Bionanocomposites for Multidimensional Brain Cell Signaling - Natural Polymers, Biopolymers, Biomaterials, and Their Composites, Blends, and IPNs - page 89

the filled cap in the center of a glass Pyrex dish. Cleaned glass slides were then posi-
tioned around the APTES filled cap and the Pyrex dish placed in an oven and baked
at 60°C for 2 hr. After this step, APTES was removed from the dish, and the dish with
slides was placed back in the oven and baked for 16 hr at 100°C. After baking, slides
were allowed to cool to room temperature and then immersed in 6% gluteraldehyde in
1X phosphate buffered saline (PBS; Invitrogen) for 2 hr at room temperature. Slides
were then washed with deionized water and blown dry using nitrogen.
For polymer printing using the Nanoenabler system (BioForceNanosciences, Inc.
USA), fluorescein isothiocyanate (FITC)-labeled poly-L-lysine (PLL) was used
(Sigma-Aldrich). To prevent premature drying, the FITC-PLL was mixed in a 1:1
ratio with a protein loading buffer (BioForceNanosciences). A stock solution of FITC-
PLL was prepared consisting of 20 µl of FITC-PLL at 10 mg/ml with 20 µl of 1x PBS.
This 40 µl of solution was then mixed with 40 µl of protein loading buffer, for a final
FITC-PLL concentration of 2.5 mg/ml. The SPT-S-C30S cantilevers (BioForceNa-
nosciences) were used for surface patterning. These cantilevers were equipped with
micro-fluidic reservoirs for holding liquids. One microliter of FITC-PLL was used to
fill the reservoir before printing.
[Ca 2+ ] i Imaging : Cytoplasmic calcium was monitored using the InCytIm 1
fluorescentsystem(Intracellular Imaging, University of Cincinnati, OH) attached to a
DVC 340 M12-bit camera after loading cells with fluo-3 (Invitrogen) as previously
described (DeCoster et al., 2002).
Primary Neuronal Rat Cell Culture : The cortical rat neuronal cells were harvested
by performing cervical disarticulation on 1-day-old rat pups. The cells were placed
in 12- and 24-multiwell plates with a 1:1 coated surface of poly-L-lysine (Sigma Al-
drich) and deionoized water (DI) to enhance cell affinity for attachment. The neuronal
cells were cultured in Neuronal Culture Media (NCM) comprised of Fetal Bovine
Serum (FBS), Horse Serum (HS), glucose solution, glutamine, antibiotics, Ham's F12-
K (ATCC) and Basal Media Eagle's as previously described (Daniel and DeCoster,
2004).
Cell Culture of RBMVECs : The RBMVECs were purchased from Cell Applica-
tions Inc., San Diego, CA. The cells were grown on plates coated with Attachment
Factor solution (Cell Applications Inc., San Diego, CA) which promoted their attach-
ment and proliferation. The plates were coated with the Attachment Factor for 30 min
at 37°C. The endothelial cells were cultured in Rat Brain Endothelial Growth Medium
(Cell Applications Inc., San Diego, CA) fully supplemented with FBS, growth factors,
trace elements, antibiotics and vascular endothelial growth factor according to the
supplier's instructions. The cells were passaged when they reached 70% confluence,
as they formed tight junctions at higher confluence and became difficult to trypsinate.
Cells were used between passages 315.
Cell culture of glioma cells : Human glioma cells were obtained from ATCC
(Manassas, VA, USA; ATCC number CRL-2020) and cultured in CRL-2020 complete
media as indicated by the vendor.
Calcein staining and confocal imaging : Calcein staining for viable glioma cells in
3D biocomposites was carried out as previously described (Xing et al., 2010).