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Titre : | Étude de lhétérogénéité cellulaire au sein de culture 3d: sphéroïde |
Auteurs : | Emilia COSTA RIBEIRO DE SEIXAS, Auteur ; O. Feron, Promoteur |
Type de document : | Travail de fin d'études |
Editeur : | Bruxelles : Institut Paul Lambin, 2020 |
Langues: | Français |
Index. décimale : | TFE - Biologie médicale |
Résumé : |
Cancer is one of the deadliest diseases in the world. A high rate of death is associated with advanced stage disease because of metastasis. Increased aggressiveness originates in part from the development of areas of hypoxia and acidosis in primary tumors where cancer cells acquired an invasive phenotype. Threedimensional cell culture (spheroid) that naturally developed tumor-like microenvironment is used in this study to understand metabolic cell adaptations leading to an aggressive phenotype. The aim of our study is to optimize a protocol to isolate and analyze cells form spheroid depending of their localization in spheroid (core: hypoxia, acidosis, and periphery: normoxia
).
Seven days-old Spheroid of two-cell lines (FaDu and HCT116) have been used; Spheroids were stained with two lipophilic fluorophores: Calcein-AM® and Rhodamine 6G in order to stain differentially the cells present in spheroid from the periphery to the core. The dissociation was performed using enzymatic activity: Trypsine or Accutase®, associated with a mechanical force. Then cells sub-population (differential staining) was identified using flow cytometry. Twice incubation of 5 min with Accutase® at room temperature lead to a complete spheroid dissociation with a high cell viability (>80%), better than Trypsine. Additionally, spheroid staining according to gradient (periphery>core) was observed using either Calcein-AM® or Rhodamine 6G at 10 μM and 1 μM,respectively. An inverse correlation between incubation time and Calcein-AM® diffusion was observed. In conclusion, spheroid dissociation protocol was optimized using Accutase® instead of Trypsine. Calcein- AM® and Rhodamine 6G are good candidates to stain the spheroid according to gradient. Further experiments are required to optimize dye concentration and incubation-time. |
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 |