The overall goal of this procedure is to verify the effect of adipose-derived stem cell sheets combined with artificial skin on wound healing for a large full-thickness skin defect in diabetic rat. This method can help answer key questions about healing of chronic wound, such as diabetic large wound with exposed bone. The main advantage of this technique is that cells are transplanted to the wound in solid sheets using cell sheet technology, and the result is a viable, stable transplant.
For a fat pad donor, plan to use eight to 33 week old Lewis rats. At the surgical site, have ready several sterile cotton-tipped applicators, gauzes, a scalpel, a needle holder, operating scissors, a pair of forceps, and 5-0 nylon suture. For temporary tissue placement, load a 100 milliliter petri dish with 10 milliliters of PBS.
Weigh the PBS-loaded dish to later find the mass of the harvested tissue. After anesthetizing the rat, position it supine on a sterile drape. Then, shave the operative area and scrub the exposed skin using several alternating scrubs of 70%ethanol.
Now, make a three centimeter skin incision in the lateral lower abdomen. Next, expose and dissect the external oblique muscle. Then, expose the epididymal adipose tissue surrounding the epididymis.
Next, carefully excise the epididymal adipose tissue. Avoid damaging the epididymis, testes, and epididymal blood vessels. Transfer the tissue into the dish of PBS.
Then, close up the donor rat and follow standard postoperative care practices. Isolate the ASCs from two to three grams of harvested adipose tissue. Digest the tissue for an hour in 0.1%type A collagenase to obtain the stromal vascular fraction, or SVF.
After purifying the SVF, suspend the SVF in 20 milliliters of media. Split the suspension into two 60 square centimeter culture dishes, and culture them for 24 hours under standard conditions. The next day, wash the plates with PBS three times to remove any unattached cells and debris.
After the final wash, add back fresh complete medium. Then, continue to passage the cells until they are nearly sub confluent on the third day of culturing. Then, using one milliliter of 0.25%trypsin EDTA in a brief incubation.
Remove the cells from the plate. Do not let the cells be exposed to active trypsin more than 10 minutes. After observing and counting the cells, subculture them at a density of 1, 700 per square centimeter.
Then, pass the cells every three days, and complete three passages before proceeding. After passing the subcultured rat ASCs for the third time, plate 150, 000 cells onto a 35 millimeter diameter temperature-responsive culture dish, and two milliliters of culture medium. Culture the dish for three days in an incubator to make a uniform cell sheet.
Three days later, change the medium, and add 16.4 micrograms per milliliter of AA.Then culture the cells, changing the AA medium every other day. After four or five days on the AA-containing medium, check the proliferation of the ASCs. Look for gaps between the cells and the contiguous cell sheet.
Once a contiguous cell sheet is established, the cells can be used for the model. When needed, collect the cell sheet by placing the plate at room temperature for 15 to 20 minutes. Because the plate is temperature sensitive, the cells will spontaneously detach as a contiguous sheet, and the sheet can be removed with forceps.
For the obesity wound healing model, use adult male ZDF rats, 16 to 18 weeks old weighing between 500 and 600 grams. Position and anesthetize rat on the surgical field, and before shaving the head, clean it using 70%ethanol. Then, shave the operative area and clean the exposed skin with alternating scrubs of 70%ethanol.
Around this time, transfer the temperature sensitive plate of adipose stem cells to the bench. Before the cell sheet detaches from the plate, remove the medium and wash the cell sheet with PBS three times. After collecting the cell sheet, soak it in PBS until needed.
Once the cells are ready, make a 15 by 10 millimeter full-thickness skin defect on the rat's head. First, remove the cutaneous tissue down to the periosteum. Then, excise the skin an cutaneous tissue with a scalpel and a pair of surgical scissors.
To complete the defect, remove the periosteum with the periosteal raspatory. Then place the cell sheet over the defect. The stem cell sheet is soft and flexible.
Use forceps to extend it to every corner of the defect. Now, cover the cell sheet and defect with a 10 by 15 millimeter patch of artificial skin soaked in saline right before use, and close the wound with approximately 10 stitches using 5-0 nylon suture. To protect the wound, use 5-0 nylon suture to secure a 15 by 20 millimeter patch of non-adhesive dressing over the artificial skin.
Plan to observe the dressing every other day, because the rat will invariably remove it, and if needed, replace the dressing promptly. In two groups of six Zucker diabetic fatty rats, the described protocol was used to create a skull defect. Experimental rats were treated with a rat adipose stem cell sheet, and the controls were not.
After 14 days, the wounds of the treatment group were significantly smaller than those of the controls. After watching this video, you should have a good understanding of how to make large wound with exposed bone in rats, and then treat that wound with adipose derived stem cells. While attempting this procedure, be sure to use strict temperature management during the entire process of using temperature responsive culture dishes.
Otherwise, cells could spontaneously detach from the dishes, delaying your experiments.
