Lorentz Center - Micro- and nanofluidics for cell biology from 18 Jan 2010 through 22 Jan 2010
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    Micro- and nanofluidics for cell biology
    from 18 Jan 2010 through 22 Jan 2010

 

Abstracts Micro- and Nanofluidics for Cell Biology

 

TALKS

 

Microdevice to capture colon crypts for in vitro studies

Nancy Allbritton

Authors: Yuli Wanga, Rahul Dhopeshwarkara, Rani Najdib, Marian Watermanb, Christopher E. Simsa, Nancy Allbrittona,c

aDepartment of Chemistry, University of North Carolina, Chapel Hill, NC 27599

bDepartment of Microbiology & Molecular Genetics, University of California, Irvine, California,

92697

cDepartment of Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599 and North Carolina State University, Raleigh, NC 27695

 

There is a need in biological research for tools designed to manipulate the environment surrounding microscopic regions of tissue.  A device for the oriented capture of an important and under-studied tissue, the colon crypt, has been designed and tested.  The objective of this work is to create a BioMEMs device for biological assays of living colonic crypts.  The end goal will be to subject the polarized tissue to user-controlled fluidic microenvironments in a manner that recapitulates the in vivo state.  Crypt surrogates, polymeric structures of similar dimensions and shape to isolated colon crypts, were used in the initial design and testing of the device.  Successful capture of crypt surrogates was accomplished on a simple device composed of an array of micron-scale capture sites that enabled individual structures to be captured with high efficiency (92 ± 3%) in an ordered and properly oriented fashion.  The device was then evaluated using colon crypts isolated from a murine animal model.  The capture efficiency attained using the biologic sample was 37 ± 5% due to the increased variability of the colon crypts compared with the surrogate structures, yet 94% ± 3% of the captured crypts were properly oriented.  A simple approach to plug the remaining capture sites in the array was performed using inert glass beads.  Blockage of unfilled capture sites is an important feature to prevent cross-contamination of the fluidic environments on the two sides of the array.  The present study demonstrates the facility and potential for rationally microengineered technologies to address the specific needs of the biologic researcher.

 

 

New type of cloned mouse embryos and embryonic stem cells after double nuclear transfer

Michele Boiani

Laboratory of Mouse Embryology, Max Planck Institute for Molecular Biomedicine

Muenster, Germany

 

The mammalian oocyte can give rise to up to 10^15 body cells of >200 tissues after fertilization. Moreover, it possesses the ability to reprogram the transplanted nuclei of somatic cells to a stem cell state. Hence, the oocyte can be considered as the mother of all stem cells. Oocyte studies provide biological understanding as well as practical tools that are of interest for regenerative medicine e.g. cloned embryos and embryonic stem (ES) cells. The risk that cloned embryos created for regenerative purposes could be misused e.g. implanted in utero for reproductive purposes is called a ‘slippery slope’, in analogy to the risk of falling down the main path’s shoulder into a ditch. My laboratory is interested in ways to make these applications of cloning independent of each other by separating pluripotency from totipotency.

            Transplantation of two somatic nuclei into mouse oocytes that have been stripped of their own nuclear material gives rise to tetraploid (4N) cloned embryos. In the female genital tract these embryos invariably fail, yet in the petri dish they give rise to 4N ES cells that express the classical markers of pluripotency over more than 20 passages. The 4N cloned ES cells rarely contributed to chimeras and never formed teratomas after subcutaneous injection in immunocompromised hosts. However in vitro they could differentiate into cardiomyocites, neurons and liver cells while maintaining the tetraploid karyotype. The rate and quality of the differentiation may be amenable to improvement by microfluidics. Altogether, these features draw a sharp line between pluripotency and totipotency in a cell reprogramming system.

 

 

Abstract Jan de Boer

 

 

Using nanostructures for biology at the single cell and single molecule level

Cees Dekker

Kavli Institute of Nanoscience, Delft University of Technology

Lorentzweg 1, 2628 CJ Delft, The Netherlands; email: c.dekker@tudelft.nl

 

In this talk I will discuss two examples that use nanofabrication of fluidic structures to address important problems in biology:

Part 1: I will report on translocation of DNA, RNA, proteins, and protein-DNA structures through solid-state nanopores1, nanometer-sized pores in an insulating membrane. We study voltage-driven translocations as well as do force measurements using an optical tweezer to hold a single DNA molecule within a nanopore. While so far most measurements have been carried out on double-strand DNA, we have recently extended the technique to also probe double and single strand RNA, RecA proteins, and RecA-coated DNA. I will report the latest status of these experiments at the meeting.

Part 2: I will report experiments where we use nanofabrication to construct landscapes for bacteria. We can use the technology to define food-rich and food-poor islands as well as channels that mutually couple island populations with a controlled strength. We study how bacteria can grow, move, and penetrate very narrow constrictions with a size comparable to or even smaller than their diameter. We observe E. coli to penetrate channels with a width that is smaller than their diameter by a factor of 2.2 Within these channels, bacteria are considerably squeezed but they still grow and divide. Tight confinement is found to exhibit a strong effect on the bacterial growth, division, and shape. We also study cooperation and cheating among bacteria in space and time. The interests of an individual may be different from those of a community. To study this conflict in bacterial populations, we mix cultures of a wild type (cooperator) and a GASP-mutant (cheater) strain E. coli bacteria in an homogeneous environment (batch culture), but also in spatially structured environments (nanofabricated patches). We find that the bacteria play the Prisoner’s Dilemma in homogeneous environments and the Snowdrift Game in heterogeneous environments.

 

1. C. Dekker, Solid state nanopores, Nature Nanotechnology 2, 209 - 215 (2007)

2. J. Mannik, R. Driessen, P. Galajda, J.E. Keymer and C. Dekker, Bacterial growth and motility in sub-micron constrictions, PNAS, 106, 14861 (2009)

 

 

Abstract Luce Dauphinot

 

 

Microfluidics to analyse the functioning of biomolecular networks

Roel van Driel

Netherlands Institute for Systems biology (NISB) and University of Amsterdam

 

Basic properties of living cells include intermediary metabolism, detection and interpretation of extracellular cues (e.g. hormones, nutrients) and regulation of large numbers of genes. We begin to understand that the systems that are responsible for this are highly complex networks of molecules that interact in time and space. Examples are metabolic networks, signal transduction networks and genetic networks. Analysis of the behaviour of biomolecular networks and unraveling of network architecture requires quantitative measurements on living cells under precisely defined static and dynamic conditions. In the past decade we have become highly proficient in high throughput measuring of metabolites, proteins and gene expression, generically known as the Omics technologies. A drawback of these biochemical technologies is that they generally average information of 105 or more cells. On the other end of the scale, modern light microscopy allows us to measure the behaviour and interactions of individual molecules inside living cells. By its very nature this is a low throughput technology, collecting data cell by cell.

