Barz, Bogdan

Flenner, Elijah

Forgacs, Gabor

Jakab, Karoly

Kosztin, Ioan

Marga, Francoise

Neagu, Adrian

Norotte, Cyrille

Elsevier - Publisher Connector 

Towards In Silico Bioprinting 

Wednesday, March 4, 2009 633a1Marine Biological Laboratory, Woods Hole, MA, USA, 2University of
Edinburgh, Edinburgh, United Kingdom.
We are exploring the use extracellular fluorescent indicators to measure analy-
tes in the microdomains between adjacent cells in tissues. Our primary initial
goal is to measure changes in Hþ activity in the intercellular space of xenograft
tumor models before and after drug treatment. It is thought that acidification in
the core of tumors protects these cancers from the primarily, weak base, che-
motherapeutic drugs. We are comparing the use of functionalized nanoparticles
and expressible indicators as putative extracellular sensors. Functionalized,
fluorescein-labeled nanoparticles were constructed that enable the particles to
adhere to the surface of schwannoma cells. However, preliminary work with
the nanoparticles showed toxic side effects, at least over a period of days.
We have also been exploring the use of expressible membrane bound Hþ indi-
cators, glycosylphosphatidylinositol (GPI)-anchored GFP mutants. A problem
with these indicators is that they localize to the plasma membrane and also the
entire exocytotic pathway. In vitro testing of the indicators was accomplished
by aggregating single cells into clusters (artificial tumors) using dielectropho-
resis (DEP) and agarose molds. Preliminary results indicated that nanoparticles
could be trapped within artificial tumors using DEP as long as electric fields are
applied. Long term (7 days) intercellular pH sensing was achieved in vitro us-
ing GPI-GFP expressing schwannoma cells in an artificial tumor crafted with
an agarose mold. While we are currently exploring the use of sensors to mea-
sure changes in pH in the intercellular space, we are also investigating the de-
velopment and testing of other sensors to detect analytes such as ATP, Cl-, Naþ,
and Kþ. This will provide valuable information regarding cellular defenses
against chemotherapeutic drugs and mechanisms of therapeutic drug action.
This project was funded by the NIH:NCRR as a national research resource
[P41 RR001395]
3264-Pos Board B311
Using Automated Cell Tracking Software to Quantifying Durokinesis and
Durotaxis in Real Time
Matthew L. Walker, David House, Margrit Betke, Joyce Y. Wong.
Boston University, Boston, MA, USA.
In vivo, there is an intimate connection between certain cellular processes and
the physical nature of the surrounding environment. Specifically, it has been
theorized that changes in the physical properties of the extra-cellular environ-
ment within the vasculature influence cellular migration which can influence
such processes as angiogenesis and occlusive vascular disease.
In order to observe and quantify the compliance directed migration of both vas-
cular smooth muscle cells and fibroblasts, we employed both polyacrylamide
substrates, in which the tensile modulus could be tuned to specific values,
and novel computer imaging software which automatically tracked cellular
movement in less then ideal optical imaging conditions.
Although both durotaxis and durokinesis have been previously observed in
large population studies, our application of computer vision software allowed
for a high throughput analysis of individual cells in real time. This method
not only standardized the data collection but also enabled us to observe and
quantify changes in speed, angular deviation, acceleration and deceleration
within a single cell’s migration track as a function of substrate stiffness and
in the presences of a compliance gradient. This detailed analysis will serve
to refine our understanding of cells respond to the physical stimuli presented
in the environment.
3265-Pos Board B312
Asynchronous Rotation as a Rapid and Sensitive Technique for Quantify-
ing Cell Growth Dynamics
Daniel W. Youngstrom, Brandon McNaughton, Pa¨ivo¨ Kinnunen,
Raoul Kopelman.
University of Michigan, Ann Arbor, MI, USA.
The ability to monitor physical parameters of cell growth dynamics on an in-
creasingly sensitive scale is of great interest. Nonlinear rotation of cell-coated
magnetic microspheres is an ex-
citing new technique for rapid
cell detection and measurement.
Previous explorations of this
approach primarily involved
bacteria and other prokaryotes,
but new methods demonstrate
it is possible to extend this
model to the world of eukary-
otes, specifically simple yeasts.
In this experiment, Saccharo-
myces cerevisiae cells were
coated in biotin and covalentlylinked to a streptavidin-coated magnetic microsphere. With their cell mem-
branes bound to the sphere, the unit was rotated asynchronously in a magnetic
