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, Ce protocole a été principalement mis au point pendant la thèse de Selma Leulmi avec l'aide de Philippe Sabon. Quelques optimisations ont été réalisées par Mélissa Morcrette et moi-même

, Allumer le MJB4 : ON / Sur la lampe : Power ON puis CP puis START, 30 min de chauffe

, Allumer les plaques chauffantes : une à 90 °C, une à 180 °C. Monter la puissance de chauffe à, pp.90-100

, Allumer la tournette en suivant les instructions, la laver avec de l'acétone, fixer le support adéquat, vérifier que le vide fonctionne, préparer 2 chronomètres sur 5 min Programmer la tournette pour la

, Dès que la tournette s'arrête, désenclencher le vide, récupérer le wafer et le déposer sur la plaque à 180°C. Déclencher de suite le chrono : 5 min

L. Dès-que, . Chrono, and . Fini,

, Programmer la tournette pour la ma-N 2403 : 4000 rotations/minute -30 secondes -2000 accélération

, Positionner le wafer, de manière centrée sur la tournette

, Déposer la ma-N 2403 : ~0,6 ml pour un wafer 2 pouces ou ~1

, Dès que la tournette s'arrête, désenclencher le vide, récupérer le wafer et le déposer sur la plaque à 90 °C. Déclencher de suite le chrono : 1 min 30

L. Dès-que, . Chrono, and . Fini,

, Positionner le masque, face chromée vers le haut, sur le porte-masque en le calant contre les 3 vis. Enclencher le vide, vérifier

, Mettre en place le porte-substrat à la bonne taille et avec le joint, le petit trou vers nous Par sécurité, descendre de 2 la molette

, Passer un coup de soufflette sur le wafer et le positionner selon les marques du porte substrat (côté droit vers nous)

, Sélectionner « WEC settings », suivre les indications. Un réglage suffit pour plusieurs wafers de même

. Desserrer-les-vis, Nettoyer le masque : mettre le bain de « Remover » dans le bac à ultra son, y plonger le masque avec la pince blanche, vérifier que le bac n'est pas en mode chauffage, allumer le bac pour 15-20min

, Sécher le masque avec la soufflette en le tenant par les bords pour ne pas laisser de traces

Q. Le,

, Une fois le process terminé, appuyer sur « LL vent ». Sortir l'échantillon quand le voyant « ATM sas » est allumé. Se débadger ? Lift-off de la ma

, De préférence, le lift-off est à faire dans la même journée que le dépôt

, Des « copeaux » de matériau se détachent, visibles à l'oeil nu. Mettre le cristallisoir dans le bac à ultrasons pour détruire les copeaux. Sortir le wafer tout en l'aspergeant d'isopropanol pour éviter que des morceaux restent accrochés

, ? Mise en suspension des particules -Lift-off de la PMMA

, Si besoin, cleaver les bords s'ils ne sont pas « propres ». Si possible, la mise en suspension doit se faire au maximum un mois après le dépôt de résines. Sinon le lift-off se fera plus difficilement

P. Le and . Dans-un-cristallisoir, Mouiller le wafer avec de l'acétone. Dès que la résine commence à se dissoudre, incliner le cristallisoir et passer le jet d'acétone sur le wafer, de haut en bas. Les particules qui descendent doivent se voir à l'oeil nu

, Le sortir, l'incliner de nouveau et aspirer tout l'acétone avec une pipette, le transvaser dans un tube de 50 ml

. /!\, Le tube n'est pas tout à fait étanche et doit rester vertical : utiliser un portoir et utiliser du parafilme

, Placer le tube Falcon sur un aimant et laisser les particules sédimenter (~30 min)

. Au-choix, Transvaser le tout dans le tube Eppendorf avec la micropipette. (facile mais long) ou Préparer un tube Eppendorf 2 ml. Plonger le cône de la micropipette au fond du Falcon. Au dernier moment, laisser tomber l'aimant et aspirer le fond du Falcon contenant les particules. Transvaser de suite dans le tube Eppendorf, avant que des gouttes ne tombent, Préparer un tube Eppendorf 2 ml

, Ne pas oublier d'annoter le tube, le placer dans le bac à ultrason pour bien disperser les particules. Annexes ? Utilisation des particules

, Pour une utilisation des particules in-vitro ou in-vivo, les étapes suivantes sont à réaliser sous PSM

, Utiliser l'aimant ou un portoir aimanté pour condenser les particules, aspirer tout l'acétone et le jeter

. En, Recommencer une nouvelle fois : condenser les particules avec l'aimant. Aspirer tout l'éthanol et le jeter

, Dans les deux cas, répéter l'opération deux fois est très important pour une bonne dispersion des particules ! Pour une utilisation dans du NaCl, ne faire l'opération qu'une seule fois et ne plus attirer les particules avec l

, Un champ magnétique rotatif à faible fréquence (20 Hz) est appliqué pour faire vibrer des particules magnétiques en contact avec les cellules cancéreuses. Les particules développées sont produites par une approche top-down en salle blanche. Les disques de permalloy utilisés présentent une configuration en vortex avec une faible rémanence et une bonne dispersion en suspension. Des particules multicouches de Co/Pt avec une anisotropie perpendiculaire et des vortex de permalloy en forme d'ellipses sont aussi étudiés. L'efficacité du TEMMP est évaluée in-vitro sur des cellules de glioblastome et les différents paramètres sont optimisés. Une forte diminution du nombre de cellules après traitement est alors observée et le comportement des cellules restantes est affecté, Résumé Le glioblastome est un cancer du cerveau très agressif dont les thérapies actuelles n'augmentent que très peu la durée de vie

, Les tissus sont peu affectés par le TEMMP comparé à une injection de particules, et une faible augmentation de la survie est observée. Pour mimer les propriétés mécaniques du cerveau de manière plus pertinente, un modèle in-vitro 3D est alors développé et validé, Conçu avec des sphéroïdes de cellules pris dans un gel d'agarose

, Mots clés : Particules magnétiques -Cancer -Glioblastome -Effet magnéto-mécanique des particules -Sphéroïde -In-vitro 3D

, Existing therapies improve only slightly the median survival. In this work, we study a new treatment by magneto-mechanical actuation of particles (TMMAP). A low frequency (20 Hz) rotating magnetic field is applied to stimulate magnetic particles localized near cancer cells

, Multilayers of Co/Pt with a perpendicular anisotropy and permalloy vortex particles with an ellipse shape are also studied. TMMAP efficiency is tested in-vitro on glioblastoma cell line and the parameters are optimized. A huge diminution of living cells and an affected behavior of the remaining cells are observed after treatment. TMMAP is then adapted to an in-vivo study on glioblastoma orthotopic model on nude mice and the intratumoral injection of the particles is developed. Few differences are observed between tissues submitted to TMMAP or injected with particles, and survival is slightly increased. To mimic mechanical properties of the brain in a more relevant model, an in-vitro 3D model is proposed and validated

, Keywords: Magnetic particles -Cancer -Glioblastoma -Magneto-mechanical actuation of particles -Spheroid -3D in-vitro

, Mots clés : Particules magnétiques -Cancer -Glioblastome -Effet magnéto-mécanique des particules -Sphéroïde -In-vitro 3D

, Keywords: Magnetic particles -Cancer -Glioblastoma -Magneto-mechanical actuation of particles -Spheroid -3D in-vitro