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Sample records for microfluidics meet cell

  1. Nanomaterials meet microfluidics.

    PubMed

    Pumera, Martin

    2011-05-28

    Nanomaterials and lab-on-a-chip platforms have undergone enormous development during the past decade. Here, we present an overview of how microfluidics benefited from the use of nanomaterials for the enhanced separation and detection of analytes. We also discuss how nanomaterials benefit from microfluidics in terms of synthesis and in terms of the simulation of environments for nanomotors and nanorobots. In our opinion, the "marriage" of nanomaterials and microfluidics is highly beneficial and is expected to solve vital challenges in related fields.

  2. Microfluidics for manipulating cells.

    PubMed

    Mu, Xuan; Zheng, Wenfu; Sun, Jiashu; Zhang, Wei; Jiang, Xingyu

    2013-01-14

    Microfluidics, a toolbox comprising methods for precise manipulation of fluids at small length scales (micrometers to millimeters), has become useful for manipulating cells. Its uses range from dynamic management of cellular interactions to high-throughput screening of cells, and to precise analysis of chemical contents in single cells. Microfluidics demonstrates a completely new perspective and an excellent practical way to manipulate cells for solving various needs in biology and medicine. This review introduces and comments on recent achievements and challenges of using microfluidics to manipulate and analyze cells. It is believed that microfluidics will assume an even greater role in the mechanistic understanding of cell biology and, eventually, in clinical applications.

  3. Stem cells in microfluidics

    PubMed Central

    Wu, Huei-Wen; Lin, Chun-Che; Lee, Gwo-Bin

    2011-01-01

    Microfluidic techniques have been recently developed for cell-based assays. In microfluidic systems, the objective is for these microenvironments to mimic in vivo surroundings. With advantageous characteristics such as optical transparency and the capability for automating protocols, different types of cells can be cultured, screened, and monitored in real time to systematically investigate their morphology and functions under well-controlled microenvironments in response to various stimuli. Recently, the study of stem cells using microfluidic platforms has attracted considerable interest. Even though stem cells have been studied extensively using bench-top systems, an understanding of their behavior in in vivo-like microenvironments which stimulate cell proliferation and differentiation is still lacking. In this paper, recent cell studies using microfluidic systems are first introduced. The various miniature systems for cell culture, sorting and isolation, and stimulation are then systematically reviewed. The main focus of this review is on papers published in recent years studying stem cells by using microfluidic technology. This review aims to provide experts in microfluidics an overview of various microfluidic systems for stem cell research. PMID:21522491

  4. 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.

  5. Cell manipulation in microfluidics.

    PubMed

    Yun, Hoyoung; Kim, Kisoo; Lee, Won Gu

    2013-06-01

    Recent advances in the lab-on-a-chip field in association with nano/microfluidics have been made for new applications and functionalities to the fields of molecular biology, genetic analysis and proteomics, enabling the expansion of the cell biology field. Specifically, microfluidics has provided promising tools for enhancing cell biological research, since it has the ability to precisely control the cellular environment, to easily mimic heterogeneous cellular environment by multiplexing, and to analyze sub-cellular information by high-contents screening assays at the single-cell level. Various cell manipulation techniques in microfluidics have been developed in accordance with specific objectives and applications. In this review, we examine the latest achievements of cell manipulation techniques in microfluidics by categorizing externally applied forces for manipulation: (i) optical, (ii) magnetic, (iii) electrical, (iv) mechanical and (v) other manipulations. We furthermore focus on history where the manipulation techniques originate and also discuss future perspectives with key examples where available.

  6. Microfluidics meet cell biology: bridging the gap by validation and application of microscale techniques for cell biological assays

    PubMed Central

    Paguirigan, Amy L.; Beebe, David J.

    2010-01-01

    Summary Microscale techniques have been applied to biological assays for nearly two decades, but haven’t been widely integrated as common tools in biological laboratories. The significant differences between several physical phenomena at the microscale versus the macroscale have been exploited to provide a variety of new types of assays (such as gradient production or spatial cell patterning). However, the use of these devices by biologists seems to be limited by issues regarding biological validation, ease of use, and the limited available readouts for assays done using microtechnology. Critical validation work has been done recently that highlights the current challenges for microfluidic methods and suggest ways in which future devices might be improved to better integrate with biological assays. With more validation and improved designs, microscale techniques hold immense promise as a platform to study aspects of cell biology that are not possible using current macroscale techniques. PMID:18693260

  7. 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.

  8. 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

  9. 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.

  10. Imaging Liquids Using Microfluidic Cells

    SciTech Connect

    Yu, Xiao-Ying; Liu, Bingwen; Yang, Li

    2013-05-10

    Chemistry occurring in the liquid and liquid surface is important in many applications. Chemical imaging of liquids using vacuum based analytical techniques is challenging due to the difficulty in working with liquids with high volatility. Recent development in microfluidics enabled and increased our capabilities to study liquid in situ using surface sensitive techniques such as electron microscopy and spectroscopy. Due to its small size, low cost, and flexibility in design, liquid cells based on microfluidics have been increasingly used in studying and imaging complex phenomena involving liquids. This paper presents a review of microfluidic cells that were developed to adapt to electron microscopes and various spectrometers for in situ chemical analysis and imaging of liquids. The following topics will be covered including cell designs, fabrication techniques, unique technical features for vacuum compatible cells, and imaging with electron microscopy and spectroscopy. Challenges are summarized and recommendations for future development priority are proposed.

  11. 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

  12. Recent developments in microfluidics for cell studies.

    PubMed

    Xiong, Bin; Ren, Kangning; Shu, Yiwei; Chen, Yin; Shen, Bo; Wu, Hongkai

    2014-08-20

    As a technique for precisely manipulating fluid at the micrometer scale, the field of microfluidics has experienced an explosive growth over the past two decades, particularly owing to the advances in device design and fabrication. With the inherent advantages associated with its scale of operation, and its flexibility in being incorporated with other microscale techniques for manipulation and detection, microfluidics has become a major enabling technology, which has introduced new paradigms in various fields involving biological cells. A microfluidic device is able to realize functions that are not easily imaginable in conventional biological analysis, such as highly parallel, sophisticated high-throughput analysis, single-cell analysis in a well-defined manner, and tissue engineering with the capability of manipulation at the single-cell level. Major advancements in microfluidic device fabrication and the growing trend of implementing microfluidics in cell studies are presented, with a focus on biological research and clinical diagnostics.

  13. 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

  14. 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

  15. A perspective on microfluidic biofuel cells.

    PubMed

    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.

  16. Microfluidic device for acoustic cell lysis

    SciTech Connect

    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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. Stem cell niche engineering through droplet microfluidics.

    PubMed

    Allazetta, Simone; Lutolf, Matthias P

    2015-12-01

    Stem cells reside in complex niches in which their behaviour is tightly regulated by various biochemical and biophysical signals. In order to unveil some of the crucial stem cell-niche interactions and expedite the implementation of stem cells in clinical and pharmaceutical applications, in vitro methodologies are being developed to reconstruct key features of stem cell niches. Recently, droplet-based microfluidics has emerged as a promising strategy to build stem cell niche models in a miniaturized and highly precise fashion. This review highlights current advances in using droplet microfluidics in stem cell biology. We also discuss recent efforts in which microgel technology has been interfaced with high-throughput analyses to engender screening paradigms with an unparalleled potential for basic and applied biological studies.

  6. 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.

  7. 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

  8. 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

  9. Cell Blebbing in Confined Microfluidic Environments

    PubMed Central

    Ibo, Markela; Srivastava, Vasudha; Robinson, Douglas N.; Gagnon, Zachary R.

    2016-01-01

    Migrating cells can extend their leading edge by forming myosin-driven blebs and F-actin-driven pseudopods. When coerced to migrate in resistive environments, Dictyostelium cells switch from using predominately pseudopods to blebs. Bleb formation has been shown to be chemotactic and can be influenced by the direction of the chemotactic gradient. In this study, we determine the blebbing responses of developed cells of Dictyostelium discoideum to cAMP gradients of varying steepness produced in microfluidic channels with different confining heights, ranging between 1.7 μm and 3.8 μm. We show that microfluidic confinement height, gradient steepness, buffer osmolarity and Myosin II activity are important factors in determining whether cells migrate with blebs or with pseudopods. Dictyostelium cells were observed migrating within the confines of microfluidic gradient channels. When the cAMP gradient steepness is increased from 0.7 nM/μm to 20 nM/μm, cells switch from moving with a mixture of blebs and pseudopods to moving only using blebs when chemotaxing in channels with confinement heights less than 2.4 μm. Furthermore, the size of the blebs increases with gradient steepness and correlates with increases in myosin-II localization at the cell cortex. Reduction of intracellular pressure by high osmolarity buffer or inhibition of myosin-II by blebbistatin leads to a decrease in bleb formation and bleb size. Together, our data reveal that the protrusion type formed by migrating cells can be influenced by the channel height and the steepness of the cAMP gradient, and suggests that a combination of confinement-induced myosin-II localization and cAMP-regulated cortical contraction leads to increased intracellular fluid pressure and bleb formation. PMID:27706201

  10. Probing cell mechanical properties with microfluidic devices

    NASA Astrophysics Data System (ADS)

    Rowat, Amy

    2012-02-01

    Exploiting flow on the micron-scale is emerging as a method to probe cell mechanical properties with 10-1000x advances in throughput over existing technologies. The mechanical properties of cells and the cell nucleus are implicated in a wide range of biological contexts: for example, the ability of white blood cells to deform is central to immune response; and malignant cells show decreased stiffness compared to benign cells. We recently developed a microfluidic device to probe cell and nucleus mechanical properties: cells are forced to deform through a narrow constrictions in response to an applied pressure; flowing cells through a series of constrictions enables us to probe the ability of hundreds of cells to deform and relax during flow. By tuning the constriction width so it is narrower than the width of the cell nucleus, we can specifically probe the effects of nuclear physical properties on whole cell deformability. We show that the nucleus is the rate-limiting step in cell passage: inducing a change in its shape to a multilobed structure results in cells that transit more quickly; increased levels of lamin A, a nuclear protein that is key for nuclear shape and mechanical stability, impairs the passage of cells through constrictions. We are currently developing a new class of microfluidic devices to simultaneously probe the deformability of hundreds of cell samples in parallel. Using the same soft lithography techniques, membranes are fabricated to have well-defined pore distribution, width, length, and tortuosity. We design the membranes to interface with a multiwell plate, enabling simultaneous measurement of hundreds of different samples. Given the wide spectrum of diseases where altered cell and nucleus mechanical properties are implicated, such a platform has great potential, for example, to screen cells based on their mechanical phenotype against a library of drugs.

  11. Large-Volume Microfluidic Cell Sorting for Biomedical Applications.

    PubMed

    Warkiani, Majid Ebrahimi; Wu, Lidan; Tay, Andy Kah Ping; Han, Jongyoon

    2015-01-01

    Microfluidic cell-separation technologies have been studied for almost two decades, but the limited throughput has restricted their impact and range of application. Recent advances in microfluidics enable high-throughput cell sorting and separation, and this has led to various novel diagnostic and therapeutic applications that previously had been impossible to implement using microfluidics technologies. In this review, we focus on recent progress made in engineering large-volume microfluidic cell-sorting methods and the new applications enabled by them. PMID:26194427

  12. 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.

  13. A membraneless microfluidic fuel cell stack

    NASA Astrophysics Data System (ADS)

    Salloum, Kamil S.; Posner, Jonathan D.

    A membraneless microfluidic fuel cell stack architecture is presented that reuses reactants from one cell to a subsequent one, analogous to PEMFC stacks. On-chip reactant reuse improves fuel utilization and power densities relative to single cells. The reactants flow separately through porous electrodes and interface with a non-reacting and conductive electrolyte which maintains their separation. The reactants remain separated downstream of the interface and are used in subsequent downstream cells. This fuel cell uses porous carbon for electrocatalysts and vanadium redox species as reactants with a sulfuric acid supporting electrolyte. The overall power density of the fuel cell increases with reactant flow rate and decreasing the separating electrolyte flow rate. The peak power, maximum fuel utilization, and efficiency nearly double when electrically connecting the cells in parallel.

  14. 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

  15. Digital microfluidic immunocytochemistry in single cells

    PubMed Central

    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

  16. 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

  17. Rare Cell Capture in Microfluidic Devices

    PubMed Central

    Pratt, Erica D.; Huang, Chao; Hawkins, Benjamin G.; Gleghorn, Jason P.; Kirby, Brian J.

    2010-01-01

    This article reviews existing methods for the isolation, fractionation, or capture of rare cells in microfluidic devices. Rare cell capture devices face the challenge of maintaining the efficiency standard of traditional bulk separation methods such as flow cytometers and immunomagnetic separators while requiring very high purity of the target cell population, which is typically already at very low starting concentrations. Two major classifications of rare cell capture approaches are covered: (1) non-electrokinetic methods (e.g., immobilization via antibody or aptamer chemistry, size-based sorting, and sheath flow and streamline sorting) are discussed for applications using blood cells, cancer cells, and other mammalian cells, and (2) electrokinetic (primarily dielectrophoretic) methods using both electrode-based and insulative geometries are presented with a view towards pathogen detection, blood fractionation, and cancer cell isolation. The included methods were evaluated based on performance criteria including cell type modeled and used, number of steps/stages, cell viability, and enrichment, efficiency, and/or purity. Major areas for improvement are increasing viability and capture efficiency/purity of directly processed biological samples, as a majority of current studies only process spiked cell lines or pre-diluted/lysed samples. Despite these current challenges, multiple advances have been made in the development of devices for rare cell capture and the subsequent elucidation of new biological phenomena; this article serves to highlight this progress as well as the electrokinetic and non-electrokinetic methods that can potentially be combined to improve performance in future studies. PMID:21532971

  18. 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

  19. Parallel single-cell analysis microfluidic platform.

    PubMed

    van den Brink, Floris T G; Gool, Elmar; Frimat, Jean-Philippe; Bomer, Johan; van den Berg, Albert; Le Gac, Séverine

    2011-11-01

    We report a PDMS microfluidic platform for parallel single-cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or non-invasive analysis schemes are performed. First, we report single-cell trapping in a fast (2-5  min) and reproducible manner with a single-cell capture yield of 85% using two cell lines (P3x63Ag8 and MCF-7), employing a protocol which is scalable and easily amenable to automation. Following this, a mixed population of P3x63Ag8 and MCF-7 cells is stained in situ using the nucleic acid probe (Hoechst) and a phycoerythrin-labeled monoclonal antibody directed at EpCAM present on the surface of the breast cancer cells MCF-7 and absent on the myeloma cells P3x63Ag8 to illustrate the potential of the device to analyze cell population heterogeneity. Next, cells are porated in situ using chemicals in a reversible (digitonin) or irreversible way (lithium dodecyl sulfate). This is visualized by the transportation of fluorescent dyes through the membrane (propidium iodide and calcein). Finally, an electrical protocol is developed for combined cell permeabilization and electroosmotic flow (EOF)-based extraction of the cell content. It is validated here using calcein-loaded cells and visualized through the progressive recovery of calcein in the side channels, indicating successful retrieval of individual cell content. PMID:22025223

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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

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

    PubMed

    He, Jie-Long; Chen, An-Te; Lee, Jyong-Huei; Fan, Shih-Kang

    2015-09-15

    The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed.

  6. 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.

  7. Droplet microfluidics--a tool for single-cell analysis.

    PubMed

    Joensson, Haakan N; Andersson Svahn, Helene

    2012-12-01

    Droplet microfluidics allows the isolation of single cells and reagents in monodisperse picoliter liquid capsules and manipulations at a throughput of thousands of droplets per second. These qualities allow many of the challenges in single-cell analysis to be overcome. Monodispersity enables quantitative control of solute concentrations, while encapsulation in droplets provides an isolated compartment for the single cell and its immediate environment. The high throughput allows the processing and analysis of the tens of thousands to millions of cells that must be analyzed to accurately describe a heterogeneous cell population so as to find rare cell types or access sufficient biological space to find hits in a directed evolution experiment. The low volumes of the droplets make very large screens economically viable. This Review gives an overview of the current state of single-cell analysis involving droplet microfluidics and offers examples where droplet microfluidics can further biological understanding.

  8. 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.

  9. Parameter screening in microfluidics based hydrodynamic single-cell trapping.

    PubMed

    Deng, B; Li, X F; Chen, D Y; You, L D; Wang, J B; Chen, J

    2014-01-01

    Microfluidic cell-based arraying technology is widely used in the field of single-cell analysis. However, among developed devices, there is a compromise between cellular loading efficiencies and trapped cell densities, which deserves further analysis and optimization. To address this issue, the cell trapping efficiency of a microfluidic device with two parallel micro channels interconnected with cellular trapping sites was studied in this paper. By regulating channel inlet and outlet status, the microfluidic trapping structure can mimic key functioning units of previously reported devices. Numerical simulations were used to model this cellular trapping structure, quantifying the effects of channel on/off status and trapping structure geometries on the cellular trapping efficiency. Furthermore, the microfluidic device was fabricated based on conventional microfabrication and the cellular trapping efficiency was quantified in experiments. Experimental results showed that, besides geometry parameters, cellular travelling velocities and sizes also affected the single-cell trapping efficiency. By fine tuning parameters, more than 95% of trapping sites were taken by individual cells. This study may lay foundation in further studies of single-cell positioning in microfluidics and push forward the study of single-cell analysis.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Nanoporous membrane-sealed microfluidic devices for improved cell viability.

    PubMed

    Masand, Shirley N; Mignone, Lindsay; Zahn, Jeffrey D; Shreiber, David I

    2011-12-01

    Cell-laden microfluidic devices have broad potential in various biomedical applications, including tissue engineering and drug discovery. However, multiple difficulties encountered while culturing cells within devices affecting cell viability, proliferation, and behavior has complicated their use. While active perfusion systems have been used to overcome the diffusive limitations associated with nutrient delivery into microchannels to support longer culture times, these systems can result in non-uniform oxygen and nutrient delivery and subject cells to shear stresses, which can affect cell behavior. Additionally, histological analysis of cell cultures within devices is generally laborious and yields inconsistent results due to difficulties in delivering labeling agents in microchannels. Herein, we describe a simple, cost-effective approach to preserve cell viability and simplify labeling within microfluidic networks without the need for active perfusion. Instead of bonding a microfluidic network to glass, PDMS, or other solid substrate, the network is bonded to a semi-permeable nanoporous membrane. The membrane-sealed devices allow free exchange of proteins, nutrients, buffers, and labeling reagents between the microfluidic channels and culture media in static culture plates under sterile conditions. The use of the semi-permeable membrane dramatically simplifies microniche cell culturing while avoiding many of the complications which arise from perfusion systems.

  15. 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.

  16. 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.

  17. 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.

  18. Electrical cell counting process characterization in a microfluidic impedance cytometer.

    PubMed

    Hassan, Umer; Bashir, Rashid

    2014-10-01

    Particle counting in microfluidic devices with coulter principle finds many applications in health and medicine. Cell enumeration using microfluidic particle counters is fast and requires small volumes of sample, and is being used for disease diagnostics in humans and animals. A complete characterization of the cell counting process is critical for accurate cell counting especially in complex systems with samples of heterogeneous population interacting with different reagents in a microfluidic device. In this paper, we have characterized the electrical cell counting process using a microfluidic impedance cytometer. Erythrocytes were lysed on-chip from whole blood and the lysing was quenched to preserve leukocytes which subsequently pass through a 15 μm × 15 μm measurement channel used to electrically count the cells. We show that cell counting over time is a non-homogeneous Poisson process and that the electrical cell counts over time show the log-normal distribution, whose skewness can be attributed to diffusion of cells in the buffer that is used to meter the blood. We further found that the heterogeneous cell population (i.e. different cell types) shows different diffusion characteristics based on the cell size. Lymphocytes spatially diffuse more as compared to granulocytes and monocytes. The time difference between the cell occurrences follows an exponential distribution and when plotted over time verifies the cell diffusion characteristics. We also characterized the probability of occurrence of more than one cell at the counter within specified time intervals using Poisson counting statistics. For high cell concentration samples, we also derived the required sample dilution based on our particle counting characterization. Buffer characterization by considering the size based particle diffusion and estimating the required dilution are critical parameters for accurate counting results.

  19. Microfluidic immunomagnetic cell separation using integrated permanent micromagnets

    PubMed Central

    Osman, O.; Toru, S.; Dumas-Bouchiat, F.; Dempsey, N. M.; Haddour, N.; Zanini, L.-F.; Buret, F.; Reyne, G.; Frénéa-Robin, M.

    2013-01-01

    In this paper, we demonstrate the possibility to trap and sort labeled cells under flow conditions using a microfluidic device with an integrated flat micro-patterned hard magnetic film. The proposed technique is illustrated using a cell suspension containing a mixture of Jurkat cells and HEK (Human Embryonic Kidney) 293 cells. Prior to sorting experiments, the Jurkat cells were specifically labeled with immunomagnetic nanoparticles, while the HEK 293 cells were unlabeled. Droplet-based experiments demonstrated that the Jurkat cells were attracted to regions of maximum stray field flux density while the HEK 293 cells settled in random positions. When the mixture was passed through a polydimethylsiloxane (PDMS) microfluidic channel containing integrated micromagnets, the labeled Jurkat cells were selectively trapped under fluid flow, while the HEK cells were eluted towards the device outlet. Increasing the flow rate produced a second eluate much enriched in Jurkat cells, as revealed by flow cytometry. The separation efficiency of this biocompatible, compact micro-fluidic separation chamber was compared with that obtained using two commercial magnetic cell separation kits. PMID:24396526

  20. 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.

  1. 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

  2. Cell research with physically modified microfluidic channels: a review.

    PubMed

    Kim, Sun Min; Lee, Sung Hoon; Suh, Kahp Yang

    2008-07-01

    An overview of the use of physically modified microfluidic channels towards cell research is presented. The physical modification can be realized either by combining embedded physical micro/nanostructures or a topographically patterned substrate at the micro- or nanoscale inside a channel. After a brief description of the background and the importance of the physically modified microfluidic system, various fabrication methods are described based on the materials and geometries of physical structures and channels. Of many operational principles for microfluidics (electrical, magnetic, optical, mechanical, and so on), this review primarily focuses on mechanical operation principles aided by structural modification of the channels. The mechanical forces are classified into (i) hydrodynamic, (ii) gravitational, (iii) capillary, (iv) wetting, and (v) adhesion forces. Throughout this review, we will specify examples where necessary and provide trends and future directions in the field.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. Microfluidic single-cell whole-transcriptome sequencing

    PubMed Central

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

    2014-01-01

    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

  10. Optical manipulation and microfluidics for studies of single cell dynamics

    NASA Astrophysics Data System (ADS)

    Eriksson, E.; Scrimgeour, J.; Granéli, A.; Ramser, K.; Wellander, R.; Enger, J.; Hanstorp, D.; Goksör, M.

    2007-08-01

    Most research on optical manipulation aims towards investigation and development of the system itself. In this paper we show how optical manipulation, imaging and microfluidics can be combined for investigations of single cells. Microfluidic systems have been fabricated and are used, in combination with optical tweezers, to enable environmental changes for single cells. The environment within the microfluidic system has been modelled to ensure control of the process. Three biological model systems have been studied with different combinations of optical manipulation, imaging techniques and microfluidics. In Saccharomyces cerevisiae, environmentally induced size modulations and spatial localization of proteins have been studied to elucidate various signalling pathways. In a similar manner the oxygenation cycle of single red blood cells was triggered and mapped using Raman spectroscopy. In the third experiment the forces between the endoplasmic reticulum and chloroplasts were studied in Pisum sativum and Arabidopsis thaliana. By combining different techniques we make advanced biological research possible, revealing information on a cellular level that is impossible to obtain with traditional techniques.

  11. 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

  12. Pharmacology on microfluidics: multimodal analysis for studying cell-cell interaction.

    PubMed

    Delamarche, Emmanuel; Tonna, Noemi; Lovchik, Robert D; Bianco, Fabio; Matteoli, Michela

    2013-10-01

    Understanding the mechanisms of cell-cell interaction is a key unanswered question in modern pharmacology, given crosstalk defects are at the basis of many pathologies. Microfluidics represents a valuable tool for analyzing intercellular communication mediated by transmission of soluble signals, as occurring for example between neurons and glial cells in neuroinflammation, or between tumor and surrounding cells in cancer. However, the use of microfluidics for studying cell behavior still encompasses many technical and biological challenges. In this review, a state of the art of successes, potentials and limitations of microfluidics applied to key biological questions in modern pharmacology is analyzed and commented.

  13. 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.

  14. 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

  15. Microfluidic impedance cytometry of tumour cells in blood.

    PubMed

    Spencer, Daniel; Hollis, Veronica; Morgan, Hywel

    2014-11-01

    The dielectric properties of tumour cells are known to differ from normal blood cells, and this difference can be exploited for label-free separation of cells. Conventional measurement techniques are slow and cannot identify rare circulating tumour cells (CTCs) in a realistic timeframe. We use high throughput single cell microfluidic impedance cytometry to measure the dielectric properties of the MCF7 tumour cell line (representative of CTCs), both as pure populations and mixed with whole blood. The data show that the MCF7 cells have a large membrane capacitance and size, enabling clear discrimination from all other leukocytes. Impedance analysis is used to follow changes in cell viability when cells are kept in suspension, a process which can be understood from modelling time-dependent changes in the dielectric properties (predominantly membrane conductivity) of the cells. Impedance cytometry is used to enumerate low numbers of MCF7 cells spiked into whole blood. Chemical lysis is commonly used to remove the abundant erythrocytes, and it is shown that this process does not alter the MCF7 cell count or change their dielectric properties. Combining impedance cytometry with magnetic bead based antibody enrichment enables MCF7 cells to be detected down to 100 MCF7 cells in 1 ml whole blood, a log 3.5 enrichment and a mean recovery of 92%. Microfluidic impedance cytometry could be easily integrated within complex cell separation systems for identification and enumeration of specific cell types, providing a fast in-line single cell characterisation method.

  16. Single-cell Genomics using Droplet-based Microfluidics

    NASA Astrophysics Data System (ADS)

    Basu, Anindita; Macosko, Evan; Shalek, Alex; McCarroll, Steven; Regev, Aviv; Weitz, Dave

    2014-03-01

    We develop a system to profile the transcriptome of mammalian cells in isolation using reverse emulsion droplet-based microfluidic techniques. This is accomplished by (a) encapsulating and lysing one cell per emulsion droplet, and (b) uniquely barcoding the RNA contents from each cell using unique DNA-barcoded microgel beads. This enables us to study the transcriptional behavior of a large number of cells at single-cell resolution. We then use these techniques to study transcriptional responses of isolated immune cells to precisely controlled chemical and pathological stimuli provided in the emulsion droplet.

  17. 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

  18. 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

  19. Cell-based microfluidic platform for mimicking human olfactory system.

    PubMed

    Lee, Seung Hwan; Oh, Eun Hae; Park, Tai Hyun

    2015-12-15

    Various attempts have been made to mimic the human olfactory system using human olfactory receptors (hORs). In particular, OR-expressed cell-based odorant detection systems mimic the smell sensing mechanism of humans, as they exploit endogenous cellular signaling pathways. However, the majority of such cell-based studies have been performed in the liquid phase to maintain cell viability, and liquid odorants were used as detection targets. Here, we present a microfluidic device for the detection of gaseous odorants which more closely mimics the human olfactory system. Cells expressing hOR were cultured on a porous membrane. The membrane was then flipped over and placed between two compartments. The upper compartment is the gaseous part where gaseous odorants are supplied, while the lower compartment is the aqueous part where viable cells are maintained in the liquid medium. Using this simple microfluidic device, we were able to detect gaseous odorant molecules by a fluorescence signal. The fluorescence signal was generated by calcium influx resulting from the interaction between odorant molecules and the hOR. The system allowed detection of gaseous odorant molecules in real-time, and the findings showed that the fluorescence responses increased dose-dependently in the range of 0-2 ppm odorant. In addition, the system can discriminate among gaseous odorant molecules. This microfluidic system closely mimics the human olfactory system in the sense that the submerged cells detect gaseous odorants.

  20. Nanopillar based electrochemical biosensor for monitoring microfluidic based cell culture

    NASA Astrophysics Data System (ADS)

    Gangadharan, Rajan

    In-vitro assays using cultured cells have been widely performed for studying many aspects of cell biology and cell physiology. These assays also form the basis of cell based sensing. Presently, analysis procedures on cell cultures are done using techniques that are not integrated with the cell culture system. This approach makes continuous and real-time in-vitro measurements difficult. It is well known that the availability of continuous online measurements for extended periods of time will help provide a better understanding and will give better insight into cell physiological events. With this motivation we developed a highly sensitive, selective and stable microfluidic electrochemical glucose biosensor to make continuous glucose measurements in cell culture media. The performance of the microfluidic biosensor was enhanced by adding 3D nanopillars to the electrode surfaces. The microfluidic glucose biosensor consisted of three electrodes---Enzyme electrode, Working electrode, and Counter electrode. All these electrodes were enhanced with nanopillars and were optimized in their respective own ways to obtain an effective and stable biosensing device in cell culture media. For example, the 'Enzyme electrode' was optimized for enzyme immobilization via either a polypyrrole-based or a self-assembled-monolayer-based immobilization method, and the 'Working electrode' was modified with Prussian Blue or electropolymerized Neutral Red to reduce the working potential and also the interference from other interacting electro-active species. The complete microfluidic biosensor was tested for its ability to monitor glucose concentration changes in cell culture media. The significance of this work is multifold. First, the developed device may find applications in continuous and real-time measurements of glucose concentrations in in-vitro cell cultures. Second, the development of a microfluidic biosensor will bring technical know-how toward constructing continuous glucose

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

    PubMed

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

    2010-11-01

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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. Microfluidic hydrogen fuel cell with a liquid electrolyte.

    PubMed

    Jayashree, Ranga S; Mitchell, Michael; Natarajan, Dilip; Markoski, Larry J; Kenis, Paul J A

    2007-06-19

    We report the design and characterization of a microfluidic hydrogen fuel cell with a flowing sulfuric acid solution instead of a Nafion membrane as the electrolyte. We studied the effect of cell resistance, hydrogen and oxygen flow rates, and electrolyte flow rate on fuel cell performance to obtain a maximum power density of 191 mW/cm2. This flowing electrolyte design avoids water management issues, including cathode flooding and anode dry out. Placing a reference electrode in the outlet stream allows for independent analysis of the polarization losses on the anode and the cathode, thereby creating an elegant catalyst characterization and optimization tool.

  7. A digital microfluidic platform for primary cell culture and analysis.

    PubMed

    Srigunapalan, Suthan; Eydelnant, Irwin A; Simmons, Craig A; Wheeler, Aaron R

    2012-01-21

    Digital microfluidics (DMF) is a technology that facilitates electrostatic manipulation of discrete nano- and micro-litre droplets across an array of electrodes, which provides the advantages of single sample addressability, automation, and parallelization. There has been considerable interest in recent years in using DMF for cell culture and analysis, but previous studies have used immortalized cell lines. We report here the first digital microfluidic method for primary cell culture and analysis. A new mode of "upside-down" cell culture was implemented by patterning the top plate of a device using a fluorocarbon liftoff technique. This method was useful for culturing three different primary cell types for up to one week, as well as implementing a fixation, permeabilization, and staining procedure for F-actin and nuclei. A multistep assay for monocyte adhesion to endothelial cells (ECs) was performed to evaluate functionality in DMF-cultured primary cells and to demonstrate co-culture using a DMF platform. Monocytes were observed to adhere in significantly greater numbers to ECs exposed to tumor necrosis factor (TNF)-α than those that were not, confirming that ECs cultured in this format maintain in vivo-like properties. The ability to manipulate, maintain, and assay primary cells demonstrates a useful application for DMF in studies involving precious samples of cells from small animals or human patients.

  8. Optical injection of mammalian cells using a microfluidic platform

    PubMed Central

    Marchington, Robert F.; Arita, Yoshihiko; Tsampoula, Xanthi; Gunn-Moore, Frank J.; Dholakia, Kishan

    2010-01-01

    The use of a focused laser beam to create a sub-micron hole in the plasma membrane of a cell (photoporation), for the selective introduction of membrane impermeable substances (optical injection) including nucleic acids (optical transfection), is a powerful technique most commonly applied to treat single cells. However, particularly for femtosecond photoporation, these studies have been limited to low throughput, small-scale studies, because they require sequential dosing of individual cells. Herein, we describe a microfluidic photoporation system for increased throughput and automated optical injection of cells. Hydrodynamic focusing is employed to direct a flow of single-file cells through a focused femtosecond laser beam for photoporation. Upon traversing the beam, a number of transient pores potentially open across the extracellular membrane, which allows the uptake of the surrounding fluid media into the cytoplasm, also containing the chosen injection agent. The process is entirely automated and a rate of 1 cell/sec could readily be obtained, enabling several thousand cells to be injected per hour using this system. The efficiency of optically injecting propidium iodide into HEK293 mammalian cells was found to be 42 ± 8%, or 28 ± 4% taking into account the requirement of post-injection viability, as tested using Calcein AM. This work now opens the way for combining photoporation with microfluidic analyses, sorting, purification or on-chip cell culture studies. PMID:21258487

  9. 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.

