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SYMPOSIUM IV
Concurrent Tracks

4:30-5:50
Saturday, September 7
 

TRACK 1

TRACK 1

NANOPROBES

T2

T3

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Symposium Chairperson
Dr. Shuming Nie, Georgia Institute of Technology and Emory University School of Medicine

4:30 Magnetodendrimers for Labeling and in Vivo Tracking of Stem/Progenitor Cells 
Dr. Jeff W.M. Bulte, Johns Hopkins University School of Medicine
During the last few years, the therapeutic use of stem and progenitor cells as a substitute for malfunctioning endogenous cells has received much attention. Unlike their use in animal models, the introduction of therapeutic cells in patients will require techniques that can monitor their tissue biodistribution noninvasively. Among the different imaging modalities, magnetic resonance (MR) imaging offers both near-cellular (i.e. 50 micron) resolution and whole-body imaging capability. In order to be visualized, cells must be labeled with an intracellular marker that can be detected by MR imaging. Superparamagnetic iron oxide nanoparticles provide currently the highest sensitivity when used as MR contrast agent. We have recently developed a new type of iron oxide nanoparticle, magnetodendrimers (MD-100), that has excellent magnetic and NMR relaxation enhancing properties [1] and, due to its coating with a dendrimer as transfection agent, is effciently taken up by a variety of mammalian cells [2]. We will demonstrate that MD-100 can be used as an efficient cellular contrast agent, allowing MR tracking of magnetically labeled progenitor cells following transplantation.

METHODS: MD-100 was synthesized with a stochiometric ratio of 100:1 of Fe:dendrimer as described [1]. Transmission electron microscopy revealed an oligocrystalline structure of 7-8 nm crystals separated by a somewhat smaller distance. MD-100 has a high saturation magnetization of 94 emu g/Fe, no magnetic hysteresis at 300 K, and exhibits T2 relaxivities of 200 mM-1s-1 (at 37 ° C), with a rapid approach to saturation at magnetic field strengths well below 1.5 Tesla. Cells were magnetically labeled by simply adding MD-100 to the culture medium at concentrations of 10-25 m g Fe/ml, and incubation of 1-2 days. This included mouse 3T3 fibroblasts, mouse C2C12 muscle progenitor cells, rat CG-4 oligodendrocyte progenitors, rat neural stem cell (NSC)-derived oligodendroglial progenitors, human HeLa cervix carcinoma cells, human GLC-28 small cell lung carcinoma cells, human endothelial progenitor cells, human NSCs, and human mesenchymal stem cells. Approximately 5 x 104 MD-100 labeled NSC-derived rat oligodendroglial progenitors were transplanted into the ventricles of neonatal (P=0) Long Evans shaker (les) rats. Cells were co-transfected with the LacZ gene (encoding for the enzyme b -galactosidase), in order to track them histochemically by incubating with the X-gal enxyme substrate. Animals were imaged on a weekly to biweekly basis using a clinical 1.5 Tesla clinical imager and a 4.7 Tesla animal imaging system.

RESULTS AND DISCUSSION: Prussian blue staining of magnetically tagged cells showed a remarkably high degree of intracellular labeling, with the cytoplasm containing large numbers of iron-containing vesicles or endosomes. All cells showed a comparable degree of uptake, demonstrating that the MD-100 uptake is non-specific and not dependent on the cell type or species. Following transplantation, migration of labeled cells into the brain parenchyma could be observed at the earliest timepoints (between 2 and 3 weeks) throughout the latest timepoints of imaging (6 weeks following transplantation). There was a good gross anatomical correlation with the macroscopic distribution of b -galactosidase-expressing cells. Moreover, the transplanted and labeled cells were also found to be able to form myelin that overlapped with the area of MR contrast. A comparison of several pulse sequences demonstrated that T2* weighted gradient echo imaging, that does not compensate for the induced dephasing of protons, was most sensitive to the presence of labeled cells. From the number of cells injected, and the overall area of contrast, we estimate that it should be possible to detect only a few cells when using T2* weighted imaging techniques. In conclusion, magnetodendrimers represent a new class of cellular contrast agents that can be used efficiently for magnetic cellular labeling and in vivo tracking of cells, regardless of animal species. The prospect of MR tracking of labeled cells appears attractive not only as a tool to perform longitudinal cell migration studies in the same animal, but also because of its potential to help guide future clinical studies involving the use of therapeutic stem and progenitor cells.

