Elucidation of the Role of Carbon Nanotube Patterns on the Development of Cultured Neuronal Cells

Carbon nanotubes (CNTs) promise various novel neural biomedical applications for interfacing neurons with electronic devices or to design appropriate biomaterials for tissue regeneration. In this study, we use a new methodology to pattern SiO₂ cell culture surfaces with double-walled carbon nanotubes (DWNTs). In contrast to homogeneous surfaces, patterned surfaces allow us to investigate new phenomena about the interactions between neural cells and CNTs. Our results demonstrate that thin layers of DWNTs can serve as effective substrates for neural cell culture. Growing neurons sense the physical and chemical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. Cells exhibit comparable adhesion and differentiation scores on homogeneous CNT layers and on a homogeneous control SiO₂ surface. Conversely, on patterned surfaces, it is found that cells preferentially grow on CNT patterns and that neurites are guided by micrometric CNT patterns. To further elucidate this observation, we investigate the interactions between CNTs and proteins that are contained in the cell culture medium by using quartz crystal microbalance measurements. Finally, we show that protein adsorption is enhanced on CNT features and that this effect is thickness dependent. CNTs seem to act as a sponge for culture medium elements, possibly explaining the selectivity in cell growth localization and differentiation

Preparation of Tethered-Lipid Bilayers on Gold Surfaces for the Incorporation of Integral Membrane Proteins Synthesized by Cell-Free Expression

There is an increasing interest to express and study membrane proteins in vitro. New techniques to produce and insert functional membrane proteins into planar lipid bilayers have to be developed. In this work, we produce a tethered lipid bilayer membrane (tBLM) to provide sufficient space for the incorporation of the integral membrane protein (IMP) Aquaporin Z (AqpZ) between the tBLM and the surface of the sensor. We use a gold (Au)-coated sensor surface compatible with mechanical sensing using a quartz crystal microbalance with dissipation monitoring (QCM-D) or optical sensing using the surface plasmon resonance (SPR) method. tBLM is produced by vesicle fusion onto a thin gold film, using phospholipid-polyethylene glycol (PEG) as a spacer. Lipid vesicles are composed of 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC) and 1,2-distearoyl-<i>sn</i>-glycero-3-phosphoethanolamine-<i>N</i>-poly­(ethyleneglycol)-2000-<i>N</i>-[3-(2-pyridyldithio)­propionate], so-called DSPE-PEG-PDP, at different molar ratios (respectively, 99.5/0.5, 97.5/2.5, and 95/5 mol %), and tBLM formation is characterized using QCM-D, SPR, and atomic force technology (AFM). We demonstrate that tBLM can be produced on the gold surface after rupture of the vesicles using an α helical (AH) peptide, derived from hepatitis C virus NS5A protein, to assist the fusion process. A cell-free expression system producing the E. coli integral membrane protein Aquaporin Z (AqpZ) is directly incubated onto the tBLMs for expression and insertion of the IMP at the upper side of tBLMs. The incorporation of AqpZ into bilayers is monitored by QCM-D and compared to a control experiment (without plasmid in the cell-free expression system). We demonstrate that an IMP such as AqpZ, produced by a cell-free expression system without any protein purification, can be incorporated into an engineered tBLM preassembled at the surface of a gold-coated sensor

Four MnCl₂ doses shown 24h post injection in marmoset brain.

<p>(A) Slices at the injection site (+6mm from bregma), and (B) slices 2 mm posterior to the injection site. Top: Raw images. Bottom: ROI of MnCl₂ hyperintensity automatically thresholded at 195 on the grey scale (256 levels). To the right of the figure, corresponding slices of the Atlas of Yuasa et al, 2010. The following structures are hyperintense: primary motor cortex M1 (Brodmann area 4), the primary sensory cortex (3a), the cingulum (23–24), the premotor cortex (6c,6d), the parietal cortex (5), corpus callosum, corona radiata, caudate (Cd), putamen (Pu), internal (IGP) and external (EGP) globus pallidus, thalamic nuclei (VL: ventral lateral, RT: reticular), the internal capsule (ic). Note that MnCl₂ follows the corpus callosum to the contralateral hemisphere most significantly with the highest doses.</p

Marmoset staircase.

<p>Picture of Hill (A) and Valley (B) staircases attached to the front of the marmoset cage. Steps are baited for left forelimb testing.</p

Statistical parametric maps of manganese in seven marmosets.

<p>Statistical parametric maps of manganese after injection (blue point) in the primary motor cortex (M1) of seven marmosets (p<0.005 uncorrected). A: Semitransparent three-dimensional MnCl₂ maps on the single brain template [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138308#pone.0138308.ref032" target="_blank">32</a>]. Animals were imaged 24h after 8nmol MnCl₂ injection. MnCl₂ induced hyper intensity on brain T1-weighted images. C-G: coronal views. H-L: Corresponding marmoset brain atlas in coronal view. The following structures are marked: M1 primary motor cortex, Cd: Caudate nucleus, Pu: putamen, GP: globus pallidus, ic: internal capsule, cp: cerebral peduncule and SN: subtantia nigra, the cingulum, the premotor cortex (Brodmann area 6c,d), the parietal cortex (Brodmann area 5), corpus callosum, corona radiata, thalamic nuclei (VL: ventral lateral thalamic nucleus, VPL: ventral posterolateral thalamic nucleus, VPM: ventral posteromedial thalamic nucleus, CM: central medial thalamic nucleus, RT: reticular thalamic nucleus). Colored lines indicate the cortico-caudate tract (orange), the corticospinal tract (green), the cortico-putaminal tract (blue), and the cortico-thalamic tract (purple).</p

Behavioral effect of MnCl₂ injection.

<p>Scores (number of pellets ± SD) at the Valley (A,C) and Hill (B,D) staircase before (white) and after (black) contralateral (C,D) and ipsilateral (A,B) MnCl₂ injection. No behavioral deficits are observed after low concentrations (80 and 8 nmol), four days after injection. However the high concentration (400 nmol) caused a decrease in valley and hill scores only in the contralateral forelimb. Baseline scores are represented in white while 4 days post-injection of MnCl₂ scores are represented in black.</p

Single marmoset’s raw image showing manganese-induced hyperintensity in the pyramids.

<p>Sagittal, coronal, and axial slices showing manganese-induced hyperintensity in the pyramids. Note the hyperintensity compared to the contralateral side. Raw images of the marmoset with MnCl2 signal reaching the pyramid with an 8 nmol injection. Three views at coordinates 1,25mm lateral; -0.2 mm AP from bregma; +3 mm dorsoventral from the interaural line. Lines show MnCl2 signal in the pyramid at the brainstem level.</p