  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. 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

  12. Microfluidic devices for measuring gene network dynamics in single cells

    PubMed Central

    Bennett, Matthew R.; Hasty, Jeff

    2010-01-01

    The dynamics governing gene regulation have an important role in determining the phenotype of a cell or organism. From processing extracellular signals to generating internal rhythms, gene networks are central to many time-dependent cellular processes. Recent technological advances now make it possible to track the dynamics of gene networks in single cells under various environmental conditions using microfluidic ‘lab-on-a-chip’ devices, and researchers are using these new techniques to analyse cellular dynamics and discover regulatory mechanisms. These technologies are expected to yield novel insights and allow the construction of mathematical models that more accurately describe the complex dynamics of gene regulation. PMID:19668248

  13. 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

  14. Enhancing the biocompatibility of microfluidics-assisted fabrication of cell-laden microgels with channel geometry.

    PubMed

    Kim, Suntae; Oh, Jonghyun; Cha, Chaenyung

    2016-11-01

    Microfluidic flow-focusing devices (FFD) are widely used to generate monodisperse droplets and microgels with controllable size, shape and composition for various biomedical applications. However, highly inconsistent and often low viability of cells encapsulated within the microgels prepared via microfluidic FFD has been a major concern, and yet this aspect has not been systematically explored. In this study, we demonstrate that the biocompatibility of microfluidic FFD to fabricate cell-laden microgels can be significantly enhanced by controlling the channel geometry. When a single emulsion ("single") microfluidic FFD is used to fabricate cell-laden microgels, there is a significant decrease and batch-to-batch variability in the cell viability, regardless of their size and composition. It is determined that during droplet generation, some of the cells are exposed to the oil phase which is shown to have a cytotoxic effect. Therefore, a microfluidic device with a sequential ('double') flow-focusing channels is employed instead, in which a secondary aqueous phase containing cells enters the primary aqueous phase, so the cells' exposure to the oil phase is minimized by directing them to the center of droplets. This microfluidic channel geometry significantly enhances the biocompatibility of cell-laden microgels, while maintaining the benefits of a typical microfluidic process. This study therefore provides a simple and yet highly effective strategy to improve the biocompatibility of microfluidic fabrication of cell-laden microgels.

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

    PubMed

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

    2004-11-01

    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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  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. 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.

  3. 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

  4. 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

  5. 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.

  6. Advances in microfluidic platforms for analyzing and regulating human pluripotent stem cells.

    PubMed

    Qian, Tongcheng; Shusta, Eric V; Palecek, Sean P

    2015-10-01

    Microfluidic devices employ submillimeter length scale control of flow to achieve high-resolution spatial and temporal control over the microenvironment, providing powerful tools to elucidate mechanisms of human pluripotent stem cell (hPSC) regulation and to elicit desired hPSC fates. In addition, microfluidics allow control of paracrine and juxtracrine signaling, thereby enabling fabrication of microphysiological systems comprised of multiple cell types organized into organs-on-a-chip. Microfluidic cell culture systems can also be integrated with actuators and sensors, permitting construction of high-density arrays of cell-based biosensors for screening applications. This review describes recent advances in using microfluidics to understand mechanisms by which the microenvironment regulates hPSC fates and applications of microfluidics to realize the potential of hPSCs for in vitro modeling and screening applications.

  7. 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

  8. Simplified fluid-structure coupled analysis of particle movement for designing of microfluidic cell sorter.

    PubMed

    Takagi, Yuto; Kotev, Vladimir; Yano, Ken'ich

    2015-01-01

    Recently, methods of the separation and selection of cells using a microfluidic device are receiving a lot of attention as the latest technology and those devices are called microfluidic cell sorter. Those methods have many advantages compared to conventional methods. There are a lot of researches on the microfluidic cell sorting but there isn't the automated design method of this device in spite of the necessary. To achieve the automated design of the microfluidic cell sorter, the analysis of the movement of cells in the microfluidic device and optimum design of the microfluidic cell sorter corresponding to kind of various cells are required. In the former case, the fluid-structure interaction analysis of fluid and cell movement is needed. However, it is very complex and needs a lot of computational time. Therefore, we focused on this problem in the fluid-structure interaction analysis for designing the microfluidic cell sorter. We assume cell is a sphere particle and propose the simplified fluid-structure coupled analysis which combines the CFD analysis with the motion equation of a sphere particle.

  9. 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

  10. Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments

    PubMed Central

    Zervantonakis, Ioannis K.; Kothapalli, Chandrasekhar R.; Chung, Seok; Sudo, Ryo; Kamm, Roger D.

    2011-01-01

    Microfluidic devices allow for precise control of the cellular and noncellular microenvironment at physiologically relevant length- and time-scales. These devices have been shown to mimic the complex in vivo microenvironment better than conventional in vitro assays, and allow real-time monitoring of homotypic or heterotypic cellular interactions. Microfluidic culture platforms enable new assay designs for culturing multiple different cell populations and∕or tissue specimens under controlled user-defined conditions. Applications include fundamental studies of cell population behaviors, high-throughput drug screening, and tissue engineering. In this review, we summarize recent developments in this field along with studies of heterotypic cell-cell interactions and tissue specimen culture in microfluidic devices from our own laboratory. PMID:21522496

  11. Screen-printed microfluidic dielectrophoresis chip for cell separation.

    PubMed

    Zhu, Hongwu; Lin, Xiaoguang; Su, Yong; Dong, Hua; Wu, Jianhua

    2015-01-15

    Dielectrophoresis (DEP), the induced motion of polarizable particles in a non-uniform electric field, has been proven as a perfect candidate to transport, accumulate, separate and characterize micro-/nano-scale bioparticles in microfluidic systems. However, conventional fabrication technologies are complex, time-consuming and relatively expensive, leading to low throughput of the DEP-based systems. In this paper, we report a novel microfluidic alternating current DEP (AC-DEP) chip fabricated via inexpensive screen printing method. The innovation of our work consists in the extreme simplicity of the fabrication procedure, i.e., the main components, including electrodes and channels, were constructed by layer-by-layer screen printing process, which is especially suitable for high-throughput mass production. Carbon paste, instead of metals, was used to print interdigitated electrodes with semi-3D structure which not only reduces dramatically the chip cost but also increases particle trapping efficiency. To test the chip performance, yeast cells, as model cells, were trapped and separated from a mixed suspension with PS microspheres. Our results show that high capture rate and separation efficiency can be achieved under optimized conditions. PMID:25127471

  12. Microfluidic devices modulate tumor cell line susceptibility to NK cell recognition.

    PubMed

    Perozziello, Gerardo; La Rocca, Rosanna; Cojoc, Gheorghe; Liberale, Carlo; Malara, Natalia; Simone, Giuseppina; Candeloro, Patrizio; Anichini, Andrea; Tirinato, Luca; Gentile, Francesco; Coluccio, Maria Laura; Carbone, Ennio; Di Fabrizio, Enzo

    2012-09-24

    This study aims to adoptively reduce the major histocompatibility complex class I (MHC-I) molecule surface expression of cancer cells by exposure to microfluid shear stress and a monoclonal antibody. A microfluidic system is developed and tumor cells are injected at different flow rates. The bottom surface of the microfluidic system is biofunctionalized with antibodies (W6/32) specific for the MHC-I molecules with a simple method based on microfluidic protocols. The antibodies promote binding between the bottom surface and the MHC-I molecules on the tumor cell membrane. The cells are injected at an optimized flow rate, then roll on the bottom surface and are subjected to shear stress. The stress is localized and enhanced on the part of the membrane where MHC-I proteins are expressed, since they stick to the antibodies of the system. The localized stress allows a stripping effect and consequent reduction of the MHC-I expression. It is shown that it is possible to specifically treat and recover eukaryotic cells without damaging the biological samples. MHC-I molecule expression on treated and control cell surfaces is measured on tumor and healthy cells. After the cell rolling treatment a clear reduction of MHC-I levels on the tumor cell membrane is observed, whereas no changes are observed on healthy cells (monocytes). The MHC-I reduction is investigated and the possibility that the developed system could induce a loss of these molecules from the tumor cell surface is addressed. The percentage of living tumor cells (viability) that remain after the treatment is measured. The changes induced by the microfluidic system are analyzed by fluorescence-activated cell sorting and confocal microscopy. Cytotoxicity tests show a relevant increased susceptibility of natural killer (NK) cells on microchip-treated tumor cells.

  13. A robust microfluidic in vitro cell perifusion system.

    PubMed

    Morris, Christina; Banks, Dylan J; Gaweda, Lukasz; Scott, Steve; Zhu, Xi Xi; Panico, Maria; Georgiou, Pantelis; Toumazou, Christofer

    2011-01-01

    We present here a robust microfluidic cell perifusion device for in vitro primary tissue cell secretion studies. This system increases the sample concentration to perifusion volume ratio by an order of magnitude compared with standard multi-well plate static incubation assays. Further, this device achieves physiologically relevant flow rates, pressures, and temperature. It has been manufactured with typical machining facilities, principally drilling and milling. No specialist clean room equipment is required to replicate it. We show its capability here with hormone perifusion experiments on primary pancreatic tissue from mice. This device can increase cell secretion concentrations by up to a factor of 20, allowing for the first time the direct measurement of islet glucagon using mass spectrometry.

  14. Suspended microfluidics.

    PubMed

    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

    2013-06-18

    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.

  15. Microfluidics for cell-based high throughput screening platforms - A review.

    PubMed

    Du, Guansheng; Fang, Qun; den Toonder, Jaap M J

    2016-01-15

    In the last decades, the basic techniques of microfluidics for the study of cells such as cell culture, cell separation, and cell lysis, have been well developed. Based on cell handling techniques, microfluidics has been widely applied in the field of PCR (Polymerase Chain Reaction), immunoassays, organ-on-chip, stem cell research, and analysis and identification of circulating tumor cells. As a major step in drug discovery, high-throughput screening allows rapid analysis of thousands of chemical, biochemical, genetic or pharmacological tests in parallel. In this review, we summarize the application of microfluidics in cell-based high throughput screening. The screening methods mentioned in this paper include approaches using the perfusion flow mode, the droplet mode, and the microarray mode. We also discuss the future development of microfluidic based high throughput screening platform for drug discovery.

  16. 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.

  17. Development of a microfluidic platform for single-cell secretion analysis using a direct photoactive cell-attaching method.

    PubMed

    Jang, Kihoon; Ngo, Hong Trang Thi; Tanaka, Yo; Xu, Yan; Mawatari, Kazuma; Kitamori, Takehiko

    2011-01-01

    A precise understanding of individual cellular processes is essential to meet the expectations of most advanced cell biology. Therefore single-cell analysis is considered to be one of possible approach to overcome any misleading of cell characteristics by averaging large groups of cells in bulk conditions. In the present work, we modified a newly designed microchip for single-cell analysis and regulated the cell-adhesive area inside a cell-chamber of the microfluidic system. By using surface-modification techniques involving a silanization compound, a photo-labile linker and the 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer were covalently bonded on the surface of a microchannel. The MPC polymer was utilized as a non-biofouling compound for inhibiting non-specific binding of the biological samples inside the microchannel, and was selectively removed by a photochemical reaction that controlled the cell attachment. To achieve the desired single-macrophage patterning and culture in the cell-chamber of the microchannel, the cell density and flow rate of the culture medium were optimized. We found that a cell density of 2.0 × 10(6) cells/ml was the appropriate condition to introduce a single cell in each cell chamber. Furthermore, the macrophage was cultured in a small size of the cell chamber in a safe way for 5 h at a flow rate of 0.2 µl/min under the medium condition. This strategy can be a powerful tool for broadening new possibilities in studies of individual cellular processes in a dynamic microfluidic device.

  18. 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

  19. 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.

  20. 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.

  1. Microfluidic cell culture system with on-chip hypoxic conditioning.

    PubMed

    Takano, Atsushi; Tanaka, Masato; Futai, Nobuyuki

    2013-01-01

    We have demonstrated a portable microfluidic cell culture system with multi-gas (CO2 and O2) incubation which we can cultivate under hypoxia without bulky peripheral apparatus such as gas tanks, regulators, and flow controllers. The system contains a chip of 26 mm × 48 mm which is capable to diffuse CO2 and absorb O2 through a gas-permeable wall of nested media reservoir. The media was water-jacketed with aqueous solution containing 0.8 M sodium bicarbonate as CO2 supply and 1 M sodium ascorbate as oxygen scavenger. The partial CO2 pressure (pCO2) in media reservoir stabilized at least 10.2% ± 0.11% for at least 72 hours. The partial O2 pressure (pO2) in the media reservoir decreased to 4.2%. Portable on-chip hypoxic culture of SV40-T2 cells for 72 h was also demonstrated. PMID:24110727

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Microfluidic device for continuous single cells analysis via Raman spectroscopy enhanced by integrated plasmonic nanodimers.

    PubMed

    Perozziello, Gerardo; Candeloro, Patrizio; De Grazia, Antonio; Esposito, Francesco; Allione, Marco; Coluccio, Maria Laura; Tallerico, Rossana; Valpapuram, Immanuel; Tirinato, Luca; Das, Gobind; Giugni, Andrea; Torre, Bruno; Veltri, Pierangelo; Kruhne, Ulrich; Della Valle, Giuseppe; Di Fabrizio, Enzo

    2016-01-25

    In this work a Raman flow cytometer is presented. It consists of a microfluidic device that takes advantages of the basic principles of Raman spectroscopy and flow cytometry. The microfluidic device integrates calibrated microfluidic channels- where the cells can flow one-by-one -, allowing single cell Raman analysis. The microfluidic channel integrates plasmonic nanodimers in a fluidic trapping region. In this way it is possible to perform Enhanced Raman Spectroscopy on single cell. These allow a label-free analysis, providing information about the biochemical content of membrane and cytoplasm of the each cell. Experiments are performed on red blood cells (RBCs), peripheral blood lymphocytes (PBLs) and myelogenous leukemia tumor cells (K562).

  10. 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.

  11. 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.

  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. Characterization of Cell Lysis Events on a Microfluidic Device for High-Throughput Single Cell Analysis

    PubMed Central

    Hargis, Amy D; Alarie, JP; Ramsey, J.M.

    2012-01-01

    A microfluidic device capable of rapidly analyzing cells in a high-throughput fashion using electrical cell lysis is further characterized. In the experiments performed, cell lysis events were studied using an EMCCD camera with high frame rate (> 100 fps) data collection. It was found that, with this microfluidic design, the path that a cell follows through the electric field affects the amount of lysate injected into the analysis channel. Elimination of variable flow paths through the electric field was achieved by coating the analysis channel with a polyamine compound to reverse the electroosmotic flow (EOF). EOF reversal forced the cells to take the same path through the electric field. The improved control of the cell trajectory will reduce device-imposed bias on the analysis and maximizes the amount of lysate injected into the analysis channel for each cell, resulting in improved analyte detection capabilities. PMID:22025127

  14. 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.

  15. Cell electroporation by CNT-featured microfluidic chip.

    PubMed

    Shahini, Mehdi; Yeow, John T W

    2013-07-01

    We present the application of carbon nanotubes (CNTs) for cell electroporation that is performed in a microfluidic device. Lab on a chip (LOC) developments have raised unique possibilities to scale down cell manipulation systems to a cellular level to achieve higher performance and accuracy. Among the systems employed for cell disruption, electroporation without chemical reagents provides many advantages but suffers from high voltage requirements. We have exploited the electric field enhancement by CNTs to realize low-voltage electroporation. A microchip with embedded aligned CNTs has been developed to test the effect of the enhanced electric field on electroporation of mammalian CHO cells. Fluorogenic Calcein AM dye is used to image the release of the intercellular medium as an indication of electroporation. The electroporation phenomenon is recorded in real-time and compared with that of a device without CNTs. The results show that at a voltage as low as 3 volts, the electroporation yield rate is increased by 72% with the incorporation of CNTs. This enhancement is a promising advancement towards integration of low-voltage electroporation with other low-voltage cell manipulation techniques.

  16. Sorting drops and cells with acoustics: acoustic microfluidic fluorescence-activated cell sorter.

    PubMed

    Schmid, Lothar; Weitz, David A; Franke, Thomas

    2014-10-01

    We describe a versatile microfluidic fluorescence-activated cell sorter that uses acoustic actuation to sort cells or drops at ultra-high rates. Our acoustic sorter combines the advantages of traditional fluorescence-activated cell (FACS) and droplet sorting (FADS) and is applicable for a multitude of objects. We sort aqueous droplets, at rates as high as several kHz, into two or even more outlet channels. We can also sort cells directly from the medium without prior encapsulation into drops; we demonstrate this by sorting fluorescently labeled mouse melanoma cells in a single phase fluid. Our acoustic microfluidic FACS is compatible with standard cell sorting cytometers, yet, at the same time, enables a rich variety of more sophisticated applications.

  17. Cell separation by an aqueous two-phase system in a microfluidic device.

    PubMed

    Tsukamoto, Masatoshi; Taira, Shu; Yamamura, Shohei; Morita, Yasutaka; Nagatani, Naoki; Takamura, Yuzuru; Tamiya, Eiichi

    2009-10-01

    We generated an aqueous two-phase laminar flow in a microfluidic chip and used the system to isolate leukocyte and erythrocyte cells from whole blood cells. The microfluidic system reduced the effect of gravity in the aqueous two-phase system (ATPS). Poly(ethylene glycol) (PEG) and dextran (Dex) solutions were used as the two phases, and the independent flow rates of the solutions were both 2 microL/min. When hydrophobic and hydrophilic polystyrene beads were introduced into the microfluidic device, the hydrophilic beads moved to the Dex layer and the hydrophobic beads to the interface between the two phases. In the case of living cells, Jurkat cells and erythrocytes moved more efficiently to the PEG and Dex layers, respectively, than they move in a conventional ATPS. When whole blood cells were inserted into the microfluidic chip, leukocytes could be separated from erythrocytes because erythrocytes moved to the Dex layer while leukocytes remained outside of this layer in the microfluidic system. The reported microfluidic chip for the whole blood cell separation can effectively be integrated into a Micro Total Analysis System designed for cell-based clinical, forensic, and environmental analyses.

  18. Monitoring the differentiation and migration patterns of neural cells derived from human embryonic stem cells using a microfluidic culture system.

    PubMed

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-06-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.

  19. 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.

  20. 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.

  1. 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

  2. Microfluidics-based devices: New tools for studying cancer and cancer stem cell migration

    PubMed Central

    Huang, Yu; Agrawal, Basheal; Sun, Dandan; Kuo, John S.; Williams, Justin C.

    2011-01-01

    Cell movement is highly sensitive to stimuli from the extracellular matrix and media. Receptors on the plasma membrane in cells can activate signal transduction pathways that change the mechanical behavior of a cell by reorganizing motion-related organelles. Cancer cells change their migration mechanisms in response to different environments more robustly than noncancer cells. Therefore, therapeutic approaches to immobilize cancer cells via inhibition of the related signal transduction pathways rely on a better understanding of cell migration mechanisms. In recent years, engineers have been working with biologists to apply microfluidics technology to study cell migration. As opposed to conventional cultures on dishes, microfluidics deals with the manipulation of fluids that are geometrically constrained to a submillimeter scale. Such small scales offer a number of advantages including cost effectiveness, low consumption of reagents, high sensitivity, high spatiotemporal resolution, and laminar flow. Therefore, microfluidics has a potential as a new platform to study cell migration. In this review, we summarized recent progress on the application of microfluidics in cancer and other cell migration researches. These studies have enhanced our understanding of cell migration and cancer invasion as well as their responses to subtle variations in their microenvironment. We hope that this review will serve as an interdisciplinary guidance for both biologists and engineers as they further develop the microfluidic toolbox toward applications in cancer research. PMID:21522502

  3. Microfluidic platforms for generating dynamic environmental perturbations to study the responses of single yeast cells.

    PubMed

    Bisaria, Anjali; Hersen, Pascal; McClean, Megan N

    2014-01-01

    Microfluidic platforms are ideal for generating dynamic temporal and spatial perturbations in extracellular environments. Single cells and organisms can be trapped and maintained in microfluidic platforms for long periods of time while their responses to stimuli are measured using appropriate fluorescence reporters and time-lapse microscopy. Such platforms have been used to study problems as diverse as C. elegans olfaction (Chronis et al. Nature Methods 4:727-731, 2007), cancer cell migration (Huang et al. Biomicrofluidics 5:13412, 2011), and E. coli chemotaxis (Ahmed et al. Integr Biol 2:604-629, 2010). In this paper we describe how to construct and use a microfluidic chip to study the response of single yeast cells to dynamic perturbations of their fluid environment. The method involves creation of a photoresist master mold followed by subsequent creation of a polydimethylsiloxane (PDMS) microfluidic chip for maintaining live yeast cells in a channel with two inputs for stimulating the cells. We emphasize simplicity and the methods discussed here are accessible to the average biological laboratory. We cover the basic toolbox for making microfluidic lab-on-a-chip devices, and the techniques discussed serve as a starting point for creating sophisticated microfluidic devices capable of implementing more complicated experimental protocols.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. A new microfluidic device for electric lysis and separation of cells.

    PubMed

    Brun, M; Frénéa-Robin, M; Chateaux, J F; Haddour, N; Deman, A L; Ferrigno, R

    2012-01-01

    This paper demonstrates the potential use of a new microfluidic device embedding thick electrodes for cell lysis and cell separation applications. The system consists of a microfluidic channel featuring conductive walls made of a polydimethylsiloxane (PDMS) matrix mixed with carbon nanoparticles. Cell lysis was performed electrically by applying square pulses across the channel width, which was monitored by fluorimetry. Lysed and unlysed cells showed different dielectrophoretic behavior under appropriate experimental conditions, which suggests that the developed device is suitable to perform both cell lysis and subsequent sorting of viable and dead cells. PMID:23367365

  9. 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

  10. Microfluidic biofunctionalisation protocols to form multi-valent interactions for cell rolling and phenotype modification investigations.

    PubMed

    Perozziello, Gerardo; Simone, Giuseppina; Malara, Natalia; La Rocca, Rosanna; Tallerico, Rossana; Catalano, Rossella; Pardeo, Francesca; Candeloro, Patrizio; Cuda, Giovanni; Carbone, Ennio; Di Fabrizio, Enzo

    2013-07-01

    In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro-fluidic protocols. Many available processes either require expensive and time-consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi-valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin-biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 μL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC-I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC-I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC-I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC-I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. A microfluidic positioning chamber for long-term live-cell imaging

    PubMed Central

    Hanson, Lindsey; Cui, Lifeng; Xie, Chong; Cui, Bianxiao

    2010-01-01

    We report a microfluidic positioning chamber (MPC) that can rapidly and repeatedly relocate the same imaging area on a microscope stage. The “roof” of the microfluidic chamber was printed with serials of coordinate numbers that act as positioning marks for mammalian cells that grow attached to the “floor” of the microfluidic chamber. MPC cell culture chamber provided a simple solution for tracking the same cell or groups of cells over days or weeks. The positioning marks were used to register time-lapse images of the same imaging area to single-pixel accuracy. Using MPC cell culture chamber, we tracked the migration, division and differentiation of individual PC12 cells for over a week using bright field and fluorescence imaging. PMID:20936672

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. Continuous cell electroporation for efficient DNA and siRNA delivery based on laminar microfluidic chips.

    PubMed

    Wei, Zewen; Li, Zhihong

    2014-01-01

    Electroporation is a high-efficiency and low-toxicity physical gene transfer method. Traditional electroporation is limited to only low volume cell samples. Here we present a continuous cell electroporation method based on commonly used microfluidic chip fabrication technology. Using easily fabricated PDMS microfluidic chip, syringe pumps, and pulse generator, we show efficient delivery of both DNA and siRNA into different cell lines. We describe the protocol of chip fabrication, apparatus setup, and cell electroporation assay. Typically, the fabrication of the devices takes 1 or 2 days and the continuous electroporation assay takes 1 h.

  2. 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

  3. 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

  4. 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.

  5. Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device.

    PubMed

    Matsumura, Taku; Tatsumi, Kazuya; Noda, Yuichiro; Nakanishi, Naoyuki; Okonogi, Atsuhito; Hirano, Kunio; Li, Liu; Osumi, Takashi; Tada, Takashi; Kotera, Hidetoshi

    2014-10-10

    The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.

  6. Using Living Radical Polymerization to Enable Facile Incorporation of Materials in Microfluidic Cell Culture Devices

    PubMed Central

    Simms, Helen M.; Bowman, Christopher M.; Anseth, Kristi S.

    2008-01-01

    High throughput screening tools are expediting cell culture studies with applications in drug discovery and tissue engineering. This contribution demonstrates a method to incorporate 3D cell culture sites into microfluidic devices and enables the fabrication of high throughput screening tools with uniquely addressable culture environments. Contact Lithographic Photopolymerization (CLiPP) was used to fabricate microfluidic devices with two types of 3D culture sites: macroporous rigid polymer cell scaffolds and poly(ethylene glycol) (PEG) encapsulated cell matrices. Cells were cultured on-device with both types of culture sites, demonstrating material cytocompatibility. Multilayer microfluidic devices were fabricated with channels passing the top and bottom sides of a series of rigid porous polymer scaffolds. Cells were seeded and cultured on-device, demonstrating the ability to deliver cells and culture cells on multiple scaffolds along the length of a single channel. Flow control through these rigid porous polymer scaffolds was demonstrated. Finally, devices were modified by grafting of PEG methacrylate from surfaces to prevent non-specific protein adsorption and ultimately cell adhesion to channel surfaces. The living radical component of this CLiPP device fabrication platform enables facile incorporation of 3D culture sites into microfluidic cell culture devices, which can be utilized for high throughput screening of cell material interactions. PMID:18294686

  7. Microfluidic platform to evaluate migration of cells from patients with DYT1 dystonia

    PubMed Central

    Kim, Edward Y.; Hettich, Jasmin; Mempel, Thorsten R.; Breakefield, Xandra O.; Irimia, Daniel

    2014-01-01

    Background Microfluidic platforms for quantitative evaluation of cell biologic processes allow low cost and time efficient research studies of biological and pathological events, such as monitoring cell migration by real-time imaging. In healthy and disease states, cell migration is crucial in development and wound healing, as well as to maintain the body's homeostasis. New Method The microfluidic chambers allow precise measurements to investigate whether fibroblasts carrying a mutation in the TOR1A gene, underlying the hereditary neurologic disease - DYT1 dystonia, have decreased migration properties when compared to control cells. Results We observed that fibroblasts from DYT1 patients showed abnormalities in basic features of cell migration, such as reduced velocity and persistence of movement. Comparison with Existing Method The microfluidic method enabled us to demonstrate reduced polarization of the nucleus and abnormal orientation of nuclei and Golgi inside the moving DYT1 patient cells compared to control cells, as well as vectorial movement of single cells. Conclusion We report here different assays useful in determining various parameters of cell migration in DYT1 patient cells as a consequence of the TOR1A gene mutation, including a microfluidic platform, which provides a means to evaluate real-time vectorial movement with single cell resolution in a three-dimensional environment. PMID:24880044

  8. Feedback control system simulator for the control of biological cells in microfluidic cross slots and integrated microfluidic systems.

    PubMed

    Curtis, Michael D; Sheard, Gregory J; Fouras, Andreas

    2011-07-21

    Control systems for lab on chip devices require careful characterisation and design for optimal performance. Traditionally, this involves either extremely computationally expensive simulations or lengthy iteration of laboratory experiments, prototype design, and manufacture. In this paper, an efficient control simulation technique, valid for typical microchannels, Computed Interpolated Flow Hydrodynamics (CIFH), is described that is over 500 times faster than conventional time integration techniques. CIFH is a hybrid approach, utilising a combination of pre-computed flows and hydrodynamic equations and allows the efficient simulation of dynamic control systems for the transport of cells through micro-fluidic devices. The speed-ups achieved by using pre-computed CFD solutions mapped to an n-dimensional control parameter space, significantly accelerate the evaluation and improvement of control strategies and chip design. Here, control strategies for a naturally unstable device geometry, the microfluidic cross-slot, have been simulated and optimal parameters have been found for proposed devices capable of trapping and sorting cells. PMID:21611664

  9. 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

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

    PubMed

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

    2013-05-01

    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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. A zero-flow microfluidics for long-term cell culture and detection

    NASA Astrophysics Data System (ADS)

    Sang, Shengbo; Tang, Xiaoliang; Feng, Qiliang; Jian, Aoqun; Zhang, Wendong

    2015-04-01

    A zero-flow microfluidic design is proposed in this paper, which can be used for long-term cell culture and detection, especially for a lab-on-chip integrated with a biosensor. It consists of two parts: a main microchannel; and a circle microchamber. The Finite Element Method (FEM) was employed to predict the fluid transport properties for a minimum fluid flow disturbance. Some commonly used microfluidic structures were also analysed systematically to prove the designed structure. Then the designed microfluidics was fabricated. Based on the simulations and experiments, this design provides a continuous flow environment, with a relatively stable and low shear stress atmosphere, similar to a zero-flow environment. Furthermore, the nutrients maintaining cells' normal growth can be taken into the chamber through the diffusion effect. It also proves that the microfluidics can realize long-term cell culture and detection. The application of the structure in the field of biological microelectromechenical systems (BioMEMS) will provide a research foundation for microfluidic technology.

  17. Optically clear alginate hydrogels for spatially controlled cell entrapment and culture at microfluidic electrode surfaces.

    PubMed

    Betz, Jordan F; Cheng, Yi; Tsao, Chen-Yu; Zargar, Amin; Wu, Hsuan-Chen; Luo, Xiaolong; Payne, Gregory F; Bentley, William E; Rubloff, Gary W

    2013-05-21

    We describe an innovation in the immobilization, culture, and imaging of cells in calcium alginate within microfluidic devices. This technique allows unprecedented optical access to the entirety of the calcium alginate hydrogel, enabling observation of growth and behavior in a chemical and mechanical environment favored by many kinds of cells.

  18. A microfluidic device to acquire high-magnification microphotographs of yeast cells

    PubMed Central

    Ohnuki, Shinsuke; Nogami, Satoru; Ohya, Yoshikazu

    2009-01-01

    Background Yeast cell morphology was investigated to reveal the molecular mechanisms of cell morphogenesis and to identify key factors of other processes such as cell cycle progression. We recently developed a semi-automatic image processing program called CalMorph, which allows us to quantitatively analyze yeast cell morphology with the 501 parameters as biological traits and uncover statistical relationships between cell morphological phenotypes and genotypes. However, the current semi-automatic method is not suitable for morphological analysis of large-scale yeast mutants for the reliable prediction of gene functions because of its low-throughput especially at the manual image-acquiring process. Results In this study, we developed a microfluidic chip designed to acquire successive microscopic images of yeast cells suitable for CalMorph image analysis. With the microfluidic chip, the morphology of living cells and morphological changes that occur during the cell cycle were successfully characterized. Conclusion The microfluidic chip enabled us to acquire the images faster than the conventional method. We speculate that the use of microfluidic chip is effective in acquiring images of large-scale for automated analysis of yeast strains. PMID:19317904

  19. 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

  20. 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…

  1. 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

  2. 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

  3. 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

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

    PubMed

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

    2011-02-15

    Microfluidic cell-based systems have enabled the study of cellular phenomena with improved spatiotemporal control of the microenvironment and at increased throughput. While poly(dimethylsiloxane) (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 lithographic 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 was validated for biological function in two different cell-based applications to demonstrate the versatility of our streamlined fabrication process.

  5. 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

  6. Development of a new microfluidic platform integrating co-cultures of intestinal and liver cell lines.

    PubMed

    Bricks, Thibault; Paullier, Patrick; Legendre, Audrey; Fleury, Marie-José; Zeller, Perrine; Merlier, Franck; Anton, Pauline M; Leclerc, Eric

    2014-08-01

    We developed a new biological model to mimic the organ-organ interactions between the intestine and the liver. We coupled polycarbonate cell culture inserts and microfluidic biochips in an integrated fluidic platform allowing dynamic co-cultures (called IIDMP for Integrated Insert in a Dynamic Microfluidic Platform). The intestinal compartment was simulated using Caco-2 TC7 cells and the liver one by HepG2 C3A. We showed that Caco-2 TC7 viability, barrier integrity and functionality (assessed by paracellular and active transport), were not altered during co-cultures in the bioreactor in comparison with the conventional insert Petri cultures. In parallel, the viability and metabolism of the HepG2 C3A cells were maintained in the microfluidic biochips. Then, as proof of concept, we used the bioreactor to follow the transport of phenacetin through the intestinal barrier and its metabolism into paracetamol by the CYP1A of the HepG2 C3A cells. Our results demonstrated the performance of this bioreactor with cell co-cultures compared to static co-culture controls in which weak biotransformation into paracetamol was detected. Our study illustrated the interest of such a bioreactor combining the advantages of a cell culture barrier and of liver microfluidic cultures in a common framework for in vitro studies.

  7. 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

  8. Detecting transforming growth factor-β release from liver cells using an aptasensor integrated with microfluidics.

    PubMed

    Matharu, Zimple; Patel, Dipali; Gao, Yandong; Haque, Amranul; Zhou, Qing; Revzin, Alexander

    2014-09-01

    We developed a cell-culture/biosensor platform consisting of aptamer-modified Au electrodes integrated with reconfigurable microfluidics for monitoring of transforming growth factor-beta 1 (TGF-β1), an important inflammatory and pro-fibrotic cytokine. Aptamers were thiolated, labeled with redox reporters, and self-assembled on gold surfaces. The biosensor was determined to be specific for TGF-β1 with an experimental detection limit of 1 ng/mL and linear range extending to 250 ng/mL. Upon determining figures of merit, aptasensor was miniaturized and integrated with human hepatic stellate cells inside microfluidic devices. Reconfigurable microfluidics were developed to ensure that seeding of "sticky" stromal cells did not foul the electrode and compromise sensor performance. This microsystem with integrated aptasensors was used to monitor TGF-β1 release from activated stellate cells over the course of 20 h. The electrochemical response went down upon infusing anti-TGF-β1 antibodies into the microfluidic devices containing activated stellate cells. To further validate aptasensor responses, stellate cells were stained for markers of activation (e.g., alpha smooth muscle actin) and were also tested for presence of TGF-β1 using enzyme linked immunosorbent assay (ELISA). Given the importance of TGF-β1 as a fibrogenic signal, a microsystem with integrated biosensors for local and continuous detection of TGF-β1 may prove to be an important tool to study fibrosis of the liver and other organs.

