structures with openings permitting media exchange. The MFDW comprises a monolithic microfluidic head, which delivers coaxial streams of cell-laden sodium alginate and calcium chloride solutions to form hydrogel fibers. Fiber diameter is controlled by adjusting the ratio of the volumetric flow rates. The MFDW head is mounted on a motorized stage, which is automatically controlled and moves at a speed synchronized with the speed of fiber fabrication. Head geometry, flow rates, and viscosity of the writing solutions were optimized to prevent the occurrence of curling and bulging. For continuous use, a highly reliable process is needed, which was accomplished with the integration of a declogging conduit supplying a solvent to dissolve the clogging gel. The MFDW was used for layer-by-layer fabrication of simple 3D structures with encapsulated cells. Assembly of 3D structures with distinct fibers is demonstrated by alternatively delivering two different alginate gel solutions. The MFDW head can be built rapidly and easily, and will allow 3D constructs for tissue engineering to be fabricated with multiple hydrogels and cell types. PMID:24590741

  10. Ultra-fast, label-free isolation of circulating tumor cells from blood using spiral microfluidics.

    PubMed

    Warkiani, Majid Ebrahimi; Khoo, Bee Luan; Wu, Lidan; Tay, Andy Kah Ping; Bhagat, Ali Asgar S; Han, Jongyoon; Lim, Chwee Teck

    2016-01-01

    Circulating tumor cells (CTCs) are rare cancer cells that are shed from primary or metastatic tumors into the peripheral blood circulation. Phenotypic and genetic characterization of these rare cells can provide important information to guide cancer staging and treatment, and thus further research into their characteristics and properties is an area of considerable interest. In this protocol, we describe detailed procedures for the production and use of a label-free spiral microfluidic device to allow size-based isolation of viable CTCs using hydrodynamic forces that are present in curvilinear microchannels. This spiral system enables us to achieve ≥ 85% recovery of spiked cells across multiple cancer cell lines and 99.99% depletion of white blood cells in whole blood. The described spiral microfluidic devices can be produced at an extremely low cost using standard microfabrication and soft lithography techniques (2-3 d), and they can be operated using two syringe pumps for lysed blood samples (7.5 ml in 12.5 min for a three-layered multiplexed chip). The fast processing time and the ability to collect CTCs from a large patient blood volume allows this technique to be used experimentally in a broad range of potential genomic and transcriptomic applications. PMID:26678083

  11. Droplet microfluidic technology for single-cell high-throughput screening

    PubMed Central

    Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J. Brian; Rothberg, Jonathan M.; Link, Darren R.; Perrimon, Norbert; Samuels, Michael L.

    2009-01-01

    We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses. PMID:19617544

  12. Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip.

    PubMed

    Hou, Hsien-San; Chang, Hui-Fang; Cheng, Ji-Yen

    2015-01-01

    The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic microfluidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies. PMID:26780080

  13. Spatio-temporal analysis of tamoxifen-induced bystander effects in breast cancer cells using microfluidics.

    PubMed

    Rios-Mondragon, Ivan; Wang, Xiang; Gerdes, Hans-Hermann

    2012-06-01

    The bystander effect in cancer therapy is the inhibition or killing of tumor cells that are adjacent to those directly affected by the agent used for treatment. In the case of chemotherapy, little is known as to how much and by which mechanisms bystander effects contribute to the elimination of tumor cells. This is mainly due to the difficulty to distinguish between targeted and bystander cells since both are exposed to the pharmaceutical compound. We here studied the interaction of tamoxifen-treated human breast cancer MCF-7 cells with their neighboring counterparts by exploiting laminar flow patterning in a microfluidic chip to ensure selective drug delivery. The spatio-temporal evolution of the bystander response in non-targeted cells was analyzed by measuring the mitochondrial membrane potential under conditions of free diffusion. Our data show that the bystander response is detectable as early as 1 hour after drug treatment and reached effective distances of at least 2.8 mm. Furthermore, the bystander effect was merely dependent on diffusible factors rather than cell contact-dependent signaling. Taken together, our study illustrates that this microfluidic approach is a promising tool for screening and optimization of putative chemotherapeutic drugs to maximize the bystander response in cancer therapy. PMID:23750189

  14. Hollow fiber integrated microfluidic platforms for in vitro Co-culture of multiple cell types.

    PubMed

    Huang, Jen-Huang; Harris, Jennifer F; Nath, Pulak; Iyer, Rashi

    2016-10-01

    This study demonstrates a rapid prototyping approach for fabricating and integrating porous hollow fibers (HFs) into microfluidic device. Integration of HF can enhance mass transfer and recapitulate tubular shapes for tissue-engineered environments. We demonstrate the integration of single or multiple HFs, which can give the users the flexibility to control the total surface area for tissue development. We also present three microfluidic designs to enable different co-culture conditions such as the ability to co-culture multiple cell types simultaneously on a flat and tubular surface, or inside the lumen of multiple HFs. Additionally, we introduce a pressurized cell seeding process that can allow the cells to uniformly adhere on the inner surface of HFs without losing their viabilities. Co-cultures of lung epithelial cells and microvascular endothelial cells were demonstrated on the different platforms for at least five days. Overall, these platforms provide new opportunities for co-culturing of multiple cell types in a single device to reconstruct native tissue micro-environment for biomedical and tissue engineering research. PMID:27613401

  15. High-throughput microfluidics to control and measure signaling dynamics in single yeast cells

    PubMed Central

    Hansen, Anders S.; Hao, Nan; O'Shea, Erin K.

    2015-01-01

    Microfluidics coupled to quantitative time-lapse fluorescence microscopy is transforming our ability to control, measure, and understand signaling dynamics in single living cells. Here we describe a pipeline that incorporates multiplexed microfluidic cell culture, automated programmable fluid handling for cell perturbation, quantitative time-lapse microscopy, and computational analysis of time-lapse movies. We illustrate how this setup can be used to control the nuclear localization of the budding yeast transcription factor Msn2. Using this protocol, we generate oscillations of Msn2 localization and measure the dynamic gene expression response of individual genes in single cells. The protocol allows a single researcher to perform up to 20 different experiments in a single day, whilst collecting data for thousands of single cells. Compared to other protocols, the present protocol is relatively easy to adopt and higher-throughput. The protocol can be widely used to control and monitor single-cell signaling dynamics in other signal transduction systems in microorganisms. PMID:26158443

  16. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation.

    PubMed

    Binder, Dennis; Probst, Christopher; Grünberger, Alexander; Hilgers, Fabienne; Loeschcke, Anita; Jaeger, Karl-Erich; Kohlheyer, Dietrich; Drepper, Thomas

    2016-01-01

    Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust

  17. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation

    PubMed Central

    Hilgers, Fabienne; Loeschcke, Anita; Jaeger, Karl-Erich; Kohlheyer, Dietrich; Drepper, Thomas

    2016-01-01

    Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust

  18. Microfluidic gradient device for studying mesothelial cell migration and the effect of chronic carbon nanotube exposure

    PubMed Central

    Zhang, Hanyuan; Lohcharoenkal, Warangkana; Sun, Jianbo; Li, Xiang; Wang, Liying; Wu, Nianqiang; Rojanasakul, Yon; Liu, Yuxin

    2016-01-01

    Cell migration is one of the crucial steps in many physiological and pathological processes, including cancer development. Our recent studies have shown that carbon nanotubes (CNTs), similarly to asbestos, can induce accelerated cell growth and invasiveness that contribute to their mesothelioma pathogenicity. Malignant mesothelioma is a very aggressive tumor that develops from cells of the mesothelium, and is most commonly caused by exposure to asbestos. CNTs have a similar structure and mode of exposure to asbestos. This has raised a concern regarding the potential carcinogenicity of CNTs, especially in the pleural area which is a key target for asbestos-related diseases. In this paper, a static microfluidic gradient device was applied to study the migration of human pleural mesothelial cells which had been through a long-term exposure (4 months) to subcytotoxic concentration (0.02 μg cm−2) of single-walled CNTs (SWCNTs). Multiple migration signatures of these cells were investigated using the microfluidic gradient device for the first time. During the migration study, we observed that cell morphologies changed from flattened shapes to spindle shapes prior to their migration after their sensing of the chemical gradient. The migration of chronically SWCNT-exposed mesothelial cells was evaluated under different fetal bovine serum (FBS) concentration gradients, and the migration speeds and number of migrating cells were extracted and compared. The results showed that chronically SWCNT-exposed mesothelial cells are more sensitive to the gradient compared to non-SWCNT-exposed cells. The method described here allows simultaneous detection of cell morphology and migration under chemical gradient conditions, and also allows for real-time monitoring of cell motility that resembles in vivo cell migration. This platform would be much needed for supporting the development of more physiologically relevant cell models for better assessment and characterization of the

  19. Microfluidic gradient device for studying mesothelial cell migration and the effect of chronic carbon nanotube exposure

    NASA Astrophysics Data System (ADS)

    Zhang, Hanyuan; Lohcharoenkal, Warangkana; Sun, Jianbo; Li, Xiang; Wang, Liying; Wu, Nianqiang; Rojanasakul, Yon; Liu, Yuxin

    2015-07-01

    Cell migration is one of the crucial steps in many physiological and pathological processes, including cancer development. Our recent studies have shown that carbon nanotubes (CNTs), similarly to asbestos, can induce accelerated cell growth and invasiveness that contribute to their mesothelioma pathogenicity. Malignant mesothelioma is a very aggressive tumor that develops from cells of the mesothelium, and is most commonly caused by exposure to asbestos. CNTs have a similar structure and mode of exposure to asbestos. This has raised a concern regarding the potential carcinogenicity of CNTs, especially in the pleural area which is a key target for asbestos-related diseases. In this paper, a static microfluidic gradient device was applied to study the migration of human pleural mesothelial cells which had been through a long-term exposure (4 months) to subcytotoxic concentration (0.02 µg cm-2) of single-walled CNTs (SWCNTs). Multiple migration signatures of these cells were investigated using the microfluidic gradient device for the first time. During the migration study, we observed that cell morphologies changed from flattened shapes to spindle shapes prior to their migration after their sensing of the chemical gradient. The migration of chronically SWCNT-exposed mesothelial cells was evaluated under different fetal bovine serum (FBS) concentration gradients, and the migration speeds and number of migrating cells were extracted and compared. The results showed that chronically SWCNT-exposed mesothelial cells are more sensitive to the gradient compared to non-SWCNT-exposed cells. The method described here allows simultaneous detection of cell morphology and migration under chemical gradient conditions, and also allows for real-time monitoring of cell motility that resembles in vivo cell migration. This platform would be much needed for supporting the development of more physiologically relevant cell models for better assessment and characterization of the

  20. Analysis of transcriptomic and proteomic profiles demonstrates improved Madin-Darby canine kidney cell function in a renal microfluidic biochip.

    PubMed

    Snouber, Leila Choucha; Letourneur, Franck; Chafey, Philippe; Broussard, Cedric; Monge, Matthieu; Legallais, Cécile; Leclerc, Eric

    2012-01-01

    We have evaluated the influence of the microfluidic environment on renal cell functionality. For that purpose, we performed a time lapse transcriptomic and proteomic analysis in which we compared gene and protein expressions of Madin-Darby canine kidney cells after 24 h and 96 h of culture in both microfluidic biochips and plates. The transcriptomic and proteomic integration revealed that the ion transporters involved in calcium, phosphate, and sodium homoeostasis and several genes involved in H(+) transporters and pH regulation were up-regulated in microfluidic biochips. Concerning drug metabolism, we found Phase I (CYP P450), Phase II enzymes (GST), various multidrug resistance genes (MRP), and Phase III transporters (SLC) were also up-regulated in the biochips. Furthermore, the study shows that those inductions were correlated with the induction of the Ahr and Nrf-2 dependent pathways, which results in a global cytoprotective response induced by the microenvironment. However, there was no apoptosis situation or cell death in the biochips. Microfluidic biochips may thus provide an important insight into exploring xenobiotic injury and transport modifications in this type of bioartificial microfluidic kidney. Finally, the investigation demonstrated that combining the transcriptomic and proteomic analyses obtained from a cell "on chip" culture would provide a pertinent new tool in the mechanistic interpretation of cellular mechanisms for predicting kidney cell toxicity and renal clearance in vitro. PMID:22095740

  1. A Bead-Based Microfluidic Approach to Integrated Single-Cell Gene Expression Analysis by Quantitative RT-PCR

    PubMed Central

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

    2015-01-01

    Gene expression analysis at the single-cell level is critical to understanding variations among cells in heterogeneous populations. Microfluidic reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is well suited to gene expression assays of single cells. We present a microfluidic approach that integrates all functional steps for RT-qPCR of a single cell, including isolation and lysis of the cell, as well as purification, reverse transcription and quantitative real-time PCR of messenger RNA in the cell lysate. In this approach, all reactions in the multi-step assay of a single lysed cell can be completed on microbeads, thereby simplifying the design, fabrication and operation of the microfluidic device, as well as facilitating the minimization of sample loss or contamination. In the microfluidic device, a single cell is isolated and lysed; mRNA in the cell lysate is then analyzed by RT-qPCR using primers immobilized on microbeads in a single microchamber whose temperature is controlled in closed loop via an integrated heater and temperature sensor. The utility of the approach was demonstrated by the analysis of the effects of the drug (methyl methanesulfonate, MMS) on the induction of the cyclin-dependent kinase inhibitor 1a (CDKN1A) in single human cancer cells (MCF-7), demonstrating the potential of our approach for efficient, integrated single-cell RT-qPCR for gene expression analysis. PMID:25883782

  2. Microfluidic Devices for Terahertz Spectroscopy of Live Cells Toward Lab-on-a-Chip Applications.

    PubMed

    Tang, Qi; Liang, Min; Lu, Yi; Wong, Pak Kin; Wilmink, Gerald J; Zhang, Donna; Xin, Hao

    2016-01-01

    THz spectroscopy is an emerging technique for studying the dynamics and interactions of cells and biomolecules, but many practical challenges still remain in experimental studies. We present a prototype of simple and inexpensive cell-trapping microfluidic chip for THz spectroscopic study of live cells. Cells are transported, trapped and concentrated into the THz exposure region by applying an AC bias signal while the chip maintains a steady temperature at 37 °C by resistive heating. We conduct some preliminary experiments on E. coli and T-cell solution and compare the transmission spectra of empty channels, channels filled with aqueous media only, and channels filled with aqueous media with un-concentrated and concentrated cells. PMID:27049392

  3. Microfluidic Devices for Terahertz Spectroscopy of Live Cells Toward Lab-on-a-Chip Applications

    PubMed Central

    Tang, Qi; Liang, Min; Lu, Yi; Wong, Pak Kin; Wilmink, Gerald J.; D. Zhang, Donna; Xin, Hao

    2016-01-01

    THz spectroscopy is an emerging technique for studying the dynamics and interactions of cells and biomolecules, but many practical challenges still remain in experimental studies. We present a prototype of simple and inexpensive cell-trapping microfluidic chip for THz spectroscopic study of live cells. Cells are transported, trapped and concentrated into the THz exposure region by applying an AC bias signal while the chip maintains a steady temperature at 37 °C by resistive heating. We conduct some preliminary experiments on E. coli and T-cell solution and compare the transmission spectra of empty channels, channels filled with aqueous media only, and channels filled with aqueous media with un-concentrated and concentrated cells. PMID:27049392

  4. A microfluidic perfusion platform for cultivation and screening study of motile microalgal cells

    PubMed Central

    Eu, Young-Jae; Park, Hye-Sun; Kim, Dong-Pyo; Wook Hong, Jong

    2014-01-01

    Systematic screening of algal cells is getting huge interest due to their capability of producing lipid-based biodiesel. Here, we introduce a new microfluidic platform composed of an array of perfusion chambers designed for long-term cultivation and preliminary screening of motile microalgal cells through loading and releasing of cells to and from the chambers. The chemical environment in each perfusion chamber was independently controlled for 5 days. The effect of nitrogen-depletion on the lipid production, phototaxis behavior in the absence of Ca2+, and cytotoxic effect of herbicide on microalgal cells was successfully monitored and compared with simultaneous control experiments on the platform. The present methodology could be extended to effective screening of algal cells and various cell lines for the production of biodiesel and other useful chemicals. PMID:24803962

  5. Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines.

    PubMed

    Szeto, Gregory Lee; Van Egeren, Debra; Worku, Hermoon; Sharei, Armon; Alejandro, Brian; Park, Clara; Frew, Kirubel; Brefo, Mavis; Mao, Shirley; Heimann, Megan; Langer, Robert; Jensen, Klavs; Irvine, Darrell J

    2015-01-01

    B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to "professional" APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale "cell squeezing" process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8(+)T-cells, and not CD4(+)T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8(+)T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8(+)T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8(+)T-cells, and decoupling of antigen uptake from B-cell activation. PMID:25999171

  6. Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines

    PubMed Central

    Lee Szeto, Gregory; Van Egeren, Debra; Worku, Hermoon; Sharei, Armon; Alejandro, Brian; Park, Clara; Frew, Kirubel; Brefo, Mavis; Mao, Shirley; Heimann, Megan; Langer, Robert; Jensen, Klavs; Irvine, Darrell J

    2015-01-01

    B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8+T-cells, and not CD4+T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8+T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8+T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8+T-cells, and decoupling of antigen uptake from B-cell activation. PMID:25999171

  7. Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines

    NASA Astrophysics Data System (ADS)

    Lee Szeto, Gregory; van Egeren, Debra; Worku, Hermoon; Sharei, Armon; Alejandro, Brian; Park, Clara; Frew, Kirubel; Brefo, Mavis; Mao, Shirley; Heimann, Megan; Langer, Robert; Jensen, Klavs; Irvine, Darrell J.

    2015-05-01

    B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8+T-cells, and not CD4+T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8+T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8+T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8+T-cells, and decoupling of antigen uptake from B-cell activation.

  8. A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

    PubMed Central

    Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

    2014-01-01

    Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. PMID:25545548

  9. Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel.

    PubMed

    Choi, Jong Seob; Piao, Yunxian; Seo, Tae Seok

    2014-01-01

    The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40% stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function. PMID:24120039

  10. Microfluidic Synthesis of Microfibers for Magnetic-Responsive Controlled Drug Release and Cell Culture

    PubMed Central

    Lin, Yung-Sheng; Huang, Keng-Shiang; Yang, Chih-Hui; Wang, Chih-Yu; Yang, Yuh-Shyong; Hsu, Hsiang-Chen; Liao, Yu-Ju; Tsai, Chia-Wen

    2012-01-01

    This study demonstrated the fabrication of alginate microfibers using a modular microfluidic system for magnetic-responsive controlled drug release and cell culture. A novel two-dimensional fluid-focusing technique with multi-inlets and junctions was used to spatiotemporally control the continuous laminar flow of alginate solutions. The diameter of the manufactured microfibers, which ranged from 211 µm to 364 µm, could be well controlled by changing the flow rate of the continuous phase. While the model drug, diclofenac, was encapsulated into microfibers, the drug release profile exhibited the characteristic of a proper and steady release. Furthermore, the diclofenac release kinetics from the magnetic iron oxide-loaded microfibers could be controlled externally, allowing for a rapid drug release by applying a magnetic force. In addition, the successful culture of glioblastoma multiforme cells in the microfibers demonstrated a good structural integrity and environment to grow cells that could be applied in drug screening for targeting cancer cells. The proposed microfluidic system has the advantages of ease of fabrication, simplicity, and a fast and low-cost process that is capable of generating functional microfibers with the potential for biomedical applications, such as drug controlled release and cell culture. PMID:22470443

  11. A hybrid microfluidic system for regulation of neural differentiation in induced pluripotent stem cells.

    PubMed

    Hesari, Zahra; Soleimani, Massoud; Atyabi, Fatemeh; Sharifdini, Meysam; Nadri, Samad; Warkiani, Majid Ebrahimi; Zare, Mehrak; Dinarvand, Rassoul

    2016-06-01

    Controlling cellular orientation, proliferation, and differentiation is valuable in designing organ replacements and directing tissue regeneration. In the present study, we developed a hybrid microfluidic system to produce a dynamic microenvironment by placing aligned PDMS microgrooves on surface of biodegradable polymers as physical guidance cues for controlling the neural differentiation of human induced pluripotent stem cells (hiPSCs). The neuronal differentiation capacity of cultured hiPSCs in the microfluidic system and other control groups was investigated using quantitative real time PCR (qPCR) and immunocytochemistry. The functionally of differentiated hiPSCs inside hybrid system's scaffolds was also evaluated on the rat hemisected spinal cord in acute phase. Implanted cell's fate was examined using tissue freeze section and the functional recovery was evaluated according to the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale. Our results confirmed the differentiation of hiPSCs to neuronal cells on the microfluidic device where the expression of neuronal-specific genes was significantly higher compared to those cultured on the other systems such as plain tissue culture dishes and scaffolds without fluidic channels. Although survival and integration of implanted hiPSCs did not lead to a significant functional recovery, we believe that combination of fluidic channels with nanofiber scaffolds provides a great microenvironment for neural tissue engineering, and can be used as a powerful tool for in situ monitoring of differentiation potential of various kinds of stem cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1534-1543, 2016. PMID:26914600

  12. High-Throughput Cytotoxicity Testing System of Acetaminophen Using a Microfluidic Device (MFD) in HepG2 Cells.

    PubMed

    Ju, Seon Min; Jang, Hyun-Jun; Kim, Kyu-Bong; Kim, Jeongyun

    2015-01-01

    A lab-on-a-chip (LOC) is a microfluidic device (MFD) that integrates several lab functions into a single chip of only millimeters in size. LOC provides several advantages, such as low fluidic volumes consumption, faster analysis, compactness, and massive parallelization. These properties enable a microfluidic-based high-throughput drug screening (HTDS) system to acquire cell-based abundant cytotoxicity results depending on linear gradient concentration of drug with only few hundreds of microliters of the drug. Therefore, a microfluidic device was developed containing an array of eight separate microchambers for cultivating HepG2 cells to be exposed to eight different concentrations of acetaminophen (APAP) through a diffusive-mixing-based concentration gradient generator. Every chamber array with eight different concentrations (0, 5.7, 11.4, 17.1, 22.8, 28.5, 34.2, or 40 mM) APAP had four replicating cell culture chambers. Consequently, 32 experimental results were acquired with a single microfluidic device experiment. The microfluidic high-throughput cytotoxicity device (μHTCD) and 96-well culture system showed comparable cytotoxicity results with increasing APAP concentration of 0 to 40 mM. The HTDS system yields progressive concentration-dependent cytotoxicity results using minimal reagent and time. Data suggest that the HTDS system may be applicable as alternative method for cytotoxicity screening for new drugs in diverse cell types. PMID:26241707

  13. Bladder cancer cells re-educate TAMs through lactate shuttling in the microfluidic cancer microenvironment

    PubMed Central

    Liu, Pengfei; Cao, Yanwei; Wang, Yonghua; Yang, Xuecheng; Xu, Xiaodong; Wang, Xinsheng; Niu, Haitao

    2015-01-01

    Background In the present study, we aimed to investigate the influence of lactate shuttling on the functional polarization and spatial distribution of transitional cell carcinoma of the bladder (TCCB) cells and macrophages. Methods We designed a microfluidic coculture chip for real-time integrative assays. The effect of lactate shuttling on the re-education of macrophages by TCCB cells was explored by measuring the levels of NO using a total NO assay kit and by evaluating the protein expression of iNOS, p-NFkB-p65, Arg-1 and HIF-1α via cell immunofluorescence and western blotting. Additionally, we examined TCCB cell viability using acridine orange/ethidium bromide (AO/EB) and MitoTracker staining. Moreover, the concentration distributions of lactate and large signaling proteins in the culture chambers were measured using 4′,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate-dextran (FITC-dextran). Furthermore, the recruitment of macrophages and the influence of macrophages on BC metastasis were observed via light microscopy. Results We confirmed that TCCB cells reprogrammed macrophages into an M2 phenotype. Moreover, lactate inhibited M1 polarization and induced M2 polarization of macrophages, but blockade of cancer cell-macrophage lactate flux significantly inhibited the re-education of macrophages by TCCB cells. In addition, lactate diffused faster and deeper than large signaling proteins in the microfluidic tumor microenvironment. Furthermore, lactate alone induced the migration of macrophages, and M1, but not M2, macrophages reduced the motility of TCCB cells. Conclusions TCCB cells reprogrammed macrophages into an M2 phenotype in a manner that depended on cancer cell-TAM lactate flux. Furthermore, the lactate shuttle may be a determinant of the density of TAMs in tumor tissue. PMID:26474279

  14. A laser-based technology for fabricating a soda-lime glass based microfluidic device for circulating tumour cell capture.

