This protocol makes it possible to develop vascularized scaffolds using kidneys in a rat model. The kidney is the most suitable organ for scaffolds of differentiated stem cells. This is a highly successful protocol for decellularization of the kidney.
It effectively removes the nuclei with minimal destruction of the ECM ultrastructure. This technique can be applied to transplantation medicine and tissue engineering. Demonstrating the procedure will be Kim Joo Hyoung, an MD/PhD associate professor of plastic surgery from my laboratory.
To begin, place the eight-week-old Sprague Dawley rat on a warming pad, and position a rectal thermometer probe in the rectum to monitor core temperature. After anesthetizing the rat, place it in a supine position, and secure the four limbs to the table with tape. Shave and clean the abdomen with germicidal soap.
Apply 2%Betadine for at least one to two minutes, then wipe it off with a 70%ethanol solution. Repeat this sequence three times. Cover the operative field with a sterile, fenestrated drape.
Make a vertical abdominal incision, and expose the left kidney, ureter, abdominal aorta, and inferior vena cava. Dissect the tissue just before cutting the pedicle. Prepare 50 milliliters of perfusion solution per rat by mixing PBS with approximately 10 units per milliliter heparin.
Mix equal volumes of 8%PFA with PBS to make the 4%PFA solution. Extend the vertical abdominal incision cranially, making sure to draw the scissors away from the organs when cutting to avoid damage. Continue the incision through the rib cage, and then cut through the diaphragm by lifting the sternum.
Retract the loose flap of skin out of the way with an Allis clamp, and free the heart by tearing any connective tissue with the forceps. Open the PBS line, and ensure that it is flowing before placing the needle into the left ventricle. Hold the heart gently with blunt forceps, and use a hemostat to control the needle.
The needle should be inserted no more than a quarter inch. While supporting the heart with the needle and hemostat, locate the right atrium, and snip through it with iridectomy scissors. Rest the hemostat on the rat's body, and make sure the needle is still positioned inside the heart.
Continue perfusing PBS for four minutes or longer if there is still blood visible in the kidney and liver. Harvest the left kidney with the abdominal aorta and inferior vena cava. Ligate the ureter, thoracic aorta, superior vena cava, and branches of the abdominal aorta.
Keep the organ hydrated in DPBS in a 10-centimeter Petri dish. Cannulate the abdominal aorta and inferior vena cava with a 23-gauge catheter. To remove residual blood, connect the cannula with a peristaltic pump, and wash the organ with 500 milliliters of DPBS and 16 units per milliliter heparin for 90 minutes at five rpm and 37 degrees Celsius.
To decellularize the kidney, perfuse it with 1%Triton X-100 for three hours and then with 0.75%SDS solution for six hours at a constant pressure of 40 milliliters of mercury. To remove residual SDS, perfuse the sample with 1%penicillin in distilled water for 18 hours and then with sterile DPBS and 16 units per milliliter heparin for 90 minutes. The gross morphology of rat kidneys was dark red.
After decellularization, the kidney became pale and translucent. Residual genomic DNA was quantified in decellularized kidney scaffolds and compared with that in native kidneys, confirming that tissue genomic DNA was almost eliminated after decellularization. From 14 cases, the average DNA contents were 115.05 nanograms per microliter for the control and 1.96 nanograms per microliter for the decellularized scaffold.
In total, 98.3%of DNA was removed. The three-dimensional structure was maintained, and acellular glomeruli were preserved in the cortical parenchyma. When inserting the needle into the left ventricle, it should be inserted no more than a quarter inch.
Any further insertion may come out the other side. We have confidence that this technique will open a new prospect in the field of tissue engineering.