  9. Sonoporation of suspension cells with a single cavitation bubble in a microfluidic confinement.

    PubMed

    Gac, Séverine Le; Zwaan, Ed; van den Berg, Albert; Ohl, Claus-Dieter

    2007-12-01

    We report here the sonoporation of HL60 (human promyelocytic leukemia) suspension cells in a microfluidic confinement using a single laser-induced cavitation bubble. Cavitation bubbles can induce membrane poration of cells located in their close vicinity. Membrane integrity of suspension cells placed in a microfluidic chamber is probed through either the calcein release out of calcein-loaded cells or the uptake of trypan blue. Cells that are located farther away than four times Rmax (maximum bubble radius) from the cavitation bubble center remain fully unaffected, while cells closer than 0.75 Rmax become porated with a probability of >75%. These results enable us to define a distance of 0.75 Rmax as a critical interaction distance of the cavitation bubble with HL60 suspension cells. These experiments suggest that flow-induced poration of suspension cells is applicable in lab-on-a-chip systems, and this might be an interesting alternative to electroporation.

  10. 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

  11. 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.

  12. 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.

  13. Droplet-based microfluidic platforms for single T cell secretion analysis of IL-10 cytokine.

    PubMed

    Konry, Tania; Dominguez-Villar, Margarita; Baecher-Allan, Clare; Hafler, David A; Yarmush, Martin L

    2011-01-15

    Here we present a microfluidic method for the analysis of single cell secretions. The method co-encapsulates cells with microspheres conjugated with capture antibodies and detection fluorescence-labeled antibodies. The secreted substance captured on the microsphere surface and detected via detection antibodies generating a localized fluorescent signal on a microsphere surface. Using this method, CD4+CD25+ regulatory T cells were encapsulated and assayed to detect IL-10 secreting cell in population.

  14. Rapid determination of cell mass and density using digitally controlled electric field in a microfluidic chip.

    PubMed

    Zhao, Yuliang; Lai, Hok Sum Sam; Zhang, Guanglie; Lee, Gwo-Bin; Li, Wen Jung

    2014-11-21

    The density of a single cell is a fundamental property of cells. Cells in the same cycle phase have similar volume, but the differences in their mass and density could elucidate each cell's physiological state. Here we report a novel technique to rapidly measure the density and mass of a single cell using an optically induced electrokinetics (OEK) microfluidic platform. Presently, single cellular mass and density measurement devices require a complicated fabrication process and their output is not scalable, i.e., it is extremely difficult to measure the mass and density of a large quantity of cells rapidly. The technique reported here operates on a principle combining sedimentation theory, computer vision, and microparticle manipulation techniques in an OEK microfluidic platform. We will show in this paper that this technique enables the measurement of single-cell volume, density, and mass rapidly and accurately in a repeatable manner. The technique is also scalable - it allows simultaneous measurement of volume, density, and mass of multiple cells. Essentially, a simple time-controlled projected light pattern is used to illuminate the selected area on the OEK microfluidic chip that contains cells to lift the cells to a particular height above the chip's surface. Then, the cells are allowed to "free fall" to the chip's surface, with competing buoyancy, gravitational, and fluidic drag forces acting on the cells. By using a computer vision algorithm to accurately track the motion of the cells and then relate the cells' motion trajectory to sedimentation theory, the volume, mass, and density of each cell can be rapidly determined. A theoretical model of micro-sized spheres settling towards an infinite plane in a microfluidic environment is first derived and validated experimentally using standard micropolystyrene beads to demonstrate the viability and accuracy of this new technique. Next, we show that the yeast cell volume, mass, and density could be rapidly

  15. A modular cell culture device for generating arrays of gradients using stacked microfluidic flows

    PubMed Central

    Sip, Christopher G.; Bhattacharjee, Nirveek; Folch, Albert

    2011-01-01

    Microfluidics has become increasingly important for the study of biochemical cues because it enables exquisite spatiotemporal control of the microenvironment. Well-characterized, stable, and reproducible generation of biochemical gradients is critical for understanding the complex behaviors involved in many biological phenomena. Although many microfluidic devices have been developed which achieve these criteria, the ongoing challenge for these platforms is to provide a suitably benign and physiologically relevant environment for cell culture in a user-friendly format. To achieve this paradigm, microfluidic designs must consider the full scope of cell culture from substrate preparation, cell seeding, and long-term maintenance to properly observe gradient sensing behavior. In addition, designs must address the challenges associated with altered culture conditions and shear forces in flow-based devices. With this consideration, we have designed and characterized a microfluidic device based on the principle of stacked flows to achieve highly stable gradients of diffusible molecules over large areas with extremely low shear forces. The device utilizes a benign vacuum sealing strategy for reversible application to pre-established cell cultures. We apply this device to an existing culture of breast cancer cells to demonstrate the negligible effect of its shear flow on migratory behavior. Lastly, we extend the stacked-flow design to demonstrate its scalable architecture with a prototype device for generating an array of combinatorial gradients. PMID:21799716

  16. A microfluidic platform for chemical stimulation and real time analysis of catecholamine secretion from neuroendocrine cells

    PubMed Central

    Ges, Igor A.; Brindley, Rebecca L.; Currie, Kevin P.M.; Baudenbacher, Franz J.

    2013-01-01

    Release of neurotransmitters and hormones by calcium-regulated exocytosis is a fundamental cellular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. As such, there is significant interest in targeting neurosecretion for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistic insight coupled with increased experimental throughput. Here, we report a simple, inexpensive, reusable, microfluidic device designed to analyze catecholamine secretion from small populations of adrenal chromaffin cells in real time, an important neuroendocrine component of the sympathetic nervous system and versatile neurosecretory model. The device is fabricated by replica molding of polydimethylsiloxane (PDMS) using patterned photoresist on silicon wafer as the master. Microfluidic inlet channels lead to an array of U-shaped “cell traps”, each capable of immobilizing single or small groups of chromaffin cells. The bottom of the device is a glass slide with patterned thin film platinum electrodes used for electrochemical detection of catecholamines in real time. We demonstrate reliable loading of the device with small populations of chromaffin cells, and perfusion / repetitive stimulation with physiologically relevant secretagogues (carbachol, PACAP, KCl) using the microfluidic network. Evoked catecholamine secretion was reproducible over multiple rounds of stimulation, and graded as expected to different concentrations of secretagogue or removal of extracellular calcium. Overall, we show this microfluidic device can be used to implement complex stimulation paradigms and analyze the amount and kinetics of catecholamine secretion from small populations of neuroendocrine cells in real time. PMID:24126415

  17. 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

  18. 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

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

    PubMed Central

    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

  20. A Nonlinear Size-Dependent Equivalent Circuit Model for Single-Cell Electroporation on Microfluidic Chips.

    PubMed

    Shagoshtasbi, Hooman; Deng, Peigang; Lee, Yi-Kuen

    2015-08-01

    Electroporation (EP) is a process of applying a pulsed intense electric field on the cell membrane to temporarily induce nanoscale electropores on the plasma membrane of biological cells. A nonlinear size-dependent equivalent circuit model of a single-cell electroporation system is proposed to investigate dynamic electromechanical behavior of cells on microfluidic chips during EP. This model consists of size-dependent electromechanical components of a cell, electrical components of poration media, and a microfluidic chip. A single-cell microfluidic EP chip with 3D microelectrode arrays along a microchannel is designed and fabricated to experimentally analyze the permeabilization of a cell. Predicted electrical current responses of the model are in good agreement (average error of 6%) with that of single-cell EP. The proposed model can successfully predict the time responses of transmembrane voltage, pore diameter, and pore density at four different stages of permeabilization. These stages are categorized based on electromechanical changes of the lipid membrane. The current-voltage characteristic curve of the cell membrane during EP is also investigated at different EP stages in detail. The model can precisely predict the electric breakdown of different cell lines at a specific critical cell membrane voltage of the target cell lines. PMID:25735616

  1. A Nonlinear Size-Dependent Equivalent Circuit Model for Single-Cell Electroporation on Microfluidic Chips.

    PubMed

    Shagoshtasbi, Hooman; Deng, Peigang; Lee, Yi-Kuen

    2015-08-01

    Electroporation (EP) is a process of applying a pulsed intense electric field on the cell membrane to temporarily induce nanoscale electropores on the plasma membrane of biological cells. A nonlinear size-dependent equivalent circuit model of a single-cell electroporation system is proposed to investigate dynamic electromechanical behavior of cells on microfluidic chips during EP. This model consists of size-dependent electromechanical components of a cell, electrical components of poration media, and a microfluidic chip. A single-cell microfluidic EP chip with 3D microelectrode arrays along a microchannel is designed and fabricated to experimentally analyze the permeabilization of a cell. Predicted electrical current responses of the model are in good agreement (average error of 6%) with that of single-cell EP. The proposed model can successfully predict the time responses of transmembrane voltage, pore diameter, and pore density at four different stages of permeabilization. These stages are categorized based on electromechanical changes of the lipid membrane. The current-voltage characteristic curve of the cell membrane during EP is also investigated at different EP stages in detail. The model can precisely predict the electric breakdown of different cell lines at a specific critical cell membrane voltage of the target cell lines.

  2. Use of Microfluidic Technology to Monitor the Differentiation and Migration of Human ESC-Derived Neural Cells.

    PubMed

    Bae, Jiwoo; Lee, Nayeon; Choi, Wankyu; Lee, Suji; Ko, Jung Jae; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2016-01-01

    Microfluidics forms the basis of unique experimental approaches that visualize the development of neural structure using micro-scale devices and aids the guidance of neurite growth in an axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stems cells (hESC). We cocultured hESC with PA6 stromal cells and isolated neural rosette-like structures, which subsequently formed neurospheres in a suspension culture. We found that Tuj1-positive neural cells but not nestin-positive neural precursor cells (NPC) were able to enter the microfluidics grooves (microchannels), suggesting a neural cell-migratory capacity that was dependent on neuronal differentiation. We also showed that bundles of axons formed and extended into the microchannels.Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells. PMID:27062598

  3. 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.

  4. 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,

    2016-07-12

    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.

  5. 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)

    SciTech Connect

    Patel, Kamlesh D; SNL,

    2012-06-01

    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.

  6. Microfluidic sieve valves

    DOEpatents

    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.

  7. Trends in the development of microfluidic cell biochips for in vitro hepatotoxicity.

    PubMed

    Baudoin, Régis; Corlu, Anne; Griscom, Laurent; Legallais, Cécile; Leclerc, Eric

    2007-06-01

    Current developments in the technological fields of liver tissue engineering, bioengineering, biomechanics, microfabrication and microfluidics have lead to highly complex and pertinent new tools called "cell biochips" for in vitro toxicology. The purpose of "cell biochips" is to mimic organ tissues in vitro in order to partially reduce the amount of in vivo testing. These "cell biochips" consist of microchambers containing engineered tissue and living cell cultures interconnected by a microfluidic network, which allows the control of microfluidic flows for dynamic cultures, by continuous feeding of nutrients to cultured cells and waste removal. Cell biochips also allow the control of physiological contact times of diluted molecules with the tissues and cells, for rapid testing of sample preparations or specific addressing. Cell biochips can be situated between in vitro and in vivo testing. These types of systems can enhance functionality of cells by mimicking the tissue architecture complexities when compared to in vitro analysis but at the same time present a more rapid and simple process when compared to in vivo testing procedures. In this paper, we first introduce the concepts of microfluidic and biochip systems based on recent progress in microfabrication techniques used to mimic liver tissue in vitro. This includes progress and understanding in biomaterials science (cell culture substrate), biomechanics (dynamic cultures conditions) and biology (tissue engineering). The development of new "cell biochips" for chronic toxicology analysis of engineered tissues can be achieved through the combination of these research domains. Combining these advanced research domains, we then present "cell biochips" that allow liver chronic toxicity analysis in vitro on engineered tissues. An extension of the "cell biochip" idea has also allowed "organ interactions on chip", which can be considered as a first step towards the replacement of animal testing using a combined liver

  8. A microfluidic systems biology approach for live single-cell mitochondrial ROS imaging.

    PubMed

    Kniss, Ariel; Lu, Hang; Jones, Dean P; Kemp, Melissa L

    2013-01-01

    Most current studies of reactive oxygen species (ROS) production report globally averaged measurements within the cell; however, ROS can be produced in distinct subcellular locations and have local effects in their immediate vicinity. A microfluidic platform for high-throughput single-cell imaging allows mitochondrial ROS production to be monitored as varying in both space and time. Using this systems biology approach, single-cell variability can be viewed within a population. We discuss single-cell monitoring of contributors to mitochondrial redox state-mitochondrial hydrogen peroxide or superoxide-through the use of a small molecule probe or targeted fluorescent reporter protein. Jurkat T lymphoma cells were stimulated with antimycin A and imaged in an arrayed microfluidic device over time. Differences in single-cell responses were observed as a function of both inhibitor concentration and type of ROS measurement used.

  9. Hydrogel microfluidics for the patterning of pluripotent stem cells.

    PubMed

    Cosson, S; Lutolf, M P

    2014-01-01

    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. PMID:24662945

  10. 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

  11. Optical reprogramming of human cells in an ultrashort femtosecond laser microfluidic transfection platform.

    PubMed

    Uchugonova, Aisada; Breunig, Hans Georg; Batista, Ana; König, Karsten

    2016-09-01

    Induced pluripotent stem cell (iPS cell) technology can be used to produce unlimited numbers of functional cells for both research and therapeutic purposes without ethical controversy. Typically, viruses are applied for efficient intracellular delivery of genes/transcription factors to generate iPS cells. However, the viral genomic integration may cause a risk of mutation as well as tumor formation therefore limits its clinical application. Here we demonstrate that spatially shaped extreme ultrashort laser pulses of sub-20 femtoseconds induce transient membrane permeabilisation which enables contamination-free transfection of cells in a microfluidic tube with multiple genes at the individual cell level in order to achieve optical reprogramming of large cell populations. We found that the ultrashort femtosecond laser-microfluidic cell transfection platform enhanced the efficacy of iPS-like colony-forming following merely a single transfection. Illustration of the spatially shaped femtosecond laser-assisted microfluidic cell transfection platform for production of iPS cell colonies. PMID:26530487

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

    PubMed

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

    2014-11-15

    Lab on a chip (LOC) systems provide interesting and low-cost solutions for key studies and applications in the biomedical field. Along with microfluidics, these microdevices make single-cell manipulation possible with high spatial and temporal resolution. In this work we have designed, fabricated and characterized a versatile and inexpensive microfluidic platform for on-chip selective single-cell trapping and treatment using laminar co-flow. The combination of co-existing laminar flow manipulation and hydrodynamic single-cell trapping for selective treatment offers a cost-effective solution for studying the effect of novel drugs on single-cells. The operation of the whole system is experimentally simple, highly adaptable and requires no specific equipment. As a proof of concept, a cytotoxicity study of ethanol in isolated hepatocytes is presented. The developed microfluidic platform controlled by means of co-flow is an attractive and multipurpose solution for the study of new substances of high interest in cell biology research. In addition, this platform will pave the way for the study of cell behavior under dynamic and controllable fluidic conditions providing information at the individual cell level. Thus, this analysis device could also hold a great potential to easily use the trapped cells as sensing elements expanding its functionalities as a cell-based biosensor with single-cell resolution. PMID:24907537

  13. 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

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

    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.

  15. 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.

  16. Clonal analysis of individual human embryonic stem cell differentiation patterns in microfluidic cultures.

    PubMed

    Sikorski, Darek J; Caron, Nicolas J; VanInsberghe, Michael; Zahn, Hans; Eaves, Connie J; Piret, James M; Hansen, Carl L

    2015-10-01

    Heterogeneity in the clonal outputs of individual human embryonic stem cells (hESCs) confounds analysis of their properties in studies of bulk populations and how to manipulate them for clinical applications. To circumvent this problem we developed a microfluidic device that supports the robust generation of colonies derived from single ESCs. This microfluidic system contains 160 individually addressable chambers equipped for perfusion culture of individual hESCs that could be shown to match the growth rates, marker expression and colony morphologies obtained in conventional cultures. Use of this microfluidic device to analyze the clonal growth kinetics of multiple individual hESCs induced to differentiation revealed variable shifts in the growth rate, area per cell and expression of OCT4 in the progeny of individual hESCs. Interestingly, low OCT4 expression, a slower growth rate and low nuclear to cytoplasmic ratios were found to be correlated responses. This study demonstrates how microfluidic systems can be used to enable large scale live-cell imaging of isolated hESCs exposed to changing culture conditions, to examine how different aspects of their variable responses are correlated. PMID:26059045

  17. Microfluidic perfusion culture of human induced pluripotent stem cells under fully defined culture conditions.

    PubMed

    Yoshimitsu, Ryosuke; Hattori, Koji; Sugiura, Shinji; Kondo, Yuki; Yamada, Rotaro; Tachikawa, Saoko; Satoh, Taku; Kurisaki, Akira; Ohnuma, Kiyoshi; Asashima, Makoto; Kanamori, Toshiyuki

    2014-05-01

    Human induced pluripotent stem cells (hiPSCs) are a promising cell source for drug screening. For this application, self-renewal or differentiation of the cells is required, and undefined factors in the culture conditions are not desirable. Microfluidic perfusion culture allows the production of small volume cultures with precisely controlled microenvironments, and is applicable to high-throughput cellular environment screening. Here, we developed a microfluidic perfusion culture system for hiPSCs that uses a microchamber array chip under defined extracellular matrix (ECM) and culture medium conditions. By screening various ECMs we determined that fibronectin and laminin are appropriate for microfluidic devices made out of the most popular material, polydimethylsiloxane (PDMS). We found that the growth rate of hiPSCs under pressure-driven perfusion culture conditions was higher than under static culture conditions in the microchamber array. We applied our new system to self-renewal and differentiation cultures of hiPSCs, and immunocytochemical analysis showed that the state of the hiPSCs was successfully controlled. The effects of three antitumor drugs on hiPSCs were comparable between microchamber array and 96-well plates. We believe that our system will be a platform technology for future large-scale screening of fully defined conditions for differentiation cultures on integrated microfluidic devices.

  18. 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

  19. 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.

  20. Microfluidic chip system for the selection and enrichment of cell binding aptamers

    PubMed Central

    Stoll, Heidi; Kiessling, Heiko; Stelzle, Martin; Wendel, Hans Peter; Schütte, Julia; Hagmeyer, Britta; Avci-Adali, Meltem

    2015-01-01

    Aptamers are promising cell targeting ligands for several applications such as for the diagnosis, therapy, and drug delivery. Especially, in the field of regenerative medicine, stem cell specific aptamers have an enormous potential. Using the combinatorial chemistry process SELEX (Systematic Evolution of Ligands by Exponential enrichment), aptamers are selected from a huge oligonucleotide library consisting of approximately 1015 different oligonucleotides. Here, we developed a microfluidic chip system that can be used for the selection of cell specific aptamers. The major drawbacks of common cell-SELEX methods are the inefficient elimination of the unspecifically bound oligonucleotides from the cell surface and the unspecific binding/uptake of oligonucleotides by dead cells. To overcome these obstacles, a microfluidic device, which enables the simultaneous performance of dielectrophoresis and electrophoresis in the same device, was designed. Using this system, viable cells can be selectively assembled by dielectrophoresis between the electrodes and then incubated with the oligonucleotides. To reduce the rate of unspecifically bound sequences, electrophoretic fields can be applied in order to draw loosely bound oligonucleotides away from the cells. Furthermore, by increasing the flow rate in the chip during the iterative rounds of SELEX, the selection pressure can be improved and aptamers with higher affinities and specificities can be obtained. This new microfluidic device has a tremendous capability to improve the cell-SELEX procedure and to select highly specific aptamers. PMID:26180568

  1. 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

  2. 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

  3. 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

  4. Microfluidic Platform for Studying Chemotaxis of Adhesive Cells Revealed a Gradient-Dependent Migration and Acceleration of Cancer Stem Cells.

    PubMed

    Zou, Heng; Yue, Wanqing; Yu, Wai-Kin; Liu, Dandan; Fong, Chi-Chun; Zhao, Jianlong; Yang, Mengsu

    2015-07-21

    Recent studies reveal that solid tumors consist of heterogeneous cells with distinct phenotypes and functions. However, it is unclear how different subtypes of cancer cells migrate under chemotaxis. Here, we developed a microfluidic device capable of generating multiple stable gradients, culturing cells on-chip, and monitoring single cell migratory behavior. The microfluidic platform was used to study gradient-induced chemotaxis of lung cancer stem cell (LCSC) and differentiated LCSC (dLCSC) in real time. Our results showed the dynamic and differential response of both LCSC and dLCSC to chemotaxis, which was regulated by the β-catenin dependent Wnt signaling pathway. The microfluidic analysis showed that LCSC and dLCSC from the same origin behaved differently in the same external stimuli, suggesting the importance of cancer cell heterogeneity. We also observed for the first time the acceleration of both LCSC and dLCSC during chemotaxis caused by increasing local concentration in different gradients, which could only be realized through the microfluidic approach. The capability to analyze single cell chemotaxis under spatially controlled conditions provides a novel analytical platform for the study of cellular microenvironments and cancer cell metastasis.

  5. Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe.

    PubMed

    Kashyap, Aditya; Cors, Julien F; Lovchik, Robert D; Kaigala, Govind V

    2016-01-01

    The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies. PMID:27685165

  6. 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

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

    PubMed

    Sun, Yung-Shin

    2016-06-15

    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.

  8. 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.

  9. 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

  10. Microfluidic perfusion culture.

    PubMed

    Hattori, Koji; Sugiura, Shinji; Kanamori, Toshiyuki

    2014-01-01

    Microfluidic perfusion culture is a novel technique to culture animal cells in a small-scale microchamber with medium perfusion. Polydimethylsiloxane (PDMS) is the most popular material to fabricate a microfluidic perfusion culture chip. Photolithography and replica molding techniques are generally used for fabrication of a microfluidic perfusion culture chip. Pressure-driven perfusion culture system is convenient technique to carry out the perfusion culture of animal cells in a microfluidic device. Here, we describe a general theory on microfluid network design, microfabrication technique, and experimental technique for pressure-driven perfusion culture in an 8 × 8 microchamber array on a glass slide-sized microchip made out of PDMS. PMID:24297421

  11. 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

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

    PubMed

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

    2013-06-21

    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

  13. 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.

  14. 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.

  15. 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.

  16. Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

    PubMed

    Park, Jae Woo; Na, Sang Cheol; Nguyen, Thanh Qua; Paik, Sang-Min; Kang, Myeongwoo; Hong, Daewha; Choi, Insung S; Lee, Jae-Hyeok; Jeon, Noo Li

    2015-03-01

    This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the "biological glue." We take advantage of hydroxyproline (Hyp)-based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm(2) . Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable. PMID:25220860

  17. 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.

  18. 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

  19. 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

  20. A microfluidic platform for probing single cell plasma membranes using optically trapped Smart Droplet Microtools (SDMs).

    PubMed

    Lanigan, Peter M P; Ninkovic, Tanja; Chan, Karen; de Mello, Andrew J; Willison, Keith R; Klug, David R; Templer, Richard H; Neil, Mark A A; Ces, Oscar

    2009-04-21

    We recently introduced a novel platform based upon optically trapped lipid coated oil droplets (Smart Droplet Microtools-SDMs) that were able to form membrane tethers upon fusion with the plasma membrane of single cells. Material transfer from the plasma membrane to the droplet via the tether was seen to occur. Here we present a customised version of the SDM approach based upon detergent coated droplets deployed within a microfluidic format. These droplets are able to differentially solubilise the plasma membrane of single cells with spatial selectivity and without forming membrane tethers. The microfluidic format facilitates separation of the target cells from the bulk SDM population and from downstream analysis modules. Material transfer from the cell to the SDM was monitored by tracking membrane localized EGFP.

  1. 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

  2. Enhancement of renal epithelial cell functions through microfluidic-based coculture with adipose-derived stem cells.

    PubMed

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

    2013-09-01

    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

  3. 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.

  4. 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

  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. 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

  7. 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

    2016-07-12

    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. 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

  9. 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.

  10. 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%.

  11. 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.

  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. 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

  14. An integrated microfluidic chip system for single-cell secretion profiling of rare circulating tumor cells.

    PubMed

    Deng, Yuliang; Zhang, Yu; Sun, Shuai; Wang, Zhihua; Wang, Minjiao; Yu, Beiqin; Czajkowsky, Daniel M; Liu, Bingya; Li, Yan; Wei, Wei; Shi, Qihui

    2014-12-16

    Genetic and transcriptional profiling, as well as surface marker identification of single circulating tumor cells (CTCs) have been demonstrated. However, quantitatively profiling of functional proteins at single CTC resolution has not yet been achieved, owing to the limited purity of the isolated CTC populations and a lack of single-cell proteomic approaches to handle and analyze rare CTCs. Here, we develop an integrated microfluidic system specifically designed for streamlining isolation, purification and single-cell secretomic profiling of CTCs from whole blood. Key to this platform is the use of photocleavable ssDNA-encoded antibody conjugates to enable a highly purified CTC population with <75 'contaminated' blood cells. An enhanced poly-L-lysine barcode pattern is created on the single-cell barcode chip for efficient capture rare CTC cells in microchambers for subsequent secreted protein profiling. This system was extensively evaluated and optimized with EpCAM-positive HCT116 cells seeded into whole blood. Patient blood samples were employed to assess the utility of the system for isolation, purification and single-cell secretion profiling of CTCs. The CTCs present in patient blood samples exhibit highly heterogeneous secretion profile of IL-8 and VEGF. The numbers of secreting CTCs are found not in accordance with CTC enumeration based on immunostaining in the parallel experiments.

  15. Quantitative comparison between microfluidic and microtiter plate formats for cell-based assays.

    PubMed

    Yin, Huabing; Pattrick, Nicola; Zhang, Xunli; Klauke, Norbert; Cordingley, Hayley C; Haswell, Steven J; Cooper, Jonathan M

    2008-01-01

    In this paper, we compare a quantitative cell-based assay measuring the intracellular Ca2+ response to the agonist uridine 5'-triphosphate in Chinese hamster ovary cells, in both microfluidic and microtiter formats. The study demonstrates that, under appropriate hydrodynamic conditions, there is an excellent agreement between traditional well-plate assays and those obtained on-chip for both suspended immobilized cells and cultured adherent cells. We also demonstrate that the on-chip assay, using adherent cells, provides the possibility of faster screening protocols with the potential for resolving subcellular information about local Ca2+ flux.

  16. High-throughput blood cell focusing and plasma isolation using spiral inertial microfluidic devices.

    PubMed

    Xiang, Nan; Ni, Zhonghua

    2015-12-01

    Herein, we explored the blood cell focusing and plasma isolation using a spiral inertial microfluidic device. First, the flow-rate and concentration effects on the migration dynamics of blood cells were systematically investigated to uncover the focusing mechanisms and steric crowding effects of cells in Dean-coupled inertial flows. A novel phenomenon that the focusing status of discoid red blood cells (RBCs) changes according to the channel height was discovered. These experimental data may provide valuable insights for the high-throughput processing of blood samples using inertial microfluidics. On the basis of the improved understandings on blood cell focusing, efficient isolation of plasma from whole blood with a 20-fold dilution was achieved at a throughput up to 700 μl/min. The purity of the isolated blood plasma was close to 100 %, and the plasma yield was calculated to be 38.5 %. As compared with previously-reported devices, our spiral inertial microfluidic device provides a balanced overall performance, and has overriding advantages in terms of processing throughput and operating efficiency.

  17. 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

  18. Slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow.

    PubMed

    Jagannadh, Veerendra Kalyan; Mackenzie, Mark D; Pal, Parama; Kar, Ajoy K; Gorthi, Sai Siva

    2016-09-19

    Three-dimensional cellular imaging techniques have become indispensable tools in biological research and medical diagnostics. Conventional 3D imaging approaches employ focal stack collection to image different planes of the cell. In this work, we present the design and fabrication of a slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow. The approach employs slanted microfluidic channels fabricated in glass using ultrafast laser inscription. The slanted nature of the microfluidic channels ensures that samples come into and go out of focus, as they pass through the microscope imaging field of view. This novel approach enables the collection of focal stacks in a straight-forward and automated manner, even with off-the-shelf microscopes that are not equipped with any motorized translation/rotation sample stages. The presented approach not only simplifies conventional focal stack collection, but also enhances the capabilities of a regular widefield fluorescence microscope to match the features of a sophisticated confocal microscope. We demonstrate the retrieval of sectioned slices of microspheres and cells, with the use of computational algorithms to enhance the signal-to-noise ratio (SNR) in the collected raw images. The retrieved sectioned images have been used to visualize fluorescent microspheres and bovine sperm cell nucleus in 3D while using a regular widefield fluorescence microscope. We have been able to achieve sectioning of approximately 200 slices per cell, which corresponds to a spatial translation of ∼ 15 nm per slice along the optical axis of the microscope. PMID:27661949

  19. High-throughput blood cell focusing and plasma isolation using spiral inertial microfluidic devices.

    PubMed

    Xiang, Nan; Ni, Zhonghua

    2015-12-01

    Herein, we explored the blood cell focusing and plasma isolation using a spiral inertial microfluidic device. First, the flow-rate and concentration effects on the migration dynamics of blood cells were systematically investigated to uncover the focusing mechanisms and steric crowding effects of cells in Dean-coupled inertial flows. A novel phenomenon that the focusing status of discoid red blood cells (RBCs) changes according to the channel height was discovered. These experimental data may provide valuable insights for the high-throughput processing of blood samples using inertial microfluidics. On the basis of the improved understandings on blood cell focusing, efficient isolation of plasma from whole blood with a 20-fold dilution was achieved at a throughput up to 700 μl/min. The purity of the isolated blood plasma was close to 100 %, and the plasma yield was calculated to be 38.5 %. As compared with previously-reported devices, our spiral inertial microfluidic device provides a balanced overall performance, and has overriding advantages in terms of processing throughput and operating efficiency. PMID:26553099

  20. Microfluidic-driven viral infection on cell cultures: Theoretical and experimental study

    PubMed Central

    Cimetta, Elisa; Franzoso, Mauro; Trevisan, Marta; Serena, Elena; Zambon, Alessandro; Giulitti, Stefano; Barzon, Luisa; Elvassore, Nicola

    2012-01-01

    Advanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the efficiency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfluidic platform used to accurately tune the virus delivery from a diffusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diffusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) transgene were used at different multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfluidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efficiency of infection. Finally, we introduced the concept of effective MOI, a more appropriate variable for microfluidic infections that considers the number of adenoviruses in solution per cell at a certain time. PMID:23734169

  1. Slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow.

    PubMed

    Jagannadh, Veerendra Kalyan; Mackenzie, Mark D; Pal, Parama; Kar, Ajoy K; Gorthi, Sai Siva

    2016-09-19

    Three-dimensional cellular imaging techniques have become indispensable tools in biological research and medical diagnostics. Conventional 3D imaging approaches employ focal stack collection to image different planes of the cell. In this work, we present the design and fabrication of a slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow. The approach employs slanted microfluidic channels fabricated in glass using ultrafast laser inscription. The slanted nature of the microfluidic channels ensures that samples come into and go out of focus, as they pass through the microscope imaging field of view. This novel approach enables the collection of focal stacks in a straight-forward and automated manner, even with off-the-shelf microscopes that are not equipped with any motorized translation/rotation sample stages. The presented approach not only simplifies conventional focal stack collection, but also enhances the capabilities of a regular widefield fluorescence microscope to match the features of a sophisticated confocal microscope. We demonstrate the retrieval of sectioned slices of microspheres and cells, with the use of computational algorithms to enhance the signal-to-noise ratio (SNR) in the collected raw images. The retrieved sectioned images have been used to visualize fluorescent microspheres and bovine sperm cell nucleus in 3D while using a regular widefield fluorescence microscope. We have been able to achieve sectioning of approximately 200 slices per cell, which corresponds to a spatial translation of ∼ 15 nm per slice along the optical axis of the microscope.

  2. 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.

  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. Measurement and validation of cell-based assays with microfluidics at the National Institute of Standards and Technology.

    PubMed

    Cooksey, Gregory A; Atencia, Javier; Forry, Samuel P

    2012-08-01

    The National Institute of Standards and Technology (NIST) is the National Metrology Institute for the USA. Our mission is to advance measurement science, standards and technology in ways that enhance economic security and improve quality of life in the USA. Due to the increased need for technologies that advance biological research and the many new and exciting innovations in microfluidics, our projects are aimed at engineering well-controlled microenvironments for quantitative measurements of cell behavior in microfluidic systems. Cell-based microfluidics at NIST is a highly multidisciplinary activity and is greatly influenced by NIST programs in biochemical sciences, materials science, engineering and information technology. Although there are many microfluidic-related activities ongoing at NIST, we will focus on projects related to cell-based measurements in this article.