    PubMed

    Nieto, Daniel; Couceiro, Ramiro; Aymerich, Maria; Lopez-Lopez, Rafael; Abal, Miguel; Flores-Arias, María Teresa

    2015-10-01

    We developed a laser-based technique for fabricating microfluidic microchips on soda-lime glass substrates. The proposed methodology combines a laser direct writing, as a manufacturing tool for the fabrication of the microfluidics structures, followed by a post-thermal treatment with a CO2 laser. This treatment will allow reshaping and improving the morphological (roughness) and optical qualities (transparency) of the generated microfluidics structures. The use of lasers commonly implemented for material processing makes this technique highly competitive when compared with other glass microstructuring approaches. The manufactured chips were tested with tumour cells (Hec 1A) after being functionalized with an epithelial cell adhesion molecule (EpCAM) antibody coating. Cells were successfully arrested on the pillars after being flown through the device giving our technology a translational application in the field of cancer research. PMID:26218523

  15. Microfluidic separation of live and dead yeast cells using reservoir-based dielectrophoresis

    PubMed Central

    Patel, Saurin; Showers, Daniel; Vedantam, Pallavi; Tzeng, Tzuen-Rong; Qian, Shizhi; Xuan, Xiangchun

    2012-01-01

    Separating live and dead cells is critical to the diagnosis of early stage diseases and to the efficacy test of drug screening, etc. This work demonstrates a novel microfluidic approach to dielectrophoretic separation of yeast cells by viability. It exploits the cell dielectrophoresis that is induced by the inherent electric field gradient at the reservoir-microchannel junction to selectively trap dead yeast cells and continuously separate them from live ones right inside the reservoir. This approach is therefore termed reservoir-based dielectrophoresis (rDEP). It has unique advantages as compared to existing dielectrophoretic approaches such as the occupation of zero channel space and the elimination of any mechanical or electrical parts inside microchannels. Such an rDEP cell sorter can be readily integrated with other components into lab-on-a-chip devices for applications to biomedical diagnostics and therapeutics. PMID:23853679

  16. Development of microfluidic-based cell collection devices for in vitro and in vivo use

    NASA Astrophysics Data System (ADS)

    Butt, Logan; Entenberg, Dave; Hemachandra, L. P. Madhubhani; Strohmayer, Matthew; Keely, Patricia; Aguirre-Ghiso, Julio; Condeelis, John S.; Castracane, James

    2016-03-01

    The NANIVID - or Nano Intravital Device - is an implantable delivery tool designed to locally affect the tumor microenvironment in vivo. This technology is being redesigned and validated as a cell collection tool for the study of metastatic cancer cells. A methodology has been developed to facilitate this transition, consisting of microfluidic analysis of the device microchannels and a series of cell-related collection experiments building up to in vivo collection. Single-chamber designs were first used to qualitatively demonstrate the feasibility of cell collection ex vivo. This was followed by the development and implementation of devices containing a second, negative-control chamber for quantitative analysis. This work sets the foundation for in vivo cancer cell migration studies utilizing the NANIVID.

  17. A Microfluidic Cell Co-Culture Platform with a Liquid Fluorocarbon Separator

    PubMed Central

    Brewer, Bryson M.; Shi, Mingjian; Edd, Jon F.; Webb, Donna J.; Li, Deyu

    2014-01-01

    A microfluidic cell co-culture platform that uses a liquid fluorocarbon oil barrier to separate cells into different culture chambers has been developed. Characterization indicates that the oil barrier could be effective for multiple days, and a maximum pressure difference between the oil barrier and aqueous media in the cell culture chamber could be as large as ∼3.43 kPa before the oil barrier fails. Biological applications have been demonstrated with the separate transfection of two groups of primary hippocampal neurons with two different fluorescent proteins and subsequent observation of synaptic contacts between the neurons. In addition, the quality of the fluidic seal provided by the oil barrier is shown to be greater than that of an alternative solid-PDMS valve barrier design by testing the ability of each device to block low molecular weight CellTracker dyes used to stain cells in the culture chambers. PMID:24420386

  18. Image processing and classification algorithm for yeast cell morphology in a microfluidic chip

    NASA Astrophysics Data System (ADS)

    Yang Yu, Bo; Elbuken, Caglar; Ren, Carolyn L.; Huissoon, Jan P.

    2011-06-01

    The study of yeast cell morphology requires consistent identification of cell cycle phases based on cell bud size. A computer-based image processing algorithm is designed to automatically classify microscopic images of yeast cells in a microfluidic channel environment. The images were enhanced to reduce background noise, and a robust segmentation algorithm is developed to extract geometrical features including compactness, axis ratio, and bud size. The features are then used for classification, and the accuracy of various machine-learning classifiers is compared. The linear support vector machine, distance-based classification, and k-nearest-neighbor algorithm were the classifiers used in this experiment. The performance of the system under various illumination and focusing conditions were also tested. The results suggest it is possible to automatically classify yeast cells based on their morphological characteristics with noisy and low-contrast images.

  19. Microfluidic separation of viruses from blood cells based on intrinsic transport processes

    PubMed Central

    Zhao, Chao; Cheng, Xuanhong

    2011-01-01

    Clinical analysis of acute viral infection in blood requires the separation of viral particles from blood cells, since the cytoplasmic enzyme inhibits the subsequent viral detection. To facilitate this procedure in settings without access to a centrifuge, we present a microfluidic device to continuously purify bionanoparticles from cells based on their different intrinsic movements on the microscale. In this device, a biological sample is layered on top of a physiological buffer, and both fluids are transported horizontally at the same flow rate in a straight channel under laminar flow. While the micron sized particles such as cells sediment to the bottom layer with a predictable terminal velocity, the nanoparticles move vertically by diffusion. As their vertical travel distances have a different dependence on time, the micro- and nanoparticles can preferentially reside in the bottom and top layers respectively after certain residence time, yielding purified viruses. We first performed numerical analysis to predicate the particle separation and then tested the theory using suspensions of synthetic particles and biological samples. The experimental results using dilute synthetic particles closely matched the numerical analysis of a two layer flow system containing different sized particles. Similar purification was achieved using diluted blood spiked with human immunodeficiency virus. However, viral purification in whole blood is compromised due to extensive bioparticle collisions. With the parallelization and automation potential offered by microfluidics, this device has the potential to function as an upstream sample preparation module to continuously provide cell depleted bio-nanoparticles for downstream analysis. PMID:22007267

  20. Glucose microfluidic fuel cell based on silver bimetallic selective catalysts for on-chip applications

    NASA Astrophysics Data System (ADS)

    Cuevas-Muñiz, F. M.; Guerra-Balcázar, M.; Esquivel, J. P.; Sabaté, N.; Arriaga, L. G.; Ledesma-García, J.

    2012-10-01

    A glucose microfluidic fuel cell with outstanding performance at zero flow condition is presented. Polarization tests showed that bimetallic materials based in silver (AuAg/C as anode, PtAg/C as cathode) exhibit tolerance to byproducts and crossover effect. This allowed achieving one of the highest power densities reported for glucose fuel cells, up to a value of 630 μW cm-2 using two separated laminar flows of reactants. Furthermore, the tolerance to crossover effect caused by the selectivity of PtAg/C to oxygen reduction reaction in presence of glucose permitted using a single flow containing a mixture of glucose/oxygen, yielding a performance as high as 270 μW cm-2. Microfluidic fuel cell was further evaluated with a simulated body fluid solution that contained salts commonly present in the human blood plasma, reaching a power of 240 μW cm-2 at zero flow. These results envisage the incorporation of this fuel cell as a portable power source in Lab-on-a-Chip devices without the need of external pumps.

  1. A microfluidic system to study cytoadhesion of Plasmodium falciparum infected erythrocytes to primary brain microvascularendothelial cells.

    PubMed

    Herricks, Thurston; Seydel, Karl B; Turner, George; Molyneux, Malcolm; Heyderman, Robert; Taylor, Terrie; Rathod, Pradipsinh K

    2011-09-01

    The cellular events leading to severe and complicated malaria in some Plasmodium falciparum infections are poorly understood. Additional tools are required to better understand the pathogenesis of this disease. In this technical report, we describe a microfluidic culture system and image processing algorithms that were developed to observe cytoadhesion interactions of P. falciparum parasitized erythrocytes rolling on primary brain microvascularendothelial cells. We isolated and cultured human primary microvascular brain endothelial cells in a closed loop microfluidic culture system where a peristaltic pump and media reservoirs were integrated onto a microscope stage insert. We developed image processing methods to enhance contrast of rolling parasitized erythrocytes on endothelial cells and to estimate the local wall shear stress. The velocity of parasitized erythrocytes rolling on primary brain microvascularendothelial cells was then measured under physiologically relevant wall shear stresses. Finally, we deployed this method successfully at a field site in Blantyre, Malawi. The method is a promising new tool for the investigation of the pathogenesis of severe malaria. PMID:21743938

  2. Numerical study of the cathode electrode in the Microfluidic Fuel Cell using agglomerate model

    NASA Astrophysics Data System (ADS)

    Moein-Jahromi, M.; Movahed, S.; Kermani, M. J.

    2015-03-01

    Simulation of the cathode electrode of a Microfluidic Fuel Cell (hereafter MFC) is performed with focus on the electrochemical reaction. Oxygen transport phenomena are modeled from the microchannel inlet to the reaction sites surface (on the platinum particles) in the catalyst layer. The dissolved oxygen in sulfuric acid and the formic acid are considered as the oxidant and the fuel, respectively. The cathode catalyst layer is modeled using the agglomerate model versus the homogenous model which is incapable of predicting concentration loss at high current densities. The results are validated versus the experiments of Choban et al. published in 2004. A set of parametric study is performed to investigate the influence of operating and structural parameters on the cell performance; at the end, a sensitivity analysis is implemented to rank the studied parameters with rank 1 for the most influential parameters. The results indicate that oxygen concentration at the inlet of microchannel within the range 0.1 M-0.7 M is the most influential parameter, and the cell performance can enhance by 2.615 W m-2 at the studied range. The results could be used by the microfluidic fuel cell manufacturers to overcome the current drawbacks of the MFCs.

  3. When cell biology meets theory

    PubMed Central

    Gonzalez-Gaitan, Marcos

    2015-01-01

    Cell biologists now have tools and knowledge to generate useful quantitative data. But how can we make sense of these data, and are we measuring the correct parameters? Moreover, how can we test hypotheses quantitatively? To answer these questions, the theory of physics is required and is essential to the future of quantitative cell biology. PMID:26416957

  4. Single cell studies of mouse embryonic stem cell (mESC) differentiation by electrical impedance measurements in a microfluidic device.

    PubMed

    Zhou, Ying; Basu, Srinjan; Laue, Ernest; Seshia, Ashwin A

    2016-07-15

    Biological populations of cells show considerable cell-to-cell variability. Study of single cells and analysis of cell heterogeneity are considered to be critical in understanding biological processes such as stem cell differentiation and cancer development. Recent advances in lab-on-a-chip techniques have allowed single-cell capture in microfluidic channels with the possibility of precise environmental control and high throughput of experiments with minimal usage of samples and reagents. In recent years, label-free techniques such as electrical impedance spectroscopy have emerged as a non-invasive approach to studying cell properties. In this study, we have designed and fabricated a microfluidic device that combines hydrodynamic trapping of single cells in pre-defined locations with the capability of running electrical impedance measurements within the same device. We have measured mouse embryonic stem cells (mESCs) at different states during differentiation (t=0h, 24h and 48h) and quantitatively analysed the changes in electrical parameters of cells during differentiation. A marked increase in the magnitude of the cell impedance is found during cell differentiation, which can be attributed to an increase in cell size. The analysis of the measurements shows that the nucleus-to-cytoplasm ratio decreases during this process. The degree of cell heterogeneity is observed to be the highest when the cells are at the transition state (24h), compare with cells at undifferentiated (0h) and fully differentiated (48h) states. The device enables highly efficient single cell trapping and provides sensitive, label-free electrical impedance measurements of individual cells, enabling the possibility of quantitatively analysing their physical state as well as studying the associated heterogeneity of a cell population. PMID:26963790

  5. Single cell studies of mouse embryonic stem cell (mESC) differentiation by electrical impedance measurements in a microfluidic device

    PubMed Central

    Zhou, Ying; Basu, Srinjan; Laue, Ernest; Seshia, Ashwin A.

    2016-01-01

    Biological populations of cells show considerable cell-to-cell variability. Study of single cells and analysis of cell heterogeneity are considered to be critical in understanding biological processes such as stem cell differentiation and cancer development. Recent advances in lab-on-a-chip techniques have allowed single-cell capture in microfluidic channels with the possibility of precise environmental control and high throughput of experiments with minimal usage of samples and reagents. In recent years, label-free techniques such as electrical impedance spectroscopy have emerged as a non-invasive approach to studying cell properties. In this study, we have designed and fabricated a microfluidic device that combines hydrodynamic trapping of single cells in pre-defined locations with the capability of running electrical impedance measurements within the same device. We have measured mouse embryonic stem cells (mESCs) at different states during differentiation (t=0 h, 24 h and 48 h) and quantitatively analysed the changes in electrical parameters of cells during differentiation. A marked increase in the magnitude of the cell impedance is found during cell differentiation, which can be attributed to an increase in cell size. The analysis of the measurements shows that the nucleus-to-cytoplasm ratio decreases during this process. The degree of cell heterogeneity is observed to be the highest when the cells are at the transition state (24 h), compare with cells at undifferentiated (0 h) and fully differentiated (48 h) states. The device enables highly efficient single cell trapping and provides sensitive, label-free electrical impedance measurements of individual cells, enabling the possibility of quantitatively analysing their physical state as well as studying the associated heterogeneity of a cell population. PMID:26963790

  6. Traceable clonal culture and chemodrug assay of heterogeneous prostate carcinoma PC3 cells in microfluidic single cell array chips

    PubMed Central

    Chung, Jaehoon; Ingram, Patrick N.; Bersano-Begey, Tom; Yoon, Euisik

    2014-01-01

    Cancer heterogeneity has received considerable attention for its role in tumor initiation and progression, and its implication for diagnostics and therapeutics in the clinic. To facilitate a cellular heterogeneity study in a low cost and highly efficient manner, we present a microfluidic platform that allows traceable clonal culture and characterization. The platform captures single cells into a microwell array and cultures them for clonal expansion, subsequently allowing on-chip characterization of clonal phenotype and response against drug treatments. Using a heterogeneous prostate cancer model, the PC3 cell line, we verified our prototype, identifying three different sub-phenotypes and correlating their clonal drug responsiveness to cell phenotype. PMID:25553180

  7. Temperature-controlled MPa-pressure ultrasonic cell manipulation in a microfluidic chip.

    PubMed

    Ohlin, Mathias; Iranmanesh, Ida; Christakou, Athanasia E; Wiklund, Martin

    2015-08-21

    We study the temperature-independent impact on cell viability of relevant physical parameters during long-term, high-acoustic-pressure ultrasonic exposure in a microfluidic chip designed for ultrasonic-standing-wave trapping and aggregation of cells. We use a light-intensity method and 5 μm polymer beads for accurate acoustic pressure calibration before injecting cells into the device, and we monitor the viability of A549 lung cancer cells trapped during one hour in an ultrasonic standing wave with 1 MPa pressure amplitude. The microfluidic chip is actuated by a novel temperature-controlled ultrasonic transducer capable of keeping the temperature stable around 37 °C with an accuracy better than ±0.2 °C, independently on the ultrasonic power and heat produced by the system, thereby decoupling any temperature effect from other relevant effects on cells caused by the high-pressure acoustic field. We demonstrate that frequency-modulated ultrasonic actuation can produce acoustic pressures of equally high magnitudes as with single-frequency actuation, and we show that A549 lung cancer cells can be exposed to 1 MPa standing-wave acoustic pressure amplitudes for one hour without compromising cell viability. At this pressure level, we also measure the acoustic streaming induced around the trapped cell aggregate, and conclude that cell viability is not affected by streaming velocities of the order of 100 μm s(-1). Our results are important when implementing acoustophoresis methods in various clinical and biomedical applications. PMID:26156858

  8. Deformability and size-based cancer cell separation using an integrated microfluidic device.

    PubMed

    Pang, Long; Shen, Shaofei; Ma, Chao; Ma, Tongtong; Zhang, Rui; Tian, Chang; Zhao, Lei; Liu, Wenming; Wang, Jinyi

    2015-11-01

    Cell sorting by filtration techniques offers a label-free approach for cell separation on the basis of size and deformability. However, filtration is always limited by the unpredictable variation of the filter hydrodynamic resistance due to cell accumulation and clogging in the microstructures. In this study, we present a new integrated microfluidic device for cell separation based on the cell size and deformability by combining the microstructure-constricted filtration and pneumatic microvalves. Using this device, the cell populations sorted by the microstructures can be easily released in real time for subsequent analysis. Moreover, the periodical sort and release of cells greatly avoided cell accumulation and clogging and improved the selectivity. Separation of cancer cells (MCF-7, MDA-MB-231 and MDA231-LM2) with different deformability showed that the mixture of the less flexible cells (MCF-7) and the flexible cells (MDA-MB-231 and MDA231-LM2) can be well separated with more than 75% purity. Moreover, the device can be used to separate cancer cells from the blood samples with more than 90% cell recovery and more than 80% purity. Compared with the current filtration methods, the device provides a new approach for cancer cell separation with high collection recovery and purity, and also, possesses practical potential to be applied as a sample preparation platform for fundamental studies and clinical applications. PMID:26366443

  9. Liver injury-on-a-chip: microfluidic co-cultures with integrated biosensors for monitoring liver cell signaling during injury.

