Hello, I'm Dr.Alaa Alshareeda from King Abdullah International Medical Research Center at the Department of Stem Cells and Regenerative Medicine. Today we will be demonstrating the effect of mesenchymal stem cells on the development of hepatocellular carcinoma on rat model by using cell sheet technology. Hello, I am Abdullah Almubarak.
I'm working in Experimental Surgery and Animal Lab King Saud University. We use the nude rat to avoid any rejections after transplanting the cell sheet. This diagram shows cell sheet transplantation procedure on nude rats.
Eight to 12 weeks old nude rats will be use in this study. First, nick down the rats and make a seven to ten centimeter cut between the skin and the underlying muscles by using scalpel in order to expose the liver. To transfer the cell sheet, use a sterile membrane.
Place the membrane over cell sheet to collect it. Then, place the cell sheet with the membrane over one lobe of the liver. Cell sheet construction.
Before seeding the cells, coat 3.5 centimeter temperature-responsive UpCell culture dishes with undiluted FBS. Coating dishes with FBS supports the growth of the cells in the membrane layer and prevents the degradation of cells. Ensure to cover all the surface of the dishes.
Incubate the dishes for 24 hours at 37 Celsius in a humidified atmosphere containing 5%carbon dioxide and 95%air. On the next day remove the excess FBS and allow the dishes to dry in room temperature for at least one hour. Seed at least one million of HepG2 cancer cells alone or co-cultured with mesenchymal stem cells into DMEM culture medium.
The medium supplemented with 10%FBS and 1%penicillin-streptomycin. The ratio used of cancer cells to mesenchymal stem cells in this study is 4:1. After adding the cells gently shake the dishes.
Incubate the cells at 37 Celsius in a humidified atmosphere containing 5%carbon dioxide and 95%air. Detachment of the cell sheet. At 100%cell confluence take the dishes out of the 37 Celsius incubator.
Now, incubate the HepG2 cancer cell sheet for one hour at room temperature, while incubate the HepG2 co-cultured with mesenchymal stem cell for 30 to 40 minute at 20 Celsius in a humidified atmosphere containing 5%carbon dioxide at 95%air. During the incubation time for the cell sheet detachment start the animal procedure. All studies on animal were approved by King Saud University ethical committee.
Performing the surgical procedure. Check the depth of anesthesia by testing the pedal withdrawal reflex. Disinfect the surgery area with iodine.
Now, apply a second scrub with iodine. Cover the animal with a sterilized drape to avoid contamination of the incision. Using a sterilized scalpel make a cut between the skin and underlying muscles, about seven to ten centimeter in size to expose the liver.
Separate the abdominal muscle from the skin with the flat back edge of the scalpel. Carefully stab the linea alba using a scalpel and extend the cut with a sharp scissor. Cell sheet transplantation on rat's liver.
Gently tap the culture dish over the bench to detach the sheet from its surface. Separate the cell sheet from the dish's surface by scraping the sheet edges in a half circle. Now, gently remove all the culture medium from the dish.
Ensure the sheet is intact without any damage, otherwise, it cannot be used. Prepare a sterile membrane to harvest and transfer the cell sheet. So first, soak the membrane with normal saline, then remove the excess saline with a sterile cotton bud.
Place the wet membrane over the cell sheet, while avoiding any air bubbles between the membrane and the cell sheet. Add few drops of normal saline over the membrane and the cell sheet to collect the material. Leave the membrane for a few seconds to ensure that the cell sheet is properly attached on the membrane, then collect it.
Now, prepare the liver for transplantation. Make sure the liver surface is dry. Place the membrane with the cell sheet over a single lobe of the rat's liver.
Add few drops of sterilized saline to detach the cell sheet from the membrane. Wait for five minutes before carefully removing the membrane. The cell sheet can be seen attached on the liver in this video.
Finally, close the underlying muscles and skin incisions with five nylon suture. After closing the incision, disinfect the surgery area with betadine. The time from anesthetic off to head up is variable, but is usually somewhere between five and 20 minutes Results.
This figure shows the developed tumor on rat's liver after one month of HepG2 cell sheet transplantation. Here, the tumor was developed after the transplantation of cell sheet fabricated HepG2 cancer cells and bone marrow mesenchymal stem cell. Whereas this one shows, the smallest developed tumor on rat's liver after transplantation of cell sheet fabricated from HepG2 cancer cells and umbilical chord mesenchymal stem cell, This figure shows H and E staining of rat's liver after one month of cell sheet transplantation.
Where A represent normal rat liver cells, and B shows cancer rat liver cells.Conclusion. So after watching this video you should be able to develop hepatocellular carcinoma model on nude rat by using cell sheet technology. Thank you for watching.