4:50 Probing the Interactions of Quantum Dots with Their Microenvironment
Dr. Warren C.W. Chan, University of California, San Diego
Advances in nanotechnology have led to the development of highly sensitive nanoprobes which may have a large impact in fundamental biology research and medical diagnostics. In this seminar, the application of quantum dots for biological detection and imaging and the interactions with their microenvironments will be discussed. Quantum dots are nanometer-size colloidal particles with dimensions smaller than the exciton bohr radius. In comparison to traditional organic probes, semiconductor quantum dots are 20 times brighter, 100 times more stable against photobleaching, and have tremendous multiplexing capabilities. Thus far, the use of quantum dots for multicolor staining, homogenous assays, and for imaging tissues have been demonstrated. This is accomplished by modifying the surface of quantum dots with hydrophilic molecules and conjugating biomolecules onto the surface. However, a fundamental understanding of the effect of solvent conditions and environments on the photophysical properties of quantum dots is important. In this talk, we demonstrate the effects of pH and temperature on the quantum efficiency of quantum dots. For example, an increase in temperature from 25oC to 80oC can decrease the photoluminescence of quantum dots by 70%. This research is important for future applications of quantum dots for in vivo cell-based experiments and PCR-related research.

5:10 Formation of Virus-Polymer Complexes Enhances the Efficiency of Gene Delivery
Dr. Joseph M. LeDoux, Georgia Institute of Technology Recombinant retroviruses and lentiviruses are promising tools for delivering genes to cells for the purposes of human gene therapy. Unfortunately, for many potential applications the efficiency of gene transfer is often too low to achieve the desired therapeutic effect. We have begun to develop a novel formulation of viruses and polymers that helps to overcome this limitation. We have found that the addition of oppositely charged polymers to virus stocks induces the formation of retrovirus-polymer complexes that rapidly sediment onto cells. The rate of sedimentation increased with increasing concentrations of polymers and correlated with the average size of the complexes. Sedimentation increased the rate of virus binding to the cells and resulted in a 2 to 3 fold increase in gene transfer. Further increases in the rate of sedimentation were achieved by centrifuging the virus-polymer complexes onto the cells. Surprisingly, we found that the virus-polymer complexes were able to complete the post-binding steps of transduction nearly as efficiently as viruses that had not been combined with the polymers. The implications of these findings to the purification, processing, and delivery to cells of recombinant retroviruses for the purposes of human gene therapy will be discussed.

5:30 Colloidal Gold Nanoparticles for Gene Chip Detection
Dr. T. Andrew Taton, University of Minnesota
We have developed a number of strategies for detecting particular DNA sequences, both individually and in parallel, using gold nanoparticles as labels. Gold nanoparticles have a number of unique physical properties, including high extinction and scattering coefficients, catalytic activity, and fluorescence quenching, that make them extremely flexible labels for DNA detection schemes. As a result, we have developed DNA array protocols whereby target hybridization to array elements can be determined by electrical conductivity, scattered light, or even absorption visible to the naked eye.

These technologies have offered a great deal of promise for simple and inexpensive, yet selective and sensitive, analysis of gene expression and mutation. However, the thiol chemistry that is commonly used to connect oligonucleotides to gold surfaces is sometimes not strong enough to survive molecular biology protocols (such as PCR). This has limited the direct impact of gold nanoparticle labels on the practice of DNA arrays. We have recently developed universal ligands that permit more stable conjugates between gold nanoparticles and biomolecules. These ligands allow enzyme reactions to be conducted directly on gold nanoparticle-bound DNA, and will further enable the labels’ direct use in biological and clinical DNA sequence analysis.