    PubMed

    Zhou, Qing; Patel, Dipali; Kwa, Timothy; Haque, Amranul; Matharu, Zimple; Stybayeva, Gulnaz; Gao, Yandong; Diehl, Anna Mae; Revzin, Alexander

    2015-12-01

    Tissue injury triggers complex communication between cells via secreted signaling molecules such as cytokines and growth factors. Discerning when and where these signals begin and how they propagate over time is very challenging with existing cell culture and analysis tools. The goal of this study was to develop new tools in the form of microfluidic co-cultures with integrated biosensors for local and continuous monitoring of secreted signals. Specifically, we focused on how alcohol injury affects TGF-β signaling between two liver cell types, hepatocytes and stellate cells. Activation of stellate cells happens early during liver injury and is at the center of liver fibrosis. We demonstrated that alcohol injury to microfluidic co-cultures caused significantly higher levels of stellate cell activation compared to conditioned media and transwell injury experiments. This highlighted the advantage of the microfluidic co-culture: placement of two cell types in close proximity to ensure high local concentrations of injury-promoting secreted signals. Next, we developed a microsystem consisting of five chambers, two for co-culturing hepatocytes with stellate cells and three additional chambers containing miniature aptamer-modified electrodes for monitoring secreted TGF-β. Importantly, the walls separating microfluidic chambers were actuatable; they could be raised or lowered to create different configurations of the device. The use of reconfigurable microfluidics and miniature biosensors revealed that alcohol injury causes hepatocytes to secrete TGF-β molecules, which diffuse over to neighboring stellate cells and trigger production of additional TGF-β from stellate cells. Our results lend credence to the emerging view of hepatocytes as active participants of liver injury. Broadly speaking, our microsystem makes it possible to monitor paracrine crosstalk between two cell types communicating via the same signaling molecule (e.g. TGF-β). PMID:26480303

  10. High-Throughput Single-Cell Cultivation on Microfluidic Streak Plates.

    PubMed

    Jiang, Cheng-Ying; Dong, Libing; Zhao, Jian-Kang; Hu, Xiaofang; Shen, Chaohua; Qiao, Yuxin; Zhang, Xinyue; Wang, Yapei; Ismagilov, Rustem F; Liu, Shuang-Jiang; Du, Wenbin

    2016-04-01

    This paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation of Escherichia coli and antimicrobial susceptibility testing of Pseudomonas aeruginosa PAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including four Mycobacterium isolates and a previously unknown fluoranthene-degrading Blastococcus species. PMID:26850294

  11. An integrated microfluidic platform for negative selection and enrichment of cancer cells

    NASA Astrophysics Data System (ADS)

    Luo, Wen-Yi; Tsai, Sung-Chi; Hsieh, Kuangwen; Lee, Gwo-Bin

    2015-08-01

    Circulating tumor cells (CTCs), tumor cells that disseminate from primary tumors to the bloodstream, have recently emerged as promising indicators for cancer diagnosis and prognosis. However, the technical difficulties in isolating and detecting rare CTCs have limited the widespread applicability of this method to date. In this work, a new integrated microfluidic system integrating micromixers and micropumps capable of performing ‘negative selection and enrichment’ of CTCs was developed. By using anti-human CD45 antibodies-coated magnetic beads, leukocytes were effectively removed by applying an external magnetic force, leaving behind an enriched target cell population. The on-chip CTC recovery rate was experimentally found to be 70   ±   5% after a single round of negative selection and enrichment. Meanwhile, CD45 depletion efficiency was 83.99   ±   1.00% and could be improved to 99.84   ±   0.04% after three consecutive rounds of depletion. Notably, on-chip negative selection and enrichment was 58% faster and the repeated depletion could be processed automatically. These promising results suggested the developed microfluidic chip is potentiated for a standardized CTC isolation platform. Preliminary results of the current paper were presented at Micro TAS 2014, San Antonio, Texas, USA, October 26-30, 2014.

  12. Acoustic micro-vortexing of fluids, particles and cells in disposable microfluidic chips.

    PubMed

    Iranmanesh, Ida; Ohlin, Mathias; Ramachandraiah, Harisha; Ye, Simon; Russom, Aman; Wiklund, Martin

    2016-08-01

    We demonstrate an acoustic platform for micro-vortexing in disposable polymer microfluidic chips with small-volume (20 μl) reaction chambers. The described method is demonstrated for a variety of standard vortexing functions, including mixing of fluids, re-suspension of a pellet of magnetic beads collected by a magnet placed on the chip, and lysis of cells for DNA extraction. The device is based on a modified Langevin-type ultrasonic transducer with an exponential horn for efficient coupling into the microfluidic chip, which is actuated by a low-cost fixed-frequency electronic driver board. The transducer is optimized by numerical modelling, and different demonstrated vortexing functions are realized by actuating the transducer for varying times; from fractions of a second for fluid mixing, to half a minute for cell lysis and DNA extraction. The platform can be operated during 1 min below physiological temperatures with the help of a PC fan, a Peltier element and an aluminum heat sink acting as the chip holder. As a proof of principle for sample preparation applications, we demonstrate on-chip cell lysis and DNA extraction within 25 s. The method is of interest for automating and chip-integrating sample preparation procedures in various biological assays. PMID:27444649

  13. Perspectives on utilizing unique features of microfluidics technology for particle and cell sorting

    PubMed Central

    Adams, Jonathan D.; Tom Soh, H.

    2009-01-01

    Sample preparation is often the most tedious and demanding step in an assay, but it also plays an essential role in determining the quality of results. As biological questions and analytical methods become increasingly sophisticated, there is a rapidly growing need for systems that can reliably and reproducibly separate cells and particles with high purity, throughput and recovery. Microfluidics technology represents a compelling approach in this regard, allowing precise control of separation forces for high performance separation in inexpensive, or even disposable, devices. In addition, microfluidics technology enables the fabrication of arrayed and integrated systems that operate either in parallel or in tandem, in a capacity that would be difficult to achieve in macro-scale systems. In this report, we use recent examples from our work to illustrate the potential of microfluidic cell- and particle-sorting devices. We demonstrate the potential of chip-based high-gradient magnetophoresis that enable high-purity separation through reversible trapping of target particles paired with high-stringency washing with minimal loss. We also describe our work in the development of devices that perform simultaneous multi-target sorting, either through precise control of magnetic and fluidic forces or through the integration of multiple actuation forces into a single monolithic device. We believe that such devices may serve as a powerful “front-end” module of highly integrated analytical platforms capable of providing actionable diagnostic information directly from crude, unprocessed samples - the success of such systems may hold the key to advancing point-of-care diagnostics and personalized medicine. PMID:20161387

  14. Microfluidic cell arrays for metabolic monitoring of stimulated cardiomyocytes.

    PubMed

    Cheng, Wei; Klauke, Norbert; Smith, Godfrey; Cooper, Jonathan M

    2010-04-01

    An array of PDMS microchambers was aligned to an array of sensor electrodes and stimulating microelectrodes, which was used for the electrochemical monitoring of the metabolic activity of single isolated adult ventricular myocytes inside the chamber array, stimulated within a transient electric field. The effect of the accumulation of metabolic byproducts in the limited extracellular volume of the picolitre chambers was demonstrated by measuring single muscle cell contraction optically, while concomitant changes in intracellular calcium transients and pH were recorded independently using fluorescent indicator dyes. Both the amplitude of the cell shortening and the magnitude of the intracellular calcium transients decreased over time and both nearly ceased after 20 min of continuous stimulation in the limited extracellullar volume. The intracellular pH decreased gradually during 20 min of continuous stimulation after which a dramatic pH drop was observed, indicating the breakdown of the intracellular buffering capacity. After continuous stimulation, intracellular lactate was released into the microchamber through cell electroporation and was detected electrochemically at a lactate microbiosensor, within the chamber. A mitochondrial uncoupler was used to mimic ischaemia and thus to enhance the cellular content of lactate. Under these circumstances, intracellular lactate concentrations were found to have risen to approximately 15 mM. This array system has the potential of simultaneous electrochemical and optical monitoring of extracellular and intracellular metabolites from single beating heart cells at a controlled metabolic state. PMID:20333720

  15. Digital microfluidic three-dimensional cell culture and chemical screening platform using alginate hydrogels

    PubMed Central

    2015-01-01

    Electro wetting-on-dielectric (EWOD) digital microfluidics (DMF) can be used to develop improved chemical screening platforms using 3-dimensional (3D) cell culture. Alginate hydrogels are one common method by which a 3D cell culture environment is created. This paper presents a study of alginate gelation on EWOD DMF and investigates designs to obtain uniform alginate hydrogels that can be repeatedly addressed by any desired liquids. A design which allows for gels to be retained in place during liquid delivery and removal without using any physical barriers or hydrophilic patterning of substrates is presented. A proof of concept screening platform is demonstrated by examining the effects of different concentrations of a test chemical on 3D cells in alginate hydrogels. In addition, the temporal effects of the various chemical concentrations on different hydrogel posts are demonstrated, thereby establishing the benefits of an EWOD DMF 3D cell culture and chemical screening platform using alginate hydrogels. PMID:25945142

  16. Learning from the Jersey Turnpike:Cell Lysis, Labeling and Washing with Microfluidic Metamaterials

    NASA Astrophysics Data System (ADS)

    Loutherback, Kevin; Morton, Keith; Inglis, David; Tsui, Opheli; Sturm, James; Chou, Stephen; Austin, Robert

    2008-03-01

    Directing objects across functional streamlines at low Reynolds number is difficult but important since this motion can be used to label, lyse, and analyze complex biological objects on-chip without cross-contamination. Here we use an asymmeteric post array to move cells across coflowing reagents and show on-chip, immunofluorescent labeling of platelets with washing and E.Coli cell lysis with simultaneous separation of bacterial chromosome from the cell contents. Furthermore, we develop the concept of a microfluidic metamaterial by using the basic asymmetric post array as a building block for complex particle handling modes. These modular array elements could be of great use for developing robust techniques for on-chip, continuous flow manipulation and analysis of cells, large bio-particles, and functional beads.

  17. Learning from the Jersey Turnpike: Cell Lysis, Labeling and Washing with Microfluidic Metamaterials

    NASA Astrophysics Data System (ADS)

    Austin, Robert

    2008-03-01

    Directing objects across functional streamlines at low Reynolds number is difficult but important since this motion can be used to label, lyse, and analyze complex biological objects on-chip without cross-contamination. Here we use an asymmeteric post array to move cells across coflowing reagents and show on-chip, immunofluorescent labeling of platelets with washing and E.Coli cell lysis with simultaneous separation of bacterial chromosome from the cell contents. Furthermore, we develop the concept of a microfluidic metamaterial by using the basic asymmetric post array as a building block for complex particle handling modes. These modular array elements could be of great use for developing robust techniques for on-chip, continuous flow manipulation and analysis of cells, large bio-particles, and functional beads.

  18. Digital microfluidic three-dimensional cell culture and chemical screening platform using alginate hydrogels.

    PubMed

    George, Subin M; Moon, Hyejin

    2015-03-01

    Electro wetting-on-dielectric (EWOD) digital microfluidics (DMF) can be used to develop improved chemical screening platforms using 3-dimensional (3D) cell culture. Alginate hydrogels are one common method by which a 3D cell culture environment is created. This paper presents a study of alginate gelation on EWOD DMF and investigates designs to obtain uniform alginate hydrogels that can be repeatedly addressed by any desired liquids. A design which allows for gels to be retained in place during liquid delivery and removal without using any physical barriers or hydrophilic patterning of substrates is presented. A proof of concept screening platform is demonstrated by examining the effects of different concentrations of a test chemical on 3D cells in alginate hydrogels. In addition, the temporal effects of the various chemical concentrations on different hydrogel posts are demonstrated, thereby establishing the benefits of an EWOD DMF 3D cell culture and chemical screening platform using alginate hydrogels. PMID:25945142

  19. Differential microfluidic sensor on printed circuit board for biological cells analysis.

    PubMed

    Shi, Dongyuan; Guo, Jinhong; Chen, Liang; Xia, Chuncheng; Yu, Zhefeng; Ai, Ye; Li, Chang Ming; Kang, Yuejun; Wang, Zhiming

    2015-08-01

    Coulter principal based resistive pulse sensor has been demonstrated as an important platform in biological cell detection and enumeration since several decades ago. Recently, the miniaturized micro-Coulter counter has attracted much attention due to its advantages in point of care diagnostics for on chip detection and enumeration of rare cells, such as circulating tumor cells. In this paper, we present a microfluidic cytometer with differential amplifier based on Coulter principle on a SU-8 coated printed circuit board substrate. The electrical current changes induced by the blockage of the microparticles in the sensing aperture are calibrated by polystyrene particles of standard size. Finally, HeLa cells are used to evaluate the performance of the proposed device for enumeration of biological samples. The proposed cytometer is built upon the cheap and widely available printed circuit board substrate and shows its great potential as personalized healthcare monitor. PMID:25735615

  20. An integrated microfluidic chip for immunomagnetic detection and isolation of rare prostate cancer cells from blood.

    PubMed

    Esmaeilsabzali, Hadi; Beischlag, Timothy V; Cox, Michael E; Dechev, Nikolai; Parameswaran, Ash M; Park, Edward J

    2016-02-01

    The quantitative and qualitative analysis of circulating tumor cells (CTCs) has the potential to improve the clinical management of several cancers, including prostate cancer. As such, there is much interest in the isolation of CTCs from the peripheral blood of cancer patients. We report the design, fabrication, and proof-of-principle testing of an integrated permalloy-based microfluidic chip for immunomagnetic isolation of blood-borne prostate cancer cells using an antibody targeting prostate surface membrane antigen (PSMA). The preliminary results using spiked blood samples indicate that the proposed device is consistently capable of isolating prostate cancer cells with high sensitivity (up to 98 %) at clinically relevant low concentrations (down to 20 cells/mL) and an acceptable throughput (100 μL/min). PMID:26876965

  1. Regulation of mesenchymal stem cell 3D microenvironment: From macro to microfluidic bioreactors.

    PubMed

    Sart, Sébastien; Agathos, Spiros N; Li, Yan; Ma, Teng

    2016-01-01

    Human mesenchymal stem cells (hMSCs) have emerged as an important cell type in cell therapy and tissue engineering. In these applications, maintaining the therapeutic properties of hMSCs requires tight control of the culture environments and the structural cell organizations. Bioreactor systems are essential tools to achieve these goals in the clinical-scale expansion and tissue engineering applications. This review summarizes how different bioreactors provide cues to regulate the structure and the chemico-mechanical microenvironment of hMSCs with a focus on 3D organization. In addition to conventional bioreactors, recent advances in microfluidic bioreactors as a novel approach to better control the hMSC microenvironment are also discussed. These advancements highlight the key role of bioreactor systems in preserving hMSC's functional properties by providing dynamic and temporal regulation of in vitro cellular microenvironment. PMID:26696441

  2. Development of microfluidic devices for in situ investigation of cells using surface-enhanced Raman spectroscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Ho, Yu-Han; Galvan, Daniel D.; Yu, Qiuming

    2016-03-01

    Surface-enhanced Raman spectroscopy (SERS) has immerged as a power analytical and sensing technique for many applications in biomedical diagnosis, life sciences, food safety, and environment monitoring because of its molecular specificity and high sensitivity. The inactive Raman scattering of water molecule makes SERS a suitable tool for studying biological systems. Microfluidic devices have also attracted a tremendous interest for the aforementioned applications. By integrating SERS-active substrates with microfluidic devices, it offers a new capability for in situ investigation of biological systems, their dynamic behaviors, and response to drugs or microenvironment changes. In this work, we designed and fabricated a microfluidic device with SERS-active substrates surrounding by cell traps in microfluidic channels for in situ study of live cells using SERS. The SERS-active substrates are quasi-3D plasmonic nanostructure array (Q3D-PNA) made in h-PDMS/PMDS with physically separated gold film with nanoholes op top and gold nanodisks at the bottom of nanowells. 3D finite-difference time-domain (3D-FDTD) electromagnetic simulations were performed to design Q3D-PNAs with the strongest local electric fields (hot spots) at the top or bottom water/Au interfaces for sensitive analysis of cells and small components, respectively. The Q3D-PNAs with the hot spots on top and bottom were placed at the up and down stream of the microfluidic channel, respectively. Each Q3D-PNA pattern was surrounded with cell trapping structures. The microfluidic device was fabricated via soft lithography. We demonstrated that normal (COS-7) and cancer (HpeG2) cells were captured on the Q3D-PNAs and investigated in situ using SERS.

  3. 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

    2015-01-01

    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

  4. Development of an Integrated Microfluidic Perfusion Cell Culture System for Real-Time Microscopic Observation of Biological Cells

    PubMed Central

    Lin, Lung; Wang, Shih-Siou; Wu, Min-Hsien; Oh-Yang, Chih-Chin

    2011-01-01

    This study reports an integrated microfluidic perfusion cell culture system consisting of a microfluidic cell culture chip, and an indium tin oxide (ITO) glass-based microheater chip for micro-scale perfusion cell culture, and its real-time microscopic observation. The system features in maintaining both uniform, and stable chemical or thermal environments, and providing a backflow-free medium pumping, and a precise thermal control functions. In this work, the performance of the medium pumping scheme, and the ITO glass microheater were experimentally evaluated. Results show that the medium delivery mechanism was able to provide pumping rates ranging from 15.4 to 120.0 μL·min−1. In addition, numerical simulation and experimental evaluation were conducted to verify that the ITO glass microheater was capable of providing a spatially uniform thermal environment, and precise temperature control with a mild variation of ±0.3 °C. Furthermore, a perfusion cell culture was successfully demonstrated, showing the cultured cells were kept at high cell viability of 95 ± 2%. In the process, the cultured chondrocytes can be clearly visualized microscopically. As a whole, the proposed cell culture system has paved an alternative route to carry out real-time microscopic observation of biological cells in a simple, user-friendly, and low cost manner. PMID:22164082

  5. Cell-based drug combination screening with a microfluidic droplet array system.

    PubMed

    Du, Guan-Sheng; Pan, Jian-Zhang; Zhao, Shi-Ping; Zhu, Ying; den Toonder, Jaap M J; Fang, Qun

    2013-07-16

    We performed cell-based drug combination screening using an integrated droplet-based microfluidic system based on the sequential operation droplet array (SODA) technique. In the system, a tapered capillary connected with a syringe pump was used for multistep droplet manipulations. An oil-covered two-dimensional droplet array chip fixed in an x-y-z translation stage was used as the platform for cell culture and analysis. Complex multistep operations for drug combination screening involving long-term cell culture, medium changing, schedule-dependent drug dosage and stimulation, and cell viability testing were achieved in parallel in the semiopen droplet array, using multiple droplet manipulations including liquid metering, aspirating, depositing, mixing, and transferring. Long-term cell culture as long as 11 days was performed in oil-covered 500 nL droplets by changing the culture medium in each droplet every 24 h. The present system was applied in parallel schedule-dependent drug combination screening for A549 nonsmall lung cancer cells with the cell cycle-dependent drug flavopiridol and two anticancer drugs of paclitaxel and 5-fluorouracil. The highest inhibition efficiency was obtained with a schedule combination of 200 nM flavopiridol followed by 100 μM 5-fluorouracil. The drug consumption for each screening test was substantially decreased to 5 ng-5 μg, corresponding to 10-1000-fold reductions compared with traditional drug screening systems with 96-well or 384-well plates. The present work provides a novel and flexible droplet-based microfluidic approach for performing cell-based screening with complex and multistep operation procedures. PMID:23786644

  6. Towards microfluidic sperm refinement: impedance-based analysis and sorting of sperm cells.

    PubMed

    de Wagenaar, B; Dekker, S; de Boer, H L; Bomer, J G; Olthuis, W; van den Berg, A; Segerink, L I

    2016-04-12

    The use of high quality semen for artificial insemination in the livestock industry is essential for successful outcome. Insemination using semen with a high number of sperm cells containing morphological defects has a negative impact on fertilization outcome. Therefore, semen with a high number of these abnormal cells is discarded in order to maintain high fertilization potential, resulting in the loss of a large number of morphologically normal sperm cells (up to 70-80% of original sample). A commonly occurring morphological sperm anomaly is the cytoplasmic droplet on the sperm flagella. Currently, no techniques are available to extract morphologically normal sperm cells from rejected samples. Therefore, we aim to develop a microfluidic setup which is able to detect and sort morphologically normal sperm cells label-free and non-invasively. In a proof-of-concept experiment, differential impedance measurements were used to detect the presence of cytoplasmic droplets on sperm flagella, which was quantified by calculating the area under the curve (AUC) of the corresponding impedance peaks. A receiver operating characteristic curve of this electrical analysis method showed the good predictive power of this analysis method (AUC value of 0.85). Furthermore, we developed a label-free cell sorting system using LabVIEW, which is capable of sorting sperm cells based on impedance. In a proof-of-concept experiment, sperm cells and 3 μm beads were sorted label-free and non-invasively using impedance detection and dielectrophoresis sorting. These experiments present our first attempt to perform sperm refinement using microfluidic technology. PMID:27025866

  7. Cancer-driven dynamics of immune cells in a microfluidic environment.

    PubMed

    Agliari, Elena; Biselli, Elena; De Ninno, Adele; Schiavoni, Giovanna; Gabriele, Lucia; Gerardino, Anna; Mattei, Fabrizio; Barra, Adriano; Businaro, Luca

    2014-01-01

    Scope of the present work is to infer the migratory ability of leukocytes by stochastic processes in order to distinguish the spontaneous organization of immune cells against an insult (namely cancer). For this purpose, spleen cells from immunodeficient mice, selectively lacking the transcription factor IRF-8 (IRF-8 knockout; IRF-8 KO), or from immunocompetent animals (wild-type; WT), were allowed to interact, alternatively, with murine B16.F10 melanoma cells in an ad hoc microfluidic environment developed on a LabOnChip technology. In this setting, only WT spleen cells were able to establish physical interactions with melanoma cells. Conversely, IRF-8 KO immune cells exhibited poor dynamical reactivity towards the neoplastic cells. In the present study, we collected data on the motility of these two types of spleen cells and built a complete set of observables that recapitulate the biological complexity of the system in these experiments. With remarkable accuracy, we concluded that the IRF-8 KO cells performed pure uncorrelated random walks, while WT splenocytes were able to make singular drifted random walks that collapsed on a straight ballistic motion for the system as a whole, hence giving rise to a highly coordinate response. These results may provide a useful system to quantitatively analyse the real time cell-cell interactions and to foresee the behavior of immune cells with tumor cells at the tissue level. PMID:25322144

  8. Numerical Analysis of Hydrodynamic Flow in Microfluidic Biochip for Single-Cell Trapping Application.

    PubMed

    Khalili, Amelia Ahmad; Ahmad, Mohd Ridzuan

    2015-01-01

    Single-cell analysis has become the interest of a wide range of biological and biomedical engineering research. It could provide precise information on individual cells, leading to important knowledge regarding human diseases. To perform single-cell analysis, it is crucial to isolate the individual cells before further manipulation is carried out. Recently, microfluidic biochips have been widely used for cell trapping and single cell analysis, such as mechanical and electrical detection. This work focuses on developing a finite element simulation model of single-cell trapping system for any types of cells or particles based on the hydrodynamic flow resistance (Rh) manipulations in the main channel and trap channel to achieve successful trapping. Analysis is carried out using finite element ABAQUS-FEA™ software. A guideline to design and optimize single-cell trapping model is proposed and the example of a thorough optimization analysis is carried out using a yeast cell model. The results show the finite element model is able to trap a single cell inside the fluidic environment. Fluid's velocity profile and streamline plots for successful and unsuccessful single yeast cell trapping are presented according to the hydrodynamic concept. The single-cell trapping model can be a significant important guideline in designing a new chip for biomedical applications. PMID:26569218

  9. Numerical Analysis of Hydrodynamic Flow in Microfluidic Biochip for Single-Cell Trapping Application

    PubMed Central

    Ahmad Khalili, Amelia; Ahmad, Mohd Ridzuan

    2015-01-01

    Single-cell analysis has become the interest of a wide range of biological and biomedical engineering research. It could provide precise information on individual cells, leading to important knowledge regarding human diseases. To perform single-cell analysis, it is crucial to isolate the individual cells before further manipulation is carried out. Recently, microfluidic biochips have been widely used for cell trapping and single cell analysis, such as mechanical and electrical detection. This work focuses on developing a finite element simulation model of single-cell trapping system for any types of cells or particles based on the hydrodynamic flow resistance (Rh) manipulations in the main channel and trap channel to achieve successful trapping. Analysis is carried out using finite element ABAQUS-FEA™ software. A guideline to design and optimize single-cell trapping model is proposed and the example of a thorough optimization analysis is carried out using a yeast cell model. The results show the finite element model is able to trap a single cell inside the fluidic environment. Fluid’s velocity profile and streamline plots for successful and unsuccessful single yeast cell trapping are presented according to the hydrodynamic concept. The single-cell trapping model can be a significant important guideline in designing a new chip for biomedical applications. PMID:26569218

  10. Single cell kinase signaling assay using pinched flow coupled droplet microfluidics

    PubMed Central

    Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S.; Tan Shao Weng, Daniel; Thakor, Nitish V.; Teck Lim, Chwee; Chen, Chia-Hung

    2014-01-01

    Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib. PMID:24926389

  11. Microfluidic application-specific integrated device for monitoring direct cell-cell communication via gap junctions between individual cell pairs

    NASA Astrophysics Data System (ADS)

    Lee, Philip J.; Hung, Paul J.; Shaw, Robin; Jan, Lily; Lee, Luke P.

