Biomedical Engineering Reference
In-Depth Information
Translational applications for this multidimensional approach include, for exam-
ple, engineered environments for assessing drug delivery to cells and tissues. For the
brain, we are also taking into consideration the added complexity of two physiological
factors. A first consideration is the bloodbrain barrier (BBB), which prevents passive
diffusion of most chemicals from the bloodstream into the brain (Weiss et al., 2009).
For this, we are including rat brain microvascular endothelial cells (RBMVECs) in
our engineered culture environments. Second, in the brain (once past the BBB), there
is a major interplay between multiple cell types, for example, between neurons and
glial cells.
We are considering the effect of the glial subtype, astrocytes on neuronal com-
munication. Using a predator-prey concept (Baxendale and Greenwood, 2010),
where astrocytes are the predator taking up glutamate, and neurons are the prey re-
leasing glutamate, we are measuring the effect of astrocytes on intracellular calcium
concentration([Ca
2+
]
i
)dynamics in these cultures. The ability to engineer the growth
environments for both of these cell types will allow us to control both the ratio and
position of these cell populations relative to each other, providing input control to the
predator-prey model, backed up with experimental data. Using multiple nanotechnol-
ogy approaches we have been able to define engineered environments that have estab-
lished single glial cells (0D), connected networks of neurons, and networks of brain
tumor cells (2D). In addition, we have recently used natural and bio-polymer com-
posites of gelatin and pulp and paper fibers to form 3D micro- and macro-structures
that guide and support the growth of brain tumor cells (Xing et al., 2010). We have
identified at least 3 major benefits of these composites for tissue engineering in 3D:
(1) the gelatin-cellulose composites are of sufficient porosity to allow for cell invasion
and penetration of nutrients into the matrix; (2) these composites allow light transmis-
sion for phase- and fluorescence-microscopy; and (3) the composites are of sufficient
strength to retain 3D structure for long-term
in vitro
studies, which in our case was up
to 17 days
in vitro
.
From the standpoint of dynamic cell communication, we utilize fluorescent calci-
um indicators such as fluo-3 combined with digital imaging to monitor changes in both
[Ca
2+
]
i
in the individual cell and changes that are intercellular, such as the synchrony or
asynchrony of [Ca2+]i
2+
]
i
oscillations. We hypothesize that the ability to engineer the spa-
tial arrangement of brain cells will aid in our analysis of these dynamics. We also an-
ticipate that defining the spatial arrangement of normal and brain tumor cells will aid
in image analysis of cell growth patterns with and without anti-cancer drug treatment.
methods
Nanofilm printing
: Glass slides for nanofilm printing were prepared by sonication
in deionized water using a Branson model 1510 sonicator for 30 min followed by
sonication in 75% ethanol for 30 min. Slides were then removed, washed once in de-
ionized water and blown dry using dry nitrogen. The newly cleaned slides were then
silanized to increase covalent bonding of polymers using vapor deposition (Mandal et
al., 2007). Silanization was accomplished by filling a micro centrifuge cap with 50 µl
of (3-aminopropyl)-triethoxysilane (APTES; Sigma-Aldrich, USA) and then placing
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