5:50 Close of Day Two

     

TRACK 2

MOLECULAR ENGINEERING AND ELECTRONICS

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T3

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Symposium Chairperson
Dr. Dan V. Nicolau, Swinburne University of Technology 

4:30 Biological Membrane Microarrays
Dr. Joydeep Lahiri, Corning, Inc. 
Membrane bound proteins constitute one of the most important families of drug targets; over 50% of current drug targets are membrane bound proteins. Despite their importance, the power and utility of microarray technology has not been extended to membrane proteins because of issues due to immobilization -- these proteins need to be embedded in a membrane environment for maintaining their native conformations. This talk will outline issues related to the fabrication of membrane microarrays, describe the fabrication of microarrays of G protein-coupled receptors, and demonstrate assays for screening of ligands on these arrays.

4:50 Using Carbon Nanotubes and Norbornyl Bridges to Communicate with Enzymes: Application to Enzyme Sensors 
Dr. J. Justin Gooding, The University of New South Wales
 
Enzyme biosensors are frequently limited by the need for a cosubstrate to complete the catalytic cycle. Direct communication between enzymes and electrodes is the key to obtaining more efficient enzyme turnover as the need for a co-substrate is obviated. To achieve direct communication effectively requires the electrode to be brought up inside the enzyme to the redox active centre. We are investigating strategies to achieving this using both norbornyl bridges and carbon nanotubes. The norbornyl bridge allows efficient electron transfer between the electrode and the redox centre of the enzyme. Electrode constructs were fabricated where the bridge is attached to a gold electrode using alkanethiol chemistry and to the other end of the bridge the redox active centre of glucose oxidase (GOD), flavin adenine dinucleotide (FAD), is attached. The apo-enzyme is then refolded over the immobilised FAD to give an active enzyme electrode.

In an analogous experiment carbon nanotubes could be used, rather than the norbornyl bridge, as the connection between the electrode and the enzyme’s active centre. We are working towards this goal using single-walled nanotubes (SWNTs) which are oxidatively shortened to give carboxylic acid moieties at the ends of the tubes. The nanotubes can be aligned onto a cysteamine modified gold electrode by forming a peptide bond. To the other end of the tube the enzyme or enzyme active centre is attached. To achieve immobilisation of enzymes on the end of the tubes requires resisting the non-specific adsorption of proteins onto the tube sides. We will discuss our approach to aligning the nanotubes, immobilising enzymes and resisting non-specific adsorption. The initial enzyme immobilised will be Microperoxidase MP-11 which has an active site close to the surface of the enzyme, thus direct electron transfer to the enzyme can easily be achieved. Finally our progress to achieving direct electron transfer where the redox active centre is located deep within the proteins will be outlined.

5:10 Surface-Induced Biomolecular Engineering of Proteins
Dr. Dan V. Nicolau
The synergy between semiconductor technology and experimental biomedical sciences has aggregated in a stream of development focused on micro- and nano-biodevices fabrication. While static biodevices, e.g. biosensors, and more recently DNA and protein arrays, are almost ‘classical’, much has to be done to develop dynamic biodevices. Of these dynamic devices, bio-micro-devices, e.g. microfluidics devices, are already in the process of maturation. On the other hand, dynamic bio-nano-devices that have potentially the highest potential of all of the biodevices mentioned above, are in the state of infancy. Protein molecular motors, both rotary and linear, are natural candidates for the fabrication of hybrid bio-nano-machines. To achieve this goal much has to be done in terms of understanding of their operation and equally their behaviour in artificial micro- and nano-confined environments.