    2005-05-01

    Direct cell-cell communication between adjacent cells is vital for the development and regulation of functional tissues. However, current biological techniques are difficult to scale up for high-throughput screening of cell-cell communication in an array format. In order to provide an effective biophysical tool for the analysis of molecular mechanisms of gap junctions that underlie intercellular communication, we have developed a microfluidic device for selective trapping of cell-pairs and simultaneous optical characterizations. Two different cell populations can be brought into membrane contact using an array of trapping channels with a 2μm by 2μm cross section. Device operation was verified by observation of dye transfer between mouse fibroblasts (NIH3T3) placed in membrane contact. Integration with lab-on-a-chip technologies offers promising applications for cell-based analytical tools such as drug screening, clinical diagnostics, and soft-state biophysical devices for the study of gap junction protein channels in cellular communications. Understanding electrical transport mechanisms via gap junctions in soft membranes will impact quantitative biomedical sciences as well as clinical applications.

  12. 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

  13. Automated microfluidic platform of bead-based electrochemical immunosensor integrated with bioreactor for continual monitoring of cell secreted biomarkers.

    PubMed

    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

  14. 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.

  15. Meeting report--Imaging the Cell.

    PubMed

    Moreau, Violaine; Cordelières, Fabrice P; Poujol, Christel; Sagot, Isabelle; Saltel, Frédéric

    2015-11-01

    Every two years, the French Society for Cell Biology (SBCF) organises an international meeting called 'Imaging the Cell'. This year, the 8th edition was held on 24-26 June 2015 at University of Bordeaux Campus Victoire in the city of Bordeaux, France, a UNESCO World Heritage site. Over the course of three days, the meeting provided a forum for experts in different areas of cell imaging. Its unique approach was to combine conventional oral presentations during morning sessions with practical workshops at hosting institutes and the Bordeaux Imaging Center during the afternoons. The meeting, co-organised by Violaine Moreau and Frédéric Saltel (both INSERM U1053, Bordeaux, France), Christel Poujol and Fabrice Cordelières (both Bordeaux Imaging Center, Bordeaux, France) and Isabelle Sagot (Institut de Biochimie et Génétique Cellulaires, Bordeaux, France), brought together about 120 scientists including 16 outstanding speakers to discuss the latest advances in cell imaging. Thanks to recent progress in imaging technologies, cell biologists are now able to visualise, follow and manipulate cellular processes with unprecedented accuracy. The meeting sessions and workshops highlighted some of the most exciting developments in the field, with sessions dedicated to optogenetics, high-content screening, in vivo and live-cell imaging, correlative light and electron microscopy, as well as super-resolution imaging. PMID:26527200

  16. Physical cell interactions with their surrounding materials: Mechanics and geometrical factors using microfluidic platforms

    NASA Astrophysics Data System (ADS)

    Lopez Garcia, Maria Del Carmen

    Microfluidics platforms are employed in: "sperm motion in a microfluidic device" and "mechanical interactions of mammary gland cells with their surrounding three dimensional extra-cellular matrix". Microfluidics has shown promise as a new platform for assisted reproduction. Sperm and fluid motion in microchannels was studied to understand the flow characteristics in the device, how sperm interacted with this flow, and how sperm-oocyte attachment occurs in the device. A threshold fluid velocity was found where sperm transition from traveling with the fluid to a regime in which they can move independently. A population of sperm remained in the inlet well area. There was also the tendency of sperm to travel along surface contours. These observations provide an improved understanding of sperm motion in microchannels and a basis for improved device designs. The effort to understand the development of breast cancer motivates the study of mammary gland cells and their interactions with the extra-cellular matrix. Mammographic density is a risk factor for breast cancer which correlates with collagen density affects cell behavior. Collagen gels with concentrations of 1.3, 2, and 3 mg/mL, were tensile tested to obtain the Young's modulus, E, at low displacement rates of 0.01, 0.1, and 1 mm/min. Local strain measurement in the gage section were used for both strain and strain rate determination. Local strain rates were on the order of cellular generated strain rate. A power law fitting described the relationship between Young's modulus and local strain rate. Mammary gland cells were seeded with collagen and fluorescent beads into microchannels and observed via four-dimensional imaging. The displacements of the beads were used to calculate strains. The Young's modulus due to the rate at which the cell was straining the collagen was obtained from the aforementioned fittings. Three-dimensional elastic theory for an isotropic material was employed to calculate the stress. The

  17. A microfluidics approach for the isolation of nucleated red blood cells (NRBCs) from the peripheral blood of pregnant women

    PubMed Central

    Huang, R.; Barber, T. A.; Schmidt, M. A.; Tompkins, R. G.; Toner, M.; Bianchi, D. W.; Kapur, R.; Flejter, W. L.

    2015-01-01

    Objective Nucleated red blood cells (NRBCs) have been identified in maternal circulation and potentially provide a resource for the monitoring and diagnosis of maternal, fetal, and neonatal health and disease. Past strategies used to isolate and enrich for NRBCs are limited to complex approaches that result in low recovery and less than optimal cell purity. Here we report the development of a high-throughput and highly efficient microfluidic device for isolating rare NRBCs from maternal blood. Material and Methods NRBCs were isolated from the peripheral blood of 58 pregnant women using a microfluidic process that consists of a microfluidic chip for size-based cell separation and a magnetic device for hemoglobin-based cell isolation. Results The microfluidic–magnetic combination removes nontarget red blood cells and white blood cells at a very high efficiency (∼99.99%). The device successfully identified NRBCs from the peripheral blood of 58/58 pretermination samples with a mean of 37.44 NRBC/mL (range 0.37–274.36 NRBC/mL). These results were compared with those from previous studies. Conclusion The microfluidic device results in an approximate 10- to 20-fold enrichment of NRBCs over methods described previously. The reliability of isolation and the purity of the NRBC product have the potential to enable the subsequent application of molecular diagnostic assays. PMID:18821715

  18. Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering.

    PubMed

    Tay, Hui Min; Yeo, David C; Wiraja, Christian; Xu, Chenjie; Hou, Han Wei

    2016-01-01

    Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (>90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving >95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (10(6) cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application. PMID:27500904

  19. Microfluidic platforms and fundamental electrocatalysis studies for fuel cell applications

    NASA Astrophysics Data System (ADS)

    Cohen, Jamie Lee

    The fabrication and testing of a planar membraneless microchannel fuel cell, based on a silicon microchannel, is described in detail. Laminar flow of fuel and oxidant streams, one on top of the other, prevents fuel crossover while allowing ionic transport at the interface between the two solutions. By employing laminar flow, the useful functions of a membrane are retained, while bypassing its inherent limitations. The planar design maximizes the anode and cathode areas, and elimination of the membrane affords broad flexibility in the choice of fuel and oxidant. Fuels including formic acid, methanol, ethanol, sodium borohydride and hydrogen were tested along with oxidants such as oxygen, hydrogen peroxide and potassium permanganate. Steps taken to improve voltage, current density, and overall power output have been addressed, including the testing of a dual electrolyte system and the use of micro-patterned electrode surfaces to enhance fuel utilization. As the complexity of the fuels studied in the microchannel fuel cell increased, it was imperative to characterize these fuels using electrochemical techniques prior to utilization in the fuel cell. The oxidation pathway of the liquid fuel methanol was studied rigorously because of its importance for micro-fuel cell applications. Activation energies for methanol oxidation at a Ptpoly surface were determined using electrochemical techniques, providing a benchmark for the comparison of activation energies of other Pt-based electrocatalysts for methanol oxidation at a given potential. A protocol to obtain Ea values was established in three different electrolytes and experimental parameters that influence the magnitude of these values are discussed in detail. The oxidation pathways of sodium borohydride were also examined at Au, Pt, and Pd surfaces using cyclic voltammetry, chronoamperometry, and rotating disk electrode voltammetry. In addition to studies on bulk Ptpoly surfaces, new bulk intermetallic catalysts were

  20. Endothelial progenitor cell recruitment in a microfluidic vascular model.

    PubMed

    Lewis, Daniel M; Abaci, Hasan E; Xu, Yu; Gerecht, Sharon

    2015-12-01

    During vessel injury, endothelial progenitors cells (EPCs) are recruited from bone marrow and directed to the hypoxic injury site. The hypoxic conditions in the damaged blood vessel promote TNF-α, which upregulates intercellular adhesion molecule-1 (ICAM-1). EPCs attach to endothelial cell lining using ICAM-1. Here we aimed to examine EPC attachment to ECs in an injured-blood vessel conditions. We first determined ICAM-1 expression in stimulated HUVECs. We stimulated HUVECs with 21% oxygen (atmospheric), atmospheric with TNF-α-supplemented media, 1% oxygen (hypoxia), and hypoxia with TNF-α-supplemented media and found the highest ECFC attachment on HUVECs stimulated with TNF-α and hypoxia, correlating with the highest ICAM-1 expression. We next designed, fabricated and tested a three-dimensional microbioreactor (3D MBR) system with precise control and monitoring of dissolve oxygen and media flow rate in the cellular environment. We utilized a step-wise seeding approach, producing monolayer of HUVECs on all four walls. When stimulated with both TNF-α and hypoxia, ECFC retention on HUVECs was significantly increased under low shear stress compared to static controls. Overall, the 3D MBR system mimics the pathological oxygen tension and shear stress in the damaged vasculature, providing a platform to model vascular-related disorders. PMID:26693599

  1. Microfluidic device with asymmetric electrodes for cell and reagent delivery

    NASA Astrophysics Data System (ADS)

    Lee, Daniel; Xu, Guolin; Tay, Hong Kiat; Yang, Chun; Ying, Jackie Y.

    2006-12-01

    We present the design and fabrication of a micro-electromechanical system (MEMS) device for cell and particle delivery using a combination of AC electrokinetic fluidic flow and negative dielectrophoresis (DEP) force. An array of interdigitated asymmetric microelectrode pairs were used in the planar device. The electrodes produced a net charge in the surrounding fluid, generating an AC electrokinetic fluidic motion. A non-uniform electric field with low actuation frequency from the microelectrode pairs resulted in a negative DEP force, which was responsible for pushing delivery particles away from sedimentation. The experimental results showed that the flow velocity increased rapidly from 267 μm/min to 394 μm/min when the applied frequency was increased from 10 kHz to 70 kHz for a cell-suspending medium buffer solution with a conductivity of 4.7 μS/cm. A maximum delivery velocity of 801 μm/min was obtained when the buffer conductivity was increased to 47 μS/cm with an actuation frequency of 100 kHz.

  2. Disposable on-chip microfluidic system for buccal cell lysis, DNA purification, and polymerase chain reaction.

    PubMed

    Cho, Woong; Maeng, Joon-Ho; Ahn, Yoomin; Hwang, Seung Yong

    2013-09-01

    This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off-chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip. PMID:23784986

  3. 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.

  4. Acoustophoretic microfluidic chip for sequential elution of surface bound molecules from beads or cells

    PubMed Central

    Augustsson, Per; Malm, Johan; Ekström, Simon

    2012-01-01

    An acoustophoresis-based microfluidic flow-chip is presented as a novel platform to facilitate analysis of proteins and peptides loosely bound to the surface of beads or cells. The chip allows for direct removal of the background surrounding the beads or cells, followed by sequential treatment and collection of a sequence of up to five different buffer conditions. During this treatment, the beads/cells are retained in a single flow by acoustic radiation force. Eluted peptides are collected from the outlets and subsequently purified by miniaturized solid-phase extraction and analyzed with matrix assisted laser desorption mass spectrometry. Fundamental parameters such as the system fluidics and dispersion are presented. The device was successfully applied for wash and sequential elution of peptides bound to the surface of microbeads and human spermatozoa, respectively. PMID:24003343

  5. Selective local lysis and sampling of live cells for nucleic acid analysis using a microfluidic probe.

    PubMed

    Kashyap, Aditya; Autebert, Julien; Delamarche, Emmanuel; Kaigala, Govind V

    2016-01-01

    Heterogeneity is inherent to biology, thus it is imperative to realize methods capable of obtaining spatially-resolved genomic and transcriptomic profiles of heterogeneous biological samples. Here, we present a new method for local lysis of live adherent cells for nucleic acid analyses. This method addresses bottlenecks in current approaches, such as dilution of analytes, one-sample-one-test, and incompatibility to adherent cells. We make use of a scanning probe technology - a microfluidic probe - and implement hierarchical hydrodynamic flow confinement (hHFC) to localize multiple biochemicals on a biological substrate in a non-contact, non-destructive manner. hHFC enables rapid recovery of nucleic acids by coupling cell lysis and lysate collection. We locally lysed ~300 cells with chemical systems adapted for DNA or RNA and obtained lysates of ~70 cells/μL for DNA analysis and ~15 cells/μL for mRNA analysis. The lysates were introduced into PCR-based workflows for genomic and transcriptomic analysis. This strategy further enabled selective local lysis of subpopulations in a co-culture of MCF7 and MDA-MB-231 cells, validated by characteristic E-cadherin gene expression in individually extracted cell types. The developed strategy can be applied to study cell-cell, cell-matrix interactions locally, with implications in understanding growth, progression and drug response of a tumor. PMID:27411740

  6. Selective local lysis and sampling of live cells for nucleic acid analysis using a microfluidic probe

    PubMed Central

    Kashyap, Aditya; Autebert, Julien; Delamarche, Emmanuel; Kaigala, Govind V.

    2016-01-01

    Heterogeneity is inherent to biology, thus it is imperative to realize methods capable of obtaining spatially-resolved genomic and transcriptomic profiles of heterogeneous biological samples. Here, we present a new method for local lysis of live adherent cells for nucleic acid analyses. This method addresses bottlenecks in current approaches, such as dilution of analytes, one-sample-one-test, and incompatibility to adherent cells. We make use of a scanning probe technology - a microfluidic probe - and implement hierarchical hydrodynamic flow confinement (hHFC) to localize multiple biochemicals on a biological substrate in a non-contact, non-destructive manner. hHFC enables rapid recovery of nucleic acids by coupling cell lysis and lysate collection. We locally lysed ~300 cells with chemical systems adapted for DNA or RNA and obtained lysates of ~70 cells/μL for DNA analysis and ~15 cells/μL for mRNA analysis. The lysates were introduced into PCR-based workflows for genomic and transcriptomic analysis. This strategy further enabled selective local lysis of subpopulations in a co-culture of MCF7 and MDA-MB-231 cells, validated by characteristic E-cadherin gene expression in individually extracted cell types. The developed strategy can be applied to study cell-cell, cell-matrix interactions locally, with implications in understanding growth, progression and drug response of a tumor. PMID:27411740

  7. Development of a microfluidic device for determination of cell osmotic behavior and membrane transport properties.

    PubMed

    Chen, Hsiu-Hung; Purtteman, Jester J P; Heimfeld, Shelly; Folch, Albert; Gao, Dayong

    2007-12-01

    An understanding of cell osmotic behavior and membrane transport properties is indispensable for cryobiology research and development of cell-type-specific, optimal cryopreservation conditions. A microfluidic perfusion system is developed here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell osmotic behavior and membrane transport properties. The system is fabricated using soft lithography and is comprised of microfluidic channels and a perfusion chamber for trapping cells. During experiments, rat basophilic leukemia (RBL-1 line) cells were injected into the inlet of the device, allowed to flow downstream, and were trapped within a perfusion chamber. The fluid continues to flow to the outlet due to suction produced by a Hamilton Syringe. Two sets of experiments have been performed: the cells were perfused by (1) hypertonic solutions with different concentrations of non-permeating solutes and (2) solutions containing a permeating cryoprotective agent (CPA), dimethylsulfoxide (Me(2)SO), plus non-permeating solute (sodium chloride (NaCl)), respectively. From experiment (1), cell osmotically inactive volume (V(b)) and the permeability coefficient of water (L(p)) for RBL cells are determined to be 41% [n=18, correlation coefficient (r(2)) of 0.903] of original/isotonic volume, and 0.32+/-0.05 microm/min/atm (n=8, r(2)>0.963), respectively, for room temperature (22 degrees C). From experiment (2), the permeability coefficient of water (L(p)) and of Me(2)SO (P(s)) for RBL cells are 0.38+/-0.09 microm/min/atm and (0.49+/-0.13) x 10(-3)cm/min (n=5, r(2)>0.86), respectively. We conclude that this device enables us to: (1) readily monitor the changes of extracellular conditions by perfusing single or a group of cells with prepared media; (2) confine cells (or a cell) within a monolayer chamber, which prevents imaging ambiguity, such as cells overlapping or moving out of the focus plane; (3) study individual cell

  8. Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

    PubMed Central

    Gruenberger, Alexander; Probst, Christopher; Heyer, Antonia; Wiechert, Wolfgang; Frunzke, Julia; Kohlheyer, Dietrich

    2013-01-01

    In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g. cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs. PMID:24336165

  9. Long-term three-dimensional cell culture and anticancer drug activity evaluation in a microfluidic chip.

    PubMed

    Ziółkowska, Karina; Stelmachowska, Agnieszka; Kwapiszewski, Radosław; Chudy, Michał; Dybko, Artur; Brzózka, Zbigniew

    2013-02-15

    In this work, we present a microfluidic array of microwells for long-term tumor spheroid cultivation and anticancer drug activity evaluation. The three-dimensional microfluidic system was obtained by double casting of poly(dimethylsiloxane). Spheroids of HT-29 human carcinoma cells were cultured in the microsystem for four weeks. After two weeks of the culture growth slowdown and stop were observed and high cell viability was determined within next two weeks. The characteristics of a homeostasis-like state were achieved. A cytostatic drug (5-fluorouracil) was introduced into the microsystem with different frequency (every day or every second day) and different concentrations. The geometry and construction of the microsystem enables flushing away of unaggregated (including dead) cells while viable spheroids remain inside microwells and decreasing spheroid diameter can be observed and measured as an indicator of decreasing cell viability. The results have shown differences in response of spheroids to different concentrations of 5-fluorouracil. It was also observed, that higher frequency of drug dosing resulted in more rapid spheroid diameter decrease. The presented microfluidic system is a solution for cell-based studies in an in vivo-like microfluidic environment. Moreover, observation of decreasing spheroid dimensions is a low-cost, label-free and easy-to-conduct mean of a quantitative determination of a 3D cellular model response to a applied drug. It is suitable for long-term observation of spheroid response, in a contrary to other viability assays requiring termination of a culture. PMID:22770829

  10. A microfluidic platform enabling single-cell RNA-seq of multigenerational lineages

    PubMed Central

    Kimmerling, Robert J.; Lee Szeto, Gregory; Li, Jennifer W.; Genshaft, Alex S.; Kazer, Samuel W.; Payer, Kristofor R.; de Riba Borrajo, Jacob; Blainey, Paul C.; Irvine, Darrell J.; Shalek, Alex K.; Manalis, Scott R.