field. As the cells grow, the viscous drag experienced by the cell-bearing micro-
sphere increases, counteracting the magnetic driving force, yielding a steady
overall increase in rotational period. The rotation rate is actively monitored
by specialized computer software using the voltage output of a laser and pho-
todiode.
Current data reinforces the success of dynamic asynchronous rotation as a valid
means for rapid and sensitive growth detection. In the future it is believed this
technique may be further extended to the study of increasingly complex organ-
isms, including mammalian cells.
3266-Pos Board B313
Detection Of Drinks Contamination Using Optical Refractometry Tech-
nique (ORT)
Christopher E. Bassey, Cynthia A. Siguenza.
Azusa Pacific University, Azusa, CA, USA.
Optical Refractometry Technique (ORT) has been used to characterize un-
known substances, check the concentration of known substances, and also de-
termine the sugar content of a given liquid. It involves the determination of
a medium’s refractive index, which is a measure of the speed of light in the me-
dium. The technique is based on the principle that the speed of light in a given
medium is a reflection of its absorption and emission characteristics. The speed
of light also depends on the physical state, composition, and molecular struc-
ture of the medium. By comparing the optical properties of pure drinks with
those of drinks tainted with foreign agents, the level of contamination can be
detected. This work examines the feasibility of using ORT to detect the con-
tamination Gatorade drink with antifreeze, which has already led to a number
of deaths. The results will enhance the development of instrumentation and
methodology for continuous monitoring and detection of possible contamina-
tion.
3267-Pos Board B314
Assessment of Cytotoxicity by Analysis of Impedance Fluctuations
Jun-Chih Lo, Daniel Opp, Chun-Min Lo.
University of South Florida, Tampa, FL, USA.
Electric cell-substrate impedance sensing (ECIS) has been used to monitor cell
behavior in tissue culture and has proven to be very sensitive to cell morpho-
logical changes and cell motility. In this method, cells are cultured on small
gold electrodes carrying weak AC signals. The impedance of these electrodes
changes dramatically when cells attach and spread on their surface, because the
cell membranes restrict the current flow. In addition, cell motion may reveal
itself as a fluctuation in the measured impedance, which is always associated
with living cells and persists even when the cells grow into a confluent layer.
The impedance fluctuation is attributed to incessant changes in the size of
the cell-substrate space as cells persistently rearrange their cell-substrate adhe-
sion sites. The magnitude of this sort of vertical motion detected by ECIS is of
the order of nanometers and referred to as micromotion. Here, we applied ECIS
to evaluate dose-dependent responses of NIH 3T3 cells exposed to cytochalasin
B, cadmium chloride, and H-7 dihydrochloride, a protein kinase C inhibitor. To
detect the alternation of cell micromotion in response to cytotoxic challenge,
time-series impedance fluctuations of cell-covered electrodes were monitored
and the values of power spectrum, variance, and variance of the increment
were calculated to verify the difference. While a dose-dependent relationship
for each chemical was generally observed from the overall resistance of the
cell monolayer, the analysis of impedance fluctuations distinguished cytocha-
lasin B, cadmium chloride, and H-7 dihydrochloride levels as low as 0.1, 10,
and 1 micromole respectively. The analytical methods used in this study can
serve as a model approach for ECIS and other electrochemical impedance bio-
sensors to investigate various aspects of cellular responses to toxins in general.
3268-Pos Board B315
Towards In Silico Bioprinting
Adrian Neagu1,2, Elijah Flenner1,3, Bogdan Barz1, Cyrille Norotte1,
Francoise Marga1, Karoly Jakab4, Ioan Kosztin1, Gabor Forgacs1.
1University of Missouri-Columbia, Columbia, MO, USA, 2Victor Babes
University of Medicine and Pharmacy, Timisoara, Romania, 3Colorado State
University, Fort Collins, CO, USA, 4University of Virginia, Charlottesville,
VA, USA.
Bioprinting is a computer-controlled procedure for building three-dimensional
tissue constructs via layer-by-layer delivery of cells and supportive hydrogels.
To describe the post-printing self-assembly of multicellular structures, we per-
formed computer simulations that incorporate a basic principle of developmen-
tal biology, the differential adhesion hypothesis (DAH). DAH states that cell
motility combined with differences in the adhesive properties of different
cell types yields tissue conformations with the largest number of strong bonds
