La migration des cellules dendritiques de surveillance utilisant l'imagerie par résonance 19F / 1H magnétique
Summary
Suivi des cellules en utilisant l'IRM a attiré l'attention remarquable dans les dernières années. Ce protocole décrit le marquage des cellules dendritiques avec le fluor (<sup> 19</sup> Particules F)-riches, l'application in vivo de ces cellules, et la surveillance de l'étendue de leur migration vers le ganglion drainant avec<sup> 19</sup> F /<sup> 1</sup> H IRM et<sup> 19</sup> F MRS.
Abstract
Progrès continus en matière modalités d'imagerie non invasives comme l'imagerie par résonance magnétique (IRM) ont grandement amélioré notre capacité à étudier les processus physiologiques ou pathologiques dans les organismes vivants. IRM s'avère également être un outil précieux pour la capture des cellules transplantées in vivo. Les premières stratégies de marquage cellulaire pour utilisation d'agents de contraste IRM qui influencent les temps de relaxation MR (T1, T2, T2 *) et conduire à une amélioration (T1) ou l'épuisement (T2 *) du signal où les cellules marquées sont présentes. T2 agents d'amélioration * tels que les agents ultrapetites d'oxyde de fer (USPIO) ont été utilisés pour étudier la migration cellulaire et certains ont également été approuvé par la FDA pour une application clinique. Un inconvénient de T2 * agents est la difficulté de distinguer l'extinction du signal créé par les cellules marquées d'autres artefacts tels que des caillots de sang, des saignements ou des micro-bulles d'air. Dans cet article, nous décrivons une nouvelle technique pour le suivi in vivo des cellules quisont basées sur le marquage des cellules avec du fluor (19 F)-particules riches. Ces particules sont préparées par émulsification d'hydrocarbures perfluorés (PFC) et de composés ensuite utilisé pour marquer les cellules, qui peuvent ensuite être visualisées par 19 F IRM. Les avantages importants de PFC pour le suivi des cellules in vivo comprennent (i) l'absence de carbone lié à 19 F in vivo, ce qui donne alors des images sans fond et complète de cellules selectivityand (ii) la possibilité de quantifier le signal de la cellule de 19 F spectroscopie MR .
Introduction
The tracking of cells in vivo is a crucial aspect in several fields of biomedicine. For this, noninvasive imaging techniques that can selectively localize cells over a period of time are extremely valuable. Prior to the development of three-dimensional magnetic resonance imaging (MRI), the tracking of immune cell migration was limited to microscopic analyses or tissue biopsies. Cell tracking with the help of MRI has gained immense attention in the past few years, not only for immunologists studying immune cell behavior in vivo, but also for clinical and stem cell researchers. During the mid-90s, the first studies on iron oxide nanoparticles 1 initiated a cascade of developments for tracking cells with MRI. Iron oxide particles shorten the MR relaxation time (T2*) of the labeled cells and thus cause signal depletion in MR images. Iron oxide particles have been employed to label macrophages 2, oligodendrocyte progenitors 3 and many other cell types. Some of these particles have also been clinically approved by the FDA for labeling cellular vaccines in melanoma patients 4. Since in vivo or ex vivo labeling of cells with iron oxide particles relies on a shortening of the T2* signal and the latter could be also brought about by in vivo susceptibility-related T2* effects such as micro bleeds, iron deposits or air bubbles, it might be difficult to identify labeled cells in vivo from other background T2* signal extinctions 5.
In this article, we describe a technique for tracking dendritic cells (DC) in vivo by employing 19F/1H magnetic resonance imaging (MRI). This cell tracking technology was only introduced in 2005 6, several years after the first recognized applications for 19F in MRI had been reported 7. One important advantage of 19F over iron oxide particle cell labeling is the low biological occurrence of 19F in tissue; this makes it possible to track cells very selectively with basically background-free images. Furthermore, it is possible to overlay the 19F MR signal from the transplanted labeled cells with anatomical images obtained from conventional 1H MRI. 19F/1H MRI is therefore considerably relevant for studies investigating cell migration in vivo. Cells studied with this method are labeled with 19F-rich particles. Synthetically-derived perfluorocarbons (PFCs) consisting primarily of carbon and fluorine atoms are commonly used to prepare the particles. These compounds are insoluble in water and need to be emulsified prior to application in vitro or in vivo. The usual size of the PFC particles that have been employed by other groups for in vivo 19F-MRI tracking experiments ranges between 100 nm and 245 nm 6,8-10. We have however shown that the efficiency in labeling dendritic cells with perfluoro-15-crown-5-ether (PFCE) particles increases with increasing particle size (>560 nm).11
Protocol
Representative Results
Discussion
Cette méthode d'employer 19 F / 1 H IRM pour suivre le mouvement de DC dans le ganglion lymphatique donne l'occasion d'étudier les schémas de migration des cellules immunitaires in vivo. Les cellules dendritiques sont d'excellents exemples de la migration rapide des cellules immunitaires qui sont capables de manœuvrer à travers des structures tridimensionnelles sans bien adhérentes à des substrats spécifiques 17. Bien que la faible résolution spatiale (?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Cette étude a été financée par la Deutsche Forschungsgemeinschaft à SW (DFG WA 2804) et une bourse universitaire pour SW à partir du Centre de recherche expérimentale et clinique, une coopération du Centre Max Delbrück de médecine moléculaire et de la faculté de médecine Charité à Berlin. Les bailleurs de fonds n'ont joué aucun rôle dans la conception de l'étude, la collecte de données et d'analyse, la décision de publier ou de la préparation du manuscrit. Nous remercions M. Robert Westphal pour le support technique lors de son stage dans notre laboratoire.
Materials
| REAGENTS | |||
| C57BL/6 mice | Charles River, Berlin | ||
| RPMI | Gibco | 21875-091 | |
| FBS Superior | Biochrom AG | S 0615 | |
| HEPES | Gibco-Invitrogen | 15630-056 | |
| Penicillin-Streptomycin | Gibco | 15140-122 | |
| L-glutamine | Gibco | 25030-024 | |
| Dulbecco’s PBS | Sigma Aldrich | D8662 | |
| PFA | Santa Cruz | sc-281692 | |
| Perfluoro-15-crown-5-ether | ChemPur | 391-1996 | |
| Pluronic F-68 | Sigma Aldrich | P5556 | |
| Petri dishes (35 x 10 mm) | VWR, Germany | 391-1996 | |
| 27 ½ G syringes | VWR, Germany | 612-0151 | |
| Nylon cell strainers (100 μm mesh) | VWR, Germany | 734-0004 | |
| NMR tubes | VWR, Germany | 634-0461 | |
| EQUIPMENT | |||
| Dissection tools | FST | ||
| CO2 incubator | Binder | ||
| Small animal MR system | Bruker Biospin | 9.4T BioSpec 94/20 USR, ParaVision Acquisition and Processing Software | |
| 1H/19F dual-tunable volume RF coil | Rapid Biomed, Würzburg, Germany | 35 mm inner diameter, 50 mm length | |
| 19F spectroscopy coil | in-house | tune/match loop coil, 4 turns, inner diameter 5 mm, 10 mm long, two capacitors for tuning and matching | |
| Isoflurane inhalation system | Föhr Medical Instruments GmbH | ||
| Animal monitoring system Model 1025 | SA Instruments Inc., New York, USA |
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