    2016-01-01

    We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multi-generational lineage tracking under controlled culture conditions. We use this platform to generate whole-transcriptome profiles of primary, activated murine CD8+ T-cell and lymphocytic leukemia cell line lineages. Here we report that both cell types have greater intra- than inter-lineage transcriptional similarity. For CD8+ T-cells, genes with functional annotation relating to lymphocyte differentiation and function—including Granzyme B—are enriched among the genes that demonstrate greater intra-lineage expression level similarity. Analysis of gene expression covariance with matched measurements of time since division reveals cell type-specific transcriptional signatures that correspond with cell cycle progression. We believe that the ability to directly measure the effects of lineage and cell cycle-dependent transcriptional profiles of single cells will be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology. PMID:26732280

  11. Continuous Flow Deformability-Based Separation of Circulating Tumor Cells Using Microfluidic Ratchets.

    PubMed

    Park, Emily S; Jin, Chao; Guo, Quan; Ang, Richard R; Duffy, Simon P; Matthews, Kerryn; Azad, Arun; Abdi, Hamidreza; Todenhöfer, Tilman; Bazov, Jenny; Chi, Kim N; Black, Peter C; Ma, Hongshen

    2016-04-01

    Circulating tumor cells (CTCs) offer tremendous potential for the detection and characterization of cancer. A key challenge for their isolation and subsequent analysis is the extreme rarity of these cells in circulation. Here, a novel label-free method is described to enrich viable CTCs directly from whole blood based on their distinct deformability relative to hematological cells. This mechanism leverages the deformation of single cells through tapered micrometer scale constrictions using oscillatory flow in order to generate a ratcheting effect that produces distinct flow paths for CTCs, leukocytes, and erythrocytes. A label-free separation of circulating tumor cells from whole blood is demonstrated, where target cells can be separated from background cells based on deformability despite their nearly identical size. In doping experiments, this microfluidic device is able to capture >90% of cancer cells from unprocessed whole blood to achieve 10(4) -fold enrichment of target cells relative to leukocytes. In patients with metastatic castration-resistant prostate cancer, where CTCs are not significantly larger than leukocytes, CTCs can be captured based on deformability at 25× greater yield than with the conventional CellSearch system. Finally, the CTCs separated using this approach are collected in suspension and are available for downstream molecular characterization. PMID:26917414

  12. Capture of esophageal and breast cancer cells with polymeric microfluidic devices for CTC isolation

    PubMed Central

    OHNAGA, TAKASHI; SHIMADA, YUTAKA; TAKATA, KOJI; OBATA, TSUTOMU; OKUMURA, TOMOYUKI; NAGATA, TAKUYA; KISHI, HIROYUKI; MURAGUCHI, ATSUSHI; TSUKADA, KAZUHIRO

    2016-01-01

    The present study evaluated the capture efficiency of esophageal and breast cancer cells with a modified ‘polymeric circulating tumor cells (CTC)-chip’ microfluidic device, which was developed for the isolation of circulating tumor cells. Esophageal cancer cell lines KYSE150, KYSE220 and KYSE510, and breast cancer cell lines MCF7, SKBR3 and MDA-MB-231 were used for evaluation. The capture efficiencies of the esophageal cancer cell lines in phosphate-buffered saline (PBS) were ~0.9, irrespective of epithelial cell adhesion molecule (EpCAM) expression, which was represented as the mean fluorescent intensity from 528 to 76. In the breast cancer cell lines, efficient capture was observed for MCF7 and SKBR3 in PBS; however, a low value of ~0.1 was obtained for MDA-MB-231. Fluorescent imaging of immunolabeled cells revealed marginal EpCAM expression in MDA-MB-231. Using whole blood, no clogging occurred in the microstructure-modified CTC-chip and efficiency of capture was successfully evaluated. Capture efficiencies for KYSE220 and MCF7 in whole blood were >0.7, but were of either equal or lesser efficiency in comparison to PBS. Therefore, the modified CTC-chip appears useful for clinical application due to its cost, practicality of use, and efficient cancer cell capture. PMID:27073672

  13. A novel microfluidic platform with stable concentration gradient for on chip cell culture and screening assays.

    PubMed

    Xu, Bi-Yi; Hu, Shan-Wen; Qian, Guang-Sheng; Xu, Jing-Juan; Chen, Hong-Yuan

    2013-09-21

    In this work a novel microfluidic platform for cell culture and assay is developed. On the chip a static cell culture region is coupled with dynamic fluidic nutrition supply structures. The cell culture unit has a sandwich structure with liquid channels on the top, the cell culture reservoir in the middle and gas channels on the bottom. Samples can be easily loaded into the reservoir and exchange constantly with the external liquid environment by diffusion. Since the flow direction is perpendicular to the liquid channel on the top of the reservoir, the cells in the reservoir are shielded from shear-force. By assembling the basic units into an array, a steady concentration gradient can be generated. Cell culture models both for continuous perfusion and one-off perfusion were established on the chip. Both adherent and suspended cells were successfully cultured on the chip in 2D and 3D culture modes. After culturing, the trapped cells were recovered for use in a later assay. As a competitive candidate for a standard cell culture and assay platform, this chip is also adaptable for cytotoxicity and cell growth assays. PMID:23884407

  14. A THREE-DIMENSIONAL MICROFLUIDIC APPROACH TO SCALING UP MICROENCAPSULATION OF CELLS

    PubMed Central

    Tendulkar, Sameer; Mirmalek-Sani, Sayed-Hadi; Childers, Charles; Saul, Justin; Opara, Emmanuel C; Ramasubramanian, Melur K.

    2013-01-01

    Current applications of the microencapsulation technique include the use of encapsulated islet cells to treat Type 1 diabetes, and encapsulated hepatocytes for providing temporary but adequate metabolic support to allow spontaneous liver regeneration, or as a bridge to liver transplantation for patients with chronic liver disease. Also, microcapsules can be used for controlled delivery of therapeutic drugs. The two most widely used devices for microencapsulation are the air-syringe pump droplet generator and the electrostatic bead generator, each of which is fitted with a single needle through which droplets of cells suspended in alginate solution are produced and cross-linked into microbeads. A major drawback in the design of these instruments is that they are incapable of producing sufficient numbers of microcapsules in a short-time period to permit mass production of encapsulated and viable cells for transplantation in large animals and humans. We present in this paper a microfluidic approach to scaling up cell and protein encapsulations. The microfluidic chip consists of a 3D air supply and multi-nozzle outlet for microcapsule generation. It has one alginate inlet and compressed air inlet. The outlet has 8 nozzles, each having 380 micrometers inner diameter, which produce hydrogel microspheres ranging from 500–700 µm in diameter. These nozzles are concentrically surrounded by air nozzles with 2mm inner diameter. There are two tubes connected at the top to allow the air to escape as the alginate solution fills up the chamber. A variable flow pump 115 V is used to pump alginate solution and Tygon® tubing is used to connect in-house air supply to the air channel and peristaltic/syringe pump to the alginate chamber. A pressure regulator is used to control the flow rate of air. We have encapsulated islets and proteins with this high throughput device, which is expected to improve product quality control in microencapsulation of cells, and hence the outcome of

  15. AuPd/polyaniline as the anode in an ethylene glycol microfluidic fuel cell operated at room temperature.

    PubMed

    Arjona, N; Palacios, A; Moreno-Zuria, A; Guerra-Balcázar, M; Ledesma-García, J; Arriaga, L G

    2014-08-01

    AuPd/polyaniline was used for the first time, for ethylene glycol (EG) electrooxidation in a novel microfluidic fuel cell (MFC) operated at room temperature. The device exhibits high electrocatalytic performance and stability for the conversion of cheap and fully available EG as fuel. PMID:24923468

  16. Microfluidic Cell Deformability Assay for Rapid and Efficient Kinase Screening with the CRISPR-Cas9 System.

    PubMed

    Han, Xin; Liu, Zongbin; Zhao, Li; Wang, Feng; Yu, Yang; Yang, Jianhua; Chen, Rui; Qin, Lidong

    2016-07-18

    Herein we report a CRISPR-Cas9-mediated loss-of-function kinase screen for cancer cell deformability and invasive potential in a high-throughput microfluidic chip. In this microfluidic cell separation platform, flexible cells with high deformability and metastatic propensity flowed out, while stiff cells remained trapped. Through deep sequencing, we found that loss of certain kinases resulted in cells becoming more deformable and invasive. High-ranking candidates identified included well-reported tumor suppressor kinases, such as chk2, IKK-α, p38 MAPKs, and DAPK2. A high-ranking candidate STK4 was chosen for functional validation and identified to play an important role in the regulation of cell deformability and tumor suppression. Collectively, we have demonstrated that CRISPR-based on-chip mechanical screening is a potentially powerful strategy to facilitate systematic genetic analyses. PMID:27258939

  17. Electrode Calibration with a Microfluidic Flow Cell for Fast-scan Cyclic Voltammetry

    PubMed Central

    Sinkala, Elly; McCutcheon, James E.; Schuck, Matt; Schmidt, Eric; Roitman, Mitchell F.; Eddington, David T.

    2012-01-01

    Fast-scan cyclic voltammetry (FSCV) is a common analytical electrochemistry tool used to measure chemical species. It has recently been adapted for measurement of neurotransmitters such as dopamine in awake and behaving animals (in vivo). Electrode calibration is an essential step in FSCV to relate observed current to concentration of a chemical species. However, existing methods require multiple components, which reduce the ease of calibrations. To this end, a microfluidic flow cell (µFC) was developed as a simple device to switch between buffer and buffer with a known concentration of the analyte of interest – in this case dopamine - in a microfluidic Y-channel. The ability to quickly switch solutions yielded electrode calibrations with faster rise times and that were more stable at peak current values. The µFC reduced the number of external electrical components and produced linear calibrations over a range of concentrations. To demonstrate this, an electrode calibrated with the µFC was used in FSCV recordings from a rat during the delivery of food reward – a stimulus that reliably evokes a brief increase in current due to the oxidation of dopamine. Using the linear calibration, dopamine concentrations were determined from the current responses evoked during the behavioral task. The µFC is able to easily and quickly calibrate FSCV electrode responses to chemical species for both in vitro and in vivo experiments. PMID:22522908

  18. Femtosecond laser machined microfluidic devices for imaging of cells during chemotaxis

    PubMed Central

    Costa, L.; Terekhov, A.; Rajput, D.; Hofmeister, W.; Jowhar, D.; Wright, G.; Janetopoulos, C.

    2013-01-01

    Microfluidic devices designed for chemotaxis assays were fabricated on fused silica substrates using femtosecond laser micromachining. These devices have built-in chemical concentration gradient forming structures and are ideally suited for establishing passive diffusion gradients over extended periods of time. Multiple gradient forming structures, with identical or distinct gradient forming characteristics, can be integrated into a single device, and migrating cells can be directly observed using an inverted microscope. In this paper, the design, fabrication, and operation of these devices are discussed. Devices with minimal structure sizes ranging from 3 to 7 lm are presented. The use of these devices to investigate the migration of Dictyostelium discoideum cells toward the chemoattractant folic acid is presented as an example of the devices' utility. PMID:24532962

  19. Adhesion assays of endothelial cells on nanopatterned surfaces within a microfluidic channel.

    PubMed

    Hwang, Se Yon; Kwon, Keon Woo; Jang, Kyung-Jin; Park, Min Cheol; Lee, Jeong Sang; Suh, Kahp Y

    2010-04-01

    We present a simple analytical method to measure adhesion of human umbilical vein endothelial cells (HUVECs) and calf pulmonary artery endothelial cells (CPAEs) using nanopatterned, biodegradable poly(lactic-co-glycolic acid) (PLGA) surfaces for potential applications to artificial tissue-engineered blood vessel. Various nanostructured PLGA surfaces (350 nm wide ridges/350 nm grooves, 350 nm ridges/700 nm grooves, 350 nm ridges/1750 nm grooves, 700 nm ridges/350 nm grooves, 1050 nm ridges/350 nm grooves, 1750 nm ridges/350 nm grooves) and flat (unpatterned) surfaces were fabricated on the bottom of polydimethylsiloxane (PDMS) microfluidic channel of 2 mm width and 60 microm height by using thermal imprinting and irreversible channel bonding. To measure adhesion strength of HUVECs and CPAEs, the cells were exposed to a range of shear stress (12, 40, and 80 dyn/cm(2)) within the channels for 20 min after a preculture for 3 days and the remaining cells were counted under each condition. The highest adhesion strength was found on the surface of 700 nm wide ridges, 350 nm wide grooves for both cell types. The enhanced adhesion on nanopatterned surfaces can be attributed to two aspects: (i) contact guidance along the line direction and (ii) clustered focal adhesions. In particular, the contact guidance induced cell alignment along the line directions, which in turn lowers wall shear stress applied to the cell surface, as supported by a simple hydrodynamic model based on cell morphology. PMID:20218573

  20. Genomic DNA extraction from cells by electroporation on an integrated microfluidic platform

    PubMed Central

    Geng, Tao; Bao, Ning; Sriranganathanw, Nammalwar; Li, Liwu; Lu, Chang

    2012-01-01

    The vast majority of genetic analysis of cells involves chemical lysis for release of DNA molecules. However, chemical reagents required in the lysis interfere with downstream molecular biology and often require removal after the step. Electrical lysis based on irreversible electroporation is a promising technique to prepare samples for genetic analysis due to its purely physical nature, fast speed, and simple operation. However, there has been no experimental confirmation on whether electrical lysis extracts genomic DNA from cells in a reproducible and efficient fashion in comparison to chemical lysis, especially for eukaryotic cells that have most of DNA enclosed in the nucleus. In this work, we construct an integrated microfluidic chip that physically traps a low number of cells, lyses the cells using electrical pulses rapidly, then purifies and concentrates genomic DNA. We demonstrate that electrical lysis offers high efficiency for DNA extraction from both eukaryotic cells (up to ~36% for Chinese hamster ovary cells) and bacterial cells (up to ~45% for Salmonella typhimurium) that is comparable to the widely-used chemical lysis. The DNA extraction efficiency has dependence on both electric parameters and relative amount of beads used for DNA adsorption. We envision that electroporation-based DNA extraction will find use in ultrasensitive assays that benefit from minimal dilution and simple procedure. PMID:23061629

  1. Investigation of Tumor Cell Behaviors on a Vascular Microenvironment-Mimicking Microfluidic Chip

    PubMed Central

    Huang, Rong; Zheng, Wenfu; Liu, Wenwen; Zhang, Wei; Long, Yunze; Jiang, Xingyu

    2015-01-01

    The extravasation of tumor cells is a key event in tumor metastasis. However, the mechanism underlying tumor cell extravasation remains unknown, mainly hindered by obstacles from the lack of complexity of biological tissues in conventional cell culture, and the costliness and ethical issues of in vivo experiments. Thus, a cheap, time and labor saving, and most of all, vascular microenvironment-mimicking research model is desirable. Herein, we report a microfluidic chip-based tumor extravasation research model which is capable of simultaneously simulating both mechanical and biochemical microenvironments of human vascular systems and analyzing their synergistic effects on the tumor extravasation. Under different mechanical conditions of the vascular system, the tumor cells (HeLa cells) had the highest viability and adhesion activity in the microenvironment of the capillary. The integrity of endothelial cells (ECs) monolayer was destroyed by tumor necrosis factor-α (TNF-α) in a hemodynamic background, which facilitated the tumor cell adhesion, this situation was recovered by the administration of platinum nanoparticles (Pt-NPs). This model bridges the gap between cell culture and animal experiments and is a promising platform for studying tumor behaviors in the vascular system. PMID:26631692

  2. A Biochip with a 3D microfluidic architecture for trapping white blood cells

    PubMed Central

    Tripathi, Anurag; Riddell, James; Chronis, Nikos

    2013-01-01

    We present a microfluidic biochip for trapping single white blood cells (WBCs). The novel biochip, microfabricated using standard surface micromachining processes, consists of an array of precisely engineered microholes that confine single cells in a tight, three dimensional space and mechanically immobilize them. A high (> 87%) trapping efficiency was achieved when WBC-containing samples were delivered to the biochip at the optimal pressure of 3 psi. The biochip can efficiently trap up to 7,500 cells, maintaining a high trapping efficiency even when the number of cells is extremely low (~200 cells). We believe that the developed biochip can be used as a standalone unit in a biology/clinical lab for trapping WBCs as well as other cell types and imaging them using a standard fluorescent microscope at the single cell level. Furthermore, it can be integrated with other miniaturized optical modules to construct a portable platform for counting a wide variety of cells and therefore it can be an excellent tool for monitoring human diseases at the point-of-care. PMID:23935241

  3. Investigation of Tumor Cell Behaviors on a Vascular Microenvironment-Mimicking Microfluidic Chip.

    PubMed

    Huang, Rong; Zheng, Wenfu; Liu, Wenwen; Zhang, Wei; Long, Yunze; Jiang, Xingyu

    2015-01-01

    The extravasation of tumor cells is a key event in tumor metastasis. However, the mechanism underlying tumor cell extravasation remains unknown, mainly hindered by obstacles from the lack of complexity of biological tissues in conventional cell culture, and the costliness and ethical issues of in vivo experiments. Thus, a cheap, time and labor saving, and most of all, vascular microenvironment-mimicking research model is desirable. Herein, we report a microfluidic chip-based tumor extravasation research model which is capable of simultaneously simulating both mechanical and biochemical microenvironments of human vascular systems and analyzing their synergistic effects on the tumor extravasation. Under different mechanical conditions of the vascular system, the tumor cells (HeLa cells) had the highest viability and adhesion activity in the microenvironment of the capillary. The integrity of endothelial cells (ECs) monolayer was destroyed by tumor necrosis factor-α (TNF-α) in a hemodynamic background, which facilitated the tumor cell adhesion, this situation was recovered by the administration of platinum nanoparticles (Pt-NPs). This model bridges the gap between cell culture and animal experiments and is a promising platform for studying tumor behaviors in the vascular system. PMID:26631692

  4. Passage times of confined cancer cells and deformable particles flowing through a microfluidic channel

    NASA Astrophysics Data System (ADS)

    Khan, Zeina; Kamyabi, Nabiollah; Hussain, Fazle; Vanapalli, Siva

    Circulating tumor cells, the primary cause of cancer metastasis, have to navigate through tight extracellular matrix and capillaries. Unfortunately, understanding of the hydrodynamic interactions between cells and narrow vessel walls is lacking. Using a microfluidic channel of rectangular cross-section, we investigate cell hydrodynamic behavior by measuring cell confinement, passage time through the microchannel, and excess pressure drop. Testing with highly and lowly aggressive cancer cells shows that passage time may not always be indicative of cancer cell aggressiveness as the relationship among passage time, friction and rheology is complex. Transport of deformable particles including droplets of varying viscosity and interfacial tension, as well as elastic particles of different elastic moduli, reveals that passage times depend on particle size and, contrary to prior claims, on viscosity but not on elastic modulus. We also find that particle viscosity and not modulus controls the friction force and lubrication film thickness, suggesting that cancer cell viscosity rather than elasticity controls cell transport on short time-scales.

  5. A microfluidic device for 2D to 3D and 3D to 3D cell navigation

    NASA Astrophysics Data System (ADS)

    Shamloo, Amir; Amirifar, Leyla

    2016-01-01

    Microfluidic devices have received wide attention and shown great potential in the field of tissue engineering and regenerative medicine. Investigating cell response to various stimulations is much more accurate and comprehensive with the aid of microfluidic devices. In this study, we introduced a microfluidic device by which the matrix density as a mechanical property and the concentration profile of a biochemical factor as a chemical property could be altered. Our microfluidic device has a cell tank and a cell culture chamber to mimic both 2D to 3D and 3D to 3D migration of three types of cells. Fluid shear stress is negligible on the cells and a stable concentration gradient can be obtained by diffusion. The device was designed by a numerical simulation so that the uniformity of the concentration gradients throughout the cell culture chamber was obtained. Adult neural cells were cultured within this device and they showed different branching and axonal navigation phenotypes within varying nerve growth factor (NGF) concentration profiles. Neural stem cells were also cultured within varying collagen matrix densities while exposed to NGF concentrations and they experienced 3D to 3D collective migration. By generating vascular endothelial growth factor concentration gradients, adult human dermal microvascular endothelial cells also migrated in a 2D to 3D manner and formed a stable lumen within a specific collagen matrix density. It was observed that a minimum absolute concentration and concentration gradient were required to stimulate migration of all types of the cells. This device has the advantage of changing multiple parameters simultaneously and is expected to have wide applicability in cell studies.

  6. Intrinsic FGF2 and FGF5 promotes angiogenesis of human aortic endothelial cells in 3D microfluidic angiogenesis system

    PubMed Central

    Seo, Ha-Rim; Jeong, Hyo Eun; Joo, Hyung Joon; Choi, Seung-Cheol; Park, Chi-Yeon; Kim, Jong-Ho; Choi, Ji-Hyun; Cui, Long-Hui; Hong, Soon Jun; Chung, Seok; Lim, Do-Sun

    2016-01-01

    The human body contains different endothelial cell types and differences in their angiogenic potential are poorly understood. We compared the functional angiogenic ability of human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using a three-dimensional (3D) microfluidic cell culture system. HAECs and HUVECs exhibited similar cellular characteristics in a 2D culture system; however, in the 3D microfluidic angiogenesis system, HAECs exhibited stronger angiogenic potential than HUVECs. Interestingly, the expression level of fibroblast growth factor (FGF)2 and FGF5 under vascular endothelial growth factor (VEGF)-A stimulation was significantly higher in HAECs than in HUVECs. Moreover, small interfering RNA-mediated knockdown of FGF2 and FGF5 more significantly attenuated vascular sprouting induced from HAECs than HUVECs. Our results suggest that HAECs have greater angiogenic potential through FGF2 and FGF5 upregulation and could be a compatible endothelial cell type to achieve robust angiogenesis. PMID:27357248

  7. Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology.

    PubMed

    Elani, Yuval

    2016-06-15

    The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved. PMID:27284034

  8. Intrinsic FGF2 and FGF5 promotes angiogenesis of human aortic endothelial cells in 3D microfluidic angiogenesis system.

    PubMed

    Seo, Ha-Rim; Jeong, Hyo Eun; Joo, Hyung Joon; Choi, Seung-Cheol; Park, Chi-Yeon; Kim, Jong-Ho; Choi, Ji-Hyun; Cui, Long-Hui; Hong, Soon Jun; Chung, Seok; Lim, Do-Sun

    2016-01-01

    The human body contains different endothelial cell types and differences in their angiogenic potential are poorly understood. We compared the functional angiogenic ability of human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using a three-dimensional (3D) microfluidic cell culture system. HAECs and HUVECs exhibited similar cellular characteristics in a 2D culture system; however, in the 3D microfluidic angiogenesis system, HAECs exhibited stronger angiogenic potential than HUVECs. Interestingly, the expression level of fibroblast growth factor (FGF)2 and FGF5 under vascular endothelial growth factor (VEGF)-A stimulation was significantly higher in HAECs than in HUVECs. Moreover, small interfering RNA-mediated knockdown of FGF2 and FGF5 more significantly attenuated vascular sprouting induced from HAECs than HUVECs. Our results suggest that HAECs have greater angiogenic potential through FGF2 and FGF5 upregulation and could be a compatible endothelial cell type to achieve robust angiogenesis. PMID:27357248

  9. Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology

    PubMed Central

    Elani, Yuval

    2016-01-01

    The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved. PMID:27284034

  10. High-throughput tracking of single yeast cells in a microfluidic imaging matrix

    PubMed Central

    Falconnet, D.; Niemistö, A.; Taylor, R.J.; Ricicova, M.; Galitski, T.; Shmulevich, I.; Hansen, C. L.