Proof of principle has been presented that microfluidics is a strong tool to quantitatively analyse single cells under precisely defined static and dynamic external conditions. This potential has to be developed further to unravel network behaviour and allow mapping of network architecture. Combining the growing spectrum of light microscopy techniques with high throughput microfluidic single cell platforms is a very promising avenue. One step further, permeabilised cell systems could be exploited to not only change the external environment, but also the intracellular composition. Also, we may start thinking about ways to integrate microscopy measurements on living or permeabilised cells with single cell quantification of metabolites, proteins and RNAs.

 

 

‘Micro-fluidics’ of immune cells in vivo: from live cell vaccines towards artificial antigen presenting cells in cancer immunotherapy

Carl G. Figdor

Department of Tumor Immunology and Medical Oncology, Nijmegen Centre for Molecular Life Sciences (NCMLS), Radboud University Nijmegen Medical Centre, Postbox 9101, 6500 HB Nijmegen (c.figdor@ncmls.ru.nl), The Netherlands.

           

To evaluate the efficacy of new forms of cellular immunotherapy, and cell based therapies in particular it is of utmost importance to develop effective monitoring tools. We exploited dendritic cells (DCs), the professional antigen presenting cells of the immune system, to vaccinate melanoma patients. When vaccinating patients we get mixed results. To really understand how these live cell vaccines behave in vivo, we developed scintigraphic as well as magnetic resonance (MR) based techniques to follow immune cells in vivo. This provided us with unique novel insights in their functional behavior, and explained differences in responses observed in cancer patients. I will illustrate what the current position is of advanced cell therapy and how new developments may ultimately result in the generation of artificial antigen presenting cells in cancer immunotherapy .

 

This work was supported by grants 1999-1950, 2000-2301, 2003-2893, 2003-2917 and 2004-3127 from the Dutch Cancer Society, and EU projects DC-THERA, and ENCITE, the TIL-foundation and the NOTK.

 

 

Neuroscience on a Chip

Albert Folch

 

Cell culture technology is falling behind in the pace of progress. As animal and bacterial genomes and proteomes are being fully probed with DNA chips and a wide array of analytical techniques, a picture of cells with dauntingly complex inner workings is emerging. Yet cell culture methodology has remained basically unchanged for almost a century: it consists essentially of the immersion of a large population of cells in a homogeneous fluid medium. This approach is becoming increasingly expensive to scale up and cannot mimic the rich biochemical and biophysical complexity of the cellular microenvironment.

 

Microtechnology offers the attractive possibility of modulating the microenvironment of single cells and, for the same price, obtain data at high throughput for a small cost.  Microfluidic or “Lab on a Chip” devices, in particular, promise to play a key role for several reasons: 1) the dimensions of microchannels can be comparable to or smaller than a single cell;  2) the unique physicochemical behavior of liquids confined to microenvironments enables new strategies for delivering compounds to cells on a subcellular level;  3) the devices consume small quantities of precious/hazardous reagents (thus reducing cost of operation/disposal);  and 4) they can be mass-produced in low-cost, portable units.  Not surprisingly, in recent years there has been an eruption of microfluidic implementations of a variety of traditional bioanalysis techniques. I will review the latest efforts of our laboratory in the development of cell-based microdevices for neurobiology studies, such as neuromuscular synaptogenesis, axon guidance, and olfaction.

 

 

Cell and Tissue Cultures in Control - From Embryos to Stem Cells

Teruo Fujii

(IIS, Univ. of Tokyo)

 

While microfluidics has already contributed largely to the development of technologies for high-throughput and comprehensive biological analyses, there must be substantial potentials to realize further more sophisticated tools for cell biology experiments with spatio-temporal controls.

 

By properly applying the device technology to biological experimentation, a novel experimental foundation can be established for automated, continuous, and quantitative measurement of cells and tissues. This opens up new possibility to understand complex systems embedded in the cells and tissues in an integrative way.

 

The key advantages of the use of microfluidics or microfluidic devices mainly lie in the realization microenvironments mimicking in vivo situation by miniaturization, compartmentalization, formation of chemical gradients, etc. Moreover, we can achieve experimental format with more controllability on the conditions of cultures by using microfluidic devices leading to more systematic analyses that could never be done by conventional experimental tools.

 

The first part of the talk will be dedicated to the overviews of the evolution and fundamental technologies of microfluidic cell and tissue culture devices. Then, some of our recent projects, which are mainly focusing on building novel microfluidic platforms for automated culture and monitoring of embryos and for controlled differentiation of stem cells, will follow.

 

Finally insights and prospects towards the future challenges and opportunities for microfluidics to further contribute to cell biology will be addressed.

 

 

Biological responses of vascular endothelial cells to wall shear stress; role of the primary cilium

Beerend P Hierck

Dept. Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands. Email: b.p.hierck@lumc.nl

 

Shear stress is exerted on the wall of blood vessels by the blood flow. Vascular endothelial cells, which line the inner surface of all blood vessels, sense shear forces and respond to changes in magnitude and patterning with activation of intracellular signaling. These signaling events can result in phenotypic and functional adaptations, for better or for worse. An interesting process in this respect is endoMT (endothelial to mesenchymal transition), which is a normal process in the embryo in e.g. heart valve formation but is a pathological process in adults in e.g. atherosclerosis. Wall shear stress turns out to be instrumental in this process.

One of the sensor mechanisms of endothelial cells is the primary cilium, which is a rod-like membrane extension into the flow compartment of a blood vessel. Cells that are exposed to low and oscillating blood flow display a cilium which renders them more responsive to wall shear stress. We have structurally characterized the endothelial primary cilium and are in the process of its functional characterization. Signaling processes include activation of the Transforming Growth Factor-ß/Krüppel-Like Factor-2 and Hedgehog signaling pathways, which partially rely on the presence of a cilium. Furthermore, we have analyzed the distribution of ciliated endothelial cells in the embryonic cardiovascular system and in adult (diseased) blood vessels. In vitro experiments show that shear stress patterning, rather than magnitude, induces a ciliated phenotype.

