Science.gov

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. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  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. 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

  6. 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

  7. 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

  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. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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.

  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. 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

  14. 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

  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. 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.

  2. 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

  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

    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 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

  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

    2016-06-01

    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.

  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

    2016-03-01

    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.

  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. 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.

  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. 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

  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. 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.

  10. 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

  11. 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

  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. 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.

  16. 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.

  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 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

  6. 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

  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. 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

  5. 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.

  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. 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…

  8. 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

  9. 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

  10. 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

  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. 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

  13. 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

  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. 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

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  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. 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.

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  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. 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.

  16. 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

  17. 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.

  18. 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.

  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

    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

  2. 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

  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. 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

  6. 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.

  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. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  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. 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

  8. 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

  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

    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