Modelo de tumor hepático murino Bioluminescent

Hello everyone. My name is Simon Regenron. I'm one of the surgical registrars in the car cancer research center here in Ireland.

Today I'll be showing you a surgical technique of inducing liver metastases. In a neuron model, the liver is a common site of metastatic spread and a major cause of cancer related mortality. Only a small percentage of patients are suitable for surgical resection with advanced liver disease.

Therefore, novel therapeutics are needed for the treatment of this condition. Many animal models of hepatic metastases do not result in disease resembling the clinical setting. The model I'm showing you today is a clinically relevant model and it is relatively easy to learn and with very good success rates.

This model involves dividing the spleen into two hemi spleens. One Hemi spleen is used for the inoculation of tumor cells. These tumor cells travel through the porter circulation into the liver.

The other Hemi spleen is left intact maintaining its immunological properties. The cell line I'm using is stably expressing luciferase, allowing for the bioluminescent analysis of tumor growth in vivo. Therefore, this model will allow for the non-invasive monitoring of therapeutics of liver disease.

Okay, let's go ahead and start this for the procedure today. The instruments that we need are dissecting scissors, needle holder, two small faucets, size 10 scalpel five oh proline suture for skin closure, four oh PIA, suture for closure of the peritoneal layer three oh Vicryl ties for dividing the spleen saline 29 gauge insulin needle for anesthetic injection, 26 gauge insulin needle for tumor suspension, injection tumor cell suspension in serum free media, injectable anesthetic, skin marker, precut sterile gauze swabs Ine. The murn model that I will use today is the Athymic MF one nude mouse.

Other models such as the C 57 and BC may be used with appropriate tumor cell lines. A heating pad is used injectable general anesthetic is used in a laboratory. The depth of anesthesia is assessed using the toe pinch.

There should not be a toe withdrawal reflex on toe pinch. The knee size mouse is properly positioned and taped, and abdomen is prepped with Betadine and draped in a sterile fashion. A subcostal incision is made, an abdominal cavity is entered, the spleen is identified and exteriorized.

The spleen is isolated from the rest of the mouse using a precut sterile gauze. Two vertical ties are passed between the two vascular pedicles and gently tied. The spleen is divided into two equal hemi spleens.

One hemi spleen is used for the inoculation of Tumor cells. Tumor cells are slowly injected into the Hemi spleen to avoid leakage. Tumor cells travel through the portal circulation and arrive at the liver.

After 10 Minutes of incubation, the injected hemi spleen is excised and the other hemi spleen is returned into the abdomen. Absorbable sutures and a running stitch are used to close the peritoneal cavity and non-absorbable five oh proline suture is used for skin closure. Recovery is carried out routinely.

The development of liver disease can be assessed over time using 2D ivus imaging. The mouse is anesthetized as before, and Lucifer substrate is given intraperitoneal. The mouse is placed on the stage in the 37 degree chamber.

Imaging is carried out for three minutes and a superimposed image of mouse with detected luminescence is produced Three weeks after treatment, abdomen is opened and diffuse liver disease is found. In conclusion, this technique permits the induction of liver localized metastatic tumor cell growth by luminescent imaging enables visualization and quantification of tumor growth and application in assessment of therapeutic responses.