In this study we are beginning to understand the interaction between local fluid forces and biological responses in blood vessels. In the end, this could have major implications for patient treatment, but also for tissue engineering of e.g. blood vessel segments and heart valves.

 

 

 

Biomimetic Microsystems Technologies: Organs-on-Chips

Donald E. Ingber, M.D.,Ph.D.

Director, Wyss Institute for Biologically Inspired Engineering at Harvard University

Judah Folkman Professor of Vascular Biology

Harvard Medical School & Children's Hospital Boston

Professor of Bioengineering, Harvard School of Engineering & Applied Sciences

 

Lab-on-a-chip technologies that contain living cells offer exciting new approaches to attack fundamental questions in biology, create smart medical devices, and positively impact human health. In this lecture, I will introduce Harvard's new Wyss Institute for Biologically Inspired Engineering that I now head. The Institute seeks to develop materials and devices that will transform healthcare and create a more sustainable world by emulating the way Nature builds. Institute Faculty and expert technical staff collaborate in high-risk research and technology development, inspired by the superior design strategies living systems use to adapt and compete for survival. Our Biomimetic Microsystems Platform uses microfabrication approaches to engineer miniaturized three-dimensional devices containing human cells, mimicking the parenchymal tissues, blood vessels, tissue-tissue interfaces and mechanical microenvironment of living organs.  Examples of these organ mimetics include an "Artificial Spleen" blood cleansing device for sepsis therapy, and a "Breathing Lung-on-a-Chip" device that provides a new way to study the biological effects, inflammatory responses and transcellular transport of environmental toxins, nanoparticles, cytokines and pathogens on lung airway epithelium and vascular endothelium under mechanical conditions that mimic physiological breathing.  These novel biomimetic microsystems technologies offer entirely new device-based approaches to accelerate drug development and toxin screening, and to enhance clinical care.

 

 

Carbon nanotube-based gene delivery systems for plant cells

Noritada Kaji

Nagoya University

 

Carbon nanotubes have attracted great interests since early 1990s due to their unique and superior chemical, physical, and mechanical properties.  Recently their application fields have been broadened from semiconductor field to biotechnology as tools for discovering biology, but few successful results are reported in the literature.  In this study, we demonstrated a novel application of carbon nanotubes as a powerful tool for gene delivery systems into plant cells. 

Since plant cells have thick cell walls, various gene transfection techniques such as particle-mediated gene gun and agrobacterium-mediated transfection have been developed to defeat the barrier of cell walls.  However, there are several drawbacks; low transfection efficiency and severe damage to plant cells especially the methods which require preparing protoplasts in prior to gene transfection.  So mild but high efficiency transfection techniques are required.  In this study, cellulase enzymes which could digest cell walls were immobilized on the surface of carbon nanotubes and used for making a nanometer-scale hole in the cell wall for DNA penetration. Several plant cells such as Arabidopsis thaliana were used for this carbon nanotube-based DNA delivery system.  Transfection efficiency was not so high (~10%) compared with conventional PEG or virus-based system, but a small quantity of DNA (~ng) was enough to transfection due to strong adsorption of DNA on the surface of carbon nanotubes.

Although we have not proved stable and permanent expression at regeneration step, this transient expression system could contribute the study of systems biology because we could observe an interacting network of genes in plant cells.

 

 

Microfluidic biochips as new supports for in vitro bioartificial organs and toxicity studies 

E.Leclerc

CNRS UMR 6600, Université de Technologie de Compiègne, Compiègne, France

 

Objectives:

Current developments in the technological fields of tissue engineering, bioengineering, biomechanics, microfabrication and microfluidics have led to highly complex and pertinent new biochips for in vitro toxicology investigations. The purpose of the biochips is to mimic organ tissues in vitro in order to partially reduce the amount of in vivo testing. These 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, continuous feeding of nutrients to cultured cells and waste removal. The biochips also allow the control of physiological contact times of diluted molecules with the tissues and cells. Cell biochips can be situated between in vitro Petri testing and in vivo testing. In this frame we present a biochip that can be applied to various cell types in order to be able to study toxicity on reconstructed tissues.

 

 

High-throughput methods to define the stem cell niche

M. Lutolf

 

The tissue-specific microenvironment, or niche, that an adult stem cell occupies is critical for its function. A complex array of instructive niche biomolecules delivered by support cells in close proximity regulates stem cell quiescence and the delicate balance between self-renewal and differentiation divisions. Despite progress in the identification of relevant niche proteins and signaling pathways in mice, many types of adult stem cells cannot be efficiently cultured in vitro without rapidly differentiating.  In order to enhance our ability to obtain adult stem cells and their progeny in sufficient and reproducible quantity for clinical use, a better molecular understanding of the factors that control stem cell fate is critical.  In this talk I will highlight some recent efforts in my laboratory to develop and utilize bioengineering strategies that allow us to biochemically and structurally deconstruct in vivo adult stem cell niches, and reconstruct them in vitro. These well-defined artificial niches are micro-arrayed in order to probe and manipulate stem cell fate in high-throughput and (if desired) at the single cell level. For example, using time-lapse microscopy in combination with quantitative image analyses we have explored the fate changes of several thousand single mouse hematopoietic stem cells (HSC) exposed to ca. 20 putative stem cell niche proteins (as single factors or in combination). Retrospective transplantation experiments in mice allowed identifying niche factors that dictate distinct HSC proliferation kinetics correlating with in vivo functions.

 

Selected references

Lutolf, M.P.*, Gilbert, P.M., Blau, H.M.* Designing materials to direct stem cell fate, Nature, 462, 433-441 (2009)

Lutolf, M.P., Doyonnas, R., Havenstrite, K., and Blau, H.M. Perturbation of single hematopoietic stem cell fate in artificial niches, Integrative Biology, 1, 59 (2009)

Cordey, M., Limacher, M., Kobel, S., Taylor, V., Lutolf, M.P., Enhancing the reliability and throughput of neurosphere culture on hydrogel microwell arrays, Stem Cells 26, 2586-2594 (2008)

 

 

Non-invasive analysis of cells and tissue with electrical impedance spectroscopy

Hywel Morgan

 

Impedance spectroscopy has been used for many years to analyse the passive dielectric properties of biological material, including tissue and cells.   Recently microfluidic technologies have enabled the development of techniques for measuring the electrical impedance of single cells at high speed.   We have developed a microfluidic single cell impedance cytometer that measures the electrical impedance and also the optical (fluorescent) properties of single cells at high speed.  The technique is being developed as a system for Point of Care label-free analysis of human whole blood, with a sample of only a few uL.  We have also developed impedance measurement methods for tissue analysis, and used this for monitoring the integrity of the epithelial barrier for cells grown within a microfluidic system.  This talk will review the basic of impedance measurements, describe how the impedance of cells and tissues is measured and describe further steps towards the development of impedance labelling methods for uniquely identifying cells expressing specific antigenic markers.