  5. Cell-based microfluidic device for screening anti-proliferative activity of drugs in vascular smooth muscle cells.

    PubMed

    Rodriguez-Rodriguez, R; Muñoz-Berbel, X; Demming, S; Büttgenbach, S; Herrera, M D; Llobera, A

    2012-12-01

    This paper presents a microfluidic device consisting of five parallel microchambers with integrated readout-grid for the screening of anti-proliferative activity of drugs in vascular smooth muscle cells (VSMC). A two-level SU-8 master was fabricated and replicated with poly(dimethylsiloxane), PDMS, using standard soft-lithographic methods. The relative small height (4-10 μm) of the integrated grid allowed the identification of single-cells or cell groups and the monitoring of their motility, morphology and size with time, without disturbing their proliferation pattern. This is of particular interest when considering VSMC which, apart of being crucial in the atherosclerotic process, do not proliferate in a single layer but in a non-homogenous hill and valley phenotype. The performance of the microfluidic device has been validated by comparison with conventional culturing methods, proving that the cell proliferation remains unaffected by the microchamber structure (with the integrated grid) and the experimental conditions. Finally, the microfluidic device was also used to evaluate the anti-proliferative activity of curcumin and colchicine in VSMC. With this cellular type, the anti-proliferative activity of curcumin (IC(50) =35 ± 5 μM) was found to be much lower than colchicine (IC(50) =3.2 ± 1.2 μM). These results demonstrate the good performance of the microfluidic device in the evaluation of the anti-proliferative activity (or cytotoxicity) of drugs.

  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. Systematic analysis of in vitro cell rolling using a multi-well plate microfluidic system.

    PubMed

    Levy, Oren; Anandakumaran, Priya; Ngai, Jessica; Karnik, Rohit; Karp, Jeffrey M

    2013-10-16

    A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-α (TNF-α), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

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

    PubMed Central

    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. A microfluidic cell culture system for monitoring of sequential changes in endothelial cells after heat stress.

    PubMed

    Tazawa, Hidekatsu; Sato, Kenjiro; Tsutiya, Atsuhiro; Tokeshi, Manabu; Ohtani-Kaneko, Ritsuko

    2015-08-01

    Endothelial damage induced by a highly elevated body temperature is crucial in some diseases including viral hemorrhagic fevers. Here, we report the heat-induced sequential changes of endothelial cells under shear stress, which were determined with a microfluidic culture system. Although live cell imaging showed only minor changes in the appearance of heat-treated cells, Hsp70 mRNA expression analysis demonstrated that the endothelial cells in channels of the system responded well to heat treatment. F-actin staining also revealed clear changes in the bundles of actin filaments after heat treatment. Well-organized bundles of actin filaments in control cells disappeared in heat-treated cells cultured in the channel. Furthermore, the system enabled detection of sequential changes in plasminogen activator inhibitor-1 (PAI-1) secretion from endothelial cells. PAI-1 concentration in the effluent solution was significantly elevated for the first 15min after initiation of heat treatment, and then decreased subsequently. This study provides fundamental information on heat-induced endothelial changes under shear stress and introduces a potent tool for analyzing endothelial secretions. PMID:26044666

  10. A microfluidic cell culture system for monitoring of sequential changes in endothelial cells after heat stress.

    PubMed

    Tazawa, Hidekatsu; Sato, Kenjiro; Tsutiya, Atsuhiro; Tokeshi, Manabu; Ohtani-Kaneko, Ritsuko

    2015-08-01

    Endothelial damage induced by a highly elevated body temperature is crucial in some diseases including viral hemorrhagic fevers. Here, we report the heat-induced sequential changes of endothelial cells under shear stress, which were determined with a microfluidic culture system. Although live cell imaging showed only minor changes in the appearance of heat-treated cells, Hsp70 mRNA expression analysis demonstrated that the endothelial cells in channels of the system responded well to heat treatment. F-actin staining also revealed clear changes in the bundles of actin filaments after heat treatment. Well-organized bundles of actin filaments in control cells disappeared in heat-treated cells cultured in the channel. Furthermore, the system enabled detection of sequential changes in plasminogen activator inhibitor-1 (PAI-1) secretion from endothelial cells. PAI-1 concentration in the effluent solution was significantly elevated for the first 15min after initiation of heat treatment, and then decreased subsequently. This study provides fundamental information on heat-induced endothelial changes under shear stress and introduces a potent tool for analyzing endothelial secretions.

  11. Electrochemical detection of catecholamine release using planar iridium oxide electrodes in nanoliter microfluidic cell culture volumes.

    PubMed

    Ges, Igor A; Currie, Kevin P M; Baudenbacher, Franz

    2012-04-15

    Release of neurotransmitters and hormones by calcium regulated exocytosis is a fundamental cellular/molecular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. Therefore, this area represents a relevant target for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistically rich data with increased throughput. Toward this goal, we have electrochemically deposited iridium oxide (IrOx) films onto planar thin film platinum electrodes (20 μm×300 μm) and utilized these for quantitative detection of catecholamine release from adrenal chromaffin cells trapped in a microfluidic network. The IrOx electrodes show a linear response to norepinephrine in the range of 0-400 μM, with a sensitivity of 23.1±0.5 mA/M mm(2). The sensitivity of the IrOx electrodes does not change in the presence of ascorbic acid, a substance commonly found in biological samples. A replica molded polydimethylsiloxane (PDMS) microfluidic device with nanoliter sensing volumes was aligned and sealed to a glass substrate with the sensing electrodes. Small populations of chromaffin cells were trapped in the microfluidic device and stimulated by rapid perfusion with high potassium (50mM) containing Tyrode's solution at a flow rate of 1 nL/s. Stimulation of the cells produced a rapid increase in current due to oxidation of the released catecholamines, with an estimated maximum concentration in the cell culture volume of ~52 μM. Thus, we demonstrate the utility of an integrated microfluidic network with IrOx electrodes for real-time quantitative detection of catecholamines released from small populations of chromaffin cells. PMID:22398270

  12. Microfluidic technologies.

    PubMed

    Bhagat, Ali Asgar S; Lim, Chwee Teck

    2012-01-01

    Presence of circulating tumor cells (CTCs) in blood is an important intermediate step in cancer metastasis, a mortal consequence of cancer. However, CTCs are extremely rare in blood with highly heterogeneous morphologies and molecular signatures, thus making their isolation technically very challenging. In the past decade, a flurry of new microfluidic-based technologies has emerged to address this compelling problem. This chapter highlights the current state of the art in microfluidic systems developed for CTCs separation and isolation. The techniques presented are broadly classified as physical- or affinity-based isolation depending on the separation principle. The performance of these techniques is evaluated based on accepted separation metrics including sensitivity, purity and processing/analysis time. Finally, further insights associated with realizing an integrated microfluidic CTC lab-on-chip system as an onco-diagnostic tool will be discussed. PMID:22527494

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

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

    PubMed

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

    2016-01-01

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

  15. 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

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

    PubMed

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

    2014-12-15

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

  18. Isolation of cells for selective treatment and analysis using a magnetic microfluidic chip

    PubMed Central

    Yassine, O.; Gooneratne, C. P.; Abu Smara, D.; Li, F.; Mohammed, H.; Merzaban, J.; Kosel, J.

    2014-01-01

    This study describes the development and testing of a magnetic microfluidic chip (MMC) for trapping and isolating cells tagged with superparamagnetic beads (SPBs) in a microfluidic environment for selective treatment and analysis. The trapping and isolation are done in two separate steps; first, the trapping of the tagged cells in a main channel is achieved by soft ferromagnetic disks and second, the transportation of the cells into side chambers for isolation is executed by tapered conductive paths made of Gold (Au). Numerical simulations were performed to analyze the magnetic flux and force distributions of the disks and conducting paths, for trapping and transporting SPBs. The MMC was fabricated using standard microfabrication processes. Experiments were performed with E. coli (K12 strand) tagged with 2.8 μm SPBs. The results showed that E. coli can be separated from a sample solution by trapping them at the disk sites, and then isolated into chambers by transporting them along the tapered conducting paths. Once the E. coli was trapped inside the side chambers, two selective treatments were performed. In one chamber, a solution with minimal nutrition content was added and, in another chamber, a solution with essential nutrition was added. The results showed that the growth of bacteria cultured in the second chamber containing nutrient was significantly higher, demonstrating that the E. coli was not affected by the magnetically driven transportation and the feasibility of performing different treatments on selectively isolated cells on a single microfluidic platform. PMID:25379074

  19. Hard top soft bottom microfluidic devices for cell culture and chemical analysis.

    PubMed

    Mehta, Geeta; Lee, Jay; Cha, Wansik; Tung, Yi-Chung; Linderman, Jennifer J; Takayama, Shuichi

    2009-05-15

    We report fabrication and characterization of microfluidic devices made of thermoplastic and elastomeric polymers. These hard-soft hybrid material devices are motivated by the combined need for large scale manufacturability, enhanced barrier properties to gas permeation and evaporation of aqueous solutions compared to poly(dimethyl siloxane) (PDMS) devices, and compatibility with deformation-based actuation. Channel features are created on rigid polymers such as polyethylene terephthalate glycol (PETG), cyclic olefin copolymer (COC), and polystyrene (PS) by hot embossing. These "hard tops" are bonded to elastomeric "soft bottoms" (polyurethane (PU) or PDMS-parylene C-PDMS) to create devices that can be used for microfluidic cell culture where deformation-based fluid actuation schemes are used to perfuse and recirculate media. The higher barrier properties of this device compared to PDMS devices enable cell culture with less evaporation and creation of hypoxic conditions. PMID:19382754

  20. Microfluidic positioning of pollen grains in lab-on-a-chip for single cell analysis.

    PubMed

    Ghanbari, Mahmood; Nezhad, Amir Sanati; Agudelo, Carlos G; Packirisamy, Muthukumaran; Geitmann, Anja

    2014-04-01

    A lab-on-a-chip device with a knot shaped microfluidic network is presented to enable trapping of single pollen grains at the entrances of a series of microchannels. This set-up serves to create identical growth conditions for serially arranged tip growing plant cells such as pollen tubes. The design consists of an inlet to introduce the pollen suspension into the chip, three outlets to evacuate excess medium or cells, a distribution chamber to guide the pollen grains toward the growth microchannels and a serial arrangement of microchannels with different geometries connected to the distribution chamber. These microchannels are to harbor the individual pollen tubes. Two different criteria were established to assess the efficiency and optimize the device: trapping probability and uniformity of fluid flow conditions within the microchannels. The performance of different geometries of the microfluidic network was numerically analyzed and experimentally tested.

  1. Combined Simulation and Experimental Study of Large Deformation of Red Blood Cells in Microfluidic Systems

    PubMed Central

    Quinn, David J.; Pivkin, Igor; Wong, Sophie Y.; Chiam, Keng-Hwee; Dao, Ming; Karniadakis, George Em; Suresh, Subra

    2011-01-01

    We investigate the biophysical characteristics of healthy human red blood cells (RBCs) traversing microfluidic channels with cross-sectional areas as small as 2.7 × 3 μm. We combine single RBC optical tweezers and flow experiments with corresponding simulations based on dissipative particle dynamics (DPD), and upon validation of the DPD model, predictive simulations and companion experiments are performed in order to quantify cell deformation and pressure–velocity relationships for different channel sizes and physiologically relevant temperatures. We discuss conditions associated with the shape transitions of RBCs along with the relative effects of membrane and cytosol viscosity, plasma environments, and geometry on flow through microfluidic systems at physiological temperatures. In particular, we identify a cross-sectional area threshold below which the RBC membrane properties begin to dominate its flow behavior at room temperature; at physiological temperatures this effect is less profound. PMID:21240637

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

    PubMed Central

    Woodruff, Kristina; Maerkl, Sebastian J.

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

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

    PubMed

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

    2011-01-21

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

  5. Microfluidic cell sorting: a review of the advances in the separation of cells from debulking to rare cell isolation.

    PubMed

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

    2015-03-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-08-16

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

  9. Development of a microfluidic device for cell concentration and blood cell-plasma separation.

    PubMed

    Maria, M Sneha; Kumar, B S; Chandra, T S; Sen, A K

    2015-12-01

    This work presents design, fabrication and test of a microfluidic device which employs Fahraeus-Lindqvist and Zweifach-Fung effects for cell concentration and blood cell-plasma separation. The device design comprises a straight main channel with a series of branched channels placed symmetrically on both sides of the main channel. The design implements constrictions before each junction (branching point) in order to direct cells that would have migrated closer to the wall (naturally or after liquid extraction at a junction) towards the centre of the main channel. Theoretical and numerical analysis are performed for design of the microchannel network to ensure that a minimum flow rate ratio (of 2.5:1, main channel-to-side channels) is maintained at each junction and predict flow rate at the plasma outlet. The dimensions and location of the constrictions were determined using numerical simulations. The effect of presence of constrictions before the junctions was demonstrated by comparing the performances of the device with and without constrictions. To demonstrate the performance of the device, initial experiments were performed with polystyrene microbeads (10 and 15 μm size) and droplets. Finally, the device was used for concentration of HL60 cells and separation of plasma and cells in diluted blood samples. The cell concentration and blood-plasma purification efficiency was quantified using Haemocytometer and Fluorescence-Activated Cell Sorter (FACS). A seven-fold cell concentration was obtained with HL60 cells and a purification efficiency of 70 % and plasma recovery of 80 % was observed for diluted (1:20) blood sample. FACS was used to identify cell lysis and the cell viability was checked using Trypan Blue test which showed that more than 99 % cells are alive indicating the suitability of the device for practical use. The proposed device has potential to be used as a sample preparation module in lab on chip based diagnostic platforms.

  10. A microfluidic localized, multiple cell culture array using vacuum actuated cell seeding: integrated anticancer drug testing.

    PubMed

    Gao, Yan; Li, Peng; Pappas, Dimitri

    2013-12-01

    In this study, we introduced a novel and convenient approach to culture multiple cells in localized arrays of microfluidic chambers using one-step vacuum actuation. In one device, we integrated 8 individually addressable regions of culture chambers, each only requiring one simple vacuum operation to seed cell lines. Four cell lines were seeded in designated regions in one device via sequential injection with high purity (99.9 %-100 %) and cultured for long-term. The on-chip simultaneous culture of HuT 78, Ramos, PC-3 and C166-GFP cells for 48 h was demonstrated with viabilities of 92 %+/-2 %, 94 %+/-4 %, 96 %+/-2 % and 97 %+/-2 %, respectively. The longest culture period for C166-GFP cells in this study was 168 h with a viability of 96 %+/-10 %. Cell proliferation in each individual side channel can be tracked. Mass transport between the main channel and side channels was achieved through diffusion and studied using fluorescein solution. The main advantage of this device is the capability to perform multiple cell-based assays on the same device for better comparative studies. After treating cells with staurosporine or anti-human CD95 for 16 h, the apoptotic cell percentage of HuT 78, CCRF-CEM, PC-3 and Ramos cells were 36 %+/-3 %, 24 %+/-4 %, 12 %+/-2 %, 18 %+/-4 % for staurosporine, and 63 %+/-2 %, 45 %+/-1 %, 3 %+/-3 %, 27 %+/-12 % for anti-human CD95, respectively. With the advantages of enhanced integration, ease of use and fabrication, and flexibility, this device will be suitable for long-term multiple cell monitoring and cell based assays.

  11. Microfluidic Device

    NASA Technical Reports Server (NTRS)

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

    2015-01-01

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

  12. Microfluidic Device

    NASA Technical Reports Server (NTRS)

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

    2016-01-01

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

  13. Microfluidic device capable of medium recirculation for non-adherent cell culture

    PubMed Central

    Dixon, Angela R.; Rajan, Shrinidhi; Kuo, Chuan-Hsien; Bersano, Tom; Wold, Rachel; Futai, Nobuyuki; Takayama, Shuichi; Mehta, Geeta

    2014-01-01

    We present a microfluidic device designed for maintenance and culture of non-adherent mammalian cells, which enables both recirculation and refreshing of medium, as well as easy harvesting of cells from the device. We demonstrate fabrication of a novel microfluidic device utilizing Braille perfusion for peristaltic fluid flow to enable switching between recirculation and refresh flow modes. Utilizing fluid flow simulations and the human promyelocytic leukemia cell line, HL-60, non-adherent cells, we demonstrate the utility of this RECIR-REFRESH device. With computer simulations, we profiled fluid flow and concentration gradients of autocrine factors and found that the geometry of the cell culture well plays a key role in cell entrapping and retaining autocrine and soluble factors. We subjected HL-60 cells, in the device, to a treatment regimen of 1.25% dimethylsulfoxide, every other day, to provoke differentiation and measured subsequent expression of CD11b on day 2 and day 4 and tumor necrosis factor-alpha (TNF-α) on day 4. Our findings display perfusion sensitive CD11b expression, but not TNF-α build-up, by day 4 of culture, with a 1:1 ratio of recirculation to refresh flow yielding the greatest increase in CD11b levels. RECIR-REFRESH facilitates programmable levels of cell differentiation in a HL-60 non-adherent cell population and can be expanded to other types of non-adherent cells such as hematopoietic stem cells. PMID:24753733

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

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

    PubMed

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

    2016-05-21

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Moore, Thomas A; Young, Edmond W K

    2016-07-01

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

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

    PubMed

    Moore, Thomas A; Young, Edmond W K

    2016-07-01

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

  19. Microfluidics for T- lymphocyte cell separation and inflammation monitoring in burn patients.

    PubMed

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

    2011-02-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    Dusny, Christian; Schmid, Andreas

    2015-06-01

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

  3. Design and evaluation of a microfluidic system for inhibition studies of yeast cell signaling

    NASA Astrophysics Data System (ADS)

    Hamngren, Charlotte; Dinér, Peter; Grøtli, Morten; Goksör, Mattias; Adiels, Caroline B.

    2012-10-01

    In cell signaling, different perturbations lead to different responses and using traditional biological techniques that result in averaged data may obscure important cell-to-cell variations. The aim of this study was to develop and evaluate a four-inlet microfluidic system that enables single-cell analysis by investigating the effect on Hog1 localization post a selective Hog1 inhibitor treatment during osmotic stress. Optical tweezers was used to position yeast cells in an array of desired size and density inside the microfluidic system. By changing the flow rates through the inlet channels, controlled and rapid introduction of two different perturbations over the cell array was enabled. The placement of the cells was determined by diffusion rates flow simulations. The system was evaluated by monitoring the subcellular localization of a fluorescently tagged kinase of the yeast "High Osmolarity Glycerol" (HOG) pathway, Hog1-GFP. By sequential treatment of the yeast cells with a selective Hog1 kinase inhibitor and sorbitol, the subcellular localization of Hog1-GFP was analysed on a single-cell level. The results showed impaired Hog1-GFP nuclear localization, providing evidence of a congenial design. The setup made it possible to remove and add an agent within 2 seconds, which is valuable for investigating the dynamic signal transduction pathways and cannot be done using traditional methods. We are confident that the features of the four-inlet microfluidic system will be a valuable tool and hence contribute significantly to unravel the mechanisms of the HOG pathway and similar dynamic signal transduction pathways.

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

    NASA Astrophysics Data System (ADS)

    Bao, Ning; Lu, Chang

    2008-05-01

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

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

    PubMed Central

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

    2016-01-01

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

  6. Contribution of aquaporins to cellular water transport observed by a microfluidic cell volume sensor.

    PubMed

    Heo, Jinseok; Meng, Fanjie; Hua, Susan Z

    2008-09-15

    Here we demonstrate that an impedance-based microfluidic cell volume sensor can be used to study the roles of aquaporin (AQP) in cellular water permeability and screen AQP-specific drugs. Human embryonic kidney (HEK-293) cells were transiently transfected with AQP3- or AQP4-encoding genes to express AQPs in plasma membranes. The swelling of cells in response to hypotonic stimulation was traced in real time using the sensor. Two time constants were obtained by fitting the swelling curves with a two-exponential function, a fast time constant associated with osmotic water permeability of AQP-expressing cells and a slow phase time constant associated mainly with water diffusion through lipid bilayers in the nontransfected cells. The AQP-expressing cells showed at least 10x faster osmotic water transport than control cells. Using the volume sensor, we examined the effects of Hg (2+) and Ni (2+) on the water transport via AQPs. Hg (2+) inhibited the water flux in AQP3-expressing cells irreversibly, while Ni (2+) blocked the AQP3 channels reversibly. Neither of the two ions blocked the AQP4 channels. The microfluidic volume sensor can sense changes in cell volume in real time, which enables perfusion of various reagents sequentially. It provides a convenient tool for studying the effect of reagents on the function and regulation mechanism of AQPs.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2009-05-01

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

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

    SciTech Connect

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

    2009-01-05

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

  11. Dynamics of a small number of droplets in microfluidic Hele-Shaw cells

    NASA Astrophysics Data System (ADS)

    Shen, B.; Leman, M.; Reyssat, M.; Tabeling, P.

    2014-05-01

    We investigate the dynamics of a small number of droplets ( N = 1, 2, 3) in microfluidic Hele-Shaw cells. We study the cases N = 1, 2, and 3 droplets and analyze the influence of the side walls. In the course of the study, we observe spontaneous alignment of droplet pairs, pair exchanges, droplet escape, multiple reflections between walls, i.e., a number of phenomena that have not been reported yet. As a whole, using pairwise far-field dipolar interactions between droplets, along with treating the walls as mirrors, allows to reproduce the observations, even though limitations in the predictability of the model are pointed out in a few cases. From a more practical prospective, the work shows that the behavior of elementary droplet assemblies can be put under acceptable experimental control in a wide variety of situations, a feature potentially interesting for self-assembly, mixing, or transport of particles in microfluidic environments.

  12. A digital microfluidic droplet generator produces self-assembled supramolecular nanoparticles for targeted cell imaging

    NASA Astrophysics Data System (ADS)

    Liu, Kan; Wang, Hao; Chen, Kuan-Ju; Guo, Feng; Lin, Wei-Yu; Chen, Yi-Chun; Linh Phung, Duy; Tseng, Hsian-Rong; K-F Shen, Clifton

    2010-11-01

    Controlling the size distribution of polymer-based nanoparticles is a challenging task due to their flexible core and surface structures. To accomplish such as task requires very precise control at the molecular level. Here we demonstrate a new approach whereby uniform-sized supramolecular nanoparticles (SNPs) can be reliably generated using a digital microfluidic droplet generator (DMDG) chip. A microfluidic environment enabled precise control over the processing parameters, and therefore high batch-to-batch reproducibility and robust production of SNPs with a very narrow size distribution could be realized. Digitally adjustment of the mixing ratios of the building blocks on the DMDG chip allowed us to rapidly scan a variety of synthesis conditions without consuming significant amounts of reagents. Nearly uniform SNPs with sizes ranging from 35 to 350 nm were obtained and characterized by transmission electron microscopy and dynamic light scattering. In addition, we could fine-tune the surface chemistry of the SNPs by incorporating an additional building block functionalized with specific ligands for targeting cells. The sizes and surface properties of these SNPs correlated strongly with their cell uptake efficiencies. This study showed a feasible method for microfluidic-assisted SNP production and provided a great means for preparing size-controlled SNPs with desired surface ligand coverage.

  13. Dielectrophoretic microfluidic device for the continuous sorting of Escherichia coli from blood cells

    PubMed Central

    Kuczenski, Robert Steven; Chang, Hsueh-Chia; Revzin, Alexander

    2011-01-01

    Microfluidic diagnostic devices promise faster disease identification by purifying and concentrating low-abundance analytes from a flowing sample. The diagnosis of sepsis, a whole body inflammatory response often caused by microbial infections of the blood, is a model system for pursuing the advantages of microfluidic devices over traditional diagnostic protocols. Traditional sepsis diagnoses require large blood samples and several days to culture and identify the low concentration microbial agent. During these long delays while culturing, the physician has little or no actionable information to treat this acute illness. We designed a microfluidic chip using dielectrophoresis to sort and concentrate the target microbe from a flowing blood sample. This design was optimized using the applicable electrokinetic and hydrodynamic theories. We quantify the sorting efficiency of this device using growth-based assays which show 30% of injected microbes are recovered viable, consistent with the electroporation of target cells by the dielectrophoretic cell sorters. Finally, the results illustrate the device is capable of a five-fold larger microbe concentration in the target analyte stream compared to the waste stream at a continuous sample flow rate of 35 μl∕h. PMID:22007268

  14. A digital microfluidic droplet generator produces self-assembled supramolecular nanoparticles for targeted cell imaging

    PubMed Central

    Liu, Kan; Wang, Hao; Chen, Kuan-Ju; Guo, Feng; Lin, Wei-Yu; Chen, Yi-Chun; Phung, Duy Linh; Tseng, Hsian-Rong; Shen, Clifton K.-F.

    2012-01-01

    Controlling the size distribution of polymer-based nanoparticles is a challenging task due to their flexible core and surface structures. To accomplish such as task requires a very precise control at the molecular level. Here, we demonstrate a new approach whereby uniform-sized supramolecular nanoparticles (SNPs) can be reliably generated using a digital microfluidic droplet generator (DMDG) chip. A microfluidic environment enabled precise control over the processing parameters and, therefore, high batch-to-batch reproducibility and robust production of SNPs with a very narrow size distribution could be realized. Digitally adjusting the mixing ratios of the building blocks on the DMDG chip allowed us to rapidly scan a variety of synthesis conditions without consuming significant amounts of reagents. Nearly uniform SNPs with sizes ranging from 35 to 350 nm were obtained and characterized by transmission electron microscopy and dynamic light scattering. In addition, we could fine-tune the surface chemistry of the SNPs by incorporating an additional building block functionalized with specific ligands for targeting cells. The sizes and surface properties of these SNPs correlated strongly with their cell uptake efficiencies. This study showed a feasible microfluidic-assisted SNP production and provided a great means for preparing size-controlled SNP with desired surface ligand coverage. PMID:20935351

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-08-12

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

  18. Microfluidics-based single-cell functional proteomics for fundamental and applied biomedical applications.

    PubMed

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

    2014-01-01

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

  19. Combined Microfluidic-Eectric Diffused Mixing of Living Cells in Continuous Flow

    NASA Astrophysics Data System (ADS)

    Ming-Wen Wang,

    2010-02-01

    The mixing process is a crucially important stage in the operation of biological and chemical microfluidic devices. If the mixing is inadequate, reactants do not fully interact with each other, and the device may not operate properly. This paper describes a simplified microfluidic mixer (different from a chaotic mixer) which can uniformly mix a buffer solution with living cells by applying an AC electric charge. Diffusion of the living cells into the buffer solution occurs rapidly following the interface of the flow stream with the electric charge; no further agitating step is needed. To accomplish this, an asymmetric pair of electrodes was integrated at the inlets of the buffer solution and the cells fluid. When the buffer solution and the cells fluid were introduced into one flow path, they remained limited to that flow stream. When the electrodes were charged, however, the cells in a short distance were efficiently moved into the solution flow, and the original fluids were mixed. The mixing efficiency depends on the polarizability of the cells, and this in turn is governed by the dielectric properties of the cells, the medium, and the solvent. This micro device, capable of efficiently mixing living cells with a buffer solution, may potentially allow biological mixing to be done outside of hospitals, in facilities without biological analyzing instruments.

  20. A microfluidic digital single-cell assay for the evaluation of anticancer drugs.

    PubMed

    Wang, Yao; Tang, Xiaolong; Feng, Xiaojun; Liu, Chao; Chen, Peng; Chen, Dongjuan; Liu, Bi-Feng

    2015-02-01

    Digital single-cell assays hold high potentials for the analysis of cell apoptosis and the evaluation of chemotherapeutic reagents for cancer therapy. In this paper, a microfluidic hydrodynamic trapping system was developed for digital single-cell assays with the capability of monitoring cellular dynamics over time. The microfluidic chip was designed with arrays of bypass structures for trapping individual cells without the need for surface modification, external electric force, or robotic equipment. After optimization of the bypass structure by both numerical simulations and experiments, a single-cell trapping efficiency of ∼90 % was achieved. We demonstrated the method as a digital single-cell assay for the evaluation of five clinically established chemotherapeutic reagents. As a result, the half maximal inhibitory concentration (IC50) values of these compounds could be conveniently determined. We further modeled the gradual decrease of active drugs over time which was often observed in vivo after an injection to investigate cell apoptosis against chemotherapeutic reagents. The developed method provided a valuable means for cell apoptotic analysis and evaluation of anticancer drugs. PMID:25433683

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

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

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

  2. Self-Digitization Microfluidic Chip for Absolute Quantification of mRNA in Single Cells

    PubMed Central

    2015-01-01

    Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques. PMID:25390242

  3. Self-digitization microfluidic chip for absolute quantification of mRNA in single cells.

    PubMed

    Thompson, Alison M; Gansen, Alexander; Paguirigan, Amy L; Kreutz, Jason E; Radich, Jerald P; Chiu, Daniel T

    2014-12-16

    Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques.

  4. Building a better cell trap: Applying Lagrangian modeling to the design of microfluidic devices for cell biology

    NASA Astrophysics Data System (ADS)

    Kim, Min-Cheol; Wang, Zhanhui; Lam, Raymond H. W.; Thorsen, Todd

    2008-02-01

    In this report, we show how computational fluid dynamics can be applied to the design of efficient hydrodynamic cell traps in microfluidic devices. Modeled hydrodynamic trap designs included a large, multiple-aperture "C-type" sieve for trapping hundreds of cells, flat single-aperture arrays for single cells, and "U-type" hydrodynamic structures with one or two apertures to confine small clusters of cells (˜10-15 cells per trap). Using 3T3 cells as a model system, the motion of each individual cell was calculated using a one-way coupled Lagrangian method. The cell was assumed to be a solid sphere, and interactions with other cells were only considered when a cell sedimented in the trap. The ordinary differential equations were solved along the cell trajectory for the three components of the velocity and location vector by using the Rosenbrock method based on an adaptive time-stepping technique. Validation of the predictive value of modeling, using 3T3 cells flowed through microfluidic devices containing "U-type sieves" under the simulation flow parameters, showed excellent agreement between experiment and simulation with respect to cell number per trap and the uniformity of cell distribution within individual microchambers. For applications such as on-chip cell culture or high-throughput screening of cell populations within a lab-on-a-chip environment, Lagrangian simulations have the potential to greatly simplify the design process.

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

    PubMed Central

    Shenoy, Aditi; Smith, Richard

    2015-01-01

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

  6. A polystyrene-based microfluidic device with three-dimensional interconnected microporous walls for perfusion cell culture

    PubMed Central

    Chan, Chung Yu; Goral, Vasiliy N.; DeRosa, Michael E.; Huang, Tony Jun

    2014-01-01

    In this article, we present a simple, rapid prototyped polystyrene-based microfluidic device with three-dimensional (3D) interconnected microporous walls for long term perfusion cell culture. Patterned 3D interconnected microporous structures were created by a chemical treatment together with a protective mask and the native hydrophobic nature of the microporous structures were selectively made hydrophilic using oxygen plasma treatment together with a protective mask. Using this polystyrene-based cell culture microfluidic device, we successfully demonstrated the support of four days perfusion cell culture of hepatocytes (C3A cells). PMID:25379110

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

    PubMed

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

    2016-04-21

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

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

    PubMed

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

    2016-04-21

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

  9. A Mechanically Tunable Microfluidic Cell-Trapping Device

    PubMed Central

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

    2015-01-01

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

  10. Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells.

    PubMed

    Benítez, Jaime J; Topolancik, Juraj; Tian, Harvey C; Wallin, Christopher B; Latulippe, David R; Szeto, Kylan; Murphy, Patrick J; Cipriany, Benjamin R; Levy, Stephen L; Soloway, Paul D; Craighead, Harold G

    2012-11-21

    We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Intact chromosomal DNA is entangled in the array, while other cellular components are washed from the channel. To demonstrate the entrapment principle, a single chromosome was hybridized to whole chromosome paints, and imaged by fluorescence microscopy. DNA extracted from a single cell and small cell populations (less than 100) was released from the device by restriction endonuclease digestion under continuous flow and collected for off-chip analysis. Quantification of the extracted material reveals that the microdevice efficiently extracts essentially all chromosomal DNA. The device described represents a novel platform to perform a variety of analyses on chromosomal DNA at the single cell level.

