The procedure is mainly composed of two parts. First, ingredient preperation methods were described. The second part is about how to generate murine orthotopic pancreatic cancer model.
1st part : Ingredient preperation.
Ketamin/Xylazine mixed solution (KX solution); KX solution was used to anesthetize mice for orthotopic tumor implantation surgery. KX solution is prepared by mixing PBS, Ketamine, and Xylazine with a ratio of 15:4:1.
Ex, for preparing 20mL of KX solution; Mix 15mL of PBS, 4mL of Ketamine Inj. (Ketamine hydrochloride solution made for injection, 50mg/mL as Ketamine and 57.68mg/mL as Ketamine hydrochloride, 유한양행) and 1mL of Xylazine (Xylazine Hydrochloride solution made for injection, 23.32mg/mL as Xylazine Hydrochloride, Elanco). Therefore, 20mL of KX solution contains 200mg of Ketamine (or 230.72mg of Ketamine hydrochloride) and 23.32 mg of Xylazine.
Pancreatic cancer cell solution; Gently resuspend 5x10^5 cells in 20-25 μL of fresh media/Matrigel (1:1) and transfer the solution into 29 guage insulin syringe.
Caution;
1. Cancer cell condition itself makes a huge difference in tumor growth in vivo, therefore researchers should keep it consistant. We recommended to change the culture media with fresh one 4-8 hrs before detaching the adherent cells from culture plate for the injection. 70% of cancer cell confluency would be a fine condition before the detachment.
2. Matrigel is a highly viscous solution and it hardens with irreversible properties when the temperature rises. Hence, it is recommended to keep the cancer cell solution at 4-celsius cold room if possible. If not, keep matrigel in ice and use pipette tips after some refrigeration. Pilot experiment for making fresh media/matrigel (1:1) solution is suggested.
3. When transferring the pancreatic cancer cell solution (Media/Matrigel, 1:1) to the 29 guage insulin syringe, cut the end of pipette tip evenly and transfer it with fine pipetting. We note that the amount of transferred cancer cells can vary greatly depending on the cutting part of tip and pipetting.
2nd part : Mouse orthotopic pancreatic tumor model generation.
1. Anesthetize the mouse using 10μL/g of KX solution via intraperitoneal injection using 29 guage insulin syringe.
2. About 10 minutes after anesthesia, make sure that the mouse is sufficiently anesthetized by applying pressure on the mouse toe with forceps.
3. Remove mouse hair under the ribs of the left flank using animal clipper.
4. Lay the mouse on Lt. decubitus position, pull the left leg and fix it with tape.
5. Spray 70% ethanol on the injection site and wipe with guaze.
6. Incise the muscle following by incision in the skin with a scissor.
7. Take out the pancreas with tissue forecep little by little from abdominal cavity of mouse and place on the gauze soaked in PBS.
8. Gently hold the pancreas with a tissue forcep and inject the cancer cell solution(Media/Matrigel, 1:1) with a syringe. A solution-filled region should be placed within the pancreatic parenchyma.
9. Slowly remove the syringe after injection.
10. After observing for 3 minutes to check hemorrhage or leage, carefully re-place the pancreas into the abdominal cavity of mouse using a tissue forcep without touching .
11. Sequentially close the abdominal muscle and the skin with 5-0 vicryl, and monitor the mouse until the anesthesia is awakened.