The last decade witnessed tremendous advances in molecular-level experimental approaches that have been used to physically probe the function of molecular motors. Of these 'direct contact' methods, the dynamic ones, such as laser trapping (e.g. Funatsu et al., 1997; Higuchi et al., 1997; Kojima et al., 1997) and Atomic Force Microscopy e.g. (Tokunaga et al., 1997), have been most successful in obtaining fundamental parameters regarding actin-myosin and kinesin-tubulin motors. In a sense, these parameters constitute 'engineering' information that is necessary for the fabrication of future nano-devices based on these motors. Despite this device-oriented attraction, and despite their relative affordability, other 'direct contact', but static probing methods, such as 'manipulation' of motility in micro-fabricated structures, e.g. channels, received much less attention. Micro- or nano-fabricated structures are more affordable and more device-oriented, but they also offer the possibility to probe the function of molecular motors at different levels of confinement.

If the laser trapping can offer '0D' confinement probing, the micro/nano-fabricated structures offer the possibility to confine the motility of motors at 1D, 2D and 3D confinement levels, i.e. flat surfaces, channels/ridges and wells/pillars. Of these static-probing structures, the 2D-confinement structures micro/nano-channels (Turner et al., 1995; Suzuki et al., 1995; Suzuki et al. 1997; Dennis et al. 1999 Riveline et al., 1998 and recently Hiratsuka et al., 2001; and Mahanivong et al., 2002) have been used most extensively for the lateral confinement of movement. In the most advanced form, arrow-shaped, micro-channels have been used by Hiratsuka and co-workers to statistically enforce the directionality of the movement of the tubulin filaments (a similar geometry had been proposed earlier by Nicolau & Cross, 2000).

For the other levels of confinement, 1D-confinement structures (i.e flat surfaces) are easiest to ‘fabricate’ and 3D-confinement structures (i.e. micro/nano-wells) would give the highest level of information regarding the fundamental understanding of molecular motors function. 1-D confinement structures have been used by Nicolau et al. 1999, who studied the movement characteristics of actin filaments on patterned areas with different hydrophobicities (and different concentrations of myosin) on flat surfaces.

To this end, we will present recent data regarding the manipulation of the nano-mechanics of linear molecular motors on/in 1D, 2D and 3D molecularly-confinement conditions.

5:30 Close of Day Two

 

TRACK 3

TISSUE ENGINEERING

T1

T2

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Symposium Chairperson
Dr. Sangeeta Bhatia, University of California, San Diego

4:30 Repairing the Retina Using Tissue Engineering
Dr. Harvey A. Fishman, Stanford Medical School
 
One of the major goals of our laboratory is to develop novel tissue engineering therapies for the treatment of age-related macular degeneration (ARMD), the leading cause of blindness in the U.S. for individuals over 65 years of age. Currently, there are no effective therapies. ARMD results in the destruction of the retinal pigment epithelial (RPE) cell and its basement membrane. Eventually, the photoreceptors degenerate resulting in blindness in those areas. A three-pronged approach for treating this disease is evaluated. First, we are developing novel materials for rebuilding the delicate basement membrane. Second, we are using soft lithography for preparing the surface of these materials for retinal stem cell transplantation. Third, we are designing microfabricated autologous material to serve as scaffolds for photoreceptor transplantation and for directing their neurite outgrowths to the adjacent retinal cell layer. These engineered materials are being investigated for their biocompatibility in the subretinal space, for their ability to preserve differentiated stem cells, and for their ease of surgical handling and implantation. A description of these new materials, the microfabrication technologies used to make them, and the results of implantation into the subretinal space of >20 rabbits will be presented.

4:50 Microtextured Cellular Habitats for Cardiac Tissue Engineering
Dr. Tejal Desai
This talk will describe fabrication schemes to create multidimensional polymeric platforms to optimize cardiomyocyte attachment, orientation, and maintain a more in vivo-like morphology of these cells in vitro, under static and cyclic mechanical stimulation. A key features of these constructs is the replication of geometries and dimensional size scales that promote cell-cell and cell-substrate contact in a systematic way. Advantages of these microtextured platforms include the high degree of reproducibility, optical clarity, and elastomeric properties suitable for dynamical mechanical stimulation. Moreover, such platforms can be designed to be biodegradable, thus facilitating their use as an in vivo construct.