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

    PubMed

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

    2016-01-01

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

  12. Measurement and analysis of Vibrio fischeri cell-based microfluidic device for personal health monitoring.

    PubMed

    Zhao, Xinyan; Dong, Tao

    2013-01-01

    The cell-based microfluidic chip was designed and fabricated as a low-cost detector to continuously monitor toxicants in drinking water or human urine samples, which is expected to be an important component of a household health monitoring system in the future. The bioluminescent bacterium, Vibrio Fischeri, was selected to validate the function of device. Water samples and Vibrio fischeri cells were mixed and encapsulated into droplets in air flow, which can guarantee sufficient oxygen supply for cells in droplets. Preliminary tests were performed using copper ion (Cu(2+)) as the model toxicant. The droplet system was measured and analyzed at various flow rates in different observation chambers. Both deionized water and human urine samples were tested in the cell-based device. Interestingly, a strong relation between the R.L.U. (Relative Luminescence Units) in the observation chamber and the minute concentration of toxicant (Cu(2+)) was found using deionized water as solvent, whereas the relation was insignificant using human urine as solvent. This study showed the Vibrio fischeri cell-based device might be reliably employed as an early-warning system for the safety of drinking water. However, Vibrio fischeri is not competent to detect dangerous materials in a complex biofluid. With the replacement of cell sensors, the microfluidic device might be functional to analyze urine samples in theory. PMID:24110218

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

    PubMed

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

    2016-10-01

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

  14. Conceptual design of integrated microfluidic system for magnetic cell separation, electroporation, and transfection.

    PubMed

    Durdík, Š; Krafčík, A; Babincová, M; Babinec, P

    2013-09-01

    For the purposes of a successful ex vivo gene therapy we have proposed and analyzed a new concept of an integrated microfluidic system for combined magnetic cell separation, electroporation, and magnetofection. For the analysis of magnetic and electric field distribution (given by Maxwell equations) as well as dynamics of magnetically labeled cell and transfection complex, we have used finite element method directly interfaced to the Matlab routine solving Newton dynamical equations of motion. Microfluidic chamber has been modeled as a channel with height and length 1 mm and 1 cm, respectively. Bottom electrode consisted of 100 parallel ferromagnetic straps and the upper electrode was plate of diamagnetic copper. From the dynamics of magnetic particle motion we have found that the characteristic time-scales for the motion of cells (mean capture time ∼ 4 s) and gene complexes (mean capture time ∼ 3 min), when permanent magnets are used, are in the range suitable for efficient cell separation and gene delivery. The largest electric field intensity (∼10 kV/m) was observed at the edges of the microelectrodes, in the close proximity of magnetically separated cells, which is optimal for subsequent cell electroporation.

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

    PubMed

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

    2015-12-29

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2009-08-25

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

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

    PubMed

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

    2014-06-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  2. Microfluidic geometric metering-based multi-reagent mixture generator for robust live cell screening array.

    PubMed

    Wang, Han; Kim, Jeongyun; Jayaraman, Arul; Han, Arum

    2014-12-01

    Microfluidic live cell arrays with integrated concentration gradient or mixture generators have been utilized in screening cellular responses to various biomolecular cues. Microfluidic network-based gradient generators that can create concentration gradients by repeatedly splitting and mixing different solutions using networks of serpentine channels are commonly used. However, in this method the generation of concentration gradients relies on the continuous flow of sample solutions at optimized flow rates, which poses challenges in maintaining the pressure and flow stability throughout the entire assay period. Here we present a microfluidic live cell screening array with an on-demand multi-reagent mixture generator where the mixing ratios, thus generated concentrations, are hard-wired into the chip itself through a geometric metering method. This platform showed significantly improved robustness and repeatability in generating concentration gradients of fluorescent dyes (average coefficient of variance C.V. = 9 %) compared to the conventional network-based gradient generators (average C.V. = 21 %). In studying the concentration dependent effects of the environmental toxicant 3-methylcholanthrene (3MC) on the activation of cytochrome P450 1A1 (Cyp 1A1) enzyme in H4IIE rat hepatoma cells, statistical variation of the Cyp 1A1 response was significantly lower (C.V. = 5 %) when using the developed mixture generator compared to that using the conventional gradient generator (C.V. = 12 %). Reduction in reagent consumption by 12-times was also achieved. This robust, accurate, and scalable multi-reagent mixture generator integrated with a cell culture array as a live cell assay platform can be readily implemented into various screening applications where repeatability, robustness, and low reagent consumptions over long periods of assay time are of importance.

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

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

    PubMed Central

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

    2014-01-01

    Systematic screening of algal cells is getting huge interest due to their capability of producing lipid-based biodiesel. Here, we introduce a new microfluidic platform composed of an array of perfusion chambers designed for long-term cultivation and preliminary screening of motile microalgal cells through loading and releasing of cells to and from the chambers. The chemical environment in each perfusion chamber was independently controlled for 5 days. The effect of nitrogen-depletion on the lipid production, phototaxis behavior in the absence of Ca2+, and cytotoxic effect of herbicide on microalgal cells was successfully monitored and compared with simultaneous control experiments on the platform. The present methodology could be extended to effective screening of algal cells and various cell lines for the production of biodiesel and other useful chemicals. PMID:24803962

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  7. Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines

    NASA Astrophysics Data System (ADS)

    Lee Szeto, Gregory; van Egeren, Debra; Worku, Hermoon; Sharei, Armon; Alejandro, Brian; Park, Clara; Frew, Kirubel; Brefo, Mavis; Mao, Shirley; Heimann, Megan; Langer, Robert; Jensen, Klavs; Irvine, Darrell J.

    2015-05-01

    B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8+T-cells, and not CD4+T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8+T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8+T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8+T-cells, and decoupling of antigen uptake from B-cell activation.

  8. A hybrid microfluidic system for regulation of neural differentiation in induced pluripotent stem cells.

    PubMed

    Hesari, Zahra; Soleimani, Massoud; Atyabi, Fatemeh; Sharifdini, Meysam; Nadri, Samad; Warkiani, Majid Ebrahimi; Zare, Mehrak; Dinarvand, Rassoul

    2016-06-01

    Controlling cellular orientation, proliferation, and differentiation is valuable in designing organ replacements and directing tissue regeneration. In the present study, we developed a hybrid microfluidic system to produce a dynamic microenvironment by placing aligned PDMS microgrooves on surface of biodegradable polymers as physical guidance cues for controlling the neural differentiation of human induced pluripotent stem cells (hiPSCs). The neuronal differentiation capacity of cultured hiPSCs in the microfluidic system and other control groups was investigated using quantitative real time PCR (qPCR) and immunocytochemistry. The functionally of differentiated hiPSCs inside hybrid system's scaffolds was also evaluated on the rat hemisected spinal cord in acute phase. Implanted cell's fate was examined using tissue freeze section and the functional recovery was evaluated according to the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale. Our results confirmed the differentiation of hiPSCs to neuronal cells on the microfluidic device where the expression of neuronal-specific genes was significantly higher compared to those cultured on the other systems such as plain tissue culture dishes and scaffolds without fluidic channels. Although survival and integration of implanted hiPSCs did not lead to a significant functional recovery, we believe that combination of fluidic channels with nanofiber scaffolds provides a great microenvironment for neural tissue engineering, and can be used as a powerful tool for in situ monitoring of differentiation potential of various kinds of stem cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1534-1543, 2016. PMID:26914600

  9. Optimization of a permeation-based microfluidic direct formic acid fuel cell (DFAFC).

    PubMed

    Erickson, Evan M; Mitrovski, Svetlana M; Gewirth, Andrew A; Nuzzo, Ralph G

    2011-04-01

    A design for a passive, air-breathing microfluidic fuel cell utilizing formic acid (FA) as a fuel is described and its performance characterized. The fuel cell integrates high surface area platinum (cathode) and palladium-platinum (anode) alloy electrodes within a PDMS microfluidic network that keeps them fully immersed in a liquid electrolyte. The polymer network that comprises the device also serves as a self-supporting membrane through which FA and oxygen are supplied to the alloy anode and cathode, respectively, by passive permeation from external sources. The cell is based on a planar form-factor and in its operation exploits FA concentration gradients that form across the PDMS membrane. These latter gradients allow the device to operate stably, producing a nearly constant limiting power density of ~0.2 mW/cm², without driven laminar flow of fluids or the incorporation of an in-channel separator between the anodic and the cathodic compartments. The power output of this elementary device in air is subject to electrolyte mass transport impacts, which can be reduced for a given design rule by decreasing the internal ohmic resistance of the cell. The results suggest that operational stability can be improved by decreasing the kinetic losses imposed on the cathode side of the cell due to FA crossover and modalities for doing so, such as by increasing the efficiency of fuel capture at the anode.

  10. Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel.

    PubMed

    Choi, Jong Seob; Piao, Yunxian; Seo, Tae Seok

    2014-01-01

    The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40% stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function.

  11. A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

    PubMed Central

    Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

    2014-01-01

    Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. PMID:25545548

  12. Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel.

    PubMed

    Choi, Jong Seob; Piao, Yunxian; Seo, Tae Seok

    2014-01-01

    The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40% stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function. PMID:24120039

  13. Microfluidic Synthesis of Microfibers for Magnetic-Responsive Controlled Drug Release and Cell Culture

    PubMed Central

    Lin, Yung-Sheng; Huang, Keng-Shiang; Yang, Chih-Hui; Wang, Chih-Yu; Yang, Yuh-Shyong; Hsu, Hsiang-Chen; Liao, Yu-Ju; Tsai, Chia-Wen

    2012-01-01

    This study demonstrated the fabrication of alginate microfibers using a modular microfluidic system for magnetic-responsive controlled drug release and cell culture. A novel two-dimensional fluid-focusing technique with multi-inlets and junctions was used to spatiotemporally control the continuous laminar flow of alginate solutions. The diameter of the manufactured microfibers, which ranged from 211 µm to 364 µm, could be well controlled by changing the flow rate of the continuous phase. While the model drug, diclofenac, was encapsulated into microfibers, the drug release profile exhibited the characteristic of a proper and steady release. Furthermore, the diclofenac release kinetics from the magnetic iron oxide-loaded microfibers could be controlled externally, allowing for a rapid drug release by applying a magnetic force. In addition, the successful culture of glioblastoma multiforme cells in the microfibers demonstrated a good structural integrity and environment to grow cells that could be applied in drug screening for targeting cancer cells. The proposed microfluidic system has the advantages of ease of fabrication, simplicity, and a fast and low-cost process that is capable of generating functional microfibers with the potential for biomedical applications, such as drug controlled release and cell culture. PMID:22470443

  14. High-Throughput Cytotoxicity Testing System of Acetaminophen Using a Microfluidic Device (MFD) in HepG2 Cells.

    PubMed

    Ju, Seon Min; Jang, Hyun-Jun; Kim, Kyu-Bong; Kim, Jeongyun

    2015-01-01

    A lab-on-a-chip (LOC) is a microfluidic device (MFD) that integrates several lab functions into a single chip of only millimeters in size. LOC provides several advantages, such as low fluidic volumes consumption, faster analysis, compactness, and massive parallelization. These properties enable a microfluidic-based high-throughput drug screening (HTDS) system to acquire cell-based abundant cytotoxicity results depending on linear gradient concentration of drug with only few hundreds of microliters of the drug. Therefore, a microfluidic device was developed containing an array of eight separate microchambers for cultivating HepG2 cells to be exposed to eight different concentrations of acetaminophen (APAP) through a diffusive-mixing-based concentration gradient generator. Every chamber array with eight different concentrations (0, 5.7, 11.4, 17.1, 22.8, 28.5, 34.2, or 40 mM) APAP had four replicating cell culture chambers. Consequently, 32 experimental results were acquired with a single microfluidic device experiment. The microfluidic high-throughput cytotoxicity device (μHTCD) and 96-well culture system showed comparable cytotoxicity results with increasing APAP concentration of 0 to 40 mM. The HTDS system yields progressive concentration-dependent cytotoxicity results using minimal reagent and time. Data suggest that the HTDS system may be applicable as alternative method for cytotoxicity screening for new drugs in diverse cell types. PMID:26241707

  15. When cell biology meets theory

    PubMed Central

    Gonzalez-Gaitan, Marcos

    2015-01-01

    Cell biologists now have tools and knowledge to generate useful quantitative data. But how can we make sense of these data, and are we measuring the correct parameters? Moreover, how can we test hypotheses quantitatively? To answer these questions, the theory of physics is required and is essential to the future of quantitative cell biology. PMID:26416957

  16. When cell biology meets theory.

    PubMed

    Gonzalez-Gaitan, Marcos; Roux, Aurélien

    2015-09-28

    Cell biologists now have tools and knowledge to generate useful quantitative data. But how can we make sense of these data, and are we measuring the correct parameters? Moreover, how can we test hypotheses quantitatively? To answer these questions, the theory of physics is required and is essential to the future of quantitative cell biology.

  17. Bladder cancer cells re-educate TAMs through lactate shuttling in the microfluidic cancer microenvironment

    PubMed Central

    Liu, Pengfei; Cao, Yanwei; Wang, Yonghua; Yang, Xuecheng; Xu, Xiaodong; Wang, Xinsheng; Niu, Haitao

    2015-01-01

    Background In the present study, we aimed to investigate the influence of lactate shuttling on the functional polarization and spatial distribution of transitional cell carcinoma of the bladder (TCCB) cells and macrophages. Methods We designed a microfluidic coculture chip for real-time integrative assays. The effect of lactate shuttling on the re-education of macrophages by TCCB cells was explored by measuring the levels of NO using a total NO assay kit and by evaluating the protein expression of iNOS, p-NFkB-p65, Arg-1 and HIF-1α via cell immunofluorescence and western blotting. Additionally, we examined TCCB cell viability using acridine orange/ethidium bromide (AO/EB) and MitoTracker staining. Moreover, the concentration distributions of lactate and large signaling proteins in the culture chambers were measured using 4′,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate-dextran (FITC-dextran). Furthermore, the recruitment of macrophages and the influence of macrophages on BC metastasis were observed via light microscopy. Results We confirmed that TCCB cells reprogrammed macrophages into an M2 phenotype. Moreover, lactate inhibited M1 polarization and induced M2 polarization of macrophages, but blockade of cancer cell-macrophage lactate flux significantly inhibited the re-education of macrophages by TCCB cells. In addition, lactate diffused faster and deeper than large signaling proteins in the microfluidic tumor microenvironment. Furthermore, lactate alone induced the migration of macrophages, and M1, but not M2, macrophages reduced the motility of TCCB cells. Conclusions TCCB cells reprogrammed macrophages into an M2 phenotype in a manner that depended on cancer cell-TAM lactate flux. Furthermore, the lactate shuttle may be a determinant of the density of TAMs in tumor tissue. PMID:26474279

  18. Concise review: microfluidic technology platforms: poised to accelerate development and translation of stem cell-derived therapies.

    PubMed

    Titmarsh, Drew M; Chen, Huaying; Glass, Nick R; Cooper-White, Justin J

    2014-01-01

    Stem cells are a powerful resource for producing a variety of cell types with utility in clinically associated applications, including preclinical drug screening and development, disease and developmental modeling, and regenerative medicine. Regardless of the type of stem cell, substantial barriers to clinical translation still exist and must be overcome to realize full clinical potential. These barriers span processes including cell isolation, expansion, and differentiation; purification, quality control, and therapeutic efficacy and safety; and the economic viability of bioprocesses for production of functional cell products. Microfluidic systems have been developed for a myriad of biological applications and have the intrinsic capability of controlling and interrogating the cellular microenvironment with unrivalled precision; therefore, they have particular relevance to overcoming such barriers to translation. Development of microfluidic technologies increasingly utilizes stem cells, addresses stem cell-relevant biological phenomena, and aligns capabilities with translational challenges and goals. In this concise review, we describe how microfluidic technologies can contribute to the translation of stem cell research outcomes, and we provide an update on innovative research efforts in this area. This timely convergence of stem cell translational challenges and microfluidic capabilities means that there is now an opportunity for both disciplines to benefit from increased interaction. PMID:24311699

  19. Concise review: microfluidic technology platforms: poised to accelerate development and translation of stem cell-derived therapies.

    PubMed

    Titmarsh, Drew M; Chen, Huaying; Glass, Nick R; Cooper-White, Justin J

    2014-01-01

    Stem cells are a powerful resource for producing a variety of cell types with utility in clinically associated applications, including preclinical drug screening and development, disease and developmental modeling, and regenerative medicine. Regardless of the type of stem cell, substantial barriers to clinical translation still exist and must be overcome to realize full clinical potential. These barriers span processes including cell isolation, expansion, and differentiation; purification, quality control, and therapeutic efficacy and safety; and the economic viability of bioprocesses for production of functional cell products. Microfluidic systems have been developed for a myriad of biological applications and have the intrinsic capability of controlling and interrogating the cellular microenvironment with unrivalled precision; therefore, they have particular relevance to overcoming such barriers to translation. Development of microfluidic technologies increasingly utilizes stem cells, addresses stem cell-relevant biological phenomena, and aligns capabilities with translational challenges and goals. In this concise review, we describe how microfluidic technologies can contribute to the translation of stem cell research outcomes, and we provide an update on innovative research efforts in this area. This timely convergence of stem cell translational challenges and microfluidic capabilities means that there is now an opportunity for both disciplines to benefit from increased interaction.

  20. A laser-based technology for fabricating a soda-lime glass based microfluidic device for circulating tumour cell capture.

    PubMed

    Nieto, Daniel; Couceiro, Ramiro; Aymerich, Maria; Lopez-Lopez, Rafael; Abal, Miguel; Flores-Arias, María Teresa

    2015-10-01

    We developed a laser-based technique for fabricating microfluidic microchips on soda-lime glass substrates. The proposed methodology combines a laser direct writing, as a manufacturing tool for the fabrication of the microfluidics structures, followed by a post-thermal treatment with a CO2 laser. This treatment will allow reshaping and improving the morphological (roughness) and optical qualities (transparency) of the generated microfluidics structures. The use of lasers commonly implemented for material processing makes this technique highly competitive when compared with other glass microstructuring approaches. The manufactured chips were tested with tumour cells (Hec 1A) after being functionalized with an epithelial cell adhesion molecule (EpCAM) antibody coating. Cells were successfully arrested on the pillars after being flown through the device giving our technology a translational application in the field of cancer research.

  1. Image processing and classification algorithm for yeast cell morphology in a microfluidic chip

    NASA Astrophysics Data System (ADS)

    Yang Yu, Bo; Elbuken, Caglar; Ren, Carolyn L.; Huissoon, Jan P.

    2011-06-01

    The study of yeast cell morphology requires consistent identification of cell cycle phases based on cell bud size. A computer-based image processing algorithm is designed to automatically classify microscopic images of yeast cells in a microfluidic channel environment. The images were enhanced to reduce background noise, and a robust segmentation algorithm is developed to extract geometrical features including compactness, axis ratio, and bud size. The features are then used for classification, and the accuracy of various machine-learning classifiers is compared. The linear support vector machine, distance-based classification, and k-nearest-neighbor algorithm were the classifiers used in this experiment. The performance of the system under various illumination and focusing conditions were also tested. The results suggest it is possible to automatically classify yeast cells based on their morphological characteristics with noisy and low-contrast images.

  2. A Microfluidic Cell Co-Culture Platform with a Liquid Fluorocarbon Separator

    PubMed Central

    Brewer, Bryson M.; Shi, Mingjian; Edd, Jon F.; Webb, Donna J.; Li, Deyu

    2014-01-01

    A microfluidic cell co-culture platform that uses a liquid fluorocarbon oil barrier to separate cells into different culture chambers has been developed. Characterization indicates that the oil barrier could be effective for multiple days, and a maximum pressure difference between the oil barrier and aqueous media in the cell culture chamber could be as large as ∼3.43 kPa before the oil barrier fails. Biological applications have been demonstrated with the separate transfection of two groups of primary hippocampal neurons with two different fluorescent proteins and subsequent observation of synaptic contacts between the neurons. In addition, the quality of the fluidic seal provided by the oil barrier is shown to be greater than that of an alternative solid-PDMS valve barrier design by testing the ability of each device to block low molecular weight CellTracker dyes used to stain cells in the culture chambers. PMID:24420386

  3. Droplet microfluidics for amplification-free genetic detection of single cells.

    PubMed

    Rane, Tushar D; Zec, Helena C; Puleo, Chris; Lee, Abraham P; Wang, Tza-Huei

    2012-09-21

    In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are tested using a synthetic target mimicking the actual expression level of 16S rRNA in single cells. The capability of the device to perform "sample-to-answer" pathogen detection of single cells is demonstrated using E. coli as a model pathogen.

  4. Development of microfluidic-based cell collection devices for in vitro and in vivo use

    NASA Astrophysics Data System (ADS)

    Butt, Logan; Entenberg, Dave; Hemachandra, L. P. Madhubhani; Strohmayer, Matthew; Keely, Patricia; Aguirre-Ghiso, Julio; Condeelis, John S.; Castracane, James

    2016-03-01

    The NANIVID - or Nano Intravital Device - is an implantable delivery tool designed to locally affect the tumor microenvironment in vivo. This technology is being redesigned and validated as a cell collection tool for the study of metastatic cancer cells. A methodology has been developed to facilitate this transition, consisting of microfluidic analysis of the device microchannels and a series of cell-related collection experiments building up to in vivo collection. Single-chamber designs were first used to qualitatively demonstrate the feasibility of cell collection ex vivo. This was followed by the development and implementation of devices containing a second, negative-control chamber for quantitative analysis. This work sets the foundation for in vivo cancer cell migration studies utilizing the NANIVID.

  5. Microfluidic housing system: a useful tool for the analysis of dye-sensitized solar cell components

    NASA Astrophysics Data System (ADS)

    Sacco, A.; Lamberti, A.; Pugliese, D.; Chiodoni, A.; Shahzad, N.; Bianco, S.; Quaglio, M.; Gazia, R.; Tresso, E.; Pirri, C. F.

    2012-11-01

    In order to understand the behavior of the different dye-sensitized solar cell (DSC) components, an in-situ analysis should give fundamental help but it is impossible to be performed without compromising the integrity of the cell. Our recently proposed novel microfluidic approach for the fabrication of DSCs is based on a reversible sealing of the two transparent electrodes and it allows the easy assembling and disassembling of the cell, making possible an analysis of the components over time. The aim of this work is not to investigate the different degradation mechanisms of a standard DSC: we want to show that, by using a microfluidic architecture, it is possible to perform a non-destructive analysis and to monitor the photoanode and the counter electrode properties during their lifetime. Morphological (field emission scanning electron microscopy), wetting (contact angle), optical (UV-visible spectroscopy) and electrical (current-voltage and electrochemical impedance spectroscopy measurements under standard AM1.5G illumination) characterizations have been performed over a period of three weeks. The results show how the variation of the wetting and morphological properties at the counter electrode and of the dye absorbance at the photoanode are strongly related to the decrease of the cell performances as evidenced by electrical characterization, thus demonstrating the effectiveness of the use of our structure in this kind of studies.

  6. A microfluidic system to study cytoadhesion of Plasmodium falciparum infected erythrocytes to primary brain microvascularendothelial cells.

    PubMed

    Herricks, Thurston; Seydel, Karl B; Turner, George; Molyneux, Malcolm; Heyderman, Robert; Taylor, Terrie; Rathod, Pradipsinh K

    2011-09-01

    The cellular events leading to severe and complicated malaria in some Plasmodium falciparum infections are poorly understood. Additional tools are required to better understand the pathogenesis of this disease. In this technical report, we describe a microfluidic culture system and image processing algorithms that were developed to observe cytoadhesion interactions of P. falciparum parasitized erythrocytes rolling on primary brain microvascularendothelial cells. We isolated and cultured human primary microvascular brain endothelial cells in a closed loop microfluidic culture system where a peristaltic pump and media reservoirs were integrated onto a microscope stage insert. We developed image processing methods to enhance contrast of rolling parasitized erythrocytes on endothelial cells and to estimate the local wall shear stress. The velocity of parasitized erythrocytes rolling on primary brain microvascularendothelial cells was then measured under physiologically relevant wall shear stresses. Finally, we deployed this method successfully at a field site in Blantyre, Malawi. The method is a promising new tool for the investigation of the pathogenesis of severe malaria.

  7. Enzyme-coated microelectrodes to monitor lactate production in a nanoliter microfluidic cell culture device.

    PubMed

    Ges, Igor A; Baudenbacher, Franz

    2010-10-15

    Monitoring the degree of anaerobic respiration of cells in high density microscale culture systems is an enabling key technology and essential for cell-based biosensors. We have fabricated and incorporated miniature amperometric lactate sensing electrodes with working areas from 3 to 5×10(-2) mm2 into a microfluidic-based microscale cell culture system to measure the lactate production rate of fibroblasts in nanoliter volumes. Planar thin film platinum electrode arrays on glass substrates were spin coated with lactate oxidase and a protective Nafion layer. The lactate electrodes had a high enzymatic activity described by a Michaelis-Menten constant of 2.6±0.1 mM, a linear response in the range 0.01-2.5 mM and a sensitivity of 7.3×10(-2) mA/mM cm2. A replica-molded polydimethylsiloxane (PDMS) microfluidic device with nanoliter sensing volumes was aligned and sealed to a glass substrate with the sensing electrodes. We trapped fibroblasts in the cell culture volume and measured the lactate production rate using a stop-flow protocol. The average lactate production rate was 0.011±0.0049 mM/min. The lactate production was suppressed with the addition of 2-deoxy-D-glucose, which binds to hexokinase. The blocking of hexokinase prevents the generation of pyruvate, the intermittent substrate required for lactate production even in the presence of glucose.

  8. Microfluidic titer plate for stratified 3D cell culture.

    PubMed

    Trietsch, Sebastiaan J; Israëls, Guido D; Joore, Jos; Hankemeier, Thomas; Vulto, Paul

    2013-09-21

    Human tissues and organs are inherently heterogeneous. Their functionality is determined by the interplay between different cell types, their secondary architecture, vascular system and gradients of signaling molecules and metabolites. Here we propose a stratified 3D cell culture platform, in which adjacent lanes of gels and liquids are patterned by phaseguides to capture this tissue heterogeneity. We demonstrate 3D cell culture of HepG2 hepatocytes under continuous perfusion, a rifampicin toxicity assay and co-culture with fibroblasts. 4T1 breast cancer cells are used to demonstrate invasion and aggregation models. The platform is incorporated in a microtiter plate format that renders it fully compatible with automation and high-content screening equipment. The extended functionality, ease of handling and full compatibility to standard equipment is an important step towards adoption of Organ-on-a-Chip technology for screening in an industrial setting.

  9. Single cell studies of mouse embryonic stem cell (mESC) differentiation by electrical impedance measurements in a microfluidic device.

    PubMed

    Zhou, Ying; Basu, Srinjan; Laue, Ernest; Seshia, Ashwin A

    2016-07-15

    Biological populations of cells show considerable cell-to-cell variability. Study of single cells and analysis of cell heterogeneity are considered to be critical in understanding biological processes such as stem cell differentiation and cancer development. Recent advances in lab-on-a-chip techniques have allowed single-cell capture in microfluidic channels with the possibility of precise environmental control and high throughput of experiments with minimal usage of samples and reagents. In recent years, label-free techniques such as electrical impedance spectroscopy have emerged as a non-invasive approach to studying cell properties. In this study, we have designed and fabricated a microfluidic device that combines hydrodynamic trapping of single cells in pre-defined locations with the capability of running electrical impedance measurements within the same device. We have measured mouse embryonic stem cells (mESCs) at different states during differentiation (t=0h, 24h and 48h) and quantitatively analysed the changes in electrical parameters of cells during differentiation. A marked increase in the magnitude of the cell impedance is found during cell differentiation, which can be attributed to an increase in cell size. The analysis of the measurements shows that the nucleus-to-cytoplasm ratio decreases during this process. The degree of cell heterogeneity is observed to be the highest when the cells are at the transition state (24h), compare with cells at undifferentiated (0h) and fully differentiated (48h) states. The device enables highly efficient single cell trapping and provides sensitive, label-free electrical impedance measurements of individual cells, enabling the possibility of quantitatively analysing their physical state as well as studying the associated heterogeneity of a cell population.

  10. Single cell studies of mouse embryonic stem cell (mESC) differentiation by electrical impedance measurements in a microfluidic device

    PubMed Central

    Zhou, Ying; Basu, Srinjan; Laue, Ernest; Seshia, Ashwin A.

    2016-01-01

    Biological populations of cells show considerable cell-to-cell variability. Study of single cells and analysis of cell heterogeneity are considered to be critical in understanding biological processes such as stem cell differentiation and cancer development. Recent advances in lab-on-a-chip techniques have allowed single-cell capture in microfluidic channels with the possibility of precise environmental control and high throughput of experiments with minimal usage of samples and reagents. In recent years, label-free techniques such as electrical impedance spectroscopy have emerged as a non-invasive approach to studying cell properties. In this study, we have designed and fabricated a microfluidic device that combines hydrodynamic trapping of single cells in pre-defined locations with the capability of running electrical impedance measurements within the same device. We have measured mouse embryonic stem cells (mESCs) at different states during differentiation (t=0 h, 24 h and 48 h) and quantitatively analysed the changes in electrical parameters of cells during differentiation. A marked increase in the magnitude of the cell impedance is found during cell differentiation, which can be attributed to an increase in cell size. The analysis of the measurements shows that the nucleus-to-cytoplasm ratio decreases during this process. The degree of cell heterogeneity is observed to be the highest when the cells are at the transition state (24 h), compare with cells at undifferentiated (0 h) and fully differentiated (48 h) states. The device enables highly efficient single cell trapping and provides sensitive, label-free electrical impedance measurements of individual cells, enabling the possibility of quantitatively analysing their physical state as well as studying the associated heterogeneity of a cell population. PMID:26963790

  11. Meeting report--Imaging the Cell.

    PubMed

    Moreau, Violaine; Cordelières, Fabrice P; Poujol, Christel; Sagot, Isabelle; Saltel, Frédéric

    2015-11-01

    Every two years, the French Society for Cell Biology (SBCF) organises an international meeting called 'Imaging the Cell'. This year, the 8th edition was held on 24-26 June 2015 at University of Bordeaux Campus Victoire in the city of Bordeaux, France, a UNESCO World Heritage site. Over the course of three days, the meeting provided a forum for experts in different areas of cell imaging. Its unique approach was to combine conventional oral presentations during morning sessions with practical workshops at hosting institutes and the Bordeaux Imaging Center during the afternoons. The meeting, co-organised by Violaine Moreau and Frédéric Saltel (both INSERM U1053, Bordeaux, France), Christel Poujol and Fabrice Cordelières (both Bordeaux Imaging Center, Bordeaux, France) and Isabelle Sagot (Institut de Biochimie et Génétique Cellulaires, Bordeaux, France), brought together about 120 scientists including 16 outstanding speakers to discuss the latest advances in cell imaging. Thanks to recent progress in imaging technologies, cell biologists are now able to visualise, follow and manipulate cellular processes with unprecedented accuracy. The meeting sessions and workshops highlighted some of the most exciting developments in the field, with sessions dedicated to optogenetics, high-content screening, in vivo and live-cell imaging, correlative light and electron microscopy, as well as super-resolution imaging.

  12. Monitoring of TGF-β 1-Induced Human Lung Adenocarcinoma A549 Cells Epithelial-Mesenchymal Transformation Process by Measuring Cell Adhesion Force with a Microfluidic Device.

    PubMed

    Li, Yuan; Gao, AnXiu; Yu, Ling

    2016-01-01

    The epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties. It is believed that EMT is associated with initiation and completion of the invasion-metastasis cascade. In this study, an economic approach was developed to fabricate a microfluidic device with less instrumentation requirement for the investigation of EMT by quantifying cell adhesion force. Fluid shear force was precisely controlled by a homemade microfluidic perfusion apparatus and interface. The adhesion capability of the human lung adenocarcinoma cell line A549 on different types of extracellular matrix protein was studied. In addition, effects of transforming growth factor-β (TGF-β) on EMT in A549 cells were investigated by characterizing the adhesion force changes and on-chip fluorescent staining. The results demonstrate that the microfluidic device is a potential tool to characterize the epithelial-mesenchymal transition process by measuring cell adhesion force.

  13. Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system.

    PubMed

    Yu, J Q; Liu, X F; Chin, L K; Liu, A Q; Luo, K Q

    2013-07-21

    To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection

  14. Traceable clonal culture and chemodrug assay of heterogeneous prostate carcinoma PC3 cells in microfluidic single cell array chips

    PubMed Central

    Chung, Jaehoon; Ingram, Patrick N.; Bersano-Begey, Tom; Yoon, Euisik

    2014-01-01

    Cancer heterogeneity has received considerable attention for its role in tumor initiation and progression, and its implication for diagnostics and therapeutics in the clinic. To facilitate a cellular heterogeneity study in a low cost and highly efficient manner, we present a microfluidic platform that allows traceable clonal culture and characterization. The platform captures single cells into a microwell array and cultures them for clonal expansion, subsequently allowing on-chip characterization of clonal phenotype and response against drug treatments. Using a heterogeneous prostate cancer model, the PC3 cell line, we verified our prototype, identifying three different sub-phenotypes and correlating their clonal drug responsiveness to cell phenotype. PMID:25553180

  15. Liver injury-on-a-chip: microfluidic co-cultures with integrated biosensors for monitoring liver cell signaling during injury.

