Forgacs, Gabor

Marga, Francoise S.

Young, Troy

Elsevier - Publisher Connector 

Tuesday, February 23, 2010 607aheparin. A set of methods was applied: Surface Plasmon Resonance (SPR) was
chosen to continuously monitor the self-assembly process of physi-sorption of
subsequent PE layers and to report about deposition efficiency, dynamics and sta-
bility of the PEM film. Atomic force microscopy (AFM) was used to visualize
and characterize the surface topology obtained after subsequent steps of the
layer-by-layer deposition. The multi-layer structure of the composite film and
its hydration were studied with neutron reflectometry. The knowledge gathered
so far on this system is now being applied for the deposition of lipid bilayers
and other biomimetic systems for subsequent biophysical studies.
3151-Pos
Analysis of Lipid Compositional Changes During Alcoholic Fermentation
in Industrial Yeast Strains with Varying Ethanol Tolerance
Clark M. Henderson, David E. Block, Marjorie L. Longo.
University of California, Davis, Davis, CA, USA.
In model lipid bilayers, ethanol is known to alter the mechanical and thermo-
dynamic properties of the membrane. The extent to which alcohol affects these
properties depends upon the lipid composition of the bilayer. Our group has
demonstrated that increased ergosterol levels in model membrane systems mit-
igate the membrane thinning effect of ethanol - a phenomenon known as inter-
digitation. Perturbations to the yeast biomembrane due to increasing ethanol
levels has been implicated in reduced sugar utilization and cell viability in Sac-
charomyces sp. However, variations in fermentation conditions and analytical
methods have not yielded a comprehensive picture of how yeast biomembranes
adapt to increasing levels of ethanol. In this work, we analyzed the partial lip-
idome of 30 industrial yeast strains at different stages of fermentation using
high-resolution mass spectrometry. Quantification of selected lipid species
was performed using high performance liquid chromatography coupled on-
line to quadrupole ion-trap mass spectroscopy. Multivariate statistical analysis
of the quantitative data was performed to determine any correlations between
changes in lipid composition and ethanol tolerance in the different yeast strains.
Information regarding how yeast biomembranes adapt to greater ethanol con-
centrations will be used to construct biophysical models to analyze the complex
physical properties of lipid biomembranes in an alcohol milieu.
3152-Pos
Automated Lipid Bilayer Formation using a PDMA Gasket
You-Hyo Baek1, Joongjin Park1, Jason L. Poulos2, Jacob J. Schmidt2,
Tae-Joon Jeon1.
1Inha University, Incheon, Republic of Korea, 2University of California,
Los Angeles, Los Angeles, CA, USA.
Planar lipid bilayer membranes(BLMs) play important roles in studying ion
channels, as well as in potential applications for drug discovery. Despite the im-
portance of many practical applications using BLMs, membrane formation is
still based on conventional methods invented by Montal and Mueller in
1960s. Although membranes can be simply reconstituted using the conven-
tional technique, membranes should be created where experiments are con-
ducted due to their mechanical instability. In our recent work a membrane for-
mation technique using high melting temperature solvent mixture was devised
by Jeon, et al. (Lab on a Chip, 8(9): 1742 (2008)) Briefly, 2:8 mixture of n-dec-
ane and hexadecane was spread over a small aperture and froze before its spon-
taneous self-assembly process to a bilayer membrane. Since the membrane pre-
cursor can be created in a central facility and shipped to any place, it can be
transported to any place and thawed when a membrane is needed, widening
the usability of artificially created lipid bilayer membranes. Nevertheless the
main drawback of the conventional technique was not completely ameliorated
in this work due to the membrane support. Since a membrane precursor was de-
posited in a small aperture on a plastic sheet, the membrane formation process
by self-assembly was unchanged, resulting in variations in membrane forma-
tion time with a range of ~30 minutes to 24 hours. In our work a PDMS gasket
was used to support membrane structure in place of a plastic sheet. Since or-
ganic solvent can be extracted into the PDMS gasket, a lipid bilayer membrane
can be formed within ~30 minutes in a controlled manner. Ion channels incor-
porated into a membrane formed in a PDMS gasket functioned as in a conven-
tional membrane. Furthermore, we will show the broad applicability of our
membrane formation technique.3153-Pos
