Ex vivo-Kultur der Patient Tissue & Prüfung von Gentransfer

Hello everyone. My name is Simon Regenron. I'm one of the surgical registrars here in the Core Cancer Research Center in Ireland.

The technique I'm showing you today involves the use of intact patient tissue for the study of gene delivery. Gene therapy offers exciting therapeutic approaches for the treatment of cancer. However, have recent clinical trials have failed to replicate results found in animal models.

The main reason, I believe, to be the variation in tissue physiology between animal and man tumor heterogeneity and differences in tumor phenotype. X vivo patient tissue model I'm demonstrating is the most stringent preclinical model available today. This model involves slicing patient tissue into thin slices and maintaining these slices in vitro.

Many gene delivery methods may be tested using this model, the viral vectors and DNA constructs I'm using express a firefly luciferase under the transcription control of the CMV promoter, allowing quantification of gene expression by bioluminescent Imaging. Okay, let's go start this. The materials that are needed for this model are fresh human tissue, vibram tissue slicing system, physical gene delivery methods to be Assessed.

An ultrasound machine, six plate tissue culture dish 27 gauge incident needle non-toxic glue gene delivery vector, which may be viral and non-viral media Containing serum and culture media. The block of tissue is attached to the slicing stage using derma bond ad heath. The material, the tissue is submerged in PBS during slicing to aid with slicing tissue Is submerged in ice and refrigerated for 30 minutes.

The required slice thickness in this case two millimeters is set and slices are cut At 20 RPM slices are cultured in a six well dish after two hours of Incubation at 37 degrees Celsius. Viral vectors are injected directly Into the slices. Slices are incubated for 24 hours in a 37 degrees incubator.

The next day, slices are analyzed for gene expression, but by a luminescent imaging.