TITLE: Subrenal capsule (SRC) implantation of tumor tissue
AUTHORS: Shubhangi     Agarwal, donna.peehl  , Renuka   Sriram

[DESCRIPTION]
This protocol describes the steps required for the successful implantation of tumor tissues under the subrenal capsule. The subrenal capsule is an optimum site for the propagation of tumor tissue as it provides an environment rich with blood vessels and growth factors. 

[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. Fire bent glass pipette: Prepare the bent pipette by exposing the pipette tip to fire and slightly bending it into an inverted C shape with the help of forceps. The image below shows an example of bent pipettes. 

  
Note: Make sure there are no sharp edges on the pipette tip as that will cause a tear in the renal capsule. 

SECTION: Preparation before surgery
4. Preparation of the PDX tissues for implantation from fresh tissue


SECTION: Preparation before surgery
2. Preparation of surgical instruments and supplies: All of the instruments and supplies should be sterilized. 
SECTION: Preparation of surgical space
6. Station 1: Fur removal


   



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

  

SECTION: Preparation of surgical space
7.1. Place a disposable sterile pad on a heating pad. 
SECTION: Preparation of surgical space
7.2. Aseptically sterilize the surgical area by spraying with 70% ethanol. 
SECTION: Preparation of surgical space
7.3. Place all the autoclaved surgical instruments within the sterilized surgical area.
SECTION: Preparation of surgical space
8. 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
9. Weigh the animal and note the weight.  
SECTION: Anesthetization and fur removal of mouse
10. 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 of anesthesia.
SECTION: Anesthetization and fur removal of mouse
11. 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
12. Determine the anesthetic depth by pinching the animal’s foot for a reflex response. 
SECTION: Anesthetization and fur removal of mouse
13. Depilate the left dorsal side of the mouse starting from the rib cage till about 4 cm below. 
SECTION: Surgical procedure
14. Move the mouse to the station 2 nose cone and place it on its abdomen. 
SECTION: Surgical procedure
15. Disinfect the surgical area on the mouse with betadine scrub, 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: Surgical procedure
16. Make a vertical incision using the straight scissors through the skin along the animal’s top curve of the spine (i.e., ~ 0.5 cm below the rib cage) towards the left side of the spine
SECTION: Surgical procedure
17. Turn the animal so that is laying on its right side. 
SECTION: Surgical procedure
18. Place right thumb on the animal’s abdomen, applying very light pressure, and with the left hand pull the skin down, so that the kidney is in view. Once the kidney is located, make a 0.5 cm incision horizontally on the animal’s left side with surgical scissors. 
SECTION: Surgical procedure
19. With the aid of the thumbs apply a little pressure on the abdomen to expose the kidney and pop it out of the body cavity. Place the kidney such that it is held by the abdominal wall. 
SECTION: Surgical procedure
19.1. If the incision is made too large, then the kidney will not stay popped out. This incision is critical to the success of this procedure. Do not try to pull on the kidney with forceps as it will cause damage to the kidney and renal vessels

  

SECTION: Surgical procedure
20. Prevent the kidney from drying out by applying sterile saline using the 1 cc syringe. 
SECTION: Surgical procedure
21. Make a small tear with dissecting scissors in the caudal-lateral aspect of the kidney capsule. With your left hand, hold and gently pull up the capsule layer using forceps. With the right hand, place the insert the bent glass pipette under the kidney capsule layer and glide it from side to side in order to make space for the tissue slice. (Note: Be careful not to puncture the kidney or tear the capsule). 
SECTION: Surgical procedure
22. Use tweezers to place the tumor tissue under the kidney capsule. Advance the tissue slice towards the cranial pole, so that it stays in place and does not leak out once the kidney is placed back in the body cavity. 

  

SECTION: Surgical procedure
23. Return the kidney to the abdominal cavity by lifting the surrounding muscle layer with forceps and letting the kidney slide back into the body cavity. 
SECTION: Surgical procedure
24. Once the kidney is in the body cavity, suture the muscle layer, using absorbable sutures. Pull the skin layer upward and clamp the skin together using a sterile wound clipper, making sure that all of the skin is closed together, and no gaps are left. 
SECTION: Surgical procedure
25. Upon completion of the surgery administer buprenorphine and lidocaine subcutaneously in the right flank. Note the time of administration
 

SECTION: Surgical procedure
26. Sterilize the surgical instruments using the bead sterilizer by placing them inside for ~ 10 secs. 
SECTION: Surgical procedure
27. Start prepping the next mouse for surgery.
SECTION: Post-Op care and monitoring
28. 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
29. Observe the animal until it has regained full consciousness and is walking around in the cage.
SECTION: Post-Op care and monitoring
30. Remove the clips using the clip remover one week from the surgery
SECTION: Post-Op care and monitoring
31. Observe the animal on a daily basis until sacrificed
SECTION: Preparation before surgery
4.1. Prepare an ice bucket with ice and a petri dish with 10 mLHBS. 





SECTION: Preparation before surgery
4.2. Euthanize the tumor-bearing mouse and extract the tumor. 
SECTION: Preparation before surgery
4.3. Blot the tumor on a Kim-wipe to get rid of excess blood.

SECTION: Preparation before surgery
4.4. Weigh the tumor and note the weight. 
SECTION: Preparation before surgery
4.5. Using a dissecting scissor, cut the tumor tissue into small sections approximately 1 mm in diameter. 

SECTION: Preparation before surgery
4.6. Place the tissue sections in a petri dish containing 10 mL HBS. 

 

5. Preparation of the PDX tissues for implantation from frozen biobank
5.1. A cryovial containing frozen tumor sections/slices should be placed on ice to thaw before the surgery.
 

5.2. Transfer the contents of the cryovial into a petri dish containing fresh HBS. 
 

SECTION: Preparation before surgery
3. Surgery record sheets