Fabricating a New Stabilized Lipid-Based Platform for Handling and
Presenting GPCRs
Yasaman Dayani, Noah Malmstadt.
Mork Family Department of Chemical Engineering and Materials Science,
University of Southern California, Los Angeles, CA, USA.
G protein-coupled receptors (GPCRs) are members of large family of signaling
molecules. They have a key function in neuronal biology and play a critical rule
in transmitting extracellular signals in eukaryotic cells. GPCRs include recep-
tors for many neurotransmitters like serotonin. The serotonin receptor 5HT1A
has an important role in mental disorders like anxiety, and is the target of
some anti anxiety drugs. Here we constructed a novel biomimetic lipid mem-
brane-based platform to be used for screening molecules that interact with
GPCRs. Since fragility and short life time limit the applicability of liposomes
that are normally used to present membrane proteins, we have developed a plat-
form based on nanoscale liposomes with enhanced longevity and stability. We
developed two approaches to make stable liposomes: liposomes containing
a UV-initiated poly(ethylene glycol) (PEG) hydrogel and conjugated hydrogel
liposomes made from lipids covalently anchored to the hydrogel network. Sta-
bility of nanoscale liposomes was confirmed by addition of high concentration
of sodium dodecyl sulfate (SDS) to the liposome suspension. The liposome/de-
tergent micelle mixture was passed over a size exclusion chromatography
(SEC) column, separating intact liposomes from micelles. Using dynamic light
scattering (DLS), we could confirm the existence of intact 160 nm liposomes.
This result was in good agreement with the SEC result for polymerized lipo-
somes without detergent. Since our goal is making GPCR-bearing liposomes,
serotonin receptor 5HT1A was incorporated into liposomes using a detergent-
mediated method. GPCR incorporation was confirmed by binding labeled
HTR1A antibody to liposomes containing biotinylated lipids and subsequently
separating them from bulk with streptavidin-coated magnetic beads. We are
currently undertaking further studies on antibody-liposome binding by fluores-
cence anisotropy to validate our approach.
3154-Pos
In Vitro Enhancement of Collagen Deposition in Fully Biological
Bioprinted Constructs
Francoise S. Marga, Troy Young, Gabor Forgacs.
University of Missouri-Columbia, Columbia, MO, USA.
Engineering new tissues, ideally from the patient’s own body cells to prevent
rejection by the immune system, is a rapidly growing field that rests on three
pillars: cells, supporting structures (or scaffold) and stimulating biological en-
vironment. As the cells grow and depose their own extracellular matrix (ECM),
the scaffold degrades slowly. After implantation, remnants of the scaffold can
trigger chronic inflammation and create mechanically weak zones by interfer-
ing with extracellular matrix assembly. Over the last few years, we developed
an alternative, scaffold-free method. Our technique exploits well-established
developmental processes (such as tissue fusion, spreading and cell sorting).
Conveniently prepared bio-ink units (multicellular spheroids or cylinders com-
posed of single or several cell types) are delivered into the bio-paper (a hydro-
gel support material) to build tubular constructs. Structure formation takes
place by the post-printing fusion of the discrete units and maturation in biore-
actor. The slow buildup of cell-produced ECM needed for adequate mechanical
strength before implantation, however remains an issue. Collagen deposition is
hindered in vitro due to the slow conversion of de novo synthesized procollagen
to collagen before its secretion.
Here we explore different strategies to enhance collagen deposition in the bio-
ink units and the post-fusion construct. Collagen deposition could be observed
throughout spheroids obtained by seeding human aortic smooth muscle cells in
96 well plates and culturing them in presence of ascorbic acid. However the
process still took 3 weeks. Boost in collagen production by the bio-ink units
was observed when negatively charged dextran sulfate (DxS, 500 kDa) was
added to the medium. After high content bio-ink units fuse into a tube, ECM
reorganization is expected under mechanical stimulation in the bioreactor.
In conclusion, DxS could fasten the production of ECM in tissue engineering
applications.
Supported by NSF 0526854.


In Vitro Enhancement of Collagen Deposition in Fully Biological Bioprinted Constructs 

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In Vitro Enhancement of Collagen Deposition in Fully Biological Bioprinted Constructs

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