    2011-01-01

    Summary Time-lapse live cell imaging is a powerful tool for studying signaling network dynamics and complexity and is uniquely suited to single cell studies of response dynamics, noise, and heritable differences. Although conventional imaging formats have the temporal and spatial resolution needed for such studies, they do not provide the simultaneous advantages of cell tracking, experimental throughput, and precise chemical control. This is particularly problematic for systems-level studies using non-adherent model organisms such as yeast, where the motion of cells complicates tracking and where large-scale analysis under a variety of genetic and chemical perturbations is desired. We present here a high-throughput microfluidic imaging system capable of tracking single cells over multiple generations in 128 simultaneous experiments with programmable and precise chemical control. High-resolution imaging and robust cell tracking is achieved through immobilization of yeast cells using a combination of mechanical clamping and polymerization in an agarose gel. The channel and valve architecture of our device allows for the formation of a matrix of 128 integrated agarose gel pads, each allowing for an independent imaging experiment with fully programmable medium exchange via diffusion. We demonstrate our system in the combinatorial and quantitative analysis of the yeast pheromone signaling response across 8 genotypes and 16 conditions, and show that lineage-dependent effects contribute to observed variability at stimulation conditions near the critical threshold for cellular decision making. PMID:21088765

  11. Metabolite profiling of microfluidic cell culture conditions for droplet based screening

    PubMed Central

    Bjork, Sara M.; Sjostrom, Staffan L.; Andersson-Svahn, Helene; Joensson, Haakan N.

    2015-01-01

    We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format. PMID:26392830

  12. Observation of reversible, rapid changes in drug susceptibility of hypoxic tumor cells in a microfluidic device.

    PubMed

    Germain, Todd; Ansari, Megan; Pappas, Dimitri

    2016-09-14

    Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 μM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target. PMID:27566353

  13. Cell death along single microfluidic channel after freeze-thaw treatments.

    PubMed

    Li, Yuhui; Wang, Fen; Wang, Hao

    2010-01-01

    Cryotherapy is a prospective green method for malignant tumor treatment. At low temperature, the cell viability relates with the cooling rate, temperature threshold, freezing interface, as well as ice formation. In clinical applications, the growth of ice ball must reach a suitable size as cells could not be all killed at the ice periphery. The cell death ratio at the ice periphery is important for the control of the freezing destruction. The mechanisms of cryoinjury around the ice periphery need thorough understanding. In this paper, a primary freeze-thaw control was carried out in a cell culture microchip. A series of directional freezing processes and cell responses was tested and discussed. The temperature in the microchip was manipulated by a thermoelectric cooler. The necrotic and apoptotic cells under different cryotreatment (duration of the freezing process, freeze-thaw cycle, postculture, etc.) were stained and distinguished by propidium iodide and fluorescein isothiocyanate (FITC)-Annexin V. The location of the ice front was recorded and a cell death boundary which was different from the ice front was observed. By controlling the cooling process in a microfluidic channel, it is possible to recreate a sketch of biological effect during the process of simulated cryosurgery. PMID:20644680

  14. Single cell swimming dynamics of Listeria monocytogenes using a nanoporous microfluidic platform

    SciTech Connect

    Wright, Evan; Neethirajan, Suresh; Warriner, Keith; Retterer, Scott T; Srijanto, Bernadeta R

    2014-01-01

    Listeria monocytogenes remains a significant foodborne pathogen due to its virulence and ability to become established in food processing facilities. The pathogen is characterized by its ability to grow over a wide temperature range and withstand a broad range of stresses. The following reports on the chemotaxis and motility of the L. monocytogenes when exposed to relatively small concentrations of acetic acid. Using the developed nanoporous microfluidic device to precisely modulate the cellular environment, we exposed the individual Listeria cells to acetic acid and, in real time and with high resolution, observed how the cells reacted to the change in their surroundings. Our results showed that concentrations of acetic acid below 10 mM had very little, if any, effect on the motility. However, when exposed to 100 mM acetic acid, the cells exhibited a sharp drop in velocity and displayed a more random pattern of motion. These results indicate that at appropriate concentrations, acetic acid has the ability to disable the flagellum of the cells, thus impairing their motility. This drop in motility has numerous effects on the cell; its main effects being the obstruction of the cell's ability to properly form biofilms and a reduction in the overall infectivity of the cells. Since these characteristics are especially useful in controlling the proliferation of L. monocytogenes, acetic acid shows potential for application in the food industry as an active compound in designing a food packaging environment and as an antimicrobial agent.

  15. 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

  16. High-Resolution Microfluidic Single-Cell Transcriptional Profiling Reveals Clinically Relevant Subtypes among Human Stem Cell Populations Commonly Utilized in Cell-Based Therapies

    PubMed Central

    Rennert, Robert C.; Schäfer, Richard; Bliss, Tonya; Januszyk, Michael; Sorkin, Michael; Achrol, Achal S.; Rodrigues, Melanie; Maan, Zeshaan N.; Kluba, Torsten; Steinberg, Gary K.; Gurtner, Geoffrey C.

    2016-01-01

    Stem cell therapies can promote neural repair and regeneration, yet controversy regarding optimal cell source and mechanism of action has slowed clinical translation, potentially due to undefined cellular heterogeneity. Single-cell resolution is needed to identify clinically relevant subpopulations with the highest therapeutic relevance. We combine single-cell microfluidic analysis with advanced computational modeling to study for the first time two common sources for cell-based therapies, human NSCs and MSCs. This methodology has the potential to logically inform cell source decisions for any clinical application. PMID:27047447

  17. A drug-compatible and temperature-controlled microfluidic device for live-cell imaging

    PubMed Central

    Chen, Tong; Gomez-Escoda, Blanca; Munoz-Garcia, Javier; Babic, Julien; Griscom, Laurent; Wu, Pei-Yun Jenny

    2016-01-01

    Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging. PMID:27512142

  18. Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry.

    PubMed

    Mellors, J Scott; Jorabchi, Kaveh; Smith, Lloyd M; Ramsey, J Michael

    2010-02-01

    A microfabricated fluidic device was developed for the automated real-time analysis of individual cells using capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS). The microfluidic structure incorporates a means for rapid lysis of single cells within a free solution electrophoresis channel, where cellular constituents were separated, and an integrated electrospray emitter for ionization of separated components. The eluent was characterized using mass spectrometry. Human erythrocytes were used as a model system for this study. In this monolithically integrated device, cell lysis occurs at a channel intersection using a combination of rapid buffer exchange and an increase in electric field strength. An electroosmotic pump is incorporated at the end of the electrophoretic separation channel to direct eluent to the integrated electrospray emitter. The dissociated heme group and the alpha and beta subunits of hemoglobin from individual erythrocytes were detected as cells continuously flowed through the device. The average analysis throughput was approximately 12 cells per minute, demonstrating the potential of this method for high-throughput single cell analysis. PMID:20058879

  19. Integrated Microfluidic Device for Automated Single Cell Analysis using Electrophoretic Separation and Electrospray Ionization Mass Spectrometry

    PubMed Central

    Mellors, J. Scott; Jorabchi, Kaveh; Smith, Lloyd M.; Ramsey, J. Michael

    2010-01-01

    A microfabricated fluidic device was developed for the automated real-time analysis of individual cells using capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS). The microfluidic structure incorporates a means for rapid lysis of single cells within a free solution electrophoresis channel where cellular constituents were separated and an integrated electrospray emitter for ionization of separated components. The eluent was characterized using mass spectrometry. Human erythrocytes were used as a model system for this study. In this monolithically integrated device, cell lysis occurs at a channel intersection using a combination of rapid buffer exchange and an increase in electric field strength. An electroosmotic pump is incorporated at the end of the electrophoretic separation channel to direct eluent to the integrated electrospray emitter. The dissociated heme group and the α and β subunits of hemoglobin from individual erythrocytes were detected as cells continuously flowed through the device. The average analysis throughput was approximately 12 cells per minute demonstrating the potential of this method for high-throughput single cell analysis. PMID:20058879

  20. Rapid quantification of live cell receptors using bioluminescence in a flow-based microfluidic device.

    PubMed

    Ramji, Ramesh; Cheong, Cheong Fook; Hirata, Hiroaki; Rahman, Abdur Rub Abdur; Lim, Chwee Teck

    2015-02-25

    The number of receptors expressed by cells plays an important role in controlling cell signaling events, thus determining its behaviour, state and fate. Current methods of quantifying receptors on cells are either laborious or do not maintain the cells in their native form. Here, a method integrating highly sensitive bioluminescence, high precision microfluidics and small footprint of lensfree optics is developed to quantify cell surface receptors. This method is safe to use, less laborious, and faster than the conventional radiolabelling and near field scanning methods. It is also more sensitive than fluorescence based assays and is ideal for high throughput screening. In quantifying β(1) adrenergic receptors expressed on the surface of H9c2 cardiomyocytes, this method yields receptor numbers from 3.12 × 10(5) to 9.36 × 10(5) receptors/cell which are comparable with current methods. This can serve as a very good platform for rapid quantification of receptor numbers in ligand/drug binding and receptor characterization studies, which is an important part of pharmaceutical and biological research. PMID:25336403

  1. A drug-compatible and temperature-controlled microfluidic device for live-cell imaging.

    PubMed

    Chen, Tong; Gomez-Escoda, Blanca; Munoz-Garcia, Javier; Babic, Julien; Griscom, Laurent; Wu, Pei-Yun Jenny; Coudreuse, Damien

    2016-08-01

    Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging. PMID:27512142

  2. Novel Carbon-based Electrode Materials for Up-scaled Microfluidic Fuel Cells

    NASA Astrophysics Data System (ADS)

    Fuerth, Dillon Adam

    In this work, a MFC fabrication procedure including two non-conventional techniques (partial baking and cap-sealing) were employed for the development of an up-scaled microfluidic fuel cell (MFC). Novel carbon-based electrode materials were employed, including carbon foam, fibre, and cloth, the results from which were compared with traditionally-employed carbon paper. The utilization of carbon cloth led to 15% of the maximum power that resulted from carbon paper; however, carbon fibre led to a 24.6% higher power density than carbon paper (normalized by electrode volume). When normalized by projected electrode area, the utilization of carbon foams resulted in power densities up to 42.5% higher than that from carbon paper. The impact of catalyst loading on MFC performance was also investigated, with an increase from 10.9 to 48.3 mgPt cm-2 resulting in a 195% increase in power density.

  3. Isolation of viable cancer cells in antibody-functionalized microfluidic devices

    PubMed Central

    Zheng, Xiangjun; Jiang, Linan; Schroeder, Joyce; Stopeck, Alison; Zohar, Yitshak

    2014-01-01

    Microfluidic devices functionalized with EpCAM antibodies were utilized for the capture of target cancer cells representing circulating tumor cells (CTCs). The fraction of cancer cells captured from homogeneous suspensions is mainly a function of flow shear rate, and can be described by an exponential function. A characteristic shear rate emerges as the most dominant parameter affecting the cell attachment ratio. Utilizing this characteristic shear rate as a scaling factor, all attachment ratio results for various combinations of receptor and ligand densities collapsed onto a single curve described by the empirical formula. The characteristic shear rate increases with both cell-receptor and surface-ligand densities, and empirical formulae featuring a product of two independent cumulative distributions described well these relationships. The minimum detection limit in isolation of target cancer cells from binary mixtures was experimentally explored utilizing microchannel arrays that allow high-throughput processing of suspensions about 0.5 ml in volume, which are clinically relevant, within a short time. Under a two-step attachment/detachment flow rate, both high sensitivity (almost 1.0) and high specificity (about 0.985) can be achieved in isolating target cancer cells from binary mixtures even for the lowest target/non-target cell concentration ratio of 1:100 000; this is a realistic ratio between CTCs and white blood cells in blood of cancer patients. Detection of CTCs from blood samples was also demonstrated using whole blood from healthy donors spiked with cancer cells. Finally, the viability of target cancer cells released after capture was confirmed by observing continuous cell growth in culture. PMID:24803968

  4. Dynamic monitoring of single cell lysis in an impedance-based microfluidic device.

    PubMed

    Zhou, Ying; Basu, Srinjan; Laue, Ernest D; Seshia, Ashwin A

    2016-08-01

    A microfluidic device that is capable of trapping and sensing dynamic variations in the electrical properties of individual cells is demonstrated. The device is applied to the real-time recording of impedance measurements of mouse embryonic stem cells (mESCs) during the process of membrane lysis, with the resulting changes in the electrical properties of cells during this process being quantitatively tracked over time. It is observed that the impedance magnitude decreases dramatically after cell membrane lysis. A significant shift in the phase spectrum is also observed during the time course of this process. By fitting experimental data to physical models, the electrical parameters of cells can be extracted and parameter variations quantified during the process. In the cell lysis experiments, the equivalent conductivity of the cell membrane is found to increase significantly due to pore formation in the membrane during lysis. An increase in the specific capacitance of the membrane is also observed. On the other hand, the conductivity of the cytoplasm is observed to decrease, which may be explained the fact that excess water enters the cell through the gradual permeabilization of the membrane during lysis. Cells can be trapped in the device for periods up to several days, and their electrical response can be monitored by real-time impedance measurements in a label-free and non-invasive manner. Furthermore, due to the highly efficient single cell trapping capacity of the device, a number of cells can be trapped and held in separate wells for concurrent parallel experiments, allowing for the possibility of stepped parametric experiments and studying cell heterogeneity by combining measurements across the array. PMID:27299468

  5. Characterization and Resolution of Evaporation-Mediated Osmolality Shifts that Constrain Microfluidic Cell Culture in Poly(dimethylsiloxane) Devices

    PubMed Central

    Heo, Yun Seok; Cabrera, Lourdes M.; Song, Jonathan W.; Futai, Nobuyuki; Tung, Yi-Chung; Smith, Gary D.; Takayama, Shuichi

    2008-01-01

    Evaporation is a critical problem when handling sub-microliter volumes of fluids. This paper characterizes this problem as it applies to microfluidic cell culture in poly(dimethylsiloxane) (PDMS) devices and provides a practical solution. Evaporation-mediated osmolality shifts through PDMS membranes with varying thicknesses (10, 1, 0.2 or 0.1 mm) were measured over 96 hours. Even in humidified cell culture incubators, evaporation through PDMS and associated shifts in the osmolality of culture media was significant and prevented mouse embryo and human endothelial cell growth and development. A simple diffusion model, where the measured diffusion coefficient for PDMS matches reported values of ~10−9 m2/s, accounts for these evaporation and osmolality shifts. To overcome this problem a PDMS-parylene-PDMS hybrid membrane was developed that greatly suppresses evaporation and osmolality shifts, yet possesses thinness and the flexibility necessary to interface with deformationbased microfluidic actuation systems, maintains the clarity for optical microscopy, and enables the successful development of single cell mouse embryos into blastocysts under static conditions and culture of human endothelial cells underdynamic recirculation of sub-microliter volumes of media. These insights and methods demonstrated specifically for embryo and endothelial cell studies will be generally useful for understanding and overcoming evaporation-associated effects in microfluidic cell cultures. PMID:17263345

  6. A method to integrate patterned electrospun fibers with microfluidic systems to generate complex microenvironments for cell culture applications.

    PubMed

    Wallin, Patric; Zandén, Carl; Carlberg, Björn; Hellström Erkenstam, Nina; Liu, Johan; Gold, Julie

    2012-06-01

    The properties of a cell's microenvironment are one of the main driving forces in cellular fate processes and phenotype expression invivo. The ability to create controlled cell microenvironments invitro becomes increasingly important for studying or controlling phenotype expression in tissue engineering and drug discovery applications. This includes the capability to modify material surface properties within well-defined liquid environments in cell culture systems. One successful approach to mimic extra cellular matrix is with porous electrospun polymer fiber scaffolds, while microfluidic networks have been shown to efficiently generate spatially and temporally defined liquid microenvironments. Here, a method to integrate electrospun fibers with microfluidic networks was developed in order to form complex cell microenvironments with the capability to vary relevant parameters. Spatially defined regions of electrospun fibers of both aligned and random orientation were patterned on glass substrates that were irreversibly bonded to microfluidic networks produced in poly-dimethyl-siloxane. Concentration gradients obtained in the fiber containing channels were characterized experimentally and compared with values obtained by computational fluid dynamic simulations. Velocity and shear stress profiles, as well as vortex formation, were calculated to evaluate the influence of fiber pads on fluidic properties. The suitability of the system to support cell attachment and growth was demonstrated with a fibroblast cell line. The potential of the platform was further verified by a functional investigation of neural stem cell alignment in response to orientation of electrospun fibers versus a microfluidic generated chemoattractant gradient of stromal cell-derived factor 1 alpha. The described method is a competitive strategy to create complex microenvironments invitro that allow detailed studies on the interplay of topography, substrate surface properties, and soluble

  7. Multivariate analysis of apoptotic markers versus cell cycle phase in living human cancer cells by microfluidic cytometry

    NASA Astrophysics Data System (ADS)

    Akagi, Jin; Skommer, Joanna; Matuszek, Anna; Takeda, Kazuo; Fujimura, Yuu; Khoshmanesh, Khashayar; Kalantar-Zadeh, Kourosh; Mitchell, Arnan; Errington, Rachel; Smith, Paul J.; Darzynkiewicz, Zbigniew; Wlodkowic, Donald

    2013-03-01

    Measurement of apoptotic markers in tumors can be directly correlated with the cell cycle phase using flow cytometry (FCM). The conventional DNA content analysis requires cell permeabilization to stain nuclei with fluorescent probes such as propidium iodide or use of a costly UV-excitation line for Hoechst 33342 probe. The access to FCM is also still limited to centralized core facilities due to its inherent high costs and complex operation. This work describes development and proof-of-concept validation of a portable and user-friendly microfluidic flow cytometer (μFCM) that can perform multivariate real time analysis on live cells using sampling volumes as small as 10 microliters. The μFCM system employs disposable microfluidic cartridges fabricated using injection molding in poly(methylmethacrylate) transparent thermoplastic. Furthermore, the dedicated and miniaturized electronic hardware interface enables up to six parameter detection using a combination of spatially separated solid-state 473 (10 mW) and 640 nm (20 mW) lasers and x-y stage for rapid laser alignment adjustment. We provide new evidence that a simple 2D flow focusing on a chip is sufficient to measure cellular DNA content in live tumor cells using a far-red DNA probe DRAQ5. The feasibility of using the μFCM system for a dose-response profiling of investigational anti-cancer agents on human hematopoietic cancer cells is also demonstrated. The data show that μFCM can provide a viable novel alternative to conventional FCM for multiparameter detection of caspase activation and dissipation of mitochondrial inner membrane potential (ΔΨm) in relation to DNA content (cell cycle phase) in live tumor cells.

  8. Multivariate analysis of apoptotic markers versus cell cycle phase in living human cancer cells by microfluidic cytometry

    PubMed Central

    Akagi, Jin; Skommer, Joanna; Matuszek, Anna; Takeda, Kazuo; Fujimura, Yuu; Khoshmanesh, Khashayar; Kalantar-Zadeh, Kourosh; Mitchell, Arnan; Errington, Rachel; Smith, Paul J; Darzynkiewicz, Zbigniew; Wlodkowic, Donald

    2013-01-01

    Measurement of apoptotic markers in tumors can be directly correlated with the cell cycle phase using flow cytometry (FCM). The conventional DNA content analysis requires cell permeabilization to stain nuclei with fluorescent probes such as propidium iodide or use of a costly UV-excitation line for Hoechst 33342 probe. The access to FCM is also still limited to centralized core facilities due to its inherent high costs and complex operation. This work describes development and proof-of-concept validation of a portable and user-friendly microfluidic flow cytometer (μFCM) that can perform multivariate real time analysis on live cells using sampling volumes as small as 10 microliters. The μFCM system employs disposable microfluidic cartridges fabricated using injection molding in poly(methylmethacrylate) transparent thermoplastic. Furthermore, the dedicated and miniaturized electronic hardware interface enables up to six parameter detection using a combination of spatially separated solid-state 473 (10 mW) and 640 nm (20 mW) lasers and x-y stage for rapid laser alignment adjustment. We provide new evidence that a simple 2D flow focusing on a chip is sufficient to measure cellular DNA content in live tumor cells using a far-red DNA probe DRAQ5. The feasibility of using the μFCM system for a dose-response profiling of investigational anti-cancer agents on human hematopoietic cancer cells is also demonstrated. The data show that μFCM can provide a viable novel alternative to conventional FCM for multiparameter detection of caspase activation and dissipation of mitochondrial inner membrane potential (ΔΨm) in relation to DNA content (cell cycle phase) in live tumor cells. PMID:24386542

  9. Microfluidic Study of Enhanced Deposition of Sickle Cells at Acute Corners

    PubMed Central

    Loiseau, Etienne; Massiera, Gladys; Mendez, Simon; Martinez, Patricia Aguilar; Abkarian, Manouk

    2015-01-01

    Sickle cell anemia is a blood disorder, known to affect the microcirculation and is characterized by painful vaso-occlusive crises in deep tissues. During the last three decades, many scenarios based on the enhanced adhesive properties of the membrane of sickle red blood cells have been proposed, all related to a final decrease in vessels lumen by cells accumulation on the vascular walls. Up to now, none of these scenarios considered the possible role played by the geometry of the flow on deposition. The question of the exact locations of occlusive events at the microcirculatory scale remains open. Here, using microfluidic devices where both geometry and oxygen levels can be controlled, we show that the flow of a suspension of sickle red blood cells around an acute corner of a triangular pillar or of a bifurcation, leads to the enhanced deposition and aggregation of cells. Thanks to our devices, we follow the growth of these aggregates in time and show that their length does not depend on oxygenation levels; instead, we find that their morphology changes dramatically to filamentous structures when using autologous plasma as a suspending fluid. We finally discuss the possible role played by such aggregates in vaso-occlusive events. PMID:26039164

  10. A microfluidic-based genetic screen to identify microbial virulence factors that inhibit dendritic cell migration

    PubMed Central

    McLaughlin, Laura M.; Xu, Hui; Carden, Sarah E.; Fisher, Samantha; Reyes, Monique; Heilshorn, Sarah C.; Monack, Denise M.