    PubMed

    Zhou, Qing; Patel, Dipali; Kwa, Timothy; Haque, Amranul; Matharu, Zimple; Stybayeva, Gulnaz; Gao, Yandong; Diehl, Anna Mae; Revzin, Alexander

    2015-12-01

    Tissue injury triggers complex communication between cells via secreted signaling molecules such as cytokines and growth factors. Discerning when and where these signals begin and how they propagate over time is very challenging with existing cell culture and analysis tools. The goal of this study was to develop new tools in the form of microfluidic co-cultures with integrated biosensors for local and continuous monitoring of secreted signals. Specifically, we focused on how alcohol injury affects TGF-β signaling between two liver cell types, hepatocytes and stellate cells. Activation of stellate cells happens early during liver injury and is at the center of liver fibrosis. We demonstrated that alcohol injury to microfluidic co-cultures caused significantly higher levels of stellate cell activation compared to conditioned media and transwell injury experiments. This highlighted the advantage of the microfluidic co-culture: placement of two cell types in close proximity to ensure high local concentrations of injury-promoting secreted signals. Next, we developed a microsystem consisting of five chambers, two for co-culturing hepatocytes with stellate cells and three additional chambers containing miniature aptamer-modified electrodes for monitoring secreted TGF-β. Importantly, the walls separating microfluidic chambers were actuatable; they could be raised or lowered to create different configurations of the device. The use of reconfigurable microfluidics and miniature biosensors revealed that alcohol injury causes hepatocytes to secrete TGF-β molecules, which diffuse over to neighboring stellate cells and trigger production of additional TGF-β from stellate cells. Our results lend credence to the emerging view of hepatocytes as active participants of liver injury. Broadly speaking, our microsystem makes it possible to monitor paracrine crosstalk between two cell types communicating via the same signaling molecule (e.g. TGF-β). PMID:26480303

  16. Microfluidic cell arrays for metabolic monitoring of stimulated cardiomyocytes.

    PubMed

    Cheng, Wei; Klauke, Norbert; Smith, Godfrey; Cooper, Jonathan M

    2010-04-01

    An array of PDMS microchambers was aligned to an array of sensor electrodes and stimulating microelectrodes, which was used for the electrochemical monitoring of the metabolic activity of single isolated adult ventricular myocytes inside the chamber array, stimulated within a transient electric field. The effect of the accumulation of metabolic byproducts in the limited extracellular volume of the picolitre chambers was demonstrated by measuring single muscle cell contraction optically, while concomitant changes in intracellular calcium transients and pH were recorded independently using fluorescent indicator dyes. Both the amplitude of the cell shortening and the magnitude of the intracellular calcium transients decreased over time and both nearly ceased after 20 min of continuous stimulation in the limited extracellullar volume. The intracellular pH decreased gradually during 20 min of continuous stimulation after which a dramatic pH drop was observed, indicating the breakdown of the intracellular buffering capacity. After continuous stimulation, intracellular lactate was released into the microchamber through cell electroporation and was detected electrochemically at a lactate microbiosensor, within the chamber. A mitochondrial uncoupler was used to mimic ischaemia and thus to enhance the cellular content of lactate. Under these circumstances, intracellular lactate concentrations were found to have risen to approximately 15 mM. This array system has the potential of simultaneous electrochemical and optical monitoring of extracellular and intracellular metabolites from single beating heart cells at a controlled metabolic state.

  17. Electrochemical Protein Cleavage in a Microfluidic Cell with Integrated Boron Doped Diamond Electrodes.

    PubMed

    van den Brink, Floris T G; Zhang, Tao; Ma, Liwei; Bomer, Johan; Odijk, Mathieu; Olthuis, Wouter; Permentier, Hjalmar P; Bischoff, Rainer; van den Berg, Albert

    2016-09-20

    Specific electrochemical cleavage of peptide bonds at the C-terminal side of tyrosine and tryptophan generates peptides amenable to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for protein identification. To this end we developed a microfluidic electrochemical cell of 160 nL volume that combines a cell geometry optimized for a high electrochemical conversion efficiency (>95%) with an integrated boron doped diamond (BDD) working electrode offering a wide potential window in aqueous solution and reduced adsorption of peptides and proteins. Efficient cleavage of the proteins bovine insulin and chicken egg white lysozyme was observed at 4 out of 4 and 7 out of 9 of the predicted cleavage sites, respectively. Chicken egg white lysozyme was identified based on 5 electrochemically generated peptides using a proteomics database searching algorithm. These results show that electrochemical peptide bond cleavage in a microfluidic cell is a novel, fully instrumental approach toward protein analysis and eventually proteomics studies in conjunction with mass spectrometry. PMID:27563730

  18. An osmotic micro-pump integrated on a microfluidic chip for perfusion cell culture.

    PubMed

    Xu, Zhang-Run; Yang, Chun-Guang; Liu, Cui-Hong; Zhou, Zhe; Fang, Jin; Wang, Jian-Hua

    2010-01-15

    A novel microfluidic chip integrating an osmosis-based micro-pump was developed and used for perfusion cell culture. The micro-pump includes two sealed chambers, i.e., the inner osmotic reagent chamber and the outer water chamber, sandwiching a semi-permeable membrane. The water in the outer chamber was forced to flow through the membrane into the inner chamber via osmosis, facilitating continuous flow of fluidic zone in the channel. An average flow rate of 0.33microLmin(-1) was obtained within 50h along with a precision of 4.3% RSD (n=51) by using a 100mgmL(-1) polyvinylpyrrolidone (PVP) solution as the osmotic driving reagent and a flow passage area of 0.98cm(2) of the semi-permeable membrane. The power-free micro-pump has been demonstrated to be pulse-free offering stable flow rates during long-term operation. The present microfluidic chip has been successfully applied for the perfusion culture of human colorectal carcinoma cell by continuously refreshing the culture medium with the osmotic micro-pump. In addition, in situ cell immunostaining was also performed on the microchip by driving all the reagent zones with the integrated micro-pump.

  19. An integrated microfluidic platform for negative selection and enrichment of cancer cells

    NASA Astrophysics Data System (ADS)

    Luo, Wen-Yi; Tsai, Sung-Chi; Hsieh, Kuangwen; Lee, Gwo-Bin

    2015-08-01

    Circulating tumor cells (CTCs), tumor cells that disseminate from primary tumors to the bloodstream, have recently emerged as promising indicators for cancer diagnosis and prognosis. However, the technical difficulties in isolating and detecting rare CTCs have limited the widespread applicability of this method to date. In this work, a new integrated microfluidic system integrating micromixers and micropumps capable of performing ‘negative selection and enrichment’ of CTCs was developed. By using anti-human CD45 antibodies-coated magnetic beads, leukocytes were effectively removed by applying an external magnetic force, leaving behind an enriched target cell population. The on-chip CTC recovery rate was experimentally found to be 70   ±   5% after a single round of negative selection and enrichment. Meanwhile, CD45 depletion efficiency was 83.99   ±   1.00% and could be improved to 99.84   ±   0.04% after three consecutive rounds of depletion. Notably, on-chip negative selection and enrichment was 58% faster and the repeated depletion could be processed automatically. These promising results suggested the developed microfluidic chip is potentiated for a standardized CTC isolation platform. Preliminary results of the current paper were presented at Micro TAS 2014, San Antonio, Texas, USA, October 26-30, 2014.

  20. High-Throughput Single-Cell Cultivation on Microfluidic Streak Plates

    PubMed Central

    Jiang, Cheng-Ying; Dong, Libing; Zhao, Jian-Kang; Hu, Xiaofang; Shen, Chaohua; Qiao, Yuxin; Zhang, Xinyue; Wang, Yapei; Ismagilov, Rustem F.

    2016-01-01

    This paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation of Escherichia coli and antimicrobial susceptibility testing of Pseudomonas aeruginosa PAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including four Mycobacterium isolates and a previously unknown fluoranthene-degrading Blastococcus species. PMID:26850294

  1. Acoustic micro-vortexing of fluids, particles and cells in disposable microfluidic chips.

    PubMed

    Iranmanesh, Ida; Ohlin, Mathias; Ramachandraiah, Harisha; Ye, Simon; Russom, Aman; Wiklund, Martin

    2016-08-01

    We demonstrate an acoustic platform for micro-vortexing in disposable polymer microfluidic chips with small-volume (20 μl) reaction chambers. The described method is demonstrated for a variety of standard vortexing functions, including mixing of fluids, re-suspension of a pellet of magnetic beads collected by a magnet placed on the chip, and lysis of cells for DNA extraction. The device is based on a modified Langevin-type ultrasonic transducer with an exponential horn for efficient coupling into the microfluidic chip, which is actuated by a low-cost fixed-frequency electronic driver board. The transducer is optimized by numerical modelling, and different demonstrated vortexing functions are realized by actuating the transducer for varying times; from fractions of a second for fluid mixing, to half a minute for cell lysis and DNA extraction. The platform can be operated during 1 min below physiological temperatures with the help of a PC fan, a Peltier element and an aluminum heat sink acting as the chip holder. As a proof of principle for sample preparation applications, we demonstrate on-chip cell lysis and DNA extraction within 25 s. The method is of interest for automating and chip-integrating sample preparation procedures in various biological assays. PMID:27444649

  2. Acoustic micro-vortexing of fluids, particles and cells in disposable microfluidic chips.

    PubMed

    Iranmanesh, Ida; Ohlin, Mathias; Ramachandraiah, Harisha; Ye, Simon; Russom, Aman; Wiklund, Martin

    2016-08-01

    We demonstrate an acoustic platform for micro-vortexing in disposable polymer microfluidic chips with small-volume (20 μl) reaction chambers. The described method is demonstrated for a variety of standard vortexing functions, including mixing of fluids, re-suspension of a pellet of magnetic beads collected by a magnet placed on the chip, and lysis of cells for DNA extraction. The device is based on a modified Langevin-type ultrasonic transducer with an exponential horn for efficient coupling into the microfluidic chip, which is actuated by a low-cost fixed-frequency electronic driver board. The transducer is optimized by numerical modelling, and different demonstrated vortexing functions are realized by actuating the transducer for varying times; from fractions of a second for fluid mixing, to half a minute for cell lysis and DNA extraction. The platform can be operated during 1 min below physiological temperatures with the help of a PC fan, a Peltier element and an aluminum heat sink acting as the chip holder. As a proof of principle for sample preparation applications, we demonstrate on-chip cell lysis and DNA extraction within 25 s. The method is of interest for automating and chip-integrating sample preparation procedures in various biological assays.

  3. Microfluidic-based patterning of embryonic stem cells for in vitro development studies.

    PubMed

    Suri, Shalu; Singh, Ankur; Nguyen, Anh H; Bratt-Leal, Andres M; McDevitt, Todd C; Lu, Hang

    2013-12-01

    In vitro recapitulation of mammalian embryogenesis and examination of the emerging behaviours of embryonic structures require both the means to engineer complexity and accurately assess phenotypes of multicellular aggregates. Current approaches to study multicellular populations in 3D configurations are limited by the inability to create complex (i.e. spatially heterogeneous) environments in a reproducible manner with high fidelity thus impeding the ability to engineer microenvironments and combinations of cells with similar complexity to that found during morphogenic processes such as development, remodelling and wound healing. Here, we develop a multicellular embryoid body (EB) fusion technique as a higher-throughput in vitro tool, compared to a manual assembly, to generate developmentally relevant embryonic patterns. We describe the physical principles of the EB fusion microfluidic device design; we demonstrate that >60 conjoined EBs can be generated overnight and emulate a development process analogous to mouse gastrulation during early embryogenesis. Using temporal delivery of bone morphogenic protein 4 (BMP4) to embryoid bodies, we recapitulate embryonic day 6.5 (E6.5) during mouse embryo development with induced mesoderm differentiation in murine embryonic stem cells leading to expression of Brachyury-T-green fluorescent protein (T-GFP), an indicator of primitive streak development and mesoderm differentiation during gastrulation. The proposed microfluidic approach could be used to manipulate hundreds or more of individual embryonic cell aggregates in a rapid fashion, thereby allowing controlled differentiation patterns in fused multicellular assemblies to generate complex yet spatially controlled microenvironments.

  4. Programmable v-type valve for cell and particle manipulation in microfluidic devices.

    PubMed

    Rho, Hoon Suk; Yang, Yoonsun; Hanke, Alexander T; Ottens, Marcel; Terstappen, Leon W M M; Gardeniers, Han

    2016-01-21

    A new microfluidic valve or a "v-type valve" which can be flexibly actuated to focus a fluid flow and block a specific area of a microchannel is demonstrated. Valves with different design parameters were fabricated by multilayer soft lithography and characterized at various operating pressures. To evaluate the functionality of the valve, single microparticles (∅ 7 μm and ∅ 15 μm) and single cells were trapped from flowing suspensions. Continuous processes of particle capture and release were achieved by controlling the actuation and deactuation of the valve. Integration of the v-type valve with poly(dimethyl siloxane) (PDMS) monolithic valves in microfluidic devices was demonstrated to illustrate the potential of the system in various applications such as the creation of a solid phase column, the isolation of a specific number of particles in reactors, and the capture and release of particles or cells in the flow of two immiscible liquids. We believe that this new valve system will be suitable for manipulating particles and cells in a broad range of applications.

  5. Perspectives on utilizing unique features of microfluidics technology for particle and cell sorting

    PubMed Central

    Adams, Jonathan D.; Tom Soh, H.

    2009-01-01

    Sample preparation is often the most tedious and demanding step in an assay, but it also plays an essential role in determining the quality of results. As biological questions and analytical methods become increasingly sophisticated, there is a rapidly growing need for systems that can reliably and reproducibly separate cells and particles with high purity, throughput and recovery. Microfluidics technology represents a compelling approach in this regard, allowing precise control of separation forces for high performance separation in inexpensive, or even disposable, devices. In addition, microfluidics technology enables the fabrication of arrayed and integrated systems that operate either in parallel or in tandem, in a capacity that would be difficult to achieve in macro-scale systems. In this report, we use recent examples from our work to illustrate the potential of microfluidic cell- and particle-sorting devices. We demonstrate the potential of chip-based high-gradient magnetophoresis that enable high-purity separation through reversible trapping of target particles paired with high-stringency washing with minimal loss. We also describe our work in the development of devices that perform simultaneous multi-target sorting, either through precise control of magnetic and fluidic forces or through the integration of multiple actuation forces into a single monolithic device. We believe that such devices may serve as a powerful “front-end” module of highly integrated analytical platforms capable of providing actionable diagnostic information directly from crude, unprocessed samples - the success of such systems may hold the key to advancing point-of-care diagnostics and personalized medicine. PMID:20161387

  6. Digital microfluidic three-dimensional cell culture and chemical screening platform using alginate hydrogels

    PubMed Central

    2015-01-01

    Electro wetting-on-dielectric (EWOD) digital microfluidics (DMF) can be used to develop improved chemical screening platforms using 3-dimensional (3D) cell culture. Alginate hydrogels are one common method by which a 3D cell culture environment is created. This paper presents a study of alginate gelation on EWOD DMF and investigates designs to obtain uniform alginate hydrogels that can be repeatedly addressed by any desired liquids. A design which allows for gels to be retained in place during liquid delivery and removal without using any physical barriers or hydrophilic patterning of substrates is presented. A proof of concept screening platform is demonstrated by examining the effects of different concentrations of a test chemical on 3D cells in alginate hydrogels. In addition, the temporal effects of the various chemical concentrations on different hydrogel posts are demonstrated, thereby establishing the benefits of an EWOD DMF 3D cell culture and chemical screening platform using alginate hydrogels. PMID:25945142

  7. Digital microfluidic three-dimensional cell culture and chemical screening platform using alginate hydrogels.

    PubMed

    George, Subin M; Moon, Hyejin

    2015-03-01

    Electro wetting-on-dielectric (EWOD) digital microfluidics (DMF) can be used to develop improved chemical screening platforms using 3-dimensional (3D) cell culture. Alginate hydrogels are one common method by which a 3D cell culture environment is created. This paper presents a study of alginate gelation on EWOD DMF and investigates designs to obtain uniform alginate hydrogels that can be repeatedly addressed by any desired liquids. A design which allows for gels to be retained in place during liquid delivery and removal without using any physical barriers or hydrophilic patterning of substrates is presented. A proof of concept screening platform is demonstrated by examining the effects of different concentrations of a test chemical on 3D cells in alginate hydrogels. In addition, the temporal effects of the various chemical concentrations on different hydrogel posts are demonstrated, thereby establishing the benefits of an EWOD DMF 3D cell culture and chemical screening platform using alginate hydrogels. PMID:25945142

  8. Differential microfluidic sensor on printed circuit board for biological cells analysis.

    PubMed

    Shi, Dongyuan; Guo, Jinhong; Chen, Liang; Xia, Chuncheng; Yu, Zhefeng; Ai, Ye; Li, Chang Ming; Kang, Yuejun; Wang, Zhiming

    2015-08-01

    Coulter principal based resistive pulse sensor has been demonstrated as an important platform in biological cell detection and enumeration since several decades ago. Recently, the miniaturized micro-Coulter counter has attracted much attention due to its advantages in point of care diagnostics for on chip detection and enumeration of rare cells, such as circulating tumor cells. In this paper, we present a microfluidic cytometer with differential amplifier based on Coulter principle on a SU-8 coated printed circuit board substrate. The electrical current changes induced by the blockage of the microparticles in the sensing aperture are calibrated by polystyrene particles of standard size. Finally, HeLa cells are used to evaluate the performance of the proposed device for enumeration of biological samples. The proposed cytometer is built upon the cheap and widely available printed circuit board substrate and shows its great potential as personalized healthcare monitor. PMID:25735615

  9. Regulation of mesenchymal stem cell 3D microenvironment: From macro to microfluidic bioreactors.

    PubMed

    Sart, Sébastien; Agathos, Spiros N; Li, Yan; Ma, Teng

    2016-01-01

    Human mesenchymal stem cells (hMSCs) have emerged as an important cell type in cell therapy and tissue engineering. In these applications, maintaining the therapeutic properties of hMSCs requires tight control of the culture environments and the structural cell organizations. Bioreactor systems are essential tools to achieve these goals in the clinical-scale expansion and tissue engineering applications. This review summarizes how different bioreactors provide cues to regulate the structure and the chemico-mechanical microenvironment of hMSCs with a focus on 3D organization. In addition to conventional bioreactors, recent advances in microfluidic bioreactors as a novel approach to better control the hMSC microenvironment are also discussed. These advancements highlight the key role of bioreactor systems in preserving hMSC's functional properties by providing dynamic and temporal regulation of in vitro cellular microenvironment.

  10. Learning from the Jersey Turnpike:Cell Lysis, Labeling and Washing with Microfluidic Metamaterials

    NASA Astrophysics Data System (ADS)

    Loutherback, Kevin; Morton, Keith; Inglis, David; Tsui, Opheli; Sturm, James; Chou, Stephen; Austin, Robert

    2008-03-01

    Directing objects across functional streamlines at low Reynolds number is difficult but important since this motion can be used to label, lyse, and analyze complex biological objects on-chip without cross-contamination. Here we use an asymmeteric post array to move cells across coflowing reagents and show on-chip, immunofluorescent labeling of platelets with washing and E.Coli cell lysis with simultaneous separation of bacterial chromosome from the cell contents. Furthermore, we develop the concept of a microfluidic metamaterial by using the basic asymmetric post array as a building block for complex particle handling modes. These modular array elements could be of great use for developing robust techniques for on-chip, continuous flow manipulation and analysis of cells, large bio-particles, and functional beads.

  11. Learning from the Jersey Turnpike: Cell Lysis, Labeling and Washing with Microfluidic Metamaterials

    NASA Astrophysics Data System (ADS)

    Austin, Robert

    2008-03-01

    Directing objects across functional streamlines at low Reynolds number is difficult but important since this motion can be used to label, lyse, and analyze complex biological objects on-chip without cross-contamination. Here we use an asymmeteric post array to move cells across coflowing reagents and show on-chip, immunofluorescent labeling of platelets with washing and E.Coli cell lysis with simultaneous separation of bacterial chromosome from the cell contents. Furthermore, we develop the concept of a microfluidic metamaterial by using the basic asymmetric post array as a building block for complex particle handling modes. These modular array elements could be of great use for developing robust techniques for on-chip, continuous flow manipulation and analysis of cells, large bio-particles, and functional beads.

  12. Differential microfluidic sensor on printed circuit board for biological cells analysis.

    PubMed

    Shi, Dongyuan; Guo, Jinhong; Chen, Liang; Xia, Chuncheng; Yu, Zhefeng; Ai, Ye; Li, Chang Ming; Kang, Yuejun; Wang, Zhiming

    2015-08-01

    Coulter principal based resistive pulse sensor has been demonstrated as an important platform in biological cell detection and enumeration since several decades ago. Recently, the miniaturized micro-Coulter counter has attracted much attention due to its advantages in point of care diagnostics for on chip detection and enumeration of rare cells, such as circulating tumor cells. In this paper, we present a microfluidic cytometer with differential amplifier based on Coulter principle on a SU-8 coated printed circuit board substrate. The electrical current changes induced by the blockage of the microparticles in the sensing aperture are calibrated by polystyrene particles of standard size. Finally, HeLa cells are used to evaluate the performance of the proposed device for enumeration of biological samples. The proposed cytometer is built upon the cheap and widely available printed circuit board substrate and shows its great potential as personalized healthcare monitor.

  13. An integrated microfluidic chip for immunomagnetic detection and isolation of rare prostate cancer cells from blood.

    PubMed

    Esmaeilsabzali, Hadi; Beischlag, Timothy V; Cox, Michael E; Dechev, Nikolai; Parameswaran, Ash M; Park, Edward J

    2016-02-01

    The quantitative and qualitative analysis of circulating tumor cells (CTCs) has the potential to improve the clinical management of several cancers, including prostate cancer. As such, there is much interest in the isolation of CTCs from the peripheral blood of cancer patients. We report the design, fabrication, and proof-of-principle testing of an integrated permalloy-based microfluidic chip for immunomagnetic isolation of blood-borne prostate cancer cells using an antibody targeting prostate surface membrane antigen (PSMA). The preliminary results using spiked blood samples indicate that the proposed device is consistently capable of isolating prostate cancer cells with high sensitivity (up to 98 %) at clinically relevant low concentrations (down to 20 cells/mL) and an acceptable throughput (100 μL/min). PMID:26876965

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

    PubMed

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

    2015-01-01

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

  15. Development of microfluidic devices for in situ investigation of cells using surface-enhanced Raman spectroscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Ho, Yu-Han; Galvan, Daniel D.; Yu, Qiuming

    2016-03-01

    Surface-enhanced Raman spectroscopy (SERS) has immerged as a power analytical and sensing technique for many applications in biomedical diagnosis, life sciences, food safety, and environment monitoring because of its molecular specificity and high sensitivity. The inactive Raman scattering of water molecule makes SERS a suitable tool for studying biological systems. Microfluidic devices have also attracted a tremendous interest for the aforementioned applications. By integrating SERS-active substrates with microfluidic devices, it offers a new capability for in situ investigation of biological systems, their dynamic behaviors, and response to drugs or microenvironment changes. In this work, we designed and fabricated a microfluidic device with SERS-active substrates surrounding by cell traps in microfluidic channels for in situ study of live cells using SERS. The SERS-active substrates are quasi-3D plasmonic nanostructure array (Q3D-PNA) made in h-PDMS/PMDS with physically separated gold film with nanoholes op top and gold nanodisks at the bottom of nanowells. 3D finite-difference time-domain (3D-FDTD) electromagnetic simulations were performed to design Q3D-PNAs with the strongest local electric fields (hot spots) at the top or bottom water/Au interfaces for sensitive analysis of cells and small components, respectively. The Q3D-PNAs with the hot spots on top and bottom were placed at the up and down stream of the microfluidic channel, respectively. Each Q3D-PNA pattern was surrounded with cell trapping structures. The microfluidic device was fabricated via soft lithography. We demonstrated that normal (COS-7) and cancer (HpeG2) cells were captured on the Q3D-PNAs and investigated in situ using SERS.

  16. Microfluidic device for DNA amplification of single cancer cells isolated from whole blood by self-seeding microwells.

    PubMed

    Yang, Yoonsun; Rho, Hoon Suk; Stevens, Michiel; Tibbe, Arjan G J; Gardeniers, Han; Terstappen, Leon W M M

    2015-11-21

    Self-seeding microwell chips can sort single cells into 6400 wells based on cell size and their identity verified by immunofluorescence staining. Here, we developed a microfluidic device in which these single cells can be placed, lysed and their DNA amplified for further interrogation. Whole blood spiked with MCF7 tumor cells was passed through the microwell chips after leukocyte depletion and 37% of the MCF7 cells were identified by epithelial cell adhesion molecule (EpCAM) staining in the microwells. Identified single cells were punched into the reaction chamber of the microfluidic device and reagents for cell lysis and DNA amplification introduced sequentially by peristaltic pumping of micro-valves. On-chip lysis and amplification was performed in 8 parallel chambers yielding a 10,000 fold amplification of DNA. Accessibility of the sample through the reaction chamber allowed for easy retrieval and interrogation of target-specific genes to characterize the tumor cells.

  17. Microfluidic growth chambers with optical tweezers for full spatial single-cell control and analysis of evolving microbes.

    PubMed

    Probst, Christopher; Grünberger, Alexander; Wiechert, Wolfgang; Kohlheyer, Dietrich

    2013-12-01

    Single-cell analysis in microfluidic systems has opened up new possibilities in biotechnological research enabling us to deal with large eukaryotic cells and even small bacteria. In particular, transient investigations in laminar flow or diffusive environments can be performed to unravel single cell behaviour. Up to now, most systems have been limited with respect to precise cell inoculation and sampling methods. Individual cell selection and manipulations have now been made possible by combining laser tweezers with microfluidic cell cultivation environments specifically tailored for micrometre-sized bacteria. Single cells were optically seeded into various micrometre-sized growth sites arranged in parallel. During cultivation, single-cell elongation, morphology and growth rates were derived from single cells and microcolonies of up to 500 cells. Growth of irradiated bacteria was not impaired by minimizing the exposed laser dosage as confirmed by exceptional growth rates. In fact, Escherichia coli exhibited doubling times of less than 20min. For the first time, a filamentous Escherichia coli WT (MG1655) was safely relocated from its growing microcolony by laser manipulations. The cell was transferred to an empty cultivation spot allowing single-cell growth and morphology investigations. Contrary to previous discussions, the filamentous E. coli exhibited normal cell morphology and division after a few generations. This combination of optical tweezers and single-cell analysis in microfluidics adds a new degree of freedom to microbial single-cell analysis. PMID:24041615

  18. Development of an Integrated Microfluidic Perfusion Cell Culture System for Real-Time Microscopic Observation of Biological Cells

    PubMed Central

    Lin, Lung; Wang, Shih-Siou; Wu, Min-Hsien; Oh-Yang, Chih-Chin

    2011-01-01

    This study reports an integrated microfluidic perfusion cell culture system consisting of a microfluidic cell culture chip, and an indium tin oxide (ITO) glass-based microheater chip for micro-scale perfusion cell culture, and its real-time microscopic observation. The system features in maintaining both uniform, and stable chemical or thermal environments, and providing a backflow-free medium pumping, and a precise thermal control functions. In this work, the performance of the medium pumping scheme, and the ITO glass microheater were experimentally evaluated. Results show that the medium delivery mechanism was able to provide pumping rates ranging from 15.4 to 120.0 μL·min−1. In addition, numerical simulation and experimental evaluation were conducted to verify that the ITO glass microheater was capable of providing a spatially uniform thermal environment, and precise temperature control with a mild variation of ±0.3 °C. Furthermore, a perfusion cell culture was successfully demonstrated, showing the cultured cells were kept at high cell viability of 95 ± 2%. In the process, the cultured chondrocytes can be clearly visualized microscopically. As a whole, the proposed cell culture system has paved an alternative route to carry out real-time microscopic observation of biological cells in a simple, user-friendly, and low cost manner. PMID:22164082

  19. Towards microfluidic sperm refinement: impedance-based analysis and sorting of sperm cells.

    PubMed

    de Wagenaar, B; Dekker, S; de Boer, H L; Bomer, J G; Olthuis, W; van den Berg, A; Segerink, L I

    2016-04-21

    The use of high quality semen for artificial insemination in the livestock industry is essential for successful outcome. Insemination using semen with a high number of sperm cells containing morphological defects has a negative impact on fertilization outcome. Therefore, semen with a high number of these abnormal cells is discarded in order to maintain high fertilization potential, resulting in the loss of a large number of morphologically normal sperm cells (up to 70-80% of original sample). A commonly occurring morphological sperm anomaly is the cytoplasmic droplet on the sperm flagella. Currently, no techniques are available to extract morphologically normal sperm cells from rejected samples. Therefore, we aim to develop a microfluidic setup which is able to detect and sort morphologically normal sperm cells label-free and non-invasively. In a proof-of-concept experiment, differential impedance measurements were used to detect the presence of cytoplasmic droplets on sperm flagella, which was quantified by calculating the area under the curve (AUC) of the corresponding impedance peaks. A receiver operating characteristic curve of this electrical analysis method showed the good predictive power of this analysis method (AUC value of 0.85). Furthermore, we developed a label-free cell sorting system using LabVIEW, which is capable of sorting sperm cells based on impedance. In a proof-of-concept experiment, sperm cells and 3 μm beads were sorted label-free and non-invasively using impedance detection and dielectrophoresis sorting. These experiments present our first attempt to perform sperm refinement using microfluidic technology. PMID:27025866

  20. Single cell kinase signaling assay using pinched flow coupled droplet microfluidics.

    PubMed

    Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S; Tan Shao Weng, Daniel; Thakor, Nitish V; Teck Lim, Chwee; Chen, Chia-Hung

    2014-05-01

    Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib. PMID:24926389

  1. Single cell kinase signaling assay using pinched flow coupled droplet microfluidics

    PubMed Central

    Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S.; Tan Shao Weng, Daniel; Thakor, Nitish V.; Teck Lim, Chwee; Chen, Chia-Hung

    2014-01-01

    Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib. PMID:24926389

  2. Numerical Analysis of Hydrodynamic Flow in Microfluidic Biochip for Single-Cell Trapping Application.

    PubMed

    Khalili, Amelia Ahmad; Ahmad, Mohd Ridzuan

    2015-01-01

    Single-cell analysis has become the interest of a wide range of biological and biomedical engineering research. It could provide precise information on individual cells, leading to important knowledge regarding human diseases. To perform single-cell analysis, it is crucial to isolate the individual cells before further manipulation is carried out. Recently, microfluidic biochips have been widely used for cell trapping and single cell analysis, such as mechanical and electrical detection. This work focuses on developing a finite element simulation model of single-cell trapping system for any types of cells or particles based on the hydrodynamic flow resistance (Rh) manipulations in the main channel and trap channel to achieve successful trapping. Analysis is carried out using finite element ABAQUS-FEA™ software. A guideline to design and optimize single-cell trapping model is proposed and the example of a thorough optimization analysis is carried out using a yeast cell model. The results show the finite element model is able to trap a single cell inside the fluidic environment. Fluid's velocity profile and streamline plots for successful and unsuccessful single yeast cell trapping are presented according to the hydrodynamic concept. The single-cell trapping model can be a significant important guideline in designing a new chip for biomedical applications. PMID:26569218

  3. Numerical Analysis of Hydrodynamic Flow in Microfluidic Biochip for Single-Cell Trapping Application

    PubMed Central

    Ahmad Khalili, Amelia; Ahmad, Mohd Ridzuan

    2015-01-01

    Single-cell analysis has become the interest of a wide range of biological and biomedical engineering research. It could provide precise information on individual cells, leading to important knowledge regarding human diseases. To perform single-cell analysis, it is crucial to isolate the individual cells before further manipulation is carried out. Recently, microfluidic biochips have been widely used for cell trapping and single cell analysis, such as mechanical and electrical detection. This work focuses on developing a finite element simulation model of single-cell trapping system for any types of cells or particles based on the hydrodynamic flow resistance (Rh) manipulations in the main channel and trap channel to achieve successful trapping. Analysis is carried out using finite element ABAQUS-FEA™ software. A guideline to design and optimize single-cell trapping model is proposed and the example of a thorough optimization analysis is carried out using a yeast cell model. The results show the finite element model is able to trap a single cell inside the fluidic environment. Fluid’s velocity profile and streamline plots for successful and unsuccessful single yeast cell trapping are presented according to the hydrodynamic concept. The single-cell trapping model can be a significant important guideline in designing a new chip for biomedical applications. PMID:26569218

  4. Cancer-driven dynamics of immune cells in a microfluidic environment.

    PubMed

    Agliari, Elena; Biselli, Elena; De Ninno, Adele; Schiavoni, Giovanna; Gabriele, Lucia; Gerardino, Anna; Mattei, Fabrizio; Barra, Adriano; Businaro, Luca

    2014-10-16

    Scope of the present work is to infer the migratory ability of leukocytes by stochastic processes in order to distinguish the spontaneous organization of immune cells against an insult (namely cancer). For this purpose, spleen cells from immunodeficient mice, selectively lacking the transcription factor IRF-8 (IRF-8 knockout; IRF-8 KO), or from immunocompetent animals (wild-type; WT), were allowed to interact, alternatively, with murine B16.F10 melanoma cells in an ad hoc microfluidic environment developed on a LabOnChip technology. In this setting, only WT spleen cells were able to establish physical interactions with melanoma cells. Conversely, IRF-8 KO immune cells exhibited poor dynamical reactivity towards the neoplastic cells. In the present study, we collected data on the motility of these two types of spleen cells and built a complete set of observables that recapitulate the biological complexity of the system in these experiments. With remarkable accuracy, we concluded that the IRF-8 KO cells performed pure uncorrelated random walks, while WT splenocytes were able to make singular drifted random walks that collapsed on a straight ballistic motion for the system as a whole, hence giving rise to a highly coordinate response. These results may provide a useful system to quantitatively analyse the real time cell-cell interactions and to foresee the behavior of immune cells with tumor cells at the tissue level.

  5. Numerical Analysis of Hydrodynamic Flow in Microfluidic Biochip for Single-Cell Trapping Application.

    PubMed

    Khalili, Amelia Ahmad; Ahmad, Mohd Ridzuan

    2015-11-09

    Single-cell analysis has become the interest of a wide range of biological and biomedical engineering research. It could provide precise information on individual cells, leading to important knowledge regarding human diseases. To perform single-cell analysis, it is crucial to isolate the individual cells before further manipulation is carried out. Recently, microfluidic biochips have been widely used for cell trapping and single cell analysis, such as mechanical and electrical detection. This work focuses on developing a finite element simulation model of single-cell trapping system for any types of cells or particles based on the hydrodynamic flow resistance (Rh) manipulations in the main channel and trap channel to achieve successful trapping. Analysis is carried out using finite element ABAQUS-FEA™ software. A guideline to design and optimize single-cell trapping model is proposed and the example of a thorough optimization analysis is carried out using a yeast cell model. The results show the finite element model is able to trap a single cell inside the fluidic environment. Fluid's velocity profile and streamline plots for successful and unsuccessful single yeast cell trapping are presented according to the hydrodynamic concept. The single-cell trapping model can be a significant important guideline in designing a new chip for biomedical applications.

