TITLE: Intrahepatic implantation of tumor cells
AUTHORS: Shubhangi   Agarwal, Robert  Bok, donna.peehl  , Renuka   Sriram

[DESCRIPTION]
This protocol describes the steps required for successful implantation of small cell neuroendocrine prostate cancer patient-derived xenograft (PDX) cells in the liver. Liver is one of the most common sites for 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 implantation of any tumor cell line in the liver. 

[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. 
3. Preparation of the PDX cells for implantation 
 

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: 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/10-20 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 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: Anesthetization and fur removal of mouse
6. 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
7. Note the start time for anesthesia.
SECTION: Anesthetization and fur removal of mouse
8. 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
9. Determine the anesthetic depth by pinching the animal’s foot for a reflex response. 
SECTION: Anesthetization and fur removal of mouse
10. Depilate the ventral side to remove fur from the abdomen. 

  

SECTION: Anesthetization and fur removal of mouse
12. The animal is now ready to be moved to station 2 for surgery. 
SECTION: Surgical procedure (Station 2)
13. 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. 
  

SECTION: Surgical procedure (Station 2)
14. Gently grab the peritoneum muscle layer and make an incision to expose the liver. 
SECTION: Surgical procedure (Station 2)
15. 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. 

  

SECTION: Surgical procedure (Station 2)
16. Transfer the cell suspension into a 1 cc syringe with a 27 G needle. 


 

 

SECTION: Surgical procedure (Station 2)
17. 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.

  

SECTION: Surgical procedure (Station 2)
18. 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. 
SECTION: Surgical procedure (Station 2)
19. 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. 
SECTION: Surgical procedure (Station 2)
20. Using absorbable sutures, suture the muscle layer and then suture the outer skin layer of the incision site. 

  

SECTION: Surgical procedure (Station 2)
21. Upon completion of the sutures administer buprenorphine and lidocaine subcutaneously. Note the time of administration. 
 

SECTION: Surgical procedure (Station 2)
22. Sterilize the surgical instruments using the bead sterilizer by placing them inside for ~ 10 secs. 
SECTION: Surgical procedure (Station 2)
23. Start prepping the next mouse for surgery.
SECTION: Post-Op care and monitoring
24. 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
25. Observe the animal until it has regained full consciousness and is walking around in the cage.
SECTION: Post-Op care and monitoring
26. Observe the animal on a daily basis until sacrificed as per your IACUC guidelines.
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: Anesthetization and fur removal of mouse
11. 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. 
SECTION: Preparation before surgery
2. Surgery record sheets