Nov 30, 2021
Intratibial implantation of tumor cells
- 1University of California, San Francisco
Protocol Citation: Shubhangi Agarwal, donna Peehl, Renuka Sriram 2021. Intratibial implantation of tumor cells . protocols.io https://dx.doi.org/10.17504/protocols.io.bvrsn56e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 11, 2021
Last Modified: November 30, 2021
Protocol Integer ID: 50706
Keywords: Intratibial, tumor implantation
Abstract
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.
Materials
| Material | Source | Catalog Number | |
| Povidone-Iodine Prep Pads | MEDLINE | MDS093918 | |
| Phosphate buffered saline | Gibco | 14040117 | |
| Ophthalmic ointment | Akorn | 59399-162-35 | |
| Lidocaine Hydrochloride 2% w/v Solution | ADVANZ Pharma | N01BB02 | |
| Buprenorphine | Covetrus | 059122 | |
| Ice bucket | Corning | 1167U68 | |
| Disposable sterile pads | Medline | MSC281224 | |
| Alcohol Prep Pads | WEBCOL | 6818 | |
| 70% Ethanol | Carolina | 861261 | |
| 1 cc syringe | BD | 309628 | |
| Isoflurane | Piramal Critical Care(RxElite) | 66794001725 | |
| BD Insulin Syringes 31 G, 0.3 ml | BD | BD-25150 | |
| Gentamicin | Gibco | 15750060 | |
| DMEM | Gibco | 11965092 | |
| Fetal bovine serum (FBS) | Global Life Sciences Solutions | SH3039603 |
Equipment
1. Isoflurane small animal anesthesia machine
Small animal anesthesia system
2. Water circulating system
3. Heating pad/Circulating water blanket/pad
4. Weighing Scale
5. Bead Sterilizer
9. Nair or any hair removal product/instrument
Preparation before surgery
Preparation before surgery
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 |
Note
This is a template for surgery records and every user should use the template according to their institute's IACUC regulations.
Preparation of the PDX cells for implantation
Note
Preparation of fresh PDX cells for implantation
Note: Follow the steps till step number 24 from the following protocol to prepare single-cell digestion from fresh tumor tissue: (dx.doi.org/10.17504/protocols.io.bvrun56w)
Preparation of cells for implantation from frozen biobank
Preparation of cells for implantation from frozen biobank
Prepare DMEM medium: Prepare fresh DMEM media supplemented with
10 % volume FBS and
100 ug/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 (9 mL DMEM per 1 mL 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 300 x g , 5 mins 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.
Note
Keep the Eppendorf tube on ice for the remainder of the procedure.
Transfer the cell suspension into a 31G 0.3 ml syringe right before beginning the surgery.
Note
Keep the syringe on the ice at all times.
Preparation of surgical space
Preparation of surgical space
Station 1: Fur removal
Station 1, Fur removal station with anesthesia tubing and nose cone, clippers for hair removal, sterile cotton tips for removal of left-over fur and kimwipe for cleaning.
Place a disposable sterile pad on a heating pad.
Aseptically sterilize the surgical area by spraying with 70% ethanol.
Station 2: Surgery
Station 2, Surgery station with 1) sterilized surgery instruments, 2) sterile dissolvable sutures, 3) lidocaine, 4) buprenorhpine 5) 70% ethanol prep pad, 6) povidone-Iodine prep pads, 7) disposable sterile pad, 8) eye ointment, 9) sterile cotton tipped applicators, 10) ice box with ice, 11) syringe on ice for intrahepatic injection of cells, 12) eppendorf tube containing cell suspension, 13) weighing scale, 14) kimwipes, 15) bead sterilizer and 16) nose cone and bain tubing.
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.
Anesthetization and fur removal of mouse
Anesthetization and fur removal of mouse
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.
Note
The concentration of buprenorphine should be kept as mentioned in the institute's IACUC protocol.
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.
Surgical procedure (Station 2)
Surgical procedure (Station 2)
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
Note
Alternatively, cells can be transferred into the syringe before step 14. Make sure to mix the cells in the syringe before injecting them into the mouse.
Note
When the cells are in the syringe, especially if you are injecting multiple mice, you must make sure the cells are resuspended and not settled to the bottom of the syringe each time you inject to inject the correct number of cells per mouse.
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.
CITATION
Note
The pre-loaded syringe with cells might get blocked due to cells being lodged into the needle, making it impossible to inject the cells. One way to avoid it would be to use an empty needle to make the insertion and then use the pre-loaded syringe to inject the cells using the same path. Relocating the same path might be difficult.
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
Note
The concentration of lidocaine should be kept as mentioned in the institute's IACUC protocol. For this protocol, 0.5% v/v solution of lidocaine was administered.
Start prepping the next mouse for surgery.
Post-Op care and monitoring
Post-Op care and monitoring
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.
Citations
Step 18
Campbell JP, Merkel AR, Masood-Campbell SK, Elefteriou F, Sterling JA. Models of bone metastasis.
https://doi.org/10.3791/4260