Ultrasound-Guided Implantation: A Technique to Implant Pancreatic Ductal Adenocarcinoma Cells into an Adult Mouse

Overview

This video describes the protocol for ultrasound-guided orthotopic implantation of murine-derived pancreatic ductal adenocarcinoma cells directly into the pancreas of the mouse. This causes less inflammation at the implantation site compared to surgical procedures.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the jove veterinary review board.

1. Pre-Surgical Preparation of Mice

NOTE: This step is recommended to be performed 24 hours prior to the procedure.

1. Place cages containing experimental mice on a warmer set to 37°C.
2. Obtain new, clean cages and place on a second warming platform set to 37°C.
3. Thoroughly clean the biological safety cabinet, the induction chamber and the ultrasound (US) stage with sterilant (see Table of Materials).
4. Turn the warming function of the US stage to 37°C.
5. Turn on the anesthesia machine: adjust the dials on the tube splitter so that airflow is restricted to the induction chamber only. Turn on the oxygen tank and set to flow rate to 1 L/min. Turn on the isoflurane vaporizer to 2-3%.
6. Place a single mouse into the induction chamber. Monitor mouse until no longer mobile and breathing has slowed.
7. Adjust the dials on the splitter so that airflow is permitted to both the induction chamber and the nose cone. Quickly move mouse from the induction chamber and lay it in ventral recumbency on the warm US stage with its muzzle in the nose cone.
8. Test the level of induction by observing any reflexive response to toe pinching. If there is none, the mouse is ready for hair removal.
9. Place a small amount of eye lubricant (see Table of Materials) on both eyes to prevent tissue dehydration. Turn the mouse so that it lays in dorsal recumbency on the stage. Gently adhere the upper and lower extremities to the stage with paper tape in order to maximize exposure of the abdomen and secure the mouse to the stage.
10. Use a sterile cotton-tip applicator to apply a generous layer depilatory cream to the upper left quadrant of the abdomen. The depilatory should be applied in the general area of the spleen and extend towards the midline. Allow to sit for about one minute.
11. Test the degree of hair removal by gently using the opposite end of the cotton tip applicator to wipe away the depilatory cream, once it removes easily, wipe the abdomen clean with a dry gauze pad. Then wet a clean layer of gauze with a small amount of warm saline and wipe the area once more to completely remove the depilatory agent. Do not exceed 2 full minutes of direct contact on mouse skin in order to reduce the chance of chemical burns.
12. Once the hair has been sufficiently removed from the abdomen, return the mouse to a new clean cage on warmer.
13. While the first animal is undergoing hair removal on the US stage, the next animal can be added to the induction chamber.
14. Before anesthetizing the next cage of mice, turn off the isoflurane and flush the induction chamber with oxygen. Clean the induction chamber and US stage/nose cone with sterilant. Repeat steps 1.5-1.14 until all cages and mice have undergone hair removal.
    NOTE: Fasting animals by temporary withdrawal of food for a period prior to implantation can be considered to minimize visual obstruction of abdominal organs due to undigested food in stomach and intestines (12-24 hours). Water restriction is not recommended. If animals are fasted, it is recommended that they are treated with an injection of 1mL warm (37°C), sterile saline following tumor injection in order to prevent dehydration.

2. Ultrasound-Guided implantation of PDA cells

NOTE: All ultrasound procedures are performed using ultrasound imaging machine and software (see Table of Materials). The transducer has a center frequency of 40 MHz and a bandwidth of 22-55 MHz.