 

 

Signaling in cellular systems

Prof dr Mathieu Noteborn

Molecular Genetics, LIC-Faculty of Sciences, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands

 

A general overview on signaling processes within and among cells, ranging from unicellular organisms up to human beings will be presented. Post-translational modifications, protein-protein, protein-lipid and/or protein-DNA/RNA interactions as well as epigenetic changes are key modulators of functional signaling processes in cellular events such as cell cycle control. Probably equally important are the dynamic cellular localizations of the various signaling molecules. Signals sensed by receptors at the outer cellular membrane can activate or inhibit the transcription of genes within the nucleus or change the activity of proteins in the cytoplasm. During development of multi-cellular organisms signaling plays a truly fascinating and often unknown role in a very dense and complicated network of timely orchestrated molecular signaling processes. Whole families of growth factors and growth factor receptors are expressed and/or down-regulated in a balanced way during early or late stages of a developing organism. Signaling processes can be of importance for cell survival or cell death. Recently, autophagy has been proven to be both of importance in cellular survival as well as for killing cells under certain conditions. Especially, such a centered dualistic cellular signaling process is intriguing to study as a model for signaling in cellular systems. Derailment of signaling processes are underlying a variety of different diseases such as cancer and neurodegenerative diseases. Fundamental as well as applied studies will be highlighted.

 

 

Meet the new meat

Mark J. Post

Marloes Langelaan, Kristel Boonen, Daisy van der Schaft and Frank Baaijens, Eindhoven University of Technology, Department of Biomedical Engineering, division Soft Tissue Engineering and Biomechanics.

 

Tissue engineering is a powerful technique that is mainly being used for regenerative medicine in a wide variety of tissues and organs. Tissue engineering of skeletal muscle in specific also has many applications, including the potential production of meat. Reasons for promoting in vitro meat production as an alternative for meat production through livestock include animal well fare, process monitoring, environmental considerations as well as efficiency of food production in terms of feedstock. In vitro meat production through stem cell technology potentially leads to a dramatic reduction in livestock. In addition, the production process can be monitored in detail in a laboratory, which could result in the elimination of food borne illnesses, such as mad cow disease or salmonella infection. Furthermore, as the world population increases, livestock increases, leading to a great augmentation in environmental burden due to intensified land usage and increases in greenhouse gas emissions. Finally, animal protein production through traditional farming is very inefficient, yielding 15% of edible animal proteins from vegetable protein input.

Tissue engineering of skeletal muscle in general has been challenging, mostly because the designated tissue stem cell, the satellite cell, has limited regenerative capacity under in vitro conditions. This regenerative capacity is imperative for successful in vitro meat production. In vivo, the satellite cells occupy a cell specific niche, which directs the cellular behaviour and comprises soluble factors such as growth factors, insoluble factors including extracellular matrix proteins, physiological factors such as neurological stimulation, and mechanical features such as dynamic stretch and matrix elasticity. It is our hypothesis that these niche components are essential to mimic the regenerative process in vitro, which is necessary to produce mature, functional muscle. 

By systematically studying the purported niche factors, we aim to optimize the use of satellite cells in regenerative and nutrition developments. In addition, we are addressing nutrional and energy requirements for these cells cultures. When the proof of principle has been generated, a new set of challenges is ahead in process technology. Achieving the ultimate goals of this program would have an enormous societal impact, justifying the risk-benefit balance.

 

 

A whole embryo lab-on-a-chip for zebrafish

Michael K. Richardson

Professor of Biology, Institute of Biology, University of Leiden, The Netherlands

  

We have designed, patented and prototyped a glass microfluidics chip for culturing zebrafish embryos. The chip is of trilaminar construction and contains 32 wells in which zebrafish embryos are fed constantly with buffer or egg water. The wells are arranged in parallel to prevent cross-contamination. Using this chip, we can cultulre zebrafish embryos for up to 5 days after fertilisation, by which time they have hatched and have most of the major organ systems present and functional. This chip is optically transpartent and is compatible with fluorescent microscopy. Compared to standard 96 well plate culture, our chip offers extremely low culture volume, thereby saving on reagents. It also provides dynamic replacement of buffer rather than the very stressful static replacement - or no replacement - of buffer in standard assays. In view of the growing demand for alternatives to mice in drug and cosmetics testing, we hope this chip will find applications in industry.

 

 

Complexity in tissues

Jeroen Rouwkema

 

Although this is still often disregarded in tissue engineering research, most tissues in the body are complex systems containing regions with different morphologies, properties and functions. Apart from that, tissues often contain supporting structures such as a vascular and a neural network. When one tries to engineer tissues in vitro, taking into account this complexity may be important, especially when the tissue will be used as a model system to test tissue responses and should thus replicate a natural tissue as closely as possible.

During this presentation I will highlight some of the implications of this complexity on cell culture and tissue engineering. Specific attention will be given to the inclusion of a vascular network in a base tissue and the remodeling of tissues due to mechanical signals.

 

 

Inertial microfluidics for high throughput cell sorting

Aman Russom

 

It is generally believed that inertial effects are not found in microfluidic systems and that they may not be useful to sort particles and cells.  Contrary to this, in recent years we and others have shown how inertia is very useful for focusing and sorting cells and particles in microfluidics. At the laminar flow conditions (i.e., low Reynolds number flow) present in microchannels—where surface tension and capillary forces dominate over gravity and macroscale forces—inertial forces can be used where particle focusing depends solely on the inherent flow characteristics of channel geometries. Notably, particles in a curvilinear channel respond to transversal forces and migrate across axial streamlines in the direction of flow resulting in particle focusing based on size. In other words, a well designed microchannel can precisely determine where across the width of the channel a particle of known size will reside. The method does not require external applied forces or mechanical parts to continuously focus, order and sort live cells. In this presentation, I will describe the inertial-induced focusing phenomenon and how it can be used to continuously order and sort blood cells at very high throughput.