634a Wednesday, March 4, 2009between cells. Metropolis Monte Carlo (MMC) simulations based on DAH pre-
dicted the emergence of long-lived structures (cell sheets, toroidal and tubular
constructs) in accord with experiments. The MMC method, however, does not
describe time evolution. We propose a kinetic Monte Carlo (KMC) approach,
where transition rates are associated with possible rearrangements of cells. The
system is represented on a lattice, with sites occupied either by cells or by vol-
ume elements of cell culture medium.We associate rates to swapping cells with
nearest neighbors of different types (cells or medium). The new approach was
tested against experiments on cell sorting within an aggregate composed of two
cell types. In quantitative studies, we determined the time evolution of the in-
terfacial area between two fusing spherical cell aggregates experimentally, an-
alytically and by KMC simulations. In the analytic approach, we used contin-
uum hydrodynamics to describe the coalescence of two identical, highly
viscous liquid droplets, and obtained good agreement with experiments on
smooth muscle cell aggregates. Apart from the early stages of fusion, the
KMC method predicted a fusion pattern similar to the experimental one. Com-
parison with measurements allowed relating KMC transition rates to experi-
mental time scales. Our results indicate that the KMC method can give an ac-
curate account of the time evolution of complex cellular structures, thus it may
be a useful tool for tissue engineering applications. Work supported by NSF-
056854.
3269-Pos Board B316
Fully Biological Bioprinted Blood Vessel Substitutes
Gabor Forgacs, Francoise Marga, Carina Poltera, Cyrille Norotte.
University of Missouri, Columbia, MO, USA.
Cardiovascular disease is a leading cause of death and often requires vascular
reconstruction. There is considerable clinical need for alternatives to the autol-
ogous vein and artery tissues used for vascular reconstructive surgeries, lower
limb bypass, arteriovenous shunts and repair of congenital defects to the pul-
monary outflow tract. 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 environment. However the use of scaffolds has often
been associated with chronic inflammation and impaired tissue-remodeling
and maturation. In this respect understanding the physical principles of biolog-
ical self-assembly is essential for developing efficient strategies to build living
tissues and organs. Here we exploit well-established developmental processes
(such as tissue fusion, spreading or sorting phenomena) to engineer small-di-
ameter blood vessels. We introduce a novel automated rapid prototyping
method (bioprinting) that allows the building of three-dimensional custom-
shaped tissue and organ modules without the use of any scaffold, thus making
the final construct fully biological, as well as structurally and functionally
closer to native tissues. Conveniently prepared bio-ink units (multicellular
spheroids or cylinders composed of single or several cell types) are delivered
into the bio-paper (a hydrogel support material) to build linear and branching
tubular structures of small diameter (down to 0.9 mm OD). Structure formation
takes place by the post-printing fusion of the discrete units. Upon removal of
the support material, the fused construct is matured in perfusion bioreactor un-
der pulsatile flow until desirable biomechanical (burst pressure, compliance)
and biochemical (e.g. ECM) properties develop. Such constructs could fulfill
the crucial need for small diameter vascular grafts and provide new strategies
for vascularization of tissues for transplantation.
3270-Pos Board B317
Amphiphilic Peptides That Self Assemble into Nanomicelles and Vesicles
Sushanth Gudlur, Matt Warner, Yasuaki Hiromasa, Takeo Iwamoto, John
M. Tomich.
Kansas State University, Manhattan, KS, USA.
Self-Assembling peptide nanovesicles are attractive candidates for drug deliv-
ery. Here we report the characterization of specific amphiphilic peptides (up to
23-amino acids in length) that un-
dergo supramolecular assembly to
form both mono- and hetero-assem-
blies that form nanomicelles and
larger vesicles (150nm) in aqueous
solutions. Depending on the pep-
tide’s composition and the pH of
the aqueous medium during assem-
bly, the peptides adopt either a mi-
cellar or vesicular structure. Simple
amphipathic sequences adopt a mi-
cellar structure (<50nm) at low to
neutral pH. When pairs of lyophi-
lized peptides with different lengthsare co-dissolved in an unbuffered Carboxyfluorescein solution at a 1:1 molar
ratio, they self-assemble into small vesicles that are visualized using a confocal
microscope. These intact vesicles average about 150nm in size (as determined