  6. Cancer-driven dynamics of immune cells in a microfluidic environment.

    PubMed

    Agliari, Elena; Biselli, Elena; De Ninno, Adele; Schiavoni, Giovanna; Gabriele, Lucia; Gerardino, Anna; Mattei, Fabrizio; Barra, Adriano; Businaro, Luca

    2014-01-01

    Scope of the present work is to infer the migratory ability of leukocytes by stochastic processes in order to distinguish the spontaneous organization of immune cells against an insult (namely cancer). For this purpose, spleen cells from immunodeficient mice, selectively lacking the transcription factor IRF-8 (IRF-8 knockout; IRF-8 KO), or from immunocompetent animals (wild-type; WT), were allowed to interact, alternatively, with murine B16.F10 melanoma cells in an ad hoc microfluidic environment developed on a LabOnChip technology. In this setting, only WT spleen cells were able to establish physical interactions with melanoma cells. Conversely, IRF-8 KO immune cells exhibited poor dynamical reactivity towards the neoplastic cells. In the present study, we collected data on the motility of these two types of spleen cells and built a complete set of observables that recapitulate the biological complexity of the system in these experiments. With remarkable accuracy, we concluded that the IRF-8 KO cells performed pure uncorrelated random walks, while WT splenocytes were able to make singular drifted random walks that collapsed on a straight ballistic motion for the system as a whole, hence giving rise to a highly coordinate response. These results may provide a useful system to quantitatively analyse the real time cell-cell interactions and to foresee the behavior of immune cells with tumor cells at the tissue level. PMID:25322144

  7. Microfluidic application-specific integrated device for monitoring direct cell-cell communication via gap junctions between individual cell pairs

    NASA Astrophysics Data System (ADS)

    Lee, Philip J.; Hung, Paul J.; Shaw, Robin; Jan, Lily; Lee, Luke P.

    2005-05-01

    Direct cell-cell communication between adjacent cells is vital for the development and regulation of functional tissues. However, current biological techniques are difficult to scale up for high-throughput screening of cell-cell communication in an array format. In order to provide an effective biophysical tool for the analysis of molecular mechanisms of gap junctions that underlie intercellular communication, we have developed a microfluidic device for selective trapping of cell-pairs and simultaneous optical characterizations. Two different cell populations can be brought into membrane contact using an array of trapping channels with a 2μm by 2μm cross section. Device operation was verified by observation of dye transfer between mouse fibroblasts (NIH3T3) placed in membrane contact. Integration with lab-on-a-chip technologies offers promising applications for cell-based analytical tools such as drug screening, clinical diagnostics, and soft-state biophysical devices for the study of gap junction protein channels in cellular communications. Understanding electrical transport mechanisms via gap junctions in soft membranes will impact quantitative biomedical sciences as well as clinical applications.

  8. Automated microfluidic platform of bead-based electrochemical immunosensor integrated with bioreactor for continual monitoring of cell secreted biomarkers

    NASA Astrophysics Data System (ADS)

    Riahi, Reza; Shaegh, Seyed Ali Mousavi; Ghaderi, Masoumeh; Zhang, Yu Shrike; Shin, Su Ryon; Aleman, Julio; Massa, Solange; Kim, Duckjin; Dokmeci, Mehmet Remzi; Khademhosseini, Ali

    2016-04-01

    There is an increasing interest in developing microfluidic bioreactors and organs-on-a-chip platforms combined with sensing capabilities for continual monitoring of cell-secreted biomarkers. Conventional approaches such as ELISA and mass spectroscopy cannot satisfy the needs of continual monitoring as they are labor-intensive and not easily integrable with low-volume bioreactors. This paper reports on the development of an automated microfluidic bead-based electrochemical immunosensor for in-line measurement of cell-secreted biomarkers. For the operation of the multi-use immunosensor, disposable magnetic microbeads were used to immobilize biomarker-recognition molecules. Microvalves were further integrated in the microfluidic immunosensor chip to achieve programmable operations of the immunoassay including bead loading and unloading, binding, washing, and electrochemical sensing. The platform allowed convenient integration of the immunosensor with liver-on-chips to carry out continual quantification of biomarkers secreted from hepatocytes. Transferrin and albumin productions were monitored during a 5-day hepatotoxicity assessment in which human primary hepatocytes cultured in the bioreactor were treated with acetaminophen. Taken together, our unique microfluidic immunosensor provides a new platform for in-line detection of biomarkers in low volumes and long-term in vitro assessments of cellular functions in microfluidic bioreactors and organs-on-chips.

  9. Automated microfluidic platform of bead-based electrochemical immunosensor integrated with bioreactor for continual monitoring of cell secreted biomarkers

    PubMed Central

    Riahi, Reza; Shaegh, Seyed Ali Mousavi; Ghaderi, Masoumeh; Zhang, Yu Shrike; Shin, Su Ryon; Aleman, Julio; Massa, Solange; Kim, Duckjin; Dokmeci, Mehmet Remzi; Khademhosseini, Ali

    2016-01-01

    There is an increasing interest in developing microfluidic bioreactors and organs-on-a-chip platforms combined with sensing capabilities for continual monitoring of cell-secreted biomarkers. Conventional approaches such as ELISA and mass spectroscopy cannot satisfy the needs of continual monitoring as they are labor-intensive and not easily integrable with low-volume bioreactors. This paper reports on the development of an automated microfluidic bead-based electrochemical immunosensor for in-line measurement of cell-secreted biomarkers. For the operation of the multi-use immunosensor, disposable magnetic microbeads were used to immobilize biomarker-recognition molecules. Microvalves were further integrated in the microfluidic immunosensor chip to achieve programmable operations of the immunoassay including bead loading and unloading, binding, washing, and electrochemical sensing. The platform allowed convenient integration of the immunosensor with liver-on-chips to carry out continual quantification of biomarkers secreted from hepatocytes. Transferrin and albumin productions were monitored during a 5-day hepatotoxicity assessment in which human primary hepatocytes cultured in the bioreactor were treated with acetaminophen. Taken together, our unique microfluidic immunosensor provides a new platform for in-line detection of biomarkers in low volumes and long-term in vitro assessments of cellular functions in microfluidic bioreactors and organs-on-chips. PMID:27098564

  10. Physical cell interactions with their surrounding materials: Mechanics and geometrical factors using microfluidic platforms

    NASA Astrophysics Data System (ADS)

    Lopez Garcia, Maria Del Carmen

    Microfluidics platforms are employed in: "sperm motion in a microfluidic device" and "mechanical interactions of mammary gland cells with their surrounding three dimensional extra-cellular matrix". Microfluidics has shown promise as a new platform for assisted reproduction. Sperm and fluid motion in microchannels was studied to understand the flow characteristics in the device, how sperm interacted with this flow, and how sperm-oocyte attachment occurs in the device. A threshold fluid velocity was found where sperm transition from traveling with the fluid to a regime in which they can move independently. A population of sperm remained in the inlet well area. There was also the tendency of sperm to travel along surface contours. These observations provide an improved understanding of sperm motion in microchannels and a basis for improved device designs. The effort to understand the development of breast cancer motivates the study of mammary gland cells and their interactions with the extra-cellular matrix. Mammographic density is a risk factor for breast cancer which correlates with collagen density affects cell behavior. Collagen gels with concentrations of 1.3, 2, and 3 mg/mL, were tensile tested to obtain the Young's modulus, E, at low displacement rates of 0.01, 0.1, and 1 mm/min. Local strain measurement in the gage section were used for both strain and strain rate determination. Local strain rates were on the order of cellular generated strain rate. A power law fitting described the relationship between Young's modulus and local strain rate. Mammary gland cells were seeded with collagen and fluorescent beads into microchannels and observed via four-dimensional imaging. The displacements of the beads were used to calculate strains. The Young's modulus due to the rate at which the cell was straining the collagen was obtained from the aforementioned fittings. Three-dimensional elastic theory for an isotropic material was employed to calculate the stress. The

  11. Microfluidic Isolation of CD34-Positive Skin Cells Enables Regeneration of Hair and Sebaceous Glands In Vivo.

    PubMed

    Zhu, Beili; Nahmias, Yaakov; Yarmush, Martin L; Murthy, Shashi K

    2014-11-01

    Skin stem cells resident in the bulge area of hair follicles and at the basal layer of the epidermis are multipotent and able to self-renew when transplanted into full-thickness defects in nude mice. Based on cell surface markers such as CD34 and the α6-integrin, skin stem cells can be extracted from tissue-derived cell suspensions for engraftment using the gold standard cell separation technique of fluorescence-activated cell sorting (FACS). This paper describes an alternative separation method using microfluidic devices coated with degradable antibody-functionalized hydrogels. The microfluidic method allows direct injection of tissue digestate (no preprocessing tagging of cells is needed), is fast (45 minutes from injected sample to purified cells), and scalable. This method is used in this study to isolate CD34-positive (CD34+) cells from murine skin tissue digestate, and the functional capability of these cells is demonstrated by transplantation into nude mice using protocols developed by other groups for FACS-sorted cells. Specifically, the transplantation of microfluidic isolated CD34+ cells along with dermal and epidermal cells was observed to generate significant levels of hair follicles and sebaceous glands consistent with those observed previously with FACS-sorted cells.

  12. Microfluidic Isolation of CD34-Positive Skin Cells Enables Regeneration of Hair and Sebaceous Glands In Vivo

    PubMed Central

    Zhu, Beili; Nahmias, Yaakov; Yarmush, Martin L.

    2014-01-01

    Skin stem cells resident in the bulge area of hair follicles and at the basal layer of the epidermis are multipotent and able to self-renew when transplanted into full-thickness defects in nude mice. Based on cell surface markers such as CD34 and the α6-integrin, skin stem cells can be extracted from tissue-derived cell suspensions for engraftment using the gold standard cell separation technique of fluorescence-activated cell sorting (FACS). This paper describes an alternative separation method using microfluidic devices coated with degradable antibody-functionalized hydrogels. The microfluidic method allows direct injection of tissue digestate (no preprocessing tagging of cells is needed), is fast (45 minutes from injected sample to purified cells), and scalable. This method is used in this study to isolate CD34-positive (CD34+) cells from murine skin tissue digestate, and the functional capability of these cells is demonstrated by transplantation into nude mice using protocols developed by other groups for FACS-sorted cells. Specifically, the transplantation of microfluidic isolated CD34+ cells along with dermal and epidermal cells was observed to generate significant levels of hair follicles and sebaceous glands consistent with those observed previously with FACS-sorted cells. PMID:25205844

  13. Rapid fabrication of microfluidic polymer electrolyte membrane fuel cell in PDMS by surface patterning of perfluorinated ion-exchange resin

    NASA Astrophysics Data System (ADS)

    Song, Yong-Ak; Batista, Candy; Sarpeshkar, Rahul; Han, Jongyoon

    In this paper we demonstrate a simple and rapid fabrication method for a microfluidic polymer electrolyte membrane (PEM) fuel cell using polydimethylsiloxane (PDMS), which has become the de facto standard material in BioMEMS. Instead of integrating a Nafion sheet film between two layers of a PDMS device in a traditional "sandwich format," we pattern a perfluorinated ion-exchange resin such as a Nafion resin on a glass substrate using a reversibly bonded PDMS microchannel to generate an ion-selective membrane between the fuel-cell electrodes. After this patterning step, the assembly of the microfluidic fuel cell is accomplished by simple oxygen plasma bonding between the PDMS chip and the glass substrate. In an example implementation, the planar PEM microfluidic fuel cell generates an open circuit voltage of 600-800 mV and delivers a maximum current output of nearly 4 μA. To enhance the power output of the fuel cell we utilize self-assembled colloidal arrays as a support matrix for the Nafion resin. Such arrays allow us to increase the thickness of the ion-selective membrane to 20 μm and increase the current output by 166%. Our novel fabrication method enables rapid prototyping of microfluidic fuel cells to study various ion-exchange resins for the polymer electrolyte membrane. Our work will facilitate the development of miniature, implantable, on-chip power sources for biomedical applications.

  14. Microfluidic platforms and fundamental electrocatalysis studies for fuel cell applications

    NASA Astrophysics Data System (ADS)

    Cohen, Jamie Lee

    The fabrication and testing of a planar membraneless microchannel fuel cell, based on a silicon microchannel, is described in detail. Laminar flow of fuel and oxidant streams, one on top of the other, prevents fuel crossover while allowing ionic transport at the interface between the two solutions. By employing laminar flow, the useful functions of a membrane are retained, while bypassing its inherent limitations. The planar design maximizes the anode and cathode areas, and elimination of the membrane affords broad flexibility in the choice of fuel and oxidant. Fuels including formic acid, methanol, ethanol, sodium borohydride and hydrogen were tested along with oxidants such as oxygen, hydrogen peroxide and potassium permanganate. Steps taken to improve voltage, current density, and overall power output have been addressed, including the testing of a dual electrolyte system and the use of micro-patterned electrode surfaces to enhance fuel utilization. As the complexity of the fuels studied in the microchannel fuel cell increased, it was imperative to characterize these fuels using electrochemical techniques prior to utilization in the fuel cell. The oxidation pathway of the liquid fuel methanol was studied rigorously because of its importance for micro-fuel cell applications. Activation energies for methanol oxidation at a Ptpoly surface were determined using electrochemical techniques, providing a benchmark for the comparison of activation energies of other Pt-based electrocatalysts for methanol oxidation at a given potential. A protocol to obtain Ea values was established in three different electrolytes and experimental parameters that influence the magnitude of these values are discussed in detail. The oxidation pathways of sodium borohydride were also examined at Au, Pt, and Pd surfaces using cyclic voltammetry, chronoamperometry, and rotating disk electrode voltammetry. In addition to studies on bulk Ptpoly surfaces, new bulk intermetallic catalysts were

  15. Biomechanical analysis of cancerous and normal cells based on bulge generation in a microfluidic device.

    PubMed

    Kim, Yu Chang; Park, Sang-Jin; Park, Je-Kyun

    2008-10-01

    This paper presents a new biomechanical analysis method for discrimination between cancerous and normal cells through compression by poly(dimethylsiloxane) (PDMS) membrane deflection in a microfluidic device. When a cell is compressed, cellular membrane will expand and then small bulges will appear on the peripheral cell membrane beyond the allowable strain. It is well known that the amount of F-actin in cancer cells is less than that of normal cells and bulges occur at the sites where cytoskeleton becomes detached from the membrane bilayer. Accordingly, we have demonstrated the difference of the bulge generation between breast cancer cells (MCF7) and normal cells (MCF10A). After excessive deformation, the bulges generated in MCF7 cells were not evenly distributed on the cell periphery. Contrary to this, the bulges of MCF10A cells showed an even distribution. In addition, the morphologies of bulges of MCF7 and MCF10A cells looked swollen protrusion and tubular protrusion, respectively. Peripheral strains at the moment of the bulge generation were also 72% in MCF7 and 46% in MCF10A. The results show that the bulge generation can be correlated with the cytoskeleton quantity inside the cell, providing the first step of a new biomechanical approach. PMID:18810292

  16. Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering.

    PubMed

    Tay, Hui Min; Yeo, David C; Wiraja, Christian; Xu, Chenjie; Hou, Han Wei

    2016-01-01

    Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (>90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving >95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (10(6) cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application. PMID:27500904

  17. Microfluidic artificial “vessels” for dynamic mechanical stimulation of mesenchymal stem cells

    PubMed Central

    Zhou, Jing; Niklason, Laura E.

    2013-01-01

    Cells in the cardiovascular system are constantly exposed to complex mechanical stimulation due to the pulsatile nature of blood flow and the haemodynamic forces that are key to the regulation of vascular development, remodeling and pathophysiology. Mechanical stretch can also modulate the differentiation of stem cells toward vascular cell lineages (i.e., vascular smooth muscle cells), and represent a critical factor in vascular tissue engineering. Here we report on the development of a microchip platform that can emulate several key aspects of the vascular mechanical environment, such as cyclic stimulation and circumferential strain. This chip consists of an array of microfluidic channels with widths ranging from 20 to 500 micrometers. These channels are covered by suspended deformable membranes, on which cells are cultured and stimulated by cyclic circumferential strain of up to 20% via hydrodynamic actuation of the fluid in the microfluidic channels, thereby mimicking the biomechanical conditions of small blood vessels. We show that human mesenchymal stem cells (MSCs) can be cultured and continuously stimulated by cyclic stretch over a period of 7 days with no evidence of device fatigue or performance degradation. We observed localization and alignment of MSCs when mechanical stretch is larger than 10%, indicating the importance of mechanical stimulation in modulating cellular behavior. We further demonstrated simultaneous detection of proteins in multiple signaling pathways, including SMAD1/SMAD2 and canonical Wnt/β-catenin. This microchip represents a generic and versatile platform for high-throughput and rapid screening of cellular responses, including signal transduction cascades, in response to mechanical cues. The system emulates the physiological conditions of blood vessels and other tissues that are subject to cyclic strain, and may have a wide range of applications in the fields of stem cell mechanobiology, vascular tissue engineering, and other areas

  18. Endothelial progenitor cell recruitment in a microfluidic vascular model.

    PubMed

    Lewis, Daniel M; Abaci, Hasan E; Xu, Yu; Gerecht, Sharon

    2015-01-01

    During vessel injury, endothelial progenitors cells (EPCs) are recruited from bone marrow and directed to the hypoxic injury site. The hypoxic conditions in the damaged blood vessel promote TNF-α, which upregulates intercellular adhesion molecule-1 (ICAM-1). EPCs attach to endothelial cell lining using ICAM-1. Here we aimed to examine EPC attachment to ECs in an injured-blood vessel conditions. We first determined ICAM-1 expression in stimulated HUVECs. We stimulated HUVECs with 21% oxygen (atmospheric), atmospheric with TNF-α-supplemented media, 1% oxygen (hypoxia), and hypoxia with TNF-α-supplemented media and found the highest ECFC attachment on HUVECs stimulated with TNF-α and hypoxia, correlating with the highest ICAM-1 expression. We next designed, fabricated and tested a three-dimensional microbioreactor (3D MBR) system with precise control and monitoring of dissolve oxygen and media flow rate in the cellular environment. We utilized a step-wise seeding approach, producing monolayer of HUVECs on all four walls. When stimulated with both TNF-α and hypoxia, ECFC retention on HUVECs was significantly increased under low shear stress compared to static controls. Overall, the 3D MBR system mimics the pathological oxygen tension and shear stress in the damaged vasculature, providing a platform to model vascular-related disorders. PMID:26693599

  19. Microfluidic module for blood cell separation for gene expression radiobiological assays

    PubMed Central

    Brengues, Muriel; Gu, Jian; Zenhausern, Frederic

    2015-01-01

    Advances in molecular techniques have improved discovery of biomarkers associated with radiation exposure. Gene expression techniques have been demonstrated as effective tools for biodosimetry, and different assay platforms with different chemistries are now available. One of the main challenges is to integrate the sample preparation processing of these assays into microfluidic platforms to be fully automated for point-of-care medical countermeasures in the case of a radiological event. Most of these assays follow the same workflow processing that comprises first the collection of blood samples followed by cellular and molecular sample preparation. The sample preparation is based on the specific reagents of the assay system and depends also on the different subsets of cells population and the type of biomarkers of interest. In this article, the authors present a module for isolation of white blood cells from peripheral blood as a prerequisite for automation of gene expression assays on a microfluidic cartridge. For each sample condition, the gene expression platform can be adapted to suit the requirements of the selected assay chemistry. PMID:25877531

  20. Disposable on-chip microfluidic system for buccal cell lysis, DNA purification, and polymerase chain reaction.

    PubMed

    Cho, Woong; Maeng, Joon-Ho; Ahn, Yoomin; Hwang, Seung Yong

    2013-09-01

    This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off-chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.

  1. A High Power-Density, Mediator-Free, Microfluidic Biophotovoltaic Device for Cyanobacterial Cells

    PubMed Central

    Bombelli, Paolo; Müller, Thomas; Herling, Therese W; Howe, Christopher J; Knowles, Tuomas P J

    2015-01-01

    Biophotovoltaics has emerged as a promising technology for generating renewable energy because it relies on living organisms as inexpensive, self-repairing, and readily available catalysts to produce electricity from an abundant resource: sunlight. The efficiency of biophotovoltaic cells, however, has remained significantly lower than that achievable through synthetic materials. Here, a platform is devised to harness the large power densities afforded by miniaturized geometries. To this effect, a soft-lithography approach is developed for the fabrication of microfluidic biophotovoltaic devices that do not require membranes or mediators. Synechocystis sp. PCC 6803 cells are injected and allowed to settle on the anode, permitting the physical proximity between cells and electrode required for mediator-free operation. Power densities of above 100 mW m-2 are demonstrated for a chlorophyll concentration of 100 μM under white light, which is a high value for biophotovoltaic devices without extrinsic supply of additional energy. PMID:26190957

  2. Selective local lysis and sampling of live cells for nucleic acid analysis using a microfluidic probe

    PubMed Central

    Kashyap, Aditya; Autebert, Julien; Delamarche, Emmanuel; Kaigala, Govind V.

    2016-01-01

    Heterogeneity is inherent to biology, thus it is imperative to realize methods capable of obtaining spatially-resolved genomic and transcriptomic profiles of heterogeneous biological samples. Here, we present a new method for local lysis of live adherent cells for nucleic acid analyses. This method addresses bottlenecks in current approaches, such as dilution of analytes, one-sample-one-test, and incompatibility to adherent cells. We make use of a scanning probe technology - a microfluidic probe - and implement hierarchical hydrodynamic flow confinement (hHFC) to localize multiple biochemicals on a biological substrate in a non-contact, non-destructive manner. hHFC enables rapid recovery of nucleic acids by coupling cell lysis and lysate collection. We locally lysed ~300 cells with chemical systems adapted for DNA or RNA and obtained lysates of ~70 cells/μL for DNA analysis and ~15 cells/μL for mRNA analysis. The lysates were introduced into PCR-based workflows for genomic and transcriptomic analysis. This strategy further enabled selective local lysis of subpopulations in a co-culture of MCF7 and MDA-MB-231 cells, validated by characteristic E-cadherin gene expression in individually extracted cell types. The developed strategy can be applied to study cell-cell, cell-matrix interactions locally, with implications in understanding growth, progression and drug response of a tumor. PMID:27411740

  3. Long-term single cell analysis of S. pombe on a microfluidic microchemostat array.

    PubMed

    Nobs, Jean-Bernard; Maerkl, Sebastian J

    2014-01-01

    Although Schyzosaccharomyces pombe is one of the principal model organisms for studying the cell cycle, surprisingly few methods have characterized S. pombe growth on the single cell level, and no methods exist capable of analyzing thousands of cells and tens of thousands of cell division events. We developed an automated microfluidic platform permitting S. pombe to be grown on-chip for several days under defined and changeable conditions. We developed an image processing pipeline to extract and quantitate several physiological parameters including cell length, time to division, and elongation rate without requiring synchronization of the culture. Over a period of 50 hours our platform analyzed over 100000 cell division events and reconstructed single cell lineages up to 10 generations in length. We characterized cell lengths and division times in a temperature shift experiment in which cells were initially grown at 30°C and transitioned to 25°C. Although cell length was identical at both temperatures at steady-state, we observed transient changes in cell length if the temperature shift took place during a critical phase of the cell cycle. We further show that cells born with normal length do divide over a wide range of cell lengths and that cell length appears to be controlled in the second generation, were large newly born cells have a tendency to divide more rapidly and thus at a normalized cell size. The platform is thus applicable to measure fine-details in cell cycle dynamics, should be a useful tool to decipher the molecular mechanism underlying size homeostasis, and will be generally applicable to study processes on the single cell level that require large numbers of precision measurements and single cell lineages.

  4. Development of a microfluidic device for determination of cell osmotic behavior and membrane transport properties.

    PubMed

    Chen, Hsiu-Hung; Purtteman, Jester J P; Heimfeld, Shelly; Folch, Albert; Gao, Dayong

    2007-12-01

    An understanding of cell osmotic behavior and membrane transport properties is indispensable for cryobiology research and development of cell-type-specific, optimal cryopreservation conditions. A microfluidic perfusion system is developed here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell osmotic behavior and membrane transport properties. The system is fabricated using soft lithography and is comprised of microfluidic channels and a perfusion chamber for trapping cells. During experiments, rat basophilic leukemia (RBL-1 line) cells were injected into the inlet of the device, allowed to flow downstream, and were trapped within a perfusion chamber. The fluid continues to flow to the outlet due to suction produced by a Hamilton Syringe. Two sets of experiments have been performed: the cells were perfused by (1) hypertonic solutions with different concentrations of non-permeating solutes and (2) solutions containing a permeating cryoprotective agent (CPA), dimethylsulfoxide (Me(2)SO), plus non-permeating solute (sodium chloride (NaCl)), respectively. From experiment (1), cell osmotically inactive volume (V(b)) and the permeability coefficient of water (L(p)) for RBL cells are determined to be 41% [n=18, correlation coefficient (r(2)) of 0.903] of original/isotonic volume, and 0.32+/-0.05 microm/min/atm (n=8, r(2)>0.963), respectively, for room temperature (22 degrees C). From experiment (2), the permeability coefficient of water (L(p)) and of Me(2)SO (P(s)) for RBL cells are 0.38+/-0.09 microm/min/atm and (0.49+/-0.13) x 10(-3)cm/min (n=5, r(2)>0.86), respectively. We conclude that this device enables us to: (1) readily monitor the changes of extracellular conditions by perfusing single or a group of cells with prepared media; (2) confine cells (or a cell) within a monolayer chamber, which prevents imaging ambiguity, such as cells overlapping or moving out of the focus plane; (3) study individual cell

  5. Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

    PubMed Central

    Gruenberger, Alexander; Probst, Christopher; Heyer, Antonia; Wiechert, Wolfgang; Frunzke, Julia; Kohlheyer, Dietrich

    2013-01-01

    In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g. cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs. PMID:24336165

  6. Microfluidic picoliter bioreactor for microbial single-cell analysis: fabrication, system setup, and operation.

    PubMed

    Gruenberger, Alexander; Probst, Christopher; Heyer, Antonia; Wiechert, Wolfgang; Frunzke, Julia; Kohlheyer, Dietrich

    2013-12-06

    In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g. cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

  7. Jetting microfluidics with size-sorting capability for single-cell protease detection.

    PubMed

    Jing, Tengyang; Ramji, Ramesh; Warkiani, Majid Ebrahimi; Han, Jongyoon; Lim, Chwee Teck; Chen, Chia-Hung

    2015-04-15

    Activated proteases such as matrix metalloproteinases (MMPs) secreted from cancer cells can degrade the extracellular matrix (ECM) and contribute to tumour formation and metastasis. Measuring MMP activity in individual cancer cells can provide important insights on cancer cell heterogeneity and disease progression. Here, we present a microfluidic platform combining a droplet jetting generator and a deterministic lateral displacement (DLD) size-sorting channel that is capable of encapsulating individual cancer cells inside picoliter droplets effectively. Droplet jetting with cell-triggered Rayleigh-Plateau instability was employed which produced large droplets capable of cell encapsulation (diameter, ~25µm) and small empty droplets (diameter, ~14µm), which were then size-separated using a DLD size-sorting channel to enrich the single-cell encapsulated droplets (~78%), regardless of the cell density of input sample solutions. The droplets containing encapsulated cancer cells were collected in an observation chamber to determine the kinetic profiles of MMP secretion and the inhibitory response in the presence of the drug doxycycline at the single-cell level to reveal their heterogeneous MMPs secretion activities. PMID:25460876

  8. High-Performance Single Cell Genetic Analysis Using Microfluidic Emulsion Generator Arrays

    PubMed Central

    Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T.; Mathies, Richard A.

    2010-01-01

    High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex PCR. Microfabricated emulsion generator array (MEGA) devices containing 4, 32 and 96 channels are developed to confer a flexible capability of generating up to 3.4 × 106 nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed, the beads are pooled and rapidly analyzed by multi-color flow cytometry. Using E. coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1:105. This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations. PMID:20192178

  9. Continuous Flow Deformability-Based Separation of Circulating Tumor Cells Using Microfluidic Ratchets.

    PubMed

    Park, Emily S; Jin, Chao; Guo, Quan; Ang, Richard R; Duffy, Simon P; Matthews, Kerryn; Azad, Arun; Abdi, Hamidreza; Todenhöfer, Tilman; Bazov, Jenny; Chi, Kim N; Black, Peter C; Ma, Hongshen

    2016-04-13

    Circulating tumor cells (CTCs) offer tremendous potential for the detection and characterization of cancer. A key challenge for their isolation and subsequent analysis is the extreme rarity of these cells in circulation. Here, a novel label-free method is described to enrich viable CTCs directly from whole blood based on their distinct deformability relative to hematological cells. This mechanism leverages the deformation of single cells through tapered micrometer scale constrictions using oscillatory flow in order to generate a ratcheting effect that produces distinct flow paths for CTCs, leukocytes, and erythrocytes. A label-free separation of circulating tumor cells from whole blood is demonstrated, where target cells can be separated from background cells based on deformability despite their nearly identical size. In doping experiments, this microfluidic device is able to capture >90% of cancer cells from unprocessed whole blood to achieve 10(4) -fold enrichment of target cells relative to leukocytes. In patients with metastatic castration-resistant prostate cancer, where CTCs are not significantly larger than leukocytes, CTCs can be captured based on deformability at 25× greater yield than with the conventional CellSearch system. Finally, the CTCs separated using this approach are collected in suspension and are available for downstream molecular characterization. PMID:26917414

  10. Microfluidic serial dilution cell-based assay for analyzing drug dose response over a wide concentration range.

    PubMed

    Sugiura, Shinji; Hattori, Koji; Kanamori, Toshiyuki

    2010-10-01

    In this paper we report a perfusion culture microchamber array chip with a serial dilution microfluidic network for analyzing drug dose response over a concentration range spanning 6 orders of magnitude, which is required for practical drug discovery applications. The microchamber array chip was equipped with a pressure-driven interface, in which medium and drug solution were added with a micropipet and delivered into the microfluidic network by pneumatic pressure. We demonstrated that the microchamber array chip could be used to estimate the 50% growth inhibitory concentration using the model anticancer drug paclitaxel and the model cancer cell line HeLa. The results obtained by using the microchamber array chip were consistent with those obtained by a conventional assay using microplates. The microchamber array chip, with its simple interface and well-designed microfluidic network, has potential as an efficient platform for high-throughput dose response assays in drug discovery applications.

  11. Rapid purification of cell encapsulated hydrogel beads from oil phase to aqueous phase in a microfluidic device.

    PubMed

    Deng, Yuliang; Zhang, Nangang; Zhao, Libo; Yu, Xiaolei; Ji, Xinghu; Liu, Wei; Guo, Shishang; Liu, Kan; Zhao, Xing-Zhong

    2011-12-01

    In this paper, we demonstrate a new type of microfluidic chip that can realize continuous-flow purification of hydrogel beads from a carrier oil into aqueous solution by using a laminar-like oil/water interface. The microfluidic chip is composed by two functional components: (1) a flow-focusing bead generation module that can control size and shape of beads, (2) a bead extraction module capable of purifying hydrogel beads from oil into aqueous solution. This module is featured with large branch channels on one side and small ones on the opposite side. Water is continuously infused into the bead extraction module through the large branch channels, resulting in a laminar-like oil/water interface between the branch junctions. Simulation and experimental data show that the efficiency of oil depletion is determined by the relative flow rates between infused water and carrier oil. By using such a microfluidic device, viable cells (HCT116, colon cancer cell line) can be encapsulated in the hydrogel beads and purified into a cell culture media. Significantly improved cell viability was achieved compared to that observed by conventional bead purification approaches. This facile microfluidic chip could be a promising candidate for sample treatment in lab-on-a-chip applications.

  12. AuPd/polyaniline as the anode in an ethylene glycol microfluidic fuel cell operated at room temperature.

    PubMed

    Arjona, N; Palacios, A; Moreno-Zuria, A; Guerra-Balcázar, M; Ledesma-García, J; Arriaga, L G

    2014-08-01

    AuPd/polyaniline was used for the first time, for ethylene glycol (EG) electrooxidation in a novel microfluidic fuel cell (MFC) operated at room temperature. The device exhibits high electrocatalytic performance and stability for the conversion of cheap and fully available EG as fuel. PMID:24923468

  13. Microfluidic Device for Electric Field-Driven Single-Cell Captureand Activation

    SciTech Connect

    Toriello, Nicholas M.; Douglas, Erik S.; Mathies, Richard A.

    2005-09-20

    A microchip that performs directed capture and chemical activation of surface-modified single-cells has been developed. The cell-capture system is comprised of interdigitated gold electrodes microfabricated on a glass substrate within PDMS channels. The cell surface is labeled with thiol functional groups using endogenous RGD receptors and adhesion to exposed gold pads on the electrodes is directed by applying a driving electric potential. Multiple cell types can thus be sequentially and selectively captured on desired electrodes. Single-cell capture efficiency is optimized by varying the duration of field application. Maximum single-cell capture is attained for the 10 min trial, with 63+-9 percent (n=30) of the electrode pad rows having a single cell. In activation studies, single M1WT3 CHO cells loaded with the calcium-sensitive dye fluo-4 AM were captured; exposure to the muscarinic agonist carbachol increased the fluorescence to 220+-74percent (n=79) of the original intensity. These results demonstrate the ability to direct the adhesion of selected living single cells on electrodes in a microfluidic device and to analyze their response to chemical stimuli.