 

Reference:

1. Russom, A.; Gupta, A.; Nagrath, S.; Dicarlo, D.; Edd, J.; Toner, M. New Journal of Physics 2009, 11 075025 (9pp)

2. Di Carlo, D.; Irimia, D.; Tompkins, R. G.; Toner, M. Proc Natl Acad Sci U S A 2007, 104, (48), 18892-7.

 

 

Tiny droplets for single-cell analysis

Valérie Taly

 

Droplet-based microfluidic has led to the developement of highly powerful systems that represent a new paradigm in High-Throughput Screening where individual assays are compartimentalized within microdroplet microreactors. The integration of such systems for series of complex individual operations on droplets offers a solution to the necessary miniaturization and automation of individual biological assays. By combining a decrease of assay volume and an increase of throughput, this technology goes beyond the capacities of conventional screening systems. Droplets (in the pL to nL range) are produced as independent microreactors that can be further actuated and analyzed at rates of the order of 1000 droplets per seconds. Added to the flexibility and versatibility of platform designs, such progress in sub-nanoliter droplet manipulation allows for a level of control that was hitherto impossible. This presentation will examplify how microfluidic systems can be used to compartimentalize various types of cells (ranging from procaryotic cells to multicellular organisms) without deleterious effects on their viability within complex and controled platforms. The application of microfluidic systems for different cell-based assays will also be demonstrated. In addition, we will present a highly efficient microfluidic fluorescence-activated droplet sorter (FADS) combining many of the advantages of microtitre-plate screening and traditional fluorescence-activated cell sorting (FACS). By allowing the study of single cells rather than populations, this technology will prove its pertinence for high-throughput and high-content quantitative cell screening.

 

 

Highthroughput whole-organism manipulation and ultrafast optics for in vivo compound/genetic discoveries for neuronal regeneration

Mehmet Fatih Yanik, Asst. Prof.

 

Massachusetts Institute of Technology

Department of Electrical Engineering and Computer Science

Program of Computational and Systems Biology

Broad Institute of Harvard and MIT

www.rle.mit.edu/Yanik

 

In recent years, the advantages of using small invertebrate animals as model systems for human diseases have become increasingly apparent, and have resulted in three Nobel Prizes in Medicine and Chemistry in 2002, 2006 and 2008 for the discoveries made using the nematode C. elegans. The availability of a wide array of genetic techniques, along with the animal’s transparency, and its ability to grow in minute volumes make C. elegans an extremely versatile model organism. However, since the first studies in the early 1960s, little has changed in how this multi-cellular organism is physically manipulated. As a result, high-throughput in vivo studies could not be performed at cellular or sub-cellular resolution. We present key technologies for complex high-throughput whole-animal genetic and drug discoveries at sub-cellular resolution. We developed high-speed microfluidic sorters, which immobilize unanesthetized animals for high-throughput in vivo imaging and manipulation of sub-cellular features using femtosecond laser techniques. We show integrated chips with hundreds of addressable incubation chambers for exposure of individual animals to biochemical compounds and high-resolution time-lapse imaging of cellular features in vivo. We show devices for delivery of compound libraries in standard multi-well plates to microfluidic devices and also for rapid dispensing of screened animals into multi-well plates. These technologies allow various high-throughput in vivo assays on small-animals to be performed with sub-cellular resolution including mutagenesis, RNAi and compound screens. Using the femtosecond laser microsurgery technique we developed for high-throughput neuronal injury, we performed in vivo neuronal regeneration screens on tens of thousands of animals, and identified compounds and genetic targets that enhance regeneration significantly following injury.

 

 

POSTERS

 

Low-Temperature, Simple and Fast Integration Technique of Microfluidic

Chips by using a UV curable adhesive

R. Arayanarakool, S. Le Gac and Albert van den Berg

BIOS, The Lab-on-a-Chip Group, MESA+ Institute for Nanotechnology; University of Twente, THE NETHERLANDS

Abstract

 

Viscosity of Water in Nano-Confinement

Nataliya Brunets, Jeroen Haneveld, Henri Jansen and Niels Tas

Transducers Science and Technology Group

MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands. Email: n.r.tas@utwente.nl

 

   Since the early 1970s there is a notion that water near polar or charged surfaces is somehow ordered or structured, leading to a so called structural component of the disjoining pressure. The first indications came from interpretation of the disjoining pressure isotherms of wetting films of water on hydrophilic substrates [1], while unambiguous experimental proof came through application of the so called Surface Forces Apparatus (SFA) [2-6] showing short range repulsion forces between charged surfaces brought in close proximity (< 5nm), in addition to the double layer repulsion and van der Waals attraction as described by the DLVO theory.

   An important question that emerges is if and how the supposed interfacial “structuring” of water affects its local viscosity. In the early 1970’s measurements of the flow velocity of liquid plugs in quartz micro-capillarities driven by a disbalance between the capillary pressure and an applied external pressure revealed an elevated viscosity of water up to 40% in capillaries of 0.04 μm in radius [7]. These results could not be reproduced in nanometer thin films in the Surface Forces Apparatus (SFA). Different SFA-based measurements indicate that nm thin films have a viscosity equal [6,8] or close to the bulk viscosity of water [9, 10]. In contrary, recent AFM experiments show orders of magnitude increase in the viscosity of water with respect to bulk water in sub-nanometer confinement [11]. In these experiments, the normal and lateral forces of a nano-sized silicon tip approaching a solid hydrophilic surface were simultaneously measured.

   In different labs it is attempted to extract data on the viscosity of water from analyzing the capillary filling dynamics of water and aqeous salt solutions in nanochannels. A significantly slower than expected filling in channels with heights in the range of 10 – 50 nm [12,13] is found in comparison with the classical Washburn model [14]. The increase of the apparent viscosity in confinement ammounts up to approx. 30% in 11 nm channels. A best fit to the experimental data assuming a highly viscous layer next to the hydrophilic channel walls shows that this immobile layer should have a  thickness of 0.9 nm at each interface, which is in correspondence with the results of ref. [11].

   Based on all experimental data available, one must conclude that the question if and how hydration forces are connected to viscosity changes of interfacial water has still not been completely answered.