    2014-01-01

    Microbial pathogens are able to modulate host cells and evade the immune system by multiple mechanisms. For example, Salmonella injects effector proteins into host cells and evades the host immune system in part by inhibiting dendritic cell (DC) migration. The identification of microbial factors that modulate normal host functions should lead to the development of new classes of therapeutics that target these pathways. Current screening methods to identify either host or pathogen genes involved in modulating migration towards a chemical signal are limited because they do not employ stable, precisely controlled chemical gradients. Here, we develop a positive selection microfluidic-based genetic screen that allows us to identify Salmonella virulence factors that manipulate DC migration within stable, linear chemokine gradients. Our screen identified 7 Salmonella effectors (SseF, SifA, SspH2, SlrP, PipB2, SpiC and SseI) that inhibit DC chemotaxis toward CCL19. This method is widely applicable for identifying novel microbial factors that influence normal host cell chemotaxis as well as revealing new mammalian genes involved in directed cell migration. PMID:24599496

  11. A microfluidic-based genetic screen to identify microbial virulence factors that inhibit dendritic cell migration.

    PubMed

    McLaughlin, Laura M; Xu, Hui; Carden, Sarah E; Fisher, Samantha; Reyes, Monique; Heilshorn, Sarah C; Monack, Denise M

    2014-04-01

    Microbial pathogens are able to modulate host cells and evade the immune system by multiple mechanisms. For example, Salmonella injects effector proteins into host cells and evades the host immune system in part by inhibiting dendritic cell (DC) migration. The identification of microbial factors that modulate normal host functions should lead to the development of new classes of therapeutics that target these pathways. Current screening methods to identify either host or pathogen genes involved in modulating migration towards a chemical signal are limited because they do not employ stable, precisely controlled chemical gradients. Here, we develop a positive selection microfluidic-based genetic screen that allows us to identify Salmonella virulence factors that manipulate DC migration within stable, linear chemokine gradients. Our screen identified 7 Salmonella effectors (SseF, SifA, SspH2, SlrP, PipB2, SpiC and SseI) that inhibit DC chemotaxis toward CCL19. This method is widely applicable for identifying novel microbial factors that influence normal host cell chemotaxis as well as revealing new mammalian genes involved in directed cell migration. PMID:24599496

  12. Microfluidic Impedimetric Cell Regeneration Assay to Monitor the Enhanced Cytotoxic Effect of Nanomaterial Perfusion

    PubMed Central

    Rothbauer, Mario; Praisler, Irene; Docter, Dominic; Stauber, Roland H.; Ertl, Peter

    2015-01-01

    In the last decade, the application of nanomaterials (NMs) in technical products and biomedicine has become a rapidly increasing market trend. As the safety and efficacy of NMs are of utmost importance, new methods are needed to study the dynamic interactions of NMs at the nano-biointerface. However, evaluation of NMs based on standard and static cell culture end-point detection methods does not provide information on the dynamics of living biological systems, which is crucial for the understanding of physiological responses. To bridge this technological gap, we here present a microfluidic cell culture system containing embedded impedance microsensors to continuously and non-invasively monitor the effects of NMs on adherent cells under varying flow conditions. As a model, the impact of silica NMs on the vitality and regenerative capacity of human lung cells after acute and chronic exposure scenarios was studied over an 18-h period following a four-hour NM treatment. Results of the study demonstrated that the developed system is applicable to reliably analyze the consequences of dynamic NM exposure to physiological cell barriers in both nanotoxicology and nanomedicine. PMID:26633532

  13. Microfluidic Impedimetric Cell Regeneration Assay to Monitor the Enhanced Cytotoxic Effect of Nanomaterial Perfusion.

    PubMed

    Rothbauer, Mario; Praisler, Irene; Docter, Dominic; Stauber, Roland H; Ertl, Peter

    2015-12-01

    In the last decade, the application of nanomaterials (NMs) in technical products and biomedicine has become a rapidly increasing market trend. As the safety and efficacy of NMs are of utmost importance, new methods are needed to study the dynamic interactions of NMs at the nano-biointerface. However, evaluation of NMs based on standard and static cell culture end-point detection methods does not provide information on the dynamics of living biological systems, which is crucial for the understanding of physiological responses. To bridge this technological gap, we here present a microfluidic cell culture system containing embedded impedance microsensors to continuously and non-invasively monitor the effects of NMs on adherent cells under varying flow conditions. As a model, the impact of silica NMs on the vitality and regenerative capacity of human lung cells after acute and chronic exposure scenarios was studied over an 18-h period following a four-hour NM treatment. Results of the study demonstrated that the developed system is applicable to reliably analyze the consequences of dynamic NM exposure to physiological cell barriers in both nanotoxicology and nanomedicine. PMID:26633532

  14. Three-Dimensional Holographic Refractive-Index Measurement of Continuously Flowing Cells in a Microfluidic Channel

    PubMed Central

    Sung, Yongjin; Lue, Niyom; Hamza, Bashar; Martel, Joseph; Irimia, Daniel; Dasari, Ramachandra R.; Choi, Wonshik; Yaqoob, Zahid; So, Peter

    2014-01-01

    Refractive index of biological specimens is a source of intrinsic contrast that can be explored without any concerns of photobleaching or harmful effects caused by extra contrast agents. In addition, RI contains rich information related to the metabolism of cells at the cellular and subcellular levels. Here, we report a no-moving parts approach that provides three-dimensional refractive index maps of biological samples continuously flowing in a microfluidic channel. Specifically, we use line illumination and off-axis digital holography to record the angular spectra of light scattered from flowing samples at high speed. Applying the scalar diffraction theory, we obtain accurate RI maps of the samples from the measured spectra. Using this method, we demonstrate label-free 3-D imaging of live RKO human colon cancer cells and RPMI8226 multiple myeloma cells, and obtain the volume, dry mass and density of these cells from the measured 3-D refractive index maps. Our results show that the reported method, alone or in combination with the existing flow cytometry techniques, promises as a quantitative tool for stain-free characterization of large number of cells. PMID:25419536

  15. Three-Dimensional Holographic Refractive-Index Measurement of Continuously Flowing Cells in a Microfluidic Channel

    NASA Astrophysics Data System (ADS)

    Sung, Yongjin; Lue, Niyom; Hamza, Bashar; Martel, Joseph; Irimia, Daniel; Dasari, Ramachandra R.; Choi, Wonshik; Yaqoob, Zahid; So, Peter

    2014-02-01

    The refractive index of biological specimens is a source of intrinsic contrast that can be explored without any concerns of photobleaching or harmful effects caused by extra contrast agents. In addition, the refractive index contains rich information related to the metabolism of cells at the cellular and subcellular levels. Here, we report a no-moving-parts approach that provides three-dimensional refractive-index maps of biological samples continuously flowing in a microfluidic channel. Specifically, we use line illumination and off-axis digital holography to record the angular spectra of light scattered from flowing samples at high speed. Applying the scalar diffraction theory, we obtain accurate refractive-index maps of the samples from the measured spectra. Using this method, we demonstrate label-free three-dimensional imaging of live RKO human colon cancer cells and RPMI8226 multiple myeloma cells, and obtain the volume, dry mass, and density of these cells from the measured three-dimensional refractive-index maps. Our results show that the reported method, alone or in combination with the existing flow cytometry techniques, shows promise as a quantitative tool for stain-free characterization of a large number of cells.

  16. A passive-flow microfluidic device for imaging latent HIV activation dynamics in single T cells

    PubMed Central

    Gearhart, Larisa M.; Miller-Jensen, Kathryn

    2015-01-01

    Quantifying cell-to-cell variability in drug response dynamics is important when evaluating therapeutic efficacy. For example, optimizing latency reversing agents (LRAs) for use in a clinical “activate-and-kill” strategy to purge the latent HIV reservoir in patients requires minimizing heterogeneous viral activation dynamics. To evaluate how heterogeneity in latent HIV activation varies across a range of LRAs, we tracked drug-induced response dynamics in single cells via live-cell imaging using a latent HIV–GFP reporter virus in a clonal Jurkat T cell line. To enable these studies in suspension cells, we designed a simple method to capture an array of single Jurkat T cells using a passive-flow microfluidic device. Our device, which does not require external pumps or tubing, can trap hundreds of cells within minutes with a high retention rate over 12 hours of imaging. Using this device, we quantified heterogeneity in viral activation stimulated by transcription factor (TF) activators and histone deacetylase (HDAC) inhibitors. Generally, TF activators resulted in both faster onset of viral activation and faster rates of production, while HDAC inhibitors resulted in more uniform onset times, but more heterogeneous rates of production. Finally, we demonstrated that while onset time of viral gene expression and rate of viral production together predict total HIV activation, rate and onset time were not correlated within the same individual cell, suggesting that these features are regulated independently. Overall, our results reveal drug-specific patterns of noisy HIV activation dynamics not previously identified in static single-cell assays, which may require consideration for the most effective activate-and-kill regime. PMID:26138068

  17. Microfluidic shear stress-regulated surfactant secretion in alveolar epithelial type II cells in vitro.

    PubMed

    Mahto, Sanjeev Kumar; Tenenbaum-Katan, Janna; Greenblum, Ayala; Rothen-Rutishauser, Barbara; Sznitman, Josué

    2014-04-01

    We investigated the role of flow-induced shear stress on the mechanisms regulating surfactant secretion in type II alveolar epithelial cells (ATII) using microfluidic models. Following flow stimulation spanning a range of wall shear stress (WSS) magnitudes, monolayers of ATII (MLE-12 and A549) cells were examined for surfactant secretion by evaluating essential steps of the process, including relative changes in the number of fusion events of lamellar bodies (LBs) with the plasma membrane (PM) and intracellular redistribution of LBs. F-actin cytoskeleton and calcium levels were analyzed in A549 cells subjected to WSS spanning 4-20 dyn/cm(2). Results reveal an enhancement in LB fusion events with the PM in MLE-12 cells upon flow stimulation, whereas A549 cells exhibit no foreseeable changes in the monitored number of fusion events for WSS levels ranging up to a threshold of ∼8 dyn/cm(2); above this threshold, we witness instead a decrease in LB fusion events in A549 cells. However, patterns of LB redistribution suggest that WSS can potentially serve as a stimulus for A549 cells to trigger the intracellular transport of LBs toward the cell periphery. This observation is accompanied by a fragmentation of F-actin, indicating that disorganization of the F-actin cytoskeleton might act as a limiting factor for LB fusion events. Moreover, we note a rise in cytosolic calcium ([Ca(2+)]c) levels following stimulation of A549 cells with WSS magnitudes ranging near or above the experimental threshold. Overall, WSS stimulation can influence key components of molecular machinery for regulated surfactant secretion in ATII cells in vitro. PMID:24487389

  18. High purity microfluidic sorting and analysis of circulating tumor cells: towards routine mutation detection.

    PubMed

    Autebert, Julien; Coudert, Benoit; Champ, Jérôme; Saias, Laure; Guneri, Ezgi Tulukcuoglu; Lebofsky, Ronald; Bidard, François-Clément; Pierga, Jean-Yves; Farace, Françoise; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis

    2015-05-01

    A new generation of the Ephesia cell capture technology optimized for CTC capture and genetic analysis is presented, characterized in depth and compared with the CellSearch system as a reference. This technology uses magnetic particles bearing tumour-cell specific EpCAM antibodies, self-assembled in a regular array in a microfluidic flow cell. 48,000 high aspect-ratio columns are generated using a magnetic field in a high throughput (>3 ml h(-1)) device and act as sieves to specifically capture the cells of interest through antibody-antigen interactions. Using this device optimized for CTC capture and analysis, we demonstrated the capture of epithelial cells with capture efficiency above 90% for concentrations as low as a few cells per ml. We showed the high specificity of capture with only 0.26% of non-epithelial cells captured for concentrations above 10 million cells per ml. We investigated the capture behavior of cells in the device, and correlated the cell attachment rate with the EpCAM expression on the cell membranes for six different cell lines. We developed and characterized a two-step blood processing method to allow for rapid processing of 10 ml blood tubes in less than 4 hours, and showed a capture rate of 70% for as low as 25 cells spiked in 10 ml blood tubes, with less than 100 contaminating hematopoietic cells. Using this device and procedure, we validated our system on patient samples using an automated cell immunostaining procedure and a semi-automated cell counting method. Our device captured CTCs in 75% of metastatic prostate cancer patients and 80% of metastatic breast cancer patients, and showed similar or better results than the CellSearch device in 10 out of 13 samples. Finally, we demonstrated the possibility of detecting cancer-related PIK3CA gene mutation in 20 cells captured in the chip with a good correlation between the cell count and the quantitation value Cq of the post-capture qPCR. PMID:25815443

  19. A method to integrate patterned electrospun fibers with microfluidic systems to generate complex microenvironments for cell culture applications

    PubMed Central

    Wallin, Patric; Zandén, Carl; Carlberg, Björn; Hellström Erkenstam, Nina; Liu, Johan; Gold, Julie

    2012-01-01

    The properties of a cell’s microenvironment are one of the main driving forces in cellular fate processes and phenotype expression invivo. The ability to create controlled cell microenvironments invitro becomes increasingly important for studying or controlling phenotype expression in tissue engineering and drug discovery applications. This includes the capability to modify material surface properties within well-defined liquid environments in cell culture systems. One successful approach to mimic extra cellular matrix is with porous electrospun polymer fiber scaffolds, while microfluidic networks have been shown to efficiently generate spatially and temporally defined liquid microenvironments. Here, a method to integrate electrospun fibers with microfluidic networks was developed in order to form complex cell microenvironments with the capability to vary relevant parameters. Spatially defined regions of electrospun fibers of both aligned and random orientation were patterned on glass substrates that were irreversibly bonded to microfluidic networks produced in poly-dimethyl-siloxane. Concentration gradients obtained in the fiber containing channels were characterized experimentally and compared with values obtained by computational fluid dynamic simulations. Velocity and shear stress profiles, as well as vortex formation, were calculated to evaluate the influence of fiber pads on fluidic properties. The suitability of the system to support cell attachment and growth was demonstrated with a fibroblast cell line. The potential of the platform was further verified by a functional investigation of neural stem cell alignment in response to orientation of electrospun fibers versus a microfluidic generated chemoattractant gradient of stromal cell-derived factor 1 alpha. The described method is a competitive strategy to create complex microenvironments invitro that allow detailed studies on the interplay of topography, substrate surface properties, and soluble

  20. Quantitative multiplexed simulated-cell identification by SERS in microfluidic devices

    NASA Astrophysics Data System (ADS)

    Hoonejani, M. R.; Pallaoro, A.; Braun, G. B.; Moskovits, M.; Meinhart, C. D.

    2015-10-01

    A reliable identification of cells on the basis of their surface markers is of great interest for diagnostic and therapeutic applications. We present a multiplexed labeling and detection strategy that is applied to four microparticle populations, each mimicking cellular or bacterial samples with varying surface concentrations of up to four epitopes, using four distinct biotags that are meant to be used in conjunction with surface enhanced Raman spectroscopy (SERS) instead of fluorescence, together with microfluidics. Four populations of 6 μm polystyrene beads were incubated with different mixtures, ``cocktails'' of four SERS biotags (SBTs), simulating the approach that one would follow when seeking to identify multiple biomarkers encountered in biological applications. Populations were flowed in a microfluidic flow-focusing device and the SERS signal from individual beads was acquired during continuous flow. The spectrally rich SERS spectra enabled us to separate confidently the populations by utilizing principal component analysis (PCA). Also, using classical least squares (CLS), we were able to calculate the contributions of each SBT to the overall signal in each of the populations, and showed that the relative SBT contributions are consistent with the nominal percentage of each marker originally designed into that bead population, by functionalizing it with a given SBT cocktail. Our results demonstrate the multiplexing capability of SBTs in potential applications such as immunophenotyping.A reliable identification of cells on the basis of their surface markers is of great interest for diagnostic and therapeutic applications. We present a multiplexed labeling and detection strategy that is applied to four microparticle populations, each mimicking cellular or bacterial samples with varying surface concentrations of up to four epitopes, using four distinct biotags that are meant to be used in conjunction with surface enhanced Raman spectroscopy (SERS) instead of

  1. An on-chip microfluidic pressure regulator that facilitates reproducible loading of cells and hydrogels into microphysiological system platforms.

    PubMed

    Wang, Xiaolin; Phan, Duc T T; Zhao, Da; George, Steven C; Hughes, Christopher C W; Lee, Abraham P

    2016-03-01

    Coculturing multiple cell types together in 3-dimensional (3D) cultures better mimics the in vivo microphysiological environment, and has become widely adopted in recent years with the development of organ-on-chip systems. However, a bottleneck in set-up of these devices arises as a result of the delivery of the gel into the microfluidic chip being sensitive to pressure fluctuations, making gel confinement at a specific region challenging, especially when manual operation is performed. In this paper, we present a novel design of an on-chip regulator module with pressure-releasing safety microvalves that can facilitate stable gel delivery into designated microchannel regions while maintaining well-controlled, non-bursting gel interfaces. This pressure regulator design can be integrated into different microfluidic chip designs and is compatible with a wide variety of gel injection apparatuses operated automatically or manually at different flow rates. The sensitivity and working range of this pressure regulator can be adjusted by changing the width of its pressure releasing safety microvalve design. The effectiveness of the design is validated by its incorporation into a microfluidic platform we have developed for generating 3D vascularized micro-organs (VMOs). Reproducible gel loading is demonstrated for both an automatic syringe pump and a manually-operated micropipettor. This design allows for rapid and reproducible loading of hydrogels into microfluidic devices without the risk of bursting gel-air interfaces. PMID:26879519

  2. Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology

    PubMed Central

    Che, James; Yu, Victor; Dhar, Manjima; Renier, Corinne; Matsumoto, Melissa; Heirich, Kyra; Garon, Edward B.; Goldman, Jonathan; Rao, Jianyu; Sledge, George W.; Pegram, Mark D.; Sheth, Shruti; Jeffrey, Stefanie S.; Kulkarni, Rajan P.; Sollier, Elodie; Di Carlo, Dino

    2016-01-01

    Circulating tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 μL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells. PMID:26863573

  3. Spatially Selective Reagent Delivery into Cancer Cells Using a Two-Layer Microfluidic Culture System

    PubMed Central

    Liu, Yan; Butler, W. Boyd; Pappas, Dimitri

    2012-01-01

    In this work, we demonstrate a two-layer microfluidic system capable of spatially selective delivery of drugs and other reagents under low shear stress. Loading occurs by hydrodynamically focusing a reagent stream over a particular region of the cell culture. The system consisted of a cell culture chamber and fluid flow channel, which were located in different layers to reduce shear stress on cells. Cells in the center of the culture chamber were exposed to parallel streams of laminar flow, which allowed fast changes to be made to the cellular environment. The shear force was reduced to 2.7 dyn/cm2 in the two-layer device (vs. 6.0 dyn/cm2 in a one-layer device). Cells in the side of the culture chamber were exposed to the side streams of buffer; the shear force was further reduced to a greater extent since the sides of the culture chamber were separated from the main fluid path. The channel shape and flow rate of the multiple streams were optimized for spatially-controlled reagent delivery. The boundaries between streams were well controlled at a flow rate of 0.1 mL/h, which was optimized for all streams. We demonstrated multi-reagent delivery to different regions of the same culture well, as well as selective treatment of cancer cells with a built in control group in the same well. In the case of apoptosis induction using staurosporine, 10% of cells remained viable after 24 hours of exposure. Cells in the same chamber, but not exposed to staurosporine, had a viability of 90%. This chip allows dynamic observation of cellular behavior immediately after drug delivery, as well as long-term drug treatment with the benefit of large cell numbers, device simplicity, and low shear stress. PMID:22882832

  4. Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology.

