12 research outputs found

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

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    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 SiO<sub>2</sub> 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 SiO<sub>2</sub> 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

    <i>In-vivo</i> labelling by intracerebral injection [<sup>18</sup>F]-FHBG after cell graft.

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    <p>1110 kBq of [<sup>18</sup>F]FHBG were injected intracerebrally one day after the graft (1 X 10<sup>6</sup> or 3 X 10<sup>6</sup> cells, n = 1 per quantity of cells)) or in the control rat (injured but not grafted n = 1). 90 and 160 min after injection, PET /CT images showed a whole body distribution of the radiotracer and cerebral retention into the grafted area (A). Image analyses were performed and the percentage dose injected per gr of tissue (% ID/g) was calculated for the different time and cell quantity(B). 90 and 160 min after injection, a higher % of ID/g was recorded in rats with 3 X 10<sup>6</sup> grafted cells than those with 1X 10<sup>6</sup> grafted cells.</p

    Four MnCl<sub>2</sub> doses shown 24h post injection in marmoset brain.

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    <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 MnCl<sub>2</sub> 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 (23ā€“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 MnCl<sub>2</sub> follows the corpus callosum to the contralateral hemisphere most significantly with the highest doses.</p

    Cell radioactivity retention.

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    <p>(A) Cell activity (dpm) or (B) Percentage [<sup>18</sup>F]FHBG cell retention as a function of the incubation time with 115 or 555 kBq/ml of [<sup>18</sup>F]FHBG. Each value represents the mean Ā± SD (* p<0.05, ** p<0.005). A significant difference was observed between the activity retained by the control cells and that retained by Neuro2A-TK cells in terms of [<sup>18</sup>F]FHBG concentrations and incubation time.</p

    Single marmosetā€™s raw image showing manganese-induced hyperintensity in the pyramids.

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    <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

    <i>In-vivo</i> cell monitoring and signal quantification in rat brain.

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    <p>Representative PET/CT images in sagittal [A,B,C], coronal [D,F,H] and axial [E,G,I] axes of rat grafted with different quantities of [<sup>18</sup>F]FHBG labelled Neuro2a-TK: 1 X 10<sup>5</sup> cells [A,D,E] 5 X 10<sup>5</sup> cells [B,F,G] and 1 X 10<sup>6</sup> cells [C,H,I]. Representative experiment with decay-corrected signal of [<sup>18</sup>F]FHBG labelled Neuro2a-TK cell-grafted rats (5 rats imaged on the same day, blue square), using the daily cell calibration range (red dots) and the average cell calibration ranges (n = 5 experiments, green dots with standard deviation) imaged with the camera (J). Colour scale intensity (Bq/ml).</p

    Generation of stably transfected Neuro2A -TK cell line.

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    <p>Neuro2A cells were transfected with the optimised mammalian vector pCpG-free-HSV1-TK. Stably transfected cells were selected by antibiotic pressure (G418, 600Ī¼gr/ml) over a 3-week period. The selection procedure was confirmed with the fluorescent liveā€“dead staining assay on stably transfected Neuro2A-TK cells (A) or Neuro2A control cells (B). Red staining indicates dead cells; green staining indicates live cells. Scale bar 30 Ī¼m.</p

    Imaging grafted cells with [<sup>18</sup>F]FHBG using an optimized HSV1-TK mammalian expression vector in a brain injury rodent model

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    <div><p>Introduction</p><p>Cell transplantation is an innovative therapeutic approach after brain injury to compensate for tissue damage. To have real-time longitudinal monitoring of intracerebrally grafted cells, we explored the feasibility of a molecular imaging approach using thymidine kinase HSV1-TK gene encoding and [<sup>18</sup>F]FHBG as a reporter probe to image enzyme expression.</p><p>Methods</p><p>A stable neuronal cell line expressing HSV1-TK was developed with an optimised mammalian expression vector to ensure long-term transgene expression. After [<sup>18</sup>F]FHBG incubation under defined parameters, calibration ranges from 1 X 10<sup>4</sup> to 3 X 10<sup>6</sup> Neuro2A-TK cells were analysed by gamma counter or by PET-camera. In parallel, grafting with different quantities of [<sup>18</sup>F]FHBG prelabelled Neuro2A-TK cells was carried out in a rat brain injury model induced by stereotaxic injection of malonate toxin. Image acquisition of the rats was then performed with PET/CT camera to study the [<sup>18</sup>F]FHBG signal of transplanted cells <i>in vivo</i>.</p><p>Results</p><p>Under the optimised incubation conditions, [<sup>18</sup>F]FHBG cell uptake rate was around 2.52%. <i>In-vitro</i> calibration range analysis shows a clear linear correlation between the number of cells and the signal intensity. The PET signal emitted into rat brain correlated well with the number of cells injected and the number of surviving grafted cells was recorded via the <i>in-vitro</i> calibration range. PET/CT acquisitions also allowed validation of the stereotaxic injection procedure. Technique sensitivity was evaluated under 5 X 10<sup>4</sup> grafted cells <i>in vivo</i>. No [<sup>18</sup>F]FHBG or [<sup>18</sup>F]metabolite release was observed showing a stable cell uptake even 2 h post-graft.</p><p>Conclusion</p><p>The development of this kind of approach will allow grafting to be controlled and ensure longitudinal follow-up of cell viability and biodistribution after intracerebral injection.</p></div

    Cell calibration range using the gamma counter or PET/CT camera.

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    <p>A 96-well plate containing different quantities of [<sup>18</sup>F]FHBG labelled Neuro2a-TK cells and control cells were imaged using a PET/CT camera (A, B). The decay-corrected signals expressed in dpm were calculated (C) for the different cell quantities using the gamma counter (blue) or the PET/CT images (red) (n = 5 experiments on separate days, p = 0.626 no significant difference was observed between average counter titration and average camera titration). Different camera calibration ranges for [<sup>18</sup>F]FHBG labelled Neuro2a-TK cells in grey (n = 5) and for control cells in green were performed (D). The percentage of [18F]FHBG cell retention was also calculated for each independent experiment (grey square).</p

    Behavioral effect of MnCl<sub>2</sub> injection.

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    <p>Scores (number of pellets Ā± SD) at the Valley (A,C) and Hill (B,D) staircase before (white) and after (black) contralateral (C,D) and ipsilateral (A,B) MnCl<sub>2</sub> 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 MnCl<sub>2</sub> scores are represented in black.</p
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