 

1.   B.V. Derjaguin, N.V. Churaev, J. Coll. Interf. Sci., 1974, 49, 249 - 255.

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3.   R.M. Pashley, J. Coll. Interf. Sci., 1981, 80, 153 - 162.

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13. J. Haneveld, N.R. Tas, N. Brunets, H.V. Jansen, M. Elwenspoek, J. Appl. Phys. 2008, 104, 014309, 1 – 6.

14. E.W. Washburn, The Physical  Review, 1921, 17, 273 – 283.

 

 

Probing Red Blood Cell Mechanics

R.C.H. van der Burgt, A.C.B. Bogaerds, P.D. Anderson, F.N. van de Vosse

 

For accurate modeling of transport in blood and its coagulation, the red blood cell (RBC) plays an important role. Because of the high volume contents in blood (hematocrit 45%), RBC mechanics play an important role in mixing and transport in plasma. Therefore, the mechanical characteristics of the RBC have to be determined accurately.

Earlier experiments involve contacts with solids, such as pipette or capillary walls, beads, and indenters. Modeling the influence of these solid interactions is required for proper mechanical analysis. We propose a method in which deformation is applied by the surrounding fluid only. The RBC can be deformed and analyzed in a silicone cross-slot geometry, if it can be positioned and kept at the stagnation point which is in the center of the cross-slot.

This situation is inherently unstable, hence continuous correction has to be performed. An optical system, in combination with valves that can vary inflow and outflow ratios, should be able to fulfill this. To test this system and to estimate the necessary outflow ratio, 2D finite element simulations with fluid-structure interaction (FSI) by means of a fictitious domain are performed. Results indicate that changing the outflow ratio as a function of the spatial position of the RBC at 100 Hz will be sufficient to position the cell. The simulations give a maximal out flow ratio of approximately 1:10, which should be physically realizable with the selected valve.

Next, 3D simulations should be performed for more accurate testing, and for the inverse analysis to determine the mechanical characteristics.

 

 

Intracellular Particle Tracking as a prospective tool in nanomedicine and mechanobiology

Yixuan Li1, Siva Vanapalli1,2, Juergen Schnekenburger3,  Andries van der Meer1,4 André Poot1,4, István Vermes1,4, Jan Feijen4, Frieder Mugele5 and Michel Duits1,5

 

1.Cell-Stress SRO, MESA+, Twente ; 2.Texas Tech. Uni.; 3, Muenster Uni.; 4, Polymer Chemistry and Biomaterials group, Twente); 5, Physics of Complex Fluids group, Twente

 

By simply tracking motions of nanometer-sized intracellular particles, one might be able to tell the status of healthy or diseased cells or understand the mechanical responses (i.e. cytoskeleton remodeling) of living cells in relationship to their physicochemical environments. In the future, these needs can be possibly fulfilled using dual-probes intracellular particle tracking (IPT) system that employs two different intracellular probe particles, ballistic injected fluorescent particles (BIP) and endogenous granules (EG).

From previous studies[1][2][3], we conclude that EG dynamics is strongly ATP sensitive and intimately connected to the dynamics of the microtubules. In contrast, BIP are mostly embedded in the actin network and display a more passive dynamics. These assertions indicate the availability of distinct microrheological probes to dissect the different contributions of actin and microtubule networks to the mechanical behavior of living cells.

To explore the potential for future application of IPT in nanomedicine we compared mechanical properties measured by both IPT and atomic force microscopy (AFM) in two pairs of tumor cells, pancreas adenocarcinoma & breast cancer cells at different metastatic potential. For both tissue types and for both probes, the mean squared displacement (MSD) function measured in the malignant cells is substantially larger than in the benign cells, indicating a softer/weaker mechanical environment in malignant tumors. However AFM could only be used to distinguish the malignant from the benign breast tumors but not for the pancreas tumors. It denotes the potential value of IPT as a simple but powerful nanomechanical tool in tumor cell characterization[4].

The ability of IPT to monitor the dynamic modification of cytoskeleton was further identified in the study of endothelial cells’ (EC) response to fluidic shear stress in a microfluidic device. EC suffered from physiological shear stress, and developed transient actin network stiffening, which  could reach a maximum 2 fold reduction in MSD of BIP in ~10 minutes of flow. Recovery from this stiffening occurred within 30 min after inducing the flow. On blocking the reception of vascular endothelial growth factor, which plays key role in the shear-induced signaling pathway of EC, this response remained absent[5]. These results corroborate IPT can also serve as a functional tool in mechanobiology studies.

 

References

[1] M.H.G. Duits, Y.Li, S.A. Vanapalli, F. Mugele, Mapping of Spatiotemporal Heterogeneous Particle Dynamics in Living Cells, Physics Review E 79, 051910 (2009)

[2] Y. Li, S.A. Vanapalli, M.H.G. Duits, Dynamics of ballistically injected latex particles in living human endothelial cells, Biorheology 46(4), 309-321 (2009).

[3] S.A. Vanapalli, Y. Li, F. Mugele and M.H.G. Duits, On the origins of the universal dynamics of endogenous granules in mammalian cells, Molecular and Cellular Biomechanics 150, 1-16 (2009).

[4] Y.Li, M.H.G. Duits, Intracellular particle tracking as a tool for tumor cell characterization. Journal of  Biomedical Optics,14, no. 6 (November 2009): 064005-7

[5] A.D. van der Meer, Y. Li, M.H.G. Duits, A.A. Poot, J. Feijen, I. Vermes; VEGFR-2-dependent, micromechanical stiffening of endothelial cells in response to fluid shear stress, submitted (2010)

http://www.mesaplus.utwente.nl/cellstress/

 

 

Spatially standardized cell biology

Jean-Philippe Frimat, Heike Menne and Jonathan West†

 

Leibniz – Institute für Analytische Wissenschaften – ISAS – e.V.,

Otto-Hahn-Str. 6b, Dortmund 44227, Germany. Contact†: jonathan.west@isas.de

 

In modern cell biology there is an onus on the development of standardized, quantitative and high throughput analysis methods. There is also great interest in the examination of single cells to discern the behaviour of individuals within the tissue population. Microfabrication techniques offer precision feature replication at cellular and tissue level length scales, and we have used these to develop microfluidic and micropatterning methods [1] for positioning cells. These spatially standardized analysis platforms are currently being used to address applications in cell biology: The research has involved the use of cellular arrays for the scalable production and analysis of 3-D tumour spheroids and as an analytical display for neurotoxicity screening [2]. In addition, juxtacrine signaling at the single cell level is currently being investigated using a microfluidic array for the heterotypic co-culture of contacting cell partners.

