TRACK 1
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
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TRACK 2
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MOLECULAR ENGINEERING AND ELECTRONICS
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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
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