by confocal images) and are capable of entrapping solutes. CD and FTIR anal-
yses of such mixtures indicated a tendency of the peptide to adopt a beta sheet
secondary structure. Isothermal Titration Calorimetry (ITC) revealed the Crit-
ical Aggregation Concentration (CAC) for the individual peptides to be less
than 1mM, which is a useful property during drug delivery. These nanostruc-
tures can be used as models for further developments.
3271-Pos Board B318
High Throughput Lipid Bilayer Technologies
Jason L. Poulos, Jacob J. Schmidt.
University of California Los Angeles, Los Angles, CA, USA.
Measurements of ion channels are important for scientific, sensing and pharma-
ceutical applications. Reconstitution of ion channels into lipid vesicles and pla-
nar lipid bilayers for measurement at the single molecule level is a laborious
and slow process incompatible with the high throughput methods and equip-
ment used for sensing and drug discovery. A recently published method of lipid
bilayer formation mechanically combines lipid monolayers self-assembled at
the interfaces of aqueous and apolar phases. We have expanded on this method
by vertically orienting these phases and using gravity as the driving force to
combine the monolayers. As this method only requires fluid dispensation, it
is trivially integrated with high throughput automated liquid-handling robotics.
In a proof-of-concept demonstration, we created over 2200 lipid bilayers in
three hours. We show single molecule measurements of technologically and
physiologically relevant ion channels incorporated into lipid bilayers formed
with this method.
3272-Pos Board B319
Construction of a Bioprinted Fully Biological Nerve Graft
Francoise S. Marga, Bradley Hubbard, Tom McEwan, Stephen Colbert,
Gabor Forgacs.
University of Missouri-Columbia, Columbia, MO, USA.
Annually over 200,000 peripheral nerve surgeries are performed in the U.S.
alone. Many of these procedures require grafts to bridge the severed nerve. Au-
tologous nerve is the gold standard for providing a temporary support across
which axons regenerate. Although successful at 80%, the autograft procedure
has several drawbacks including the limited number of available nerves, the
loss of function and /or sensation at the donor site and the need of multiple sur-
geries. Synthetic or natural nerve guidance hollow tubes have been used suc-
cessfully as scaffolds for small gaps (<3cm) but regeneration fails over longer
distances. As a result, tissue engineering has emerged as a promising alterna-
tive. Based on recent studies, synthetic and autologous tubes failure has been
linked to low density of supporting cells such as Schwann cells and the lack
of longitudinally-oriented structural features, which favor Bunger’s bands-
like formation and axonal growth by mimicking endoneural architecture. In ad-
dition, axonal growth can be impaired by inflammatory and immunological re-
sponses triggered by the implanted scaffold.
We present here a novel tissue engineering technology that is based on princi-
ples of developmental biology and employs bioprinting, This automated rapid
prototyping method allows for creating well-defined architectural features,
without any scaffold, thus making the final construct completely biological
as well as structurally and functionally closer to native tissues. Spherical or cy-
lindrical bio-ink units (composed of Schwann and bone marrow stem cells) are
delivered according to a computer scrypt together with agarose rods, as support
material units. Structure formation takes place by the post-printing fusion of the
discrete units. The geometrical parameters of those tubes, such as wall thick-
ness, diameter, and number of lumens can easily be controlled. Such constructs
could fulfill the crucial need for larger nerve grafts.
Supported by NSF 0526854.
3273-Pos Board B320
Influenza A Nucleoprotein Detection by a Novel Immuno-Interferometric
Sensor
Leslie R. Farris, Wenhui Wang, A. Clarizia Lisa-Jo, Xingwei Wang,
Melisenda J. McDonald.
UMASS Lowell, Lowell, MA, USA.
Rapid detection and identification is imperative to combat known or emerging
infectious agents. This novel immuno-interferometric sensor utilizes the spec-
ificity and selectivity of antibody-antigen interactions to detect and identify
a specific influenza viral component in a label-free manner. Primary antibodies
to the nucleoprotein of Influenza A (IFA) were oriented utilizing a previously
reported polymer- protein interaction system of poly(methyl methacrylate)
(PMMA, CAS# 9011-14-7) and biolinker protein G0. This unique noncovalent
adsorption method resulted in increased primary antibody orientation, and


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Towards In Silico Bioprinting

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