 

[1]        J.-P. Frimat, et al, Analytical and Bioanalytical Chemistry, 2009, 395(3), 601–609

[2]        J.-P. Frimat, et al, Lab on a Chip, 2010, DOI: 10.1039/b922193j

 

 

Microfluidic platform for simultaneous generation of four independent gradients:

Towards high throughput screening of trace elements for bone tissue engineering

Björn Harink

Abstract

 

Microfluidic channels for studying vascular biology

Andries van der Meer, André Poot, Jan Feijen, István Vermes

MIRA Institute for Biomedical Technology and Technical Medicine

University of Twente, The Netherlands

 

Introduction

The effects of shear stress on endothelial cells are studied in a lot of laboratories world-wide. In the majority of cases, cells are subjected to shear stress in parallel plate flow chambers or with cone-and-plate systems. There are a number of difficulties associated with these set-ups: relatively large amounts of cells, reagents and media are needed to perform the experiments, it is difficult to combine shear stress experiments with live cell microscopy and only limited amounts of samples and conditions can be tested in one experiment. Microfluidic channels – channels with dimensions in the micrometer range – are well-suited to overcome these challenges. We have developed these types of channels and tested them in a number of different studies related to endothelial cells and their response to shear stress.

 

Methods

Microfluidic channels were produced by soft lithography. In short, a mixture of poly(dimethysiloxane) (PDMS) oligomers and crosslinkers were poured on top of a silicon mould with plastic structures. The PDMS mixture was allowed to react for 2 hours at 60°C, after which the solid slab of PDMS was peeled off of the mould. Holes were punctured to reach the channel structures, and the PDMS was bound to a glass coverslip by activating both surfaces with oxygen plasma and pressing them together. Subsequently, fibronectin was used to coat the channels and a highly concentrated suspension of human endothelial cells was pipetted into them. After a monolayer was formed, the channels were connected to a syringe pump and the microfluidic set-up was ready to be used. Several experiments were performed using this microfluidic system: assaying actin filament reorganization in response to shear stress by staining with phalloidin-FITC, monitoring particle uptake by adding fluorescently labelled lipoplexes or low density lipoprotein and following nitric oxide release by pre-treating the cells with DAF-FM.

 

Results

We show that the microfluidic channels in this study are a versatile tool in fundamental research of endothelial cells and their response to shear stress. In all experiments, low amounts of reagents were needed. This is especially advantageous when expensive inhibitor drugs or growth factors are used. The channels are compatible with fluorescent microscopy on living cells, as illustrated by the nitric oxide release assay, or on fixated cells, as has been used in the actin filament assay. Because of the relative ease of assembling the microfluidic set-up, a lot of experiments could be performed in relatively short amounts of time.

 

Conclusions

Microfluidic channels are a valuable addition to the in vitro research tools that are available to researchers interested in studying the endothelial response to shear stress. The field of microfluidics is expanding rapidly, which will lead to useful additions to the basic microfluidic set-up described here.

 

 

Micro-PIV as research tool for in vivo and in vitro studies

Christian Poelma and Jerry Westerweel

Laboratory for Aero & Hydrodynamics (3ME-P&E), Delft University of Technology

 

We present recent research from our group utilizing micro Particle Image Velocimetry; the two main applications are (1) detailed in vivo flow and wall shear stress measurements in chicken embryos and (2) local wall shear stress measurements on endothelial cells in flow chambers combined with gene expression measurements using fluorescent imaging.

 

 

Mechanical screening and selection of circulating cells

Agnese Ravetto, Carlijn Bouten, Jaap den Toonder, Patrick Anderson

Abstract                                                                                    

 

 

Systems biology approaches to unravel the complexity and dynamics of G protein-coupled receptor signalling networks

Eric Reiter

BIOS group, INRA, CNRS, Université de Tours, UMR6175 Physiologie de la Reproduction et des Comportements, 37380 Nouzilly, France.

 

Intracellular signalling pathways are organized as coordinated communication networks in which multi-protein complexes process and integrate signal fluxes. One important challenge in cell biology is to understand the behaviour of these intertwined networks in order to decipher the “language” of the cell. G protein-coupled receptors (GPCR) expressed at the plasma membrane represent the largest class of membrane receptors. They are capable of binding a wide diversity of molecules regulating most physiological processes and are targeted by up to 50% of currently marketed drugs. It is therefore of paramount importance to understand how they signal. Classically, upon ligand binding, GPCRs undergo a conformational change that leads to heterotrimeric G protein recruitment and activation, followed by the generation of diffusible second messengers such as cAMP (cyclic Adenosine Mono-Phosphate), calcium or phosphoinositides. However, GPCRs also activate multiple pathways which form intricate networks. For instance, some GPCRs have the ability to couple to multiple G protein subtypes while many GPCRs directly interact with non-G protein signalling effectors through specific protein-protein interaction domains. Among these GPCR interactors, G protein-coupled receptor kinases (GRKs) and -arrestins have long been associated with the desensitisation and internalisation/recycling of most receptors. Recently, however, GPCRs have also been demonstrated to elicit signals, independently of heterotrimeric G protein coupling, through interaction with -arrestins and GRKs. For instance, -arrestins have been shown to act as multifunctional scaffolds and activators for a growing number of signalling proteins including ERK, p38, JNK, I-B, Akt and RhoA.

Adding to this complexity is the spatial and temporal encoding of GPCR-induced signals. For instance, G protein-mediated ERK activation is rapid (taking ~2-5 min to reach a maximum), transient and is followed by translocation to the nucleus. In contrast, the ERK activation via -arrestins is slower in onset (taking ~5-10 min to reach a maximum), very persistent (t1/2 > 1 hour) and ERK is sequestered in the cytosol.

A system-level understanding of GPCR-mediated signalling networks would be a significant asset to rationalize and speed-up the discovery of new “pathway-selective” drugs. To achieve this goal, it is crucial to produce new large scale data sets both in control or perturbed condition. The ability to tightly control the inputs (i.e. agonist stimulation) both in time and concentration using a microfluidic approach would represent a powerful mean to tightly control agonist exposure to the cells compared to conventional experimentation set ups. The potential is also there to closely mimic stimulation patterns which are encountered in vivo.

