TITLE: Intratibial implantation of tumor cells 
AUTHORS: Shubhangi     Agarwal, donna.peehl  , Renuka   Sriram

[DESCRIPTION]
This protocol describes the steps required for the successful implantation of small cell neuroendocrine prostate cancer patient-derived xenograft (PDX) cells in the bone. Bone is one of the most common sites for the development of metastatic prostate cancer and its study is important for evaluating the tumor characteristics and response to therapy.
This protocol can be used for the implantation of any tumor cell line in the bone. 

[GUIDELINES]
Species: Mouse, Male. 

Strain: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ

Age: 6-8 weeks. 

Vender: Jackson Laboratory

Housing: House animals within the university-specific IACUC approved housing mouse colony facility at a weight ranging 14-19 grams. 

[STEPS]
SECTION: Preparation before surgery
1. Preparation of surgical instruments and supplies: All of the instruments and supplies should be sterilized. 
SECTION: Preparation before surgery
3. Preparation of the PDX cells for implantation
 

SECTION: Preparation of cells for implantation from frozen biobank
3.1. Prepare DMEM medium: Prepare fresh DMEM media supplemented with 
10 % volume FBS and 
100 ug/ml Gentamicin. 
SECTION: Preparation of cells for implantation from frozen biobank
3.3. Perform live/dead assay using trypan blue and note the live and total cell count. 


SECTION: Preparation of cells for implantation from frozen biobank
3.4. Centrifuge the 15 ml tube containing cells at 300 x g  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. 

 

SECTION: Preparation of cells for implantation from frozen biobank
3.5. Transfer the cell suspension into a 31G 0.3 ml syringe right before beginning the surgery. 
 

SECTION: Preparation of surgical space
4. Station 1: Fur removal


  


SECTION: Preparation of surgical space
4.1. Place a disposable sterile pad on a heating pad. 
SECTION: Preparation of surgical space
4.2. Aseptically sterilize the surgical area by spraying with 70% ethanol. 
SECTION: Preparation of surgical space
5. Station 2: Surgery

   

SECTION: Preparation of surgical space
5.1. Place a disposable sterile pad on a heating pad. 
SECTION: Preparation of surgical space
5.2. Aseptically sterilize the surgical area by spraying with 70% ethanol. 
SECTION: Preparation of surgical space
5.3. Place all the autoclaved surgical instruments within the sterilized surgical area.
SECTION: Preparation of surgical space
6. 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.
SECTION: Anesthetization and fur removal of mouse
7. 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. 
SECTION: Anesthetization and fur removal of mouse
8. Note the start time for anesthesia.
SECTION: Anesthetization and fur removal of mouse
9. 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. 
SECTION: Anesthetization and fur removal of mouse
10. Determine the anesthetic depth by pinching the animal’s foot for a reflex response. 
SECTION: Anesthetization and fur removal of mouse
11. Depilate right or left knee. 
SECTION: Anesthetization and fur removal of mouse
12. Administer buprenorphine subcutaneously. Note the time of administration. 
 

SECTION: Anesthetization and fur removal of mouse
14. The animal is now ready to be moved to station 2 for surgery. 
SECTION: Surgical procedure (Station 2)
15. 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. 
SECTION: Surgical procedure (Station 2)
16. 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. 
SECTION: Surgical procedure (Station 2)
18. 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. 
 


 

SECTION: Surgical procedure (Station 2)
17. Transfer the cell suspension into a syringe
 

 

SECTION: Surgical procedure (Station 2)
19. When inserting the needle into the tibia, guide carefully through the growth plate using steady, firm pressure with slight drilling action.
SECTION: Surgical procedure (Station 2)
20. The needle will encounter markedly less resistance upon penetration of the tibial growth plate. 
SECTION: Surgical procedure (Station 2)
21. 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.
SECTION: Surgical procedure (Station 2)
22. Slowly depress the plunger to inject 10 μl of cell solution. Little to no resistance should be felt at this point.
SECTION: Surgical procedure (Station 2)
23. Slowly extract the needle.
SECTION: Surgical procedure (Station 2)
24. Administer lidocaine in the flank. Note the time of administration
 

SECTION: Surgical procedure (Station 2)
25. Start prepping the next mouse for surgery.
SECTION: Post-Op care and monitoring
26. Place the animal in a clean cage on a warm heating pad to aid in regaining its body temperature.
SECTION: Post-Op care and monitoring
27. Observe the animal until it has regained full consciousness and is walking around in the cage
SECTION: Post-Op care and monitoring
28. Observe the animal on a daily basis until sacrificed.
SECTION: Preparation of cells for implantation from frozen biobank
3.2. 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 (9 mL DMEM per 1 mL of the cell mixture) and mix gently. 
SECTION: Anesthetization and fur removal of mouse
13. 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. 
SECTION: Preparation before surgery
2. Surgery record sheets