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Titre : | Optimisation dun protocole de transparisation tissulaire et dimmunomarquages sur une culture cellulaire 3D : Visualisation par microscopie de fluorescence à feuillet de lumière. |
Auteurs : | Sounia BENBOUCHTA, Auteur ; C. Bouzin, Promoteur |
Type de document : | Travail de fin d'études |
Editeur : | Bruxelles : Institut Paul Lambin, 2020 |
Langues: | Français |
Index. décimale : | TFE - Biologie médicale |
Résumé : | Three-dimensional cultures of cancer cells, also known as tumor spheroids, are of large interest in cancer research. Two-dimensional cancerous cell cultures are limited in mimicking the in vivo tumor microenvironment whereas spheroids recapitulate some of the characteristics encountered in the in vivo tumors microenvironment such as acidosis, hypoxia and the metabolic switch. Those features can be highlighted by specific markers like pHlip for acidosis, carbonic anhydrase IX (CAIX) for hypoxia, and glucose and lactate transporters for the metabolic activity. Although microscopic imaging of two-dimensional cell culture is well established, the imaging of three-dimensional cell structures like spheroids is however limited by the refraction of the light making the cell layers that are distant (tens of microns) from the surface opaque. Hence, the laboratory adapted a tissue clearing protocol for the spheroids to prevent the light refraction using the CUBIC method which is well known and described in literature. The purpose of this study was to set up a fluorescent immunostaining method, within a pre-established tissue clearing protocol, on a three-dimensional cell culture. This work was focused 1) on the optimization of the nuclear staining, and 2) on the optimization of the immunofluorescent labeling of the tumor microenvironment markers (acidosis and hypoxia). One of the major challenges of this work was to define the best immunolabeling conditions on a three-dimensional cell structure ( |
Accès : | Identifiez-vous avant d'accéder au document électronique |
Disponible en ligne : | Oui |
Lieu du stage : | UCL / IREC / FATH |
Département : | Biologie médicale |
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TFE biologie médicale Adobe Acrobat PDF |