 

 

Self-remodeling microfabricated tissues as templates for stereotyped angiogenesis

N.C. Rivron1, E. J. Vrij1, R. Truckenmüller1, J. Rouwkema2, S. Legac3, A. van den Berg3, C.A. van Blitterswijk1

1: Department of Tissue Regeneration, MIRA

2: Department of Biomechanical Engineering, MIRA

3: BIOS - the Lab-on-a-Chip group, MESA+ -Institute of Nanotechnology

 

Developing tissues remodel and organize based on complex interplays of biological and physical cues. Recapitulating these interplays in microfabricated multicellular systems allows for the study of simplified cases of tissue organization. We demonstrate the microfabrication of millimeter-scale tissue units, with simple geometries, which are prone to self-remodeling and self-organization. Tissues were built by sequentially assembling cells into condensed microscale clusters then assembled into 3D geometric millimeter-scale tissues. The free-floating tissues compacted overtime and deformed according to their initial geometries thus adopting various shapes. When formed by human mesenchymal stem cells and human umbilical vein endothelial cells, tissues reproducibly self-organized with internal regions of cellular density and with stereotyped patterns angiogenesis. Patterns of angiogenesis formed due to compaction and to a local changes of the microenvironment including compression, the formation of a VEGF gradient and the upregulation of VEGFR2. We show that self-organization is a powerful engineering process for the fabrication of heterogeneous tissues with internal patterned capillary structures.

 

 

A simple microfluidic connection

J de Sonneville, Sylvia Le Dévédec, RCT Jorand, E van Stapele, MHM Noteborn, JP Abrahams, B van de Water and ME Kuil

 

Recent advances in microfluidic research have shown many advantages of miniaturized cell biology analysis platforms Currently a method to use these platforms in an automated fashion is lacking. We present a connection technique that allows the high throughput use of microfluidic analysis in

(cell) biology.

Connecting microfluidic chips using injection of a beveled capillary tube is demonstrated in three different applications. Connecting microfluidic flow cells from the side as opposed to top side connecting allowed us to accurately monitor the cellular processes. The shear stress induced by laminar flow in renal epithelial PK1 cells over-expressing Green Fluorescent Protein (GFP)-actin was monitored with Differential Interference Contrast (DIC), confocal and Total Internal Reflection Fluorescence (TIRF) microscopy.

In a second application the flow cell was connected, filled and disconnected automatically from the top using a modified pipetting robot.

In a third setup a glass pipette tip was used to fill the microfluidic flow cell. Using pipette tips to directly fill microfluidic channels paves the way to automate microfluidic research by using the same pipetting robots that have been developed and employed to fill micro-titre plates.

 

 

Fluid flow and kidney development

Aleksandr Vasilyev, Yan Liu, Sudha Mudumana, Steve Mangos, Pui-Ying Lam, Arindam Mujumdar, Jinhua Zhao, Kar-Lai Poon, Igor Kondrychyn, Vladimir Korzh, Iain A. Drummond

 

Metazoan development is traditionally viewed as a stereotypical unfolding of a genetic developmental program. However, organ development occurs in concert with the emergence of organ function, which in turn modifies developmental process and leads to further refinement of organ shape and function. In our studies of kidney development using the zebrafish model, we uncovered an interaction between luminal fluid flow and epithelial morphogenesis. We demonstrated that pronephric epithelia engage in directed collective migration towards the glomerulus in response to pronephric fluid flow. This directed epithelial migration, in turn, leads to a shift in the position of kidney segments and also to the convolution of the proximal tubule. The  flow-guided collective migration also results in a compensatory increase in cell proliferation in the distal nephron. Thus, we demonstrated that pronephric fluid flow governs multiple complex cellular processes within the developing kidney and modulates kidney morphogenesis.

 

 

Pdms microsystem exploiting polymer membranes for improved experimental control in metabolism studies using intestinal and liver slices

Sabeth Verpoorte

Abstract

 

 

in vivo Imaging of Stem Cells using Quantum Dots for Stem Cell Therapy

M. Watanabe1, H. Yukawa2, Y. Kagami1, N. Kaji1,3, Y. Okamoto1,3,

Y. Miyamoto2, H. Noguchi4, M. Tokeshi1,3, S. Hayashi2,and Y. Baba1,3,5

 

1 Department of Applied Chemistry, Graduate School of Engineering, Nagoya University

2 Department of Advanced Medicine in Biotechnology and Robotics, Graduate School of Medicine, Nagoya University

3 MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University

4 Baylor Institute for Immunology Research, Baylor Research Institute

5 Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST)

 

Adipose tissue-derived stem cells(ASCs) can be induced to differentiate into various mesenchymal cells including chondrocytes, adipocytes, osteoblasts. Also, ASCs secrete many growth factors such as VEGF, HGF, bFGF and others. ASCs have a great potential for regenerative medical application because adipose tissue can be harvested by a lower invasive procedure, and is available in large number of stem cells at harvest. Recently, there was a report that the transplantation of ASCs with heparin was effectively treat acute liver failure. However, in vivo behaviors of the transplanted ASCs have not been investigated. Therefore, it is desirable to label ASCs for tracing the transplanted cells in vivo.

The purpose of this study is to reveal spatiotemporal distribution of the transplanted ASCs by tracing the fluorescence-labeled ASCs in vivo. In this study, Quantum Dots(QDs) are used for fluorescence labeling because QDs have unique optical properties including size-tunable emission, higher quantum efficiency, broader absorption spectra, narrower emission spectra, and resistance to photobleaching. Negatively charged QDs were used with octaarginine(R8), a kind of cationic cell penetrating peptides(CPPs).

First, ASCs were non-toxically labeled using QDs-R8 complex under suitable concentration condition. Secondly, subcutaneously injected QDs-labeled ASCs could be imaged clearly with in vivo fluorescence imaging system. Furthermore, we found that the injected QDs-labeled ASCs via tail vein were mainly accumulated in the lung.

In conclusion, ASCs could be labeled with QDs-R8 complex and be imaged for tracing the transplanted ASCs with in vivo fluorescence imaging system. These results suggested that the QDs-R8 complex can be effectively used to label ASCs and will be useful for studies of tracing the transplanted cells in vivo.

 

 



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