  14. Electrode Calibration with a Microfluidic Flow Cell for Fast-scan Cyclic Voltammetry

    PubMed Central

    Sinkala, Elly; McCutcheon, James E.; Schuck, Matt; Schmidt, Eric; Roitman, Mitchell F.; Eddington, David T.

    2012-01-01

    Fast-scan cyclic voltammetry (FSCV) is a common analytical electrochemistry tool used to measure chemical species. It has recently been adapted for measurement of neurotransmitters such as dopamine in awake and behaving animals (in vivo). Electrode calibration is an essential step in FSCV to relate observed current to concentration of a chemical species. However, existing methods require multiple components, which reduce the ease of calibrations. To this end, a microfluidic flow cell (µFC) was developed as a simple device to switch between buffer and buffer with a known concentration of the analyte of interest – in this case dopamine - in a microfluidic Y-channel. The ability to quickly switch solutions yielded electrode calibrations with faster rise times and that were more stable at peak current values. The µFC reduced the number of external electrical components and produced linear calibrations over a range of concentrations. To demonstrate this, an electrode calibrated with the µFC was used in FSCV recordings from a rat during the delivery of food reward – a stimulus that reliably evokes a brief increase in current due to the oxidation of dopamine. Using the linear calibration, dopamine concentrations were determined from the current responses evoked during the behavioral task. The µFC is able to easily and quickly calibrate FSCV electrode responses to chemical species for both in vitro and in vivo experiments. PMID:22522908

  15. Microfluidic Cell Deformability Assay for Rapid and Efficient Kinase Screening with the CRISPR-Cas9 System

    PubMed Central

    Han, Xin; Liu, Zongbin; Zhao, Li; Wang, Feng; Yu, Yang; Yang, Jianhua; Chen, Rui

    2016-01-01

    Herein we report a CRISPR-Cas9-mediated loss-of-function kinase screen for cancer cell deformability and invasive potential in a high-throughput microfluidic chip. In this microfluidic cell separation platform, flexible cells with high deformability and metastatic propensity flowed out, while stiff cells remained trapped. Through deep sequencing, we found that loss of certain kinases resulted in cells becoming more deformable and invasive. High-ranking candidates identified included well-reported tumor suppressor kinases, such as chk2, IKK-α, p38 MAPKs, and DAPK2. A high-ranking candidate STK4 was chosen for functional validation and identified to play an important role in the regulation of cell deformability and tumor suppression. Collectively, we have demonstrated that CRISPR-based on-chip mechanical screening is a potentially powerful strategy to facilitate systematic genetic analyses. PMID:27258939

  16. Microfluidic Cell Deformability Assay for Rapid and Efficient Kinase Screening with the CRISPR-Cas9 System.

    PubMed

    Han, Xin; Liu, Zongbin; Zhao, Li; Wang, Feng; Yu, Yang; Yang, Jianhua; Chen, Rui; Qin, Lidong

    2016-07-18

    Herein we report a CRISPR-Cas9-mediated loss-of-function kinase screen for cancer cell deformability and invasive potential in a high-throughput microfluidic chip. In this microfluidic cell separation platform, flexible cells with high deformability and metastatic propensity flowed out, while stiff cells remained trapped. Through deep sequencing, we found that loss of certain kinases resulted in cells becoming more deformable and invasive. High-ranking candidates identified included well-reported tumor suppressor kinases, such as chk2, IKK-α, p38 MAPKs, and DAPK2. A high-ranking candidate STK4 was chosen for functional validation and identified to play an important role in the regulation of cell deformability and tumor suppression. Collectively, we have demonstrated that CRISPR-based on-chip mechanical screening is a potentially powerful strategy to facilitate systematic genetic analyses.

  17. High frequency fluidic and microfluidic sensors for contactless dielectric and in vitro cell culture measurement applications

    NASA Astrophysics Data System (ADS)

    Nacke, T.; Barthel, A.; Cahill, B. P.; Meister, M.; Zaikou, Y.

    2013-04-01

    There is a widespread need for highly-sensitive robust sensors that operate without direct contact to the fluid for analysis of fluids in bioprocess technology. Measuring the variation of dielectric properties (conductivity and permittivity) in the microwave frequency band can be used as an approach to investigate biological and chemical matter and processes such as, cell growth, cell metabolism and the concentration of large aqueous based molecules. In comparison to measurement at lower frequencies, DC conductivity (σ) effects on material properties (permittivity ε) can be neglected with increasing of the frequency. This presentation describes a high frequency sensor, which combines detection in macro- or microfluidic networks with quick and precise analysis. It is composed of a fluidic channel placed contactless between a micro-strip line waveguide combined with resonant properties.

  18. A simple microfluidic dispenser for single-microparticle and cell samples

    PubMed Central

    Kasukurti, A.; Eggleton, C.D.; Desai, S.A.; Disharoon, D. I.; Marr, D.W.M.

    2014-01-01

    Non-destructive isolation of single-cells has become an important need for many biology research laboratories; however, there is a lack of easily employed and inexpensive tools. Here, we present a single-particle sample delivery approach fabricated from simple, economical components that may address this need. In this, we employ unique flow and timing strategies to bridge the significant force and length scale differences inherent in transitioning from single particle isolation to delivery. Demonstrating this approach, we use an optical trap to isolate individual microparticles and red blood cells that are dispensed within separate 50 µl droplets off a microfluidic chip for collection into microscope slides or microtiter plates. PMID:25316326

  19. Microfluidity mapping using fluorescence correlation spectroscopy: a new way to investigate plasma membrane microorganization of living cells.

    PubMed

    Winckler, Pascale; Cailler, Aurélie; Deturche, Régis; Jeannesson, Pierre; Morjani, Hamid; Jaffiol, Rodolphe

    2012-11-01

    Diffusion time distribution analysis has been employed to highlight the microfluidity fingerprint of plasma membrane of living cells. Diffusion time measurements were obtained through fluorescence correlation spectroscopy performed at the single cell level, over various eukaryotic cell lines (MCF7, LR73, KB3.1, MESSA and MDCKII). The nonsymmetric profile of the diffusion time distributions established experimentally, is discussed according to Monte Carlo simulations, which reproduce the diffusion of the fluorescent probe in heterogeneous membrane.

  20. Adhesion assays of endothelial cells on nanopatterned surfaces within a microfluidic channel.

    PubMed

    Hwang, Se Yon; Kwon, Keon Woo; Jang, Kyung-Jin; Park, Min Cheol; Lee, Jeong Sang; Suh, Kahp Y

    2010-04-01

    We present a simple analytical method to measure adhesion of human umbilical vein endothelial cells (HUVECs) and calf pulmonary artery endothelial cells (CPAEs) using nanopatterned, biodegradable poly(lactic-co-glycolic acid) (PLGA) surfaces for potential applications to artificial tissue-engineered blood vessel. Various nanostructured PLGA surfaces (350 nm wide ridges/350 nm grooves, 350 nm ridges/700 nm grooves, 350 nm ridges/1750 nm grooves, 700 nm ridges/350 nm grooves, 1050 nm ridges/350 nm grooves, 1750 nm ridges/350 nm grooves) and flat (unpatterned) surfaces were fabricated on the bottom of polydimethylsiloxane (PDMS) microfluidic channel of 2 mm width and 60 microm height by using thermal imprinting and irreversible channel bonding. To measure adhesion strength of HUVECs and CPAEs, the cells were exposed to a range of shear stress (12, 40, and 80 dyn/cm(2)) within the channels for 20 min after a preculture for 3 days and the remaining cells were counted under each condition. The highest adhesion strength was found on the surface of 700 nm wide ridges, 350 nm wide grooves for both cell types. The enhanced adhesion on nanopatterned surfaces can be attributed to two aspects: (i) contact guidance along the line direction and (ii) clustered focal adhesions. In particular, the contact guidance induced cell alignment along the line directions, which in turn lowers wall shear stress applied to the cell surface, as supported by a simple hydrodynamic model based on cell morphology. PMID:20218573

  1. Passage times of confined cancer cells and deformable particles flowing through a microfluidic channel

    NASA Astrophysics Data System (ADS)

    Khan, Zeina; Kamyabi, Nabiollah; Hussain, Fazle; Vanapalli, Siva

    Circulating tumor cells, the primary cause of cancer metastasis, have to navigate through tight extracellular matrix and capillaries. Unfortunately, understanding of the hydrodynamic interactions between cells and narrow vessel walls is lacking. Using a microfluidic channel of rectangular cross-section, we investigate cell hydrodynamic behavior by measuring cell confinement, passage time through the microchannel, and excess pressure drop. Testing with highly and lowly aggressive cancer cells shows that passage time may not always be indicative of cancer cell aggressiveness as the relationship among passage time, friction and rheology is complex. Transport of deformable particles including droplets of varying viscosity and interfacial tension, as well as elastic particles of different elastic moduli, reveals that passage times depend on particle size and, contrary to prior claims, on viscosity but not on elastic modulus. We also find that particle viscosity and not modulus controls the friction force and lubrication film thickness, suggesting that cancer cell viscosity rather than elasticity controls cell transport on short time-scales.

  2. Microfluidic devices for label-free separation of cells through transient interaction with asymmetric receptor patterns

    NASA Astrophysics Data System (ADS)

    Bose, S.; Singh, R.; Hollatz, M. H.; Lee, C.-H.; Karp, J.; Karnik, R.

    2012-02-01

    Cell sorting serves an important role in clinical diagnosis and biological research. Most of the existing microscale sorting techniques are either non-specific to antigen type or rely on capturing cells making sample recovery difficult. We demonstrate a simple; yet effective technique for isolating cells in an antigen specific manner by using transient interactions of the cell surface antigens with asymmetric receptor patterned surface. Using microfluidic devices incorporating P-selectin patterns we demonstrate separation of HL60 cells from K562 cells. We achieved a sorting purity above 90% and efficiency greater than 85% with this system. We also present a mathematical model incorporating flow mediated and adhesion mediated transport of cells in the microchannel that can be used to predict the performance of these devices. Lastly, we demonstrate the clinical significance of the method by demonstrating single step separation of neutrophils from whole blood. When whole blood is introduced in the device, the granulocyte population gets separated exclusively yielding neutrophils of high purity (<10% RBC contamination). To our knowledge, this is the first ever demonstration of continuous label free sorting of neutrophils from whole blood. We believe this technology will be useful in developing point-of-care diagnostic devices and also for a host of cell sorting applications.

  3. Self-loading and cell culture in one layer microfluidic devices.

    PubMed

    Wang, Li; Ni, Xiao-Fang; Luo, Chun-Xiong; Zhang, Zhi-Ling; Pang, Dai-Wen; Chen, Yong

    2009-06-01

    We report on a simple method for self loading and culture of mammalian cells in microfluidic multi-chambers for high throughput screening. The device was obtained by using one layer soft lithography with polydimethylsiloxane (PDMS) and thermal bonding on a glass slide. Self loading of cell suspension could be possible after degassing of the PDMS device for 30 min. Both cell loading efficiency and cell proliferation behaviors have been analyzed with triangle chambers of different sizes, all connected to the main flow channels with small entrances. We found that the number of cells loaded into the micro-chamber increased with the side length of the triangle, showing well size dependence and that self loading at a single cell level was possible for small chambers. For large chambers, the cell area density after loading and proliferation is however quite heterogeneous. For demonstration, HeLa cell growth behavior has been followed for 11 days until the total area of the largest chambers was fully filled.

  4. Investigation of Tumor Cell Behaviors on a Vascular Microenvironment-Mimicking Microfluidic Chip

    PubMed Central

    Huang, Rong; Zheng, Wenfu; Liu, Wenwen; Zhang, Wei; Long, Yunze; Jiang, Xingyu

    2015-01-01

    The extravasation of tumor cells is a key event in tumor metastasis. However, the mechanism underlying tumor cell extravasation remains unknown, mainly hindered by obstacles from the lack of complexity of biological tissues in conventional cell culture, and the costliness and ethical issues of in vivo experiments. Thus, a cheap, time and labor saving, and most of all, vascular microenvironment-mimicking research model is desirable. Herein, we report a microfluidic chip-based tumor extravasation research model which is capable of simultaneously simulating both mechanical and biochemical microenvironments of human vascular systems and analyzing their synergistic effects on the tumor extravasation. Under different mechanical conditions of the vascular system, the tumor cells (HeLa cells) had the highest viability and adhesion activity in the microenvironment of the capillary. The integrity of endothelial cells (ECs) monolayer was destroyed by tumor necrosis factor-α (TNF-α) in a hemodynamic background, which facilitated the tumor cell adhesion, this situation was recovered by the administration of platinum nanoparticles (Pt-NPs). This model bridges the gap between cell culture and animal experiments and is a promising platform for studying tumor behaviors in the vascular system. PMID:26631692

  5. A microfluidic device for 2D to 3D and 3D to 3D cell navigation

    NASA Astrophysics Data System (ADS)

    Shamloo, Amir; Amirifar, Leyla

    2016-01-01

    Microfluidic devices have received wide attention and shown great potential in the field of tissue engineering and regenerative medicine. Investigating cell response to various stimulations is much more accurate and comprehensive with the aid of microfluidic devices. In this study, we introduced a microfluidic device by which the matrix density as a mechanical property and the concentration profile of a biochemical factor as a chemical property could be altered. Our microfluidic device has a cell tank and a cell culture chamber to mimic both 2D to 3D and 3D to 3D migration of three types of cells. Fluid shear stress is negligible on the cells and a stable concentration gradient can be obtained by diffusion. The device was designed by a numerical simulation so that the uniformity of the concentration gradients throughout the cell culture chamber was obtained. Adult neural cells were cultured within this device and they showed different branching and axonal navigation phenotypes within varying nerve growth factor (NGF) concentration profiles. Neural stem cells were also cultured within varying collagen matrix densities while exposed to NGF concentrations and they experienced 3D to 3D collective migration. By generating vascular endothelial growth factor concentration gradients, adult human dermal microvascular endothelial cells also migrated in a 2D to 3D manner and formed a stable lumen within a specific collagen matrix density. It was observed that a minimum absolute concentration and concentration gradient were required to stimulate migration of all types of the cells. This device has the advantage of changing multiple parameters simultaneously and is expected to have wide applicability in cell studies.

  6. Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology

    PubMed Central

    Elani, Yuval

    2016-01-01

    The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved. PMID:27284034

  7. Ductular reaction-on-a-chip: Microfluidic co-cultures to study stem cell fate selection during liver injury

    PubMed Central

    Haque, Amranul; Gheibi, Pantea; Stybayeva, Gulnaz; Gao, Yandong; Torok, Natalie; Revzin, Alexander

    2016-01-01

    Liver injury modulates local microenvironment, triggering production of signals that instruct stem cell fate choices. In this study, we employed a microfluidic co-culture system to recreate important interactions in the liver stem cell niche, those between adult hepatocytes and liver progenitor cells (LPCs). We demonstrate that pluripotent stem cell-derived LPCs choose hepatic fate when cultured next to healthy hepatocytes but begin biliary differentiation program when co-cultured with injured hepatocytes. We connect this fate selection to skewing in production of hepatocyte growth factor (HGF) and transforming growth factor (TGF)-β1 caused by injury. Significantly, biliary fate selection of LPCs was not observed in the absence of hepatocytes nor did it happen in the presence of TGF-β inhibitors. Our study demonstrates that microfluidic culture systems may offer an interesting new tool for dissecting cellular interactions leading to aberrant stem cell differentiation during injury. PMID:27796316

  8. Intrinsic FGF2 and FGF5 promotes angiogenesis of human aortic endothelial cells in 3D microfluidic angiogenesis system

    PubMed Central

    Seo, Ha-Rim; Jeong, Hyo Eun; Joo, Hyung Joon; Choi, Seung-Cheol; Park, Chi-Yeon; Kim, Jong-Ho; Choi, Ji-Hyun; Cui, Long-Hui; Hong, Soon Jun; Chung, Seok; Lim, Do-Sun

    2016-01-01

    The human body contains different endothelial cell types and differences in their angiogenic potential are poorly understood. We compared the functional angiogenic ability of human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using a three-dimensional (3D) microfluidic cell culture system. HAECs and HUVECs exhibited similar cellular characteristics in a 2D culture system; however, in the 3D microfluidic angiogenesis system, HAECs exhibited stronger angiogenic potential than HUVECs. Interestingly, the expression level of fibroblast growth factor (FGF)2 and FGF5 under vascular endothelial growth factor (VEGF)-A stimulation was significantly higher in HAECs than in HUVECs. Moreover, small interfering RNA-mediated knockdown of FGF2 and FGF5 more significantly attenuated vascular sprouting induced from HAECs than HUVECs. Our results suggest that HAECs have greater angiogenic potential through FGF2 and FGF5 upregulation and could be a compatible endothelial cell type to achieve robust angiogenesis. PMID:27357248

  9. Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology.

    PubMed

    Elani, Yuval

    2016-06-15

    The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved. PMID:27284034

  10. Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology.

    PubMed

    Elani, Yuval

    2016-06-15

    The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved.

  11. Metabolic monitoring of the electrically stimulated single heart cell within a microfluidic platform.

    PubMed

    Cheng, Wei; Klauke, Norbert; Sedgwick, Helen; Smith, Godfrey L; Cooper, Jonathan M

    2006-11-01

    A device based on five individually addressable microelectrodes, fully integrated within a microfluidic system, has been fabricated to enable the real-time measurement of ionic and metabolic fluxes from electrically active, beating single heart cells. The electrode array comprised one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an electrochemical lactate microbiosensor, that were used to measure the amounts of lactate produced by the heart cell. The device also allowed simultaneous in-situ microscopy, enabling optical measurements of cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+. Initial experiments aimed to create a metabolic profile of the beating heart cell, and results show well defined excitation-contraction (EC) coupling at different rates. Ca2+ transients and extracellular pH measurements were obtained from continually paced single myocytes, both as a function of the rate of cell contraction. Finally, the relative amounts of intra- and extra-cellular lactate produced during field stimulation were determined, using cell electroporation where necessary.

  12. Single cell swimming dynamics of Listeria monocytogenes using a nanoporous microfluidic platform

    SciTech Connect

    Wright, Evan; Neethirajan, Suresh; Warriner, Keith; Retterer, Scott T; Srijanto, Bernadeta R

    2014-01-01

    Listeria monocytogenes remains a significant foodborne pathogen due to its virulence and ability to become established in food processing facilities. The pathogen is characterized by its ability to grow over a wide temperature range and withstand a broad range of stresses. The following reports on the chemotaxis and motility of the L. monocytogenes when exposed to relatively small concentrations of acetic acid. Using the developed nanoporous microfluidic device to precisely modulate the cellular environment, we exposed the individual Listeria cells to acetic acid and, in real time and with high resolution, observed how the cells reacted to the change in their surroundings. Our results showed that concentrations of acetic acid below 10 mM had very little, if any, effect on the motility. However, when exposed to 100 mM acetic acid, the cells exhibited a sharp drop in velocity and displayed a more random pattern of motion. These results indicate that at appropriate concentrations, acetic acid has the ability to disable the flagellum of the cells, thus impairing their motility. This drop in motility has numerous effects on the cell; its main effects being the obstruction of the cell's ability to properly form biofilms and a reduction in the overall infectivity of the cells. Since these characteristics are especially useful in controlling the proliferation of L. monocytogenes, acetic acid shows potential for application in the food industry as an active compound in designing a food packaging environment and as an antimicrobial agent.

  13. Cell death along single microfluidic channel after freeze-thaw treatments

    PubMed Central

    Li, Yuhui; Wang, Fen; Wang, Hao

    2010-01-01

    Cryotherapy is a prospective green method for malignant tumor treatment. At low temperature, the cell viability relates with the cooling rate, temperature threshold, freezing interface, as well as ice formation. In clinical applications, the growth of ice ball must reach a suitable size as cells could not be all killed at the ice periphery. The cell death ratio at the ice periphery is important for the control of the freezing destruction. The mechanisms of cryoinjury around the ice periphery need thorough understanding. In this paper, a primary freeze-thaw control was carried out in a cell culture microchip. A series of directional freezing processes and cell responses was tested and discussed. The temperature in the microchip was manipulated by a thermoelectric cooler. The necrotic and apoptotic cells under different cryotreatment (duration of the freezing process, freeze-thaw cycle, postculture, etc.) were stained and distinguished by propidium iodide and fluorescein isothiocyanate (FITC)-Annexin V. The location of the ice front was recorded and a cell death boundary which was different from the ice front was observed. By controlling the cooling process in a microfluidic channel, it is possible to recreate a sketch of biological effect during the process of simulated cryosurgery. PMID:20644680

  14. Observation of reversible, rapid changes in drug susceptibility of hypoxic tumor cells in a microfluidic device.

    PubMed

    Germain, Todd; Ansari, Megan; Pappas, Dimitri

    2016-09-14

    Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 μM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target. PMID:27566353

  15. Metabolite profiling of microfluidic cell culture conditions for droplet based screening

    PubMed Central

    Bjork, Sara M.; Sjostrom, Staffan L.; Andersson-Svahn, Helene; Joensson, Haakan N.

    2015-01-01

    We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format. PMID:26392830

  16. A label-free microfluidic biosensor for activity detection of single microalgae cells based on chlorophyll fluorescence.

    PubMed

    Wang, Junsheng; Sun, Jinyang; Song, Yongxin; Xu, Yongyi; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

    2013-11-26

    Detection of living microalgae cells is very important for ballast water treatment and analysis. Chlorophyll fluorescence is an indicator of photosynthetic activity and hence the living status of plant cells. In this paper, we developed a novel microfluidic biosensor system that can quickly and accurately detect the viability of single microalgae cells based on chlorophyll fluorescence. The system is composed of a laser diode as an excitation light source, a photodiode detector, a signal analysis circuit, and a microfluidic chip as a microalgae cell transportation platform. To demonstrate the utility of this system, six different living and dead algae samples (Karenia mikimotoi Hansen, Chlorella vulgaris, Nitzschia closterium, Platymonas subcordiformis, Pyramidomonas delicatula and Dunaliella salina) were tested. The developed biosensor can distinguish clearly between the living microalgae cells and the dead microalgae cells. The smallest microalgae cells that can be detected by using this biosensor are 3 μm ones. Even smaller microalgae cells could be detected by increasing the excitation light power. The developed microfluidic biosensor has great potential for in situ ballast water analysis.

  17. Cross talk between cancer and immune cells: exploring complex dynamics in a microfluidic environment.

    PubMed

    Businaro, Luca; De Ninno, Adele; Schiavoni, Giovanna; Lucarini, Valeria; Ciasca, Gabriele; Gerardino, Annamaria; Belardelli, Filippo; Gabriele, Lucia; Mattei, Fabrizio

    2013-01-21

    The reconstitution of a complex microenvironment on microfluidic chips is one of the cornerstones to demonstrate the improved flexibility of these devices with respect to macroscale in vitro approaches. In this work, we realised an on-chip model to investigate the interactions between cancer and immune system. To this end, we exploited mice deficient (Knock Out, KO) for interferon regulatory factor 8 (IRF-8), a transcription factor essential for the induction of competent immune responses, to investigate how IRF-8 gene expression contributes to regulate immune and melanoma cells crosstalk. In vivo, IRF-8 KO mice are highly permissive to B16 melanoma growth due to failure of immune cells to properly exert immunosurveillance. B16 cells and immune cells isolated from the spleen of wild type (WT) and IRF-8 KO mice were co-cultured for one week in a PDMS platform and monitored by fluorescence microscopy and time-lapse recordings. We observed that WT spleen cells migrated through microchannels connecting the culturing chambers towards B16 cells and tightly interacted with tumor cells, forming clusters of activation. In contrast, IRF-8 KO immune cells poorly interacted with melanoma cells. In parallel, B16 cells were more attracted towards microchannels, acquiring a more invasive behaviour in the presence of IRF-8 KO spleen cells, with respect to WT cells. Our results strongly confirm the in vivo observations and highlight the value of on-chip co-culture systems as a useful in vitro tool to elucidate the reciprocal interactions between cancer cells and host immune system, with relevant impact in the development of more effective anti-tumor therapeutic strategies. PMID:23108434

  18. Novel Carbon-based Electrode Materials for Up-scaled Microfluidic Fuel Cells

    NASA Astrophysics Data System (ADS)

    Fuerth, Dillon Adam

    In this work, a MFC fabrication procedure including two non-conventional techniques (partial baking and cap-sealing) were employed for the development of an up-scaled microfluidic fuel cell (MFC). Novel carbon-based electrode materials were employed, including carbon foam, fibre, and cloth, the results from which were compared with traditionally-employed carbon paper. The utilization of carbon cloth led to 15% of the maximum power that resulted from carbon paper; however, carbon fibre led to a 24.6% higher power density than carbon paper (normalized by electrode volume). When normalized by projected electrode area, the utilization of carbon foams resulted in power densities up to 42.5% higher than that from carbon paper. The impact of catalyst loading on MFC performance was also investigated, with an increase from 10.9 to 48.3 mgPt cm-2 resulting in a 195% increase in power density.

  19. A drug-compatible and temperature-controlled microfluidic device for live-cell imaging

    PubMed Central

    Chen, Tong; Gomez-Escoda, Blanca; Munoz-Garcia, Javier; Babic, Julien; Griscom, Laurent; Wu, Pei-Yun Jenny

    2016-01-01

    Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging. PMID:27512142

  20. A drug-compatible and temperature-controlled microfluidic device for live-cell imaging.

    PubMed

    Chen, Tong; Gomez-Escoda, Blanca; Munoz-Garcia, Javier; Babic, Julien; Griscom, Laurent; Wu, Pei-Yun Jenny; Coudreuse, Damien

    2016-08-01

    Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging. PMID:27512142

  1. Geometric correction factor for transepithelial electrical resistance measurements in transwell and microfluidic cell cultures

    NASA Astrophysics Data System (ADS)

    Yeste, J.; Illa, X.; Gutiérrez, C.; Solé, M.; Guimerà, A.; Villa, R.

    2016-09-01

    Transepithelial electrical resistance (TEER) measurements are regularly used in in vitro models to quantitatively evaluate the cell barrier function. Although it would be expected that TEER values obtained with the same cell type and experimental setup were comparable, values reported in the literature show a large dispersion for unclear reasons. This work highlights a possible error in a widely used formula to calculate the TEER, in which it may be erroneously assumed that the entire cell culture area contributes equally to the measurement. In this study, we have numerically calculated this error in some cell cultures previously reported. In particular, we evidence that some TEER measurements resulted in errors when measuring low TEERs, especially when using Transwell inserts 12 mm in diameter or microfluidic systems that have small chamber heights. To correct this error, we propose the use of a geometric correction factor (GCF) for calculating the TEER. In addition, we describe a simple method to determine the GCF of a particular measurement system, so that it can be applied retrospectively. We have also experimentally validated an interdigitated electrodes (IDE) configuration where the entire cell culture area contributes equally to the measurement, and it also implements minimal electrode coverage so that the cells can be visualized alongside TEER analysis.

  2. High-Resolution Microfluidic Single-Cell Transcriptional Profiling Reveals Clinically Relevant Subtypes among Human Stem Cell Populations Commonly Utilized in Cell-Based Therapies.

    PubMed

    Rennert, Robert C; Schäfer, Richard; Bliss, Tonya; Januszyk, Michael; Sorkin, Michael; Achrol, Achal S; Rodrigues, Melanie; Maan, Zeshaan N; Kluba, Torsten; Steinberg, Gary K; Gurtner, Geoffrey C

    2016-01-01

    Stem cell therapies can promote neural repair and regeneration, yet controversy regarding optimal cell source and mechanism of action has slowed clinical translation, potentially due to undefined cellular heterogeneity. Single-cell resolution is needed to identify clinically relevant subpopulations with the highest therapeutic relevance. We combine single-cell microfluidic analysis with advanced computational modeling to study for the first time two common sources for cell-based therapies, human NSCs and MSCs. This methodology has the potential to logically inform cell source decisions for any clinical application. PMID:27047447

  3. Dynamic monitoring of single cell lysis in an impedance-based microfluidic device.

    PubMed

    Zhou, Ying; Basu, Srinjan; Laue, Ernest D; Seshia, Ashwin A

    2016-08-01

    A microfluidic device that is capable of trapping and sensing dynamic variations in the electrical properties of individual cells is demonstrated. The device is applied to the real-time recording of impedance measurements of mouse embryonic stem cells (mESCs) during the process of membrane lysis, with the resulting changes in the electrical properties of cells during this process being quantitatively tracked over time. It is observed that the impedance magnitude decreases dramatically after cell membrane lysis. A significant shift in the phase spectrum is also observed during the time course of this process. By fitting experimental data to physical models, the electrical parameters of cells can be extracted and parameter variations quantified during the process. In the cell lysis experiments, the equivalent conductivity of the cell membrane is found to increase significantly due to pore formation in the membrane during lysis. An increase in the specific capacitance of the membrane is also observed. On the other hand, the conductivity of the cytoplasm is observed to decrease, which may be explained the fact that excess water enters the cell through the gradual permeabilization of the membrane during lysis. Cells can be trapped in the device for periods up to several days, and their electrical response can be monitored by real-time impedance measurements in a label-free and non-invasive manner. Furthermore, due to the highly efficient single cell trapping capacity of the device, a number of cells can be trapped and held in separate wells for concurrent parallel experiments, allowing for the possibility of stepped parametric experiments and studying cell heterogeneity by combining measurements across the array.

  4. Characterization and Resolution of Evaporation-Mediated Osmolality Shifts that Constrain Microfluidic Cell Culture in Poly(dimethylsiloxane) Devices

    PubMed Central

    Heo, Yun Seok; Cabrera, Lourdes M.; Song, Jonathan W.; Futai, Nobuyuki; Tung, Yi-Chung; Smith, Gary D.; Takayama, Shuichi

    2008-01-01

    Evaporation is a critical problem when handling sub-microliter volumes of fluids. This paper characterizes this problem as it applies to microfluidic cell culture in poly(dimethylsiloxane) (PDMS) devices and provides a practical solution. Evaporation-mediated osmolality shifts through PDMS membranes with varying thicknesses (10, 1, 0.2 or 0.1 mm) were measured over 96 hours. Even in humidified cell culture incubators, evaporation through PDMS and associated shifts in the osmolality of culture media was significant and prevented mouse embryo and human endothelial cell growth and development. A simple diffusion model, where the measured diffusion coefficient for PDMS matches reported values of ~10−9 m2/s, accounts for these evaporation and osmolality shifts. To overcome this problem a PDMS-parylene-PDMS hybrid membrane was developed that greatly suppresses evaporation and osmolality shifts, yet possesses thinness and the flexibility necessary to interface with deformationbased microfluidic actuation systems, maintains the clarity for optical microscopy, and enables the successful development of single cell mouse embryos into blastocysts under static conditions and culture of human endothelial cells underdynamic recirculation of sub-microliter volumes of media. These insights and methods demonstrated specifically for embryo and endothelial cell studies will be generally useful for understanding and overcoming evaporation-associated effects in microfluidic cell cultures. PMID:17263345

  5. Multivariate analysis of apoptotic markers versus cell cycle phase in living human cancer cells by microfluidic cytometry

    NASA Astrophysics Data System (ADS)

    Akagi, Jin; Skommer, Joanna; Matuszek, Anna; Takeda, Kazuo; Fujimura, Yuu; Khoshmanesh, Khashayar; Kalantar-Zadeh, Kourosh; Mitchell, Arnan; Errington, Rachel; Smith, Paul J.; Darzynkiewicz, Zbigniew; Wlodkowic, Donald

    2013-03-01

    Measurement of apoptotic markers in tumors can be directly correlated with the cell cycle phase using flow cytometry (FCM). The conventional DNA content analysis requires cell permeabilization to stain nuclei with fluorescent probes such as propidium iodide or use of a costly UV-excitation line for Hoechst 33342 probe. The access to FCM is also still limited to centralized core facilities due to its inherent high costs and complex operation. This work describes development and proof-of-concept validation of a portable and user-friendly microfluidic flow cytometer (μFCM) that can perform multivariate real time analysis on live cells using sampling volumes as small as 10 microliters. The μFCM system employs disposable microfluidic cartridges fabricated using injection molding in poly(methylmethacrylate) transparent thermoplastic. Furthermore, the dedicated and miniaturized electronic hardware interface enables up to six parameter detection using a combination of spatially separated solid-state 473 (10 mW) and 640 nm (20 mW) lasers and x-y stage for rapid laser alignment adjustment. We provide new evidence that a simple 2D flow focusing on a chip is sufficient to measure cellular DNA content in live tumor cells using a far-red DNA probe DRAQ5. The feasibility of using the μFCM system for a dose-response profiling of investigational anti-cancer agents on human hematopoietic cancer cells is also demonstrated. The data show that μFCM can provide a viable novel alternative to conventional FCM for multiparameter detection of caspase activation and dissipation of mitochondrial inner membrane potential (ΔΨm) in relation to DNA content (cell cycle phase) in live tumor cells.

  6. Microfluidics and microbial engineering.

    PubMed

    Kou, Songzi; Cheng, Danhui; Sun, Fei; Hsing, I-Ming

    2016-02-01

    The combination of m