5:10 The Tools and Biology Towards Three Dimensional Tissue Engineering
Dr. Lowell R. Matthews, Sciperio Inc.
The specific goal for molecular medicine is to channel multipotent human cells with high proliferative capacity into specified differentiation programs within the body. Realizing this goal quickly requires the development of a tool to leverage the biological learnings of the importance of three-dimensionality and cell micro-environment to achieve normal tissue morphogenesis, vascularization and organ functions.

A particlular tool, named the Biological Architectural Tool (BAT), is a direct-write deposition machine that has been able to deposit materials with a wide range of viscosities, e.g., from 1 to 1,000,000 centipoise. It has the potential to optimize the juxtaposition and geospatial relationships between the cells, growth factors, scaffolds and biocompatible constituents via a directed self-assembly process. The tool provides a directed-assembly process, the biology takes over with self-assembly.

The wide range of viscosities has enabled the tool to handle materials from biodegradable scaffolds to cellular suspensions. The deposition process to create 3D tissue engineered constructs employs the use of various dispensing nozzles from micro-dispense through-nozzles to capillary and quill pen designs. For the flow-through nozzle, dispensing is accomplished by using pressure to drive a viscous fluid through a small orifice. The through-nozzle process has been successfully demonstrated for component features ranging from several hundred micrometers to as small as 50 µm in width and deposits as small as a picoliter of material. These feature sizes are compatible with tissue engineering. The primary hurdle in any fine-detail dispensing process is developing the ability to start and stop the flow of fluid through the dispensing nozzle in a highly controlled fashion. Significant research effort has been focused on fluid valving and pumping control strategies to accomplish this task. The constructs are built in a layer-by-layer construction process. Furthermore, it is possible to build constructs in a conformal manner using a sensoric real-time z sensing architecture.

As one example to demonstrate the efficacy of the dispensing system, experiments with human white blood cells (WBC’s) were performed in pursuit of the objective of creating high-cell-density slides for the detection and analysis of rare cancer cells with the Harvard Rare Event Imaging System (REIS). For these purposes, it proved critically important to demonstrate the capability of a mechanical xyz-translation deposition system to create reliably highly populated, monodisperse cell slides. But more importantly, it shows the controlled environments and delicacy with which cells can be handled through the deposition process. In fact, a primary advantage of the BAT over either dynamic or static tissue-culture systems is its low-shear deposition environment as predicted from computational fluid dynamic calculations. Other examples will be shown as well.

5:30 Microfabrication and Photoactive Polymers for Tissue Engineering
Dr. Sangeeta Bhatia
 
Recent advances in tissue engineering have combined advances in polymer chemistry and cell biology to develop biomimetic materials. For example, photopolymerizable biomaterials have been developed that can be used to photoencapsulate cells in peptide-derivatized hydrogel networks. While these materials have been useful in bone, cartilage and vascular tissue engineering, they have limited applicability to more complex tissues that are characterized by precise cell and tissue organization (e.g. liver, microcirculation). Typically, the tissue shape is defined by the container used for photopolymerization and the cellular organization consists of random dispersal within the hydrogel network. In this presentation, we describe the use of microfabrication tools to broaden the capability of photopolymerizable biomaterials by inclusion of structural features within the cell/hydrogel network. Specifically, we describe the development of a photopatterning technique that allows localized photoencapsulation of live mammalian cells to control the tissue shape as well as the use of dielectrophoretic forces (induced dipoles) to organize cells within hydrogel networks. The combination of microfabrication approaches with photopolymerizable biomaterials may have implications in drug delivery, cell encapsulation and tissue engineering.

5:50 Close of Day Two

 

 


 

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