    PubMed

    Che, James; Yu, Victor; Dhar, Manjima; Renier, Corinne; Matsumoto, Melissa; Heirich, Kyra; Garon, Edward B; Goldman, Jonathan; Rao, Jianyu; Sledge, George W; Pegram, Mark D; Sheth, Shruti; Jeffrey, Stefanie S; Kulkarni, Rajan P; Sollier, Elodie; Di Carlo, Dino

    2016-03-15

    Circulating tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 μL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells. PMID:26863573

  5. A microfluidic device for the hydrodynamic immobilisation of living fission yeast cells for super-resolution imaging☆

    PubMed Central

    Bell, Laurence; Seshia, Ashwin; Lando, David; Laue, Ernest; Palayret, Matthieu; Lee, Steven F.; Klenerman, David

    2014-01-01

    We describe a microfluidic device designed specifically for the reversible immobilisation of Schizosaccharomyces pombe (Fission Yeast) cells to facilitate live cell super-resolution microscopy. Photo-Activation Localisation Microscopy (PALM) is used to create detailed super-resolution images within living cells with a modal accuracy of >25 nm in the lateral dimensions. The novel flow design captures and holds cells in a well-defined array with minimal effect on the normal growth kinetics. Cells are held over several hours and can continue to grow and divide within the device during fluorescence imaging. PMID:25844024

  6. Automated Chemotactic Sorting and Single-cell Cultivation of Microbes using Droplet Microfluidics

    PubMed Central

    Dong, Libing; Chen, Dong-Wei; Liu, Shuang-Jiang; Du, Wenbin

    2016-01-01

    We report a microfluidic device for automated sorting and cultivation of chemotactic microbes from pure cultures or mixtures. The device consists of two parts: in the first part, a concentration gradient of the chemoeffector was built across the channel for inducing chemotaxis of motile cells; in the second part, chemotactic cells from the sample were separated, and mixed with culture media to form nanoliter droplets for encapsulation, cultivation, enumeration, and recovery of single cells. Chemotactic responses were assessed by imaging and statistical analysis of droplets based on Poisson distribution. An automated procedure was developed for rapid enumeration of droplets with cell growth, following with scale-up cultivation on agar plates. The performance of the device was evaluated by the chemotaxis assays of Escherichia coli (E. coli) RP437 and E. coli RP1616. Moreover, enrichment and isolation of non-labelled Comamonas testosteroni CNB-1 from its 1:10 mixture with E. coli RP437 was demonstrated. The enrichment factor reached 36.7 for CNB-1, based on its distinctive chemotaxis toward 4-hydroxybenzoic acid. We believe that this device can be widely used in chemotaxis studies without necessarily relying on fluorescent labelling, and isolation of functional microbial species from various environments. PMID:27074762

  7. Air-breathing direct formic acid microfluidic fuel cell with an array of cylinder anodes

    NASA Astrophysics Data System (ADS)

    Zhu, Xun; Zhang, Biao; Ye, Ding-Ding; Li, Jun; Liao, Qiang

    2014-02-01

    An air-breathing direct formic acid membraneless microfluidic fuel cell using graphite cylinder arrays as the anode is proposed. The three dimensional anode volumetrically extends the reactive surface area and improves fuel utilization. The effects of spacer configuration, fuel and electrolyte concentration as well as reactant flow rate on the species transport and cell performance are investigated. The dynamic behavior of generated CO2 bubbles is visualized and its effect on current generation is discussed. The results show that the absence of two spacers adjacent to the cathode surface improves the cell performance by reducing the proton transfer resistance. The CO2 gas bubbles are constrained within the anode array and expelled by the fluid flow periodically. Proper reactant concentration and flow rate are crucial for cell operation. At optimum conditions, a maximum current density of 118.3 mA cm-3 and a peak power density of 21.5 mW cm-3 are obtained. In addition, benefit from the volumetrically stacked anodes and enhanced fuel transfer, the maximum single pass fuel utilization rate reaches up to 87.6% at the flow rate of 1 mL h-1.

  8. Raman tweezers in microfluidic systems for analysis and sorting of living cells

    NASA Astrophysics Data System (ADS)

    Pilát, Zdenëk; Ježek, Jan; Kaňka, Jan; Zemánek, Pavel

    2014-03-01

    We have devised an analytical and sorting system combining optical trapping with Raman spectroscopy in microfluidic environment in order to identify and sort biological objects, such as living cells of various prokaryotic and eukaryotic organisms. Our main objective was to create a robust and universal platform for non-contact sorting of microobjects based on their Raman spectral properties. This approach allowed us to collect information about the chemical composition of the objects, such as the presence and composition of lipids, proteins, or nucleic acids without using artificial chemical probes such as fluorescent markers. The non-destructive and non-contact nature of this optical analysis and manipulation allowed us to separate individual living cells of our interest in a sterile environment and provided the possibility to cultivate the selected cells for further experiments. We used differently treated cells of algae to test and demonstrate the function of our analytical and sorting system. The devised system could find its use in many medical, biotechnological, and biological applications.

  9. 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

  10. Raman tweezers in microfluidic systems for analysis and sorting of living cells

    NASA Astrophysics Data System (ADS)

    Pilát, Zdeněk.; Ježek, Jan; Kaňka, Jan; Zemánek, Pavel

    2014-12-01

    We have devised an analytical and sorting system combining optical trapping with Raman spectroscopy in microfluidic environment, dedicated to identification and sorting of biological objects, such as living cells of various unicellular organisms. Our main goal was to create a robust and universal platform for non-destructive and non-contact sorting of micro-objects based on their Raman spectral properties. This approach allowed us to collect spectra containing information about the chemical composition of the objects, such as the presence and composition of pigments, lipids, proteins, or nucleic acids, avoiding artificial chemical probes such as fluorescent markers. The non-destructive nature of this optical analysis and manipulation allowed us to separate individual living cells of our interest in a sterile environment and provided the possibility to cultivate the selected cells for further experiments. We used a mixture of polystyrene micro-particles and algal cells to test and demonstrate the function of our analytical and sorting system. The devised system could find its use in many medical, biotechnological, and biological applications.

  11. Automated Chemotactic Sorting and Single-cell Cultivation of Microbes using Droplet Microfluidics

    NASA Astrophysics Data System (ADS)

    Dong, Libing; Chen, Dong-Wei; Liu, Shuang-Jiang; Du, Wenbin

    2016-04-01

    We report a microfluidic device for automated sorting and cultivation of chemotactic microbes from pure cultures or mixtures. The device consists of two parts: in the first part, a concentration gradient of the chemoeffector was built across the channel for inducing chemotaxis of motile cells; in the second part, chemotactic cells from the sample were separated, and mixed with culture media to form nanoliter droplets for encapsulation, cultivation, enumeration, and recovery of single cells. Chemotactic responses were assessed by imaging and statistical analysis of droplets based on Poisson distribution. An automated procedure was developed for rapid enumeration of droplets with cell growth, following with scale-up cultivation on agar plates. The performance of the device was evaluated by the chemotaxis assays of Escherichia coli (E. coli) RP437 and E. coli RP1616. Moreover, enrichment and isolation of non-labelled Comamonas testosteroni CNB-1 from its 1:10 mixture with E. coli RP437 was demonstrated. The enrichment factor reached 36.7 for CNB-1, based on its distinctive chemotaxis toward 4-hydroxybenzoic acid. We believe that this device can be widely used in chemotaxis studies without necessarily relying on fluorescent labelling, and isolation of functional microbial species from various environments.

  12. Spatial Engineering of Osteochondral Tissue Constructs Through Microfluidically Directed Differentiation of Mesenchymal Stem Cells.

    PubMed

    Goldman, Stephen M; Barabino, Gilda A

    2016-01-01

    The development of tissue engineered osteochondral units has been slowed by a number of technical hurdles associated with recapitulating their heterogeneous nature ex vivo. Subsequently, numerous approaches with respect to cell sourcing, scaffolding composition, and culture media formulation have been pursued, which have led to high variability in outcomes and ultimately the lack of a consensus bioprocessing strategy. As such, the objective of this study was to standardize the design process by focusing on differentially supporting formation of cartilaginous and bony matrix by a single cell source in a spatially controlled manner within a single material system. A cell-polymer solution of bovine mesenchymal stem cells and agarose was cast against micromolds of a serpentine network and stacked to produce tissue constructs containing two independent microfluidic networks. Constructs were fluidically connected to two controlled flow loops and supplied with independently tuned differentiation parameters for chondrogenic and osteogenic induction, respectively. Constructs receiving inductive media showed differential gene expression of both chondrogenic and osteogenic markers in opposite directions along the thickness of the construct that was recapitulated at the protein level with respect to collagens I, II, and X. A control group receiving noninductive media showed homogeneous expression of these biomarkers measured in lower concentrations at both the mRNA and protein level. This work represents an important step in the rational design of engineered osteochondral units through establishment of an enabling technology for further optimization of scaffolding formulations and bioprocessing conditions toward the production of commercially viable osteochondral tissue products. PMID:27190700

  13. Spatial Engineering of Osteochondral Tissue Constructs Through Microfluidically Directed Differentiation of Mesenchymal Stem Cells

    PubMed Central

    Goldman, Stephen M.; Barabino, Gilda A.

    2016-01-01

    Abstract The development of tissue engineered osteochondral units has been slowed by a number of technical hurdles associated with recapitulating their heterogeneous nature ex vivo. Subsequently, numerous approaches with respect to cell sourcing, scaffolding composition, and culture media formulation have been pursued, which have led to high variability in outcomes and ultimately the lack of a consensus bioprocessing strategy. As such, the objective of this study was to standardize the design process by focusing on differentially supporting formation of cartilaginous and bony matrix by a single cell source in a spatially controlled manner within a single material system. A cell-polymer solution of bovine mesenchymal stem cells and agarose was cast against micromolds of a serpentine network and stacked to produce tissue constructs containing two independent microfluidic networks. Constructs were fluidically connected to two controlled flow loops and supplied with independently tuned differentiation parameters for chondrogenic and osteogenic induction, respectively. Constructs receiving inductive media showed differential gene expression of both chondrogenic and osteogenic markers in opposite directions along the thickness of the construct that was recapitulated at the protein level with respect to collagens I, II, and X. A control group receiving noninductive media showed homogeneous expression of these biomarkers measured in lower concentrations at both the mRNA and protein level. This work represents an important step in the rational design of engineered osteochondral units through establishment of an enabling technology for further optimization of scaffolding formulations and bioprocessing conditions toward the production of commercially viable osteochondral tissue products. PMID:27190700

  14. Quantification of the specific membrane capacitance of single cells using a microfluidic device and impedance spectroscopy measurement.

    PubMed

    Tan, Qingyuan; Ferrier, Graham A; Chen, Brandon K; Wang, Chen; Sun, Yu

    2012-09-01

    The specific membrane capacitance (SMC) is an electrical parameter that correlates with both the electrical activity and morphology of the plasma membrane, which are physiological markers for cellular phenotype and health. We have developed a microfluidic device that enables impedance spectroscopy measurements of the SMC of single biological cells. Impedance spectra induced by single cells aspirated into the device are captured over a moderate frequency range (5 kHz-1 MHz). Maximum impedance sensitivity is achieved using a tapered microfluidic channel, which effectively routes electric fields across the cell membranes. The SMC is extracted by curve-fitting impedance spectra to an equivalent circuit model. From our measurement, acute myeloid leukemia (AML) cells are found to exhibit larger SMC values in hypertonic solutions as compared with those in isotonic solutions. In addition, AML cell phenotypes (AML2 and NB4) exhibiting varying metastatic potential yield distinct SMC values (AML2: 16.9 ± 1.9 mF/m(2) (n = 23); NB4: 22.5 ± 4.7 mF/m(2) (n = 23)). Three-dimensional finite element simulations of the microfluidic device confirm the feasibility of this approach. PMID:23940502

  15. Interstitial flows promote an amoeboid over mesenchymal motility of breast cancer cells revealed by a three dimensional microfluidic model

    PubMed Central

    Huang, Yu Ling; Tung, Chih-kuan; Zheng, Anqi; Kim, Beum Jun; Wu, Mingming

    2015-01-01

    Malignant tumors are often associated with an elevated fluid pressure due to the abnormal growth of vascular vessels, and thus an increased interstitial flow out of the tumor. Recent in vitro work revealed that interstitial flows critically regulated tumor cell migration within a three dimensional biomatrix, and breast cancer cell migration behavior depended sensitively on the cell seeding density, chemokine availability and flow rates. In this paper, we focus on roles of interstitial flows in modulating heterogeneity of cancer cell motility phenotype within a three dimensional biomatrix. Using a newly developed microfluidic model, we show that breast cancer cells (MDA-MB-231) embedded in a 3D type I collagen matrix exhibit both an amoeboid and a mesenchymal motility, and interstitial flows promote the cell population towards the amoeboid motility phenotype. Furthermore, the addition of exogenous adhesion molecules (fibronectin) within the extracellular matrix (type I collagen) partially rescues the mesenchymal phenotype in the presence of the flow. Quantitative analysis of cell tracks and cell shape shows distinct differential migration characteristics of amoeboid and mesenchymal cells. Notably, the fastest moving cells belong to the subpopulation of amoeboid cells. Together, these findings highlight the important roles of biophysical forces in modulating tumor cell migration heterogeneity and plasticity, as well as the suitability of microfluidic models in interrogating tumor cell dynamics at single-cell and subpopulation level. PMID:26235230

  16. Interstitial flows promote amoeboid over mesenchymal motility of breast cancer cells revealed by a three dimensional microfluidic model.

    PubMed

    Huang, Yu Ling; Tung, Chih-Kuan; Zheng, Anqi; Kim, Beum Jun; Wu, Mingming

    2015-11-01

    Malignant tumors are often associated with an elevated fluid pressure due to the abnormal growth of vascular vessels, and thus an increased interstitial flow out of the tumors. Recent in vitro works revealed that interstitial flows critically regulated tumor cell migration within a three dimensional biomatrix, and breast cancer cell migration behavior depended sensitively on the cell seeding density, chemokine availability and flow rates. In this paper, we focus on the role of interstitial flows in modulating the heterogeneity of cancer cell motility phenotype within a three dimensional biomatrix. Using a newly developed microfluidic model, we show that breast cancer cells (MDA-MB-231) embedded in a 3D type I collagen matrix exhibit both amoeboid and mesenchymal motility, and interstitial flows promote the cell population towards the amoeboid motility phenotype. Furthermore, the addition of exogenous adhesion molecules (fibronectin) within the extracellular matrix (type I collagen) partially rescues the mesenchymal phenotype in the presence of the flow. Quantitative analysis of cell tracks and cell shapes shows distinct differential migration characteristics of amoeboid and mesenchymal cells. Notably, the fastest moving cells belong to the subpopulation of amoeboid cells. Together, these findings highlight the important role of biophysical forces in modulating tumor cell migration heterogeneity and plasticity, as well as the suitability of microfluidic models in interrogating tumor cell dynamics at single-cell and subpopulation level. PMID:26235230

  17. Real-time direct cell concentration and viability determination using a fully automated microfluidic platform for standalone process monitoring.

    PubMed

    Nunes, P S; Kjaerulff, S; Dufva, M; Mogensen, K B

    2015-06-21

    The industrial production of cells has a large unmet need for greater process monitoring, in addition to the standard temperature, pH and oxygen concentration determination. Monitoring the cell health by a vast range of fluorescence cell-based assays can greatly improve the feedback control and thereby ensure optimal cell production, by prolonging the fermentation cycle and increasing the bioreactor output. In this work, we report on the development of a fully automated microfluidic system capable of extracting samples directly from a bioreactor, diluting the sample, staining the cells, and determining the total cell and dead cells concentrations, within a time frame of 10.3 min. The platform consists of custom made stepper motor actuated peristaltic pumps and valves, fluidic interconnections, sample to waste liquid management and image cytometry-based detection. The total concentration of cells is determined by brightfield microscopy, while fluorescence detection is used to detect propidium iodide stained non-viable cells. This method can be incorporated into facilities with bioreactors to monitor the cell concentration and viability during the cultivation process. Here, we demonstrate the microfluidic system performance by monitoring in real time the cell concentration and viability of yeast extracted directly from an in-house made bioreactor. This is the first demonstration of using the Dean drag force, generated due to the implementation of a curved microchannel geometry in conjunction with high flow rates, to promote passive mixing of cell samples and thus homogenization of the diluted cell plug. The autonomous operation of the fluidics furthermore allows implementation of intelligent protocols for administering air bubbles from the bioreactor in the microfluidic system, so that these will be guided away from the imaging region, thereby significantly improving both the robustness of the system and the quality of the data. PMID:25923294

  18. A radial flow microfluidic device for ultra-high-throughput affinity-based isolation of circulating tumor cells.

    PubMed

    Murlidhar, Vasudha; Zeinali, Mina; Grabauskiene, Svetlana; Ghannad-Rezaie, Mostafa; Wicha, Max S; Simeone, Diane M; Ramnath, Nithya; Reddy, Rishindra M; Nagrath, Sunitha

    2014-12-10

    Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer-related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time-consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs radial flow that introduces a varying shear profile across the device, enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h(-1) in contrast to a flow rate of 1 mL h(-1) standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs. PMID:25074448

  19. Detection of Infertility-related Neutralizing Antibodies with a Cell-free Microfluidic Method

    NASA Astrophysics Data System (ADS)

    Eyer, Klaus; Root, Katharina; Verboket, Pascal E.; Dittrich, Petra S.

    2015-11-01

    The unwanted emergence of neutralizing antibodies (nAbs) against an endogenous or a therapeutic protein can result in deficiency diseases or therapy failure. Here, we developed a cell-free microfluidic method for the sensitive detection and quantification of nAbs in human serum that are associated with infertility. We used cell-derived vesicles containing the luteinizing hormone (LH)/choriogonadotropin receptor (LHHCGR) to detect nAbs against LH. The method exploits the entire cellular signal amplification mechanism, and facilitates the detection of as little as 0.44 nM of LH-nAb (Kd 1.5 nM) in human serum matrix within only 15 minutes. In addition, dose-response curves can be generated in less than 2 hours to evaluate the nAB concentration and dissociation constant. The developed system is devoid of problems associated with cell-based assays and we believe that this simple effect-directed analysis can be used in clinical environments, and is adaptable to other hormones or cytokines and their respective nAbs.

  20. Alterations in Red Blood Cell Deformability during Storage: A Microfluidic Approach

    PubMed Central

    Cluitmans, Judith C. A.; Chokkalingam, Venkatachalam; Janssen, Arno M.; Brock, Roland; Huck, Wilhelm T. S.; Bosman, Giel J. C. G. M.

    2014-01-01

    Red blood cells (RBCs) undergo extensive deformation when travelling through the microcapillaries. Deformability, the combined result of properties of the membrane-cytoskeleton complex, the surface area-to-volume ratio, and the hemoglobin content, is a critical determinant of capillary blood flow. During blood bank storage and in many pathophysiological conditions, RBC morphology changes, which has been suggested to be associated with decreased deformability and removal of RBC. While various techniques provide information on the rheological properties of stored RBCs, their clinical significance is controversial. We developed a microfluidic approach for evaluating RBC deformability in a physiologically meaningful and clinically significant manner. Unlike other techniques, our method enables a high-throughput determination of changes in deformation capacity to provide statistically significant data, while providing morphological information at the single-cell level. Our data show that, under conditions that closely mimic capillary dimensions and flow, the capacity to deform and the capacity to relax are not affected during storage in the blood bank. Our data also show that altered cell morphology by itself does not necessarily affect deformability. PMID:25295273

  1. Charge injection through nanocomposite electrode in microfluidic channel for electrical lysis of biological cells

    NASA Astrophysics Data System (ADS)

    Mishra, Madhusmita; Krishna, Anil; Chandra, Aman; Shenoy, B. M.; Hegde, G. M.; Mahapatra, D. Roy

    2013-03-01

    Several concepts have been developed in the recent years for nanomaterial based integrated MEMS platform in order to accelerate the process of biological sample preparation followed by selective screening and identification of target molecules. In this context, there exist several challenges which need to be addressed in the process of electrical lysis of biological cells. These are due to (i) low resource settings while achieving maximal lysis (ii) high throughput of target molecules to be detected (iii) automated extraction and purification of relevant molecules such as DNA and protein from extremely small volume of sample (iv) requirement of fast, accurate and yet scalable methods (v) multifunctionality toward process monitoring and (vi) downward compatibility with already existing diagnostic protocols. This paper reports on the optimization of electrical lysis process based on various different nanocomposite coated electrodes placed in a microfluidic channel. The nanocomposites are synthesized using different nanomaterials like Zinc nanorod dispersion in polymer. The efficiency of electrical lysis with various different electrode coatings has been experimentally verified in terms of DNA concentration, amplification and protein yield. The influence of the coating thickness on the injection current densities has been analyzed. We further correlate experimentally the current density vs. voltage relationship with the extent of bacterial cell lysis. A coupled multiphysics based simulation model is used to predict the cell trajectories and lysis efficiencies under various electrode boundary conditions as estimated from experimental results. Detailed in-situ fluorescence imaging and spectroscopy studies are performed to validate various hypotheses.

  2. Detection of Infertility-related Neutralizing Antibodies with a Cell-free Microfluidic Method

    PubMed Central

    Eyer, Klaus; Root, Katharina; Verboket, Pascal E.; Dittrich, Petra S.

    2015-01-01

    The unwanted emergence of neutralizing antibodies (nAbs) against an endogenous or a therapeutic protein can result in deficiency diseases or therapy failure. Here, we developed a cell-free microfluidic method for the sensitive detection and quantification of nAbs in human serum that are associated with infertility. We used cell-derived vesicles containing the luteinizing hormone (LH)/choriogonadotropin receptor (LHHCGR) to detect nAbs against LH. The method exploits the entire cellular signal amplification mechanism, and facilitates the detection of as little as 0.44 nM of LH-nAb (Kd 1.5 nM) in human serum matrix within only 15 minutes. In addition, dose-response curves can be generated in less than 2 hours to evaluate the nAB concentration and dissociation constant. The developed system is devoid of problems associated with cell-based assays and we believe that this simple effect-directed analysis can be used in clinical environments, and is adaptable to other hormones or cytokines and their respective nAbs. PMID:26585778

  3. Computational modeling of alkaline air-breathing microfluidic fuel cells with an array of cylinder anodes

    NASA Astrophysics Data System (ADS)

    Ye, Ding-Ding; Zhang, Biao; Zhu, Xun; Sui, Pang-Chieh; Djilali, Ned; Liao, Qiang

    2015-08-01

    A three-dimensional computational model is developed for an alkaline air-breathing microfluidic fuel cell (AMFC) with an array of cylinder anodes. The model is validated against experimental data from an in-house prototype AMFC. The distributions of fluid velocity, fuel concentration and current density of the fuel cell are analyzed in detail. The effect of reactant flow rate on the cell performance and electrode potentials is also studied. The model results suggest that fuel crossover is minimized by the fast electrolyte flow in the vicinity of the cathode. The current production of each anode is uneven and is well correlated with internal ohmic resistance. Fuel transfer limitation occurs at low flow rates (<100 μL min-1) but diminishes at high flow rates. The model results also indicate