The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.
Because they assume positive effect on tissue healing ASC sheets have been tested in many disease model, such as, myocardial infarction. This experiment show that ASC sheets can be used intra-abdominally and apply to intestine. The main advantage of ASC sheets is that no synthetic materials are needed.
In addition, ASC sheet transplantation is relatively easy to perform, with only a small amount of practice. This novel application of ASC sheet transplantation was developed to prevent leakage after bowel resection, and offers a first step to which a new therapeutic strategy. ASC sheets applied to intestinal anastomosis have been shown to be promising in preventing leakage.
ASC sheets may also be applied to other intra-abdominal organs, such as, the urinary tract. Begin by dissecting human subcutaneous abdominal adipose tissue into 0.5 cm pieces, with a size 10 sterile surgical blade, Adson-Brown tissue forceps and Metzenbaum scissors. Weigh the total amount of dissected tissue in a sterile glass bottle.
Add 30 ml of isolation medium per bottle. And digest the dissected adipose tissue in a shaker incubator at 150 rpm and 37 degrees Celsius for one hour. After an hour divide the digested tissue solution into 50 ml tubes, and centrifuge the tubes for 10 minutes at 390 G.Following the centrifugation remove the supernatant.
Add medium to resuspend the cell pellets, and add more culture medium, up to 45 ml. Centrifuge the combined cells for five minutes at 390 G and remove the supernatant. Resuspend the cell pellet with 10 ml of culture medium.
And filter the cell suspension through a 100 micron filter. Add 50 l of 3%acidic acid methylene blue solution, to 50 l of cell suspension, to lice red blood cells. After mixing the solution, pipette 10 l onto a hemocytometer, and count the cells.
Plate the cells at a density of 40000 cells per square cm, in a T175 flask containing culture medium. And incubate the cells at 37 degrees Celsius in a humid atmosphere with 5%CO2 for 24 hours. The next day, wash the cells with warm phosphate buffered saline to remove cell debris.
Then, replace the medium with 10%FBS culture medium, plus freshly added ascorbic acid-2-phosphate and human recombinant fibroblast growth factor two. Incubate until the cells reach 90%confluence. Subculture the adipose stem cells at 90%confluence, using standard 0.25%Trypsin-EDTA solution.
After three to five minutes neutralize the 0.25%Trypsin-EDTA solution with 10 ml of culture medium. Transfer to a centrifuge tube, and centrifuge the cells for eight minutes at 250 G.Remove the supernatant, and resuspend the cells in 1 ml of culture medium. After counting the cells with a hemocytometer, plate the cells in T175 culture flasks, at a density of 8000 cells per square cm.
Freeze the remaining ASCs in liquid nitrogen, with 10%dimethyl sulfoxide in culture medium before further use. Precoat 3.5 cm diameter thermo-responsive culture dishes, with 1 ml of fetal bovine serum. Then place the dishes in an incubator at 37 degrees Celsius, for at least 30 minutes before seeding.
To assist ASC adhesion to the thermo-responsive dish, and reduce the risk of premature ASC sheet detachment in culture, the thermo-responsive dish should be precoat with fetal bovine serum before cell seeding. Trypsinize ASCs as before, if there are any cell clumps after trypsinization, filter the cells through a 100 micron filter before counting. Remove the dishes from the incubator, and transfer to a warming plate at 37 degrees Celsius.
Remove FBS from the dish. After resuspending the cell pellet in LG DMEM, with 10%FBS, and counting the cells, dilute 3.52 times 10 to the 6th ASCs in 2 ml of culture medium per dish, to seed 400000 cells per square cm in each 3.5 cm dish. Carefully distribute the cells as evenly as possible, by gently swinging the dish.
Then, culture ASC sheets for 48 hours at 37 degrees Celsius in a humid atmosphere with 5%CO2. On the day of transplantation, allow the culture dishes to cool to room temperature 40 minutes before in vivo transplantation to facilitate ASC sheet detachment. Once cooled, remove the culture medium, and replace it with 1 ml of serum-free LG DMEM.
To transplant the cell sheet gently grab the edge of ASC sheet with atraumatic forceps and place the dish side of the ASC sheet on top of the anastomotic line. Gentle sheet handling and correct sheet placement are essential to ensure successful intact sheet transplantation. Carefully stretch out the ASC sheet to approximately 0.25 cm above and below the anastomotic line.
Wrap the sheet around the colon, lifting the colon, to wrap the ASC sheet around the dorsal side. Control animals, that did not receive transplanted ASC sheets, exhibit fecal leakage near the anastomotic line, indicated by the white arrow at post-operative day three. Those animals with transplanted ASC sheets, indicated by the black arrow, showed reduced fecal leakage and abscess formation at post-operative day three.
The same result was observed seven days after surgery. This image shows a representative cross-section of the colorectal anastomosis site in a transplanted animal stained with H and E.The sheet structure could be identified at the anastomosis site, up to seven days post-operatively. Once mastered, this technique can be done in a few minutes if it is performed properly.
ASC sheet will be spontaneously adherent to the zero cell surface of the colon, there is no need for using glue or suture to fit the ASC sheet. After watching this video you should have a good understanding of how to prepare and transplant ASC sheets onto colorectal anastomosis in rats, for preventing leakage after partial colectomy.