1. Adjust the ultrasound platform such that the platform surface is parallel to the floor and the investigator faces the left side of the animal, with the animal's head to the right. Adjust the transducer position so that a transverse abdominal image will be obtained (Figure 1A).
2. Anesthetize the mouse to be injected as described in steps 1.1-1.8. Stabilize the mouse on the US platform as described in step 1.9.
3. Apply a generous amount of warm (37°C) ultrasound gel to the hairless section of the abdomen.
4. Gently lower the transducer to contact the mouse abdomen. Adjust the transducer as needed until the pancreas is clearly visible. Locate the left kidney and spleen in order to provide an accurate orientation of the abdominal cavity.
   NOTE: Because the transducer position has been changed to allow access to the left side of the abdomen, the X and Y axis on the stage controls are now inverted.
5. Load a 29G x ½" insulin syringe (see Table of Materials) with 25µL of tumor cell suspension. Wipe needle tip with sterile alcohol prep pad prior to injection in order to minimize tumor cell seeding in the abdominal wall.
6. Using blunt-edge forceps, grasp the skin and peritoneal wall to increase tension at the desired injection site. Holding the syringe at approximately a 25°-45° angle to the ultrasound platform surface, slowly advance the needle through the skin and the peritoneal wall. Confirm the needle has punctured through the peritoneal wall before proceeding to the next step. A small pop should be felt as the needle pierces the peritoneal wall.
7. Under ultrasound visualization, guide the needle directly into the pancreas (Figure 1B-C). Confirm the needle is within pancreas tissue by gently moving the syringe barrel up and down. If placement is correct, the needle tip will remain within the pancreas tissue while the syringe barrel is moving.
8. Slowly inject the tumor cells and confirm the cells are being implanted in the desired location by the formation of a fluid bolus within the pancreas (visible on the ultrasound screen, Figure 1D).
   NOTE: Some resistance should be felt while depressing plunger. Be careful not to pierce pancreas multiple times as this increases the likelihood of leakage into the abdominal cavity.
9. Once the full volume of suspension has been injected and a fluid bolus can be seen in the pancreas, keep needle very still for several seconds. Slowly retract needle from mouse abdomen, taking great care not to disturb the injected cells.
10. Place the mouse in a clean, warm cage and ensure that the mouse fully recovers from anesthesia. Repeat this process for all animals.
11. Before anesthetizing the next mouse, clean the induction chamber and US stage/nose cone with sterilant.
12. Repeat steps 2.2-2.11 until all mice have been injected.

Representative Results

Figure 1: Ultrasound-guided implantation of PDA cells into the murine pancreas. (A) Orientation of mouse, ultrasound stage, and ultrasound probe used to obtain a high-resolution image of abdominal organs. Note the stage and platform have been turned 90° from the standard orientation to allow easy access to the upper left quadrant of mouse abdomen. (B) Ultrasonographic image depicting the identification of kidney, spleen and pancreas. Here the injection needle is positioned against the mouse abdomen. (C) Ultrasonographic image depicting the needle within the mouse pancreas. (D) Ultrasonographic image depicting the bubble at the injection site (outlined in blue) following controlled injection of tumor cells into the pancreas. (E) Laparotomy revealing the fluid bolus containing PDA cells in the pancreas after ultrasound guided injection. Please click here to view a larger version of this figure.

Materials

Name	Company	Catalog Number	Comments
50 mL Conicals	Thomas Scientific	2602A26
Blunt edged forceps	Fine Science Tools	11000-12
Cell Dissociation Buffer	Thermo-Fisher	13151014
Cotton Tipped swabs	Thermo-Fisher	19062614
Covidien Monoject 3/10mL, 29G X 1/2"	Thermo-Fisher	8881600145
Depilatory Agent	Amazon	Nair Body Lotion
Forceps (blunt edge)	Fine Science Tools	11000-12
Gauze	Fisher	13-761-52
Gentamicin	Thermo-Fisher	15750060
Induction Chamber	VetEquip	941444
Isofluorane	Penn Vet Supply	VED1350
Isofluorane Vaporizer	VetEquip	911103
L-glutamine	Thermo-Fisher	25030081
Optixcare	MidWest Veterinary Supply	052.50310.3
Paper Tape	Medline	MMM1530Z5
PBS	Thermo-Fisher	14-190-250
Slide warmer	C&A Scientific	XH-2001
Sterilant (Clidox-S)	Fisher Scientific	NC0332382 (activator) NC9189926 (base)	Needs to be combined according to manufacturer's instructions
Sterile Alcohol prep pad	Covidien	6818
Trypsin	Thermo-Fisher	15090046
Ultrasound gel	Thermo-Fisher	03-34-1LT
Visualsonics Ultrasound Vevo 2100	Visual Sonics	Vevo 2100