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

<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 [¹⁸F]FHBG labelled Neuro2a-TK: 1 X 10⁵ cells [A,D,E] 5 X 10⁵ cells [B,F,G] and 1 X 10⁶ cells [C,H,I]. Representative experiment with decay-corrected signal of [¹⁸F]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

Cell radioactivity retention.

<p>(A) Cell activity (dpm) or (B) Percentage [¹⁸F]FHBG cell retention as a function of the incubation time with 115 or 555 kBq/ml of [¹⁸F]FHBG. Each value represents the mean ± 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 [¹⁸F]FHBG concentrations and incubation time.</p

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

<p>1110 kBq of [¹⁸F]FHBG were injected intracerebrally one day after the graft (1 X 10⁶ or 3 X 10⁶ 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 10⁶ grafted cells than those with 1X 10⁶ grafted cells.</p

Generation of stably transfected Neuro2A -TK cell line.

<p>Neuro2A cells were transfected with the optimised mammalian vector pCpG-free-HSV1-TK. Stably transfected cells were selected by antibiotic pressure (G418, 600μgr/ml) over a 3-week period. The selection procedure was confirmed with the fluorescent live–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 μm.</p

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

<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 [¹⁸F]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 [¹⁸F]FHBG incubation under defined parameters, calibration ranges from 1 X 10⁴ to 3 X 10⁶ Neuro2A-TK cells were analysed by gamma counter or by PET-camera. In parallel, grafting with different quantities of [¹⁸F]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 [¹⁸F]FHBG signal of transplanted cells <i>in vivo</i>.</p><p>Results</p><p>Under the optimised incubation conditions, [¹⁸F]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 10⁴ grafted cells <i>in vivo</i>. No [¹⁸F]FHBG or [¹⁸F]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.

<p>A 96-well plate containing different quantities of [¹⁸F]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 [¹⁸F]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