This article discusses the preparation of decellularized whole-organ scaffolds, specifically focusing on the rat kidney. These scaffolds maintain tissue composition and functional integrity, which are crucial for organ bioengineering applications.
Decellularized whole-organ scaffolds consist of a three-dimensional extracellular matrix with an intact vascular network. These scaffolds retain specific tissue composition and functional integrity and play an essential role in organ bioengineering.
To