Intrahepatic implantation of tumor cells

Preparation of surgical instruments and supplies: All of the instruments and supplies should be sterilized.

Surgery record sheets

A	B	C
PI
Personnel
Date
Procedure name	Hepatic Tumor Implantation
Protocol #
Type	Survival Surgery
Species	Mouse
Experimental agents administered	Tumor cells
Anesthetics	Isoflurane	Dosage: (1-5 % or mg/kg; mL)
Analgesics	(1) Lidocaine	Dosage
	(2)Buprenorphine	Dosage

A	B	C
Mouse ID
Mouse weight
Anesthesia start time
Analgesics	(1) Lidocaine	Time administered:
	(2) Buprenorphine	Time administered:
Tumor cells administration time
Anesthesia end time

Preparation of the PDX cells for implantation

Prepare DMEM medium: Prepare fresh DMEM media supplemented with

10% volume FBS and

100ug/ml Gentamicin.

Retrieve cryovials containing cells from liquid nitrogen storage. Thaw the cell-containing cryovial by placing it in 37 degrees water bath. Move the vial into a BSL2 hood and transfer the contents of the vial in a 15 ml conical tube containing fresh DMEM media (9mL DMEM per 1mL of the cell mixture) and mix gently.

Perform live/dead assay using trypan blue and note the live and total cell count.

Centrifuge the 15 ml tube containing cells at 300x g,0h 0m 0s and aspirate the supernatant. Resuspend the cells in fresh DMEM to a final concentration of 1M live cells/10-20 ul and transfer this mixture into an eppendorf tube.

Transfer the cell suspension into a syringe right before beginning the surgery.

Station 1: Fur removal

Place a disposable sterile pad on a heating pad.

Aseptically sterilize the surgical area by spraying with 70% ethanol.

Station 2: Surgery

Place a disposable sterile pad on a heating pad.

Aseptically sterilize the surgical area by spraying with 70% ethanol.

Place all the autoclaved surgical instruments within the sterilized surgical area.

Place the animal in a knock-down box circulating with a gas mixture of Isoflurane @ 1.4-2.0% and O₂ @ 1-1.2 lt/min inhalant, maintained via a bain-closed system.

Note the start time for anesthesia.

Move the animal to the station 1 nose cone and apply ophthalmic ointment on the animal’s eyes to prevent them from drying out during the procedure.

Determine the anesthetic depth by pinching the animal’s foot for a reflex response.

Depilate the ventral side to remove fur from the abdomen.

Disinfect the surgical area by rubbing the area in a circular motion with a povidone-Iodine prep pad followed by a 70% alcohol pad. Repeat this process 2 more times.

The animal is now ready to be moved to station 2 for surgery.

Gently grab the skin with iris forceps 1 cm under the xiphoid process and make a small (1–2 cm) vertical incision using dissecting scissors in the skin, perpendicular to the linea alba.

Gently grab the peritoneum muscle layer and make an incision to expose the liver.

Pull the muscle layer with iris forceps to adequately expose the liver for needle access. Two hemostats instead of forceps can be used to hold both sides of the muscle layer apart.

Transfer the cell suspension into a 1 cc syringe with a 27 G needle.

While keeping the liver in view with forceps or hemostats, puncture the liver with the pre-loaded syringe and advance the needle a few millimeters along the subcapsular place.

Inject 10-20 ul of cells suspension from the syringe into the liver. Hold the syringe in place for 20-30 secs to minimize any leakage of cells from the liver.

Pull the needle slowly while applying pressure with sterile cotton-tipped applicators in order to minimize leakage of the cells. The injection site should turn white upon successful injection.

Using absorbable sutures, suture the muscle layer and then suture the outer skin layer of the incision site.

Upon completion of the sutures administer buprenorphine and lidocaine subcutaneously. Note the time of administration.

Sterilize the surgical instruments using the bead sterilizer by placing them inside for ~ 10 secs.

Start prepping the next mouse for surgery.

Place the animal in a clean cage on a warm heating pad to aid in regaining its body temperature.

Observe the animal until it has regained full consciousness and is walking around in the cage.

Observe the animal on a daily basis until sacrificed as per your IACUC guidelines.