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	Intratibial 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 ul and transfer this mixture into an eppendorf tube.

Transfer the cell suspension into a 31G 0.3 ml 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.

Heat: Animal should be kept on a heating pad or circulating water blanket/pad during the entire procedure and, after the surgery is over for approx. 2-4 hrs.

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 right or left knee.

Administer buprenorphine subcutaneously. Note the time of administration.

Prep the shaved area with povidone-Iodine prep pads, rubbing in a circular motion. Apply 70% alcohol pads to the surgical area to remove the betadine and loose hair. Repeat this process 2 or more times.

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

Gently grasp lateral malleolus, medial malleolus, and lower half of tibia with forefinger and thumb, then bend leg such that the knee is visible and accessible.

Wet the skin with 70% ethanol to increase the visibility of the underlying patellar ligament, which should be visible as a distinct, thick, white line.

Transfer the cell suspension into a syringe

While firmly grasping the ankle/leg of the mouse insert the syringe with cells suspension under the patella, through the middle of the patellar ligament, and into the anterior intercondylar area in the top of the tibia.

When inserting the needle into the tibia, guide carefully through the growth plate using steady, firm pressure with slight drilling action.

The needle will encounter markedly less resistance upon penetration of the tibial growth plate.

Use a gentle, lateral movement of the needle to ensure the needle is in the tibia and through the growth plate. The movement will be limited if the needle is in the proper place within the tibia.

Slowly depress the plunger to inject 10 μl of cell solution. Little to no resistance should be felt at this point.

Slowly extract the needle.

Administer lidocaine in the flank. Note the time of administration

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.