Nov 30, 2021
Intrahepatic implantation of tumor cells
- Shubhangi Agarwal1,
- Robert Bok1,
- donna Peehl1,
- Renuka Sriram1
- 1University of California, San Francisco
Protocol Citation: Shubhangi Agarwal, Robert Bok, donna Peehl, Renuka Sriram 2021. Intrahepatic implantation of tumor cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bvrtn56n
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: 50707
Keywords: Intrahepatic, tumor implantation,
Abstract
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.
Materials
| Materials | Source | Catalog Number | |
| Surgical Instruments | Kent Scientific | INMOUSEKIT | |
| Sterile cotton tipped applicators | Puritan | 25-806 10WC | |
| Povidone-Iodine Prep Pads | MEDLINE | MDS093918 | |
| Phosphate buffered saline | Gibco | 14040117 | |
| Petri dishes | Millipore Sigma | P5481 | |
| Pasteur pipette | Corning | 7095D-9 | |
| 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 | |
| Disposable scalpels | Exel international | 29550 | |
| Buprenorphine | |||
| Alcohol Prep Pads | WEBCOL | 6818 | |
| Absorbable sutures | CP Medical | 421A – VISORB® 5/0 FS-2 30″ | |
| 70% Ethanol | Carolina | 861261 | |
| 1 cc syringe | BD | 309628 | |
| Isoflurane | Piramal Critical Care(RxElite) | 66794001725 | |
| 27 G Needle | BD | BD-305109 | |
| Gentamicin | Gibco | 15750060 | |
| DMEM | Gibco | 11965092 | |
| Fetal bovine serum (FBS) | Global Life Sciences Solutions | SH3039603 |
quipment
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 | Hepatic Tumor 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/10-20 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 syringe right before beginning the surgery.
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 and 4) buprenorphine, 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.
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
Heat: Animal should be kept on a heating pad or circulating water blanket/pad during the entire procedure and, until the animal has fully recovered from anesthesia/is mobile
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 the ventral side to remove fur from the abdomen.
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.
The animal is now ready to be moved to station 2 for surgery.
Surgical procedure (Station 2)
Surgical procedure (Station 2)
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.
Gently grab the peritoneum muscle layer and make an incision to expose the liver.
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.
Transfer the cell suspension into a 1 cc syringe with a 27 G needle.
Note
Do not leave the liver exposed for too long. This step should be completed in less than 30 secs. Alternatively, cells can be transferred into the syringe before step 13.
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 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.
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.
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.
Using absorbable sutures, suture the muscle layer and then suture the outer skin layer of the incision site.
Upon completion of the sutures administer buprenorphine and lidocaine subcutaneously. Note the time of administration.
Note
The concentration of lidocaine and buprenorphine should be kept as mentioned in the institute's IACUC protocol. For this protocol, 0.5% v/v solution of lidocaine was administered.
Sterilize the surgical instruments using the bead sterilizer by placing them inside for ~ 10 secs.
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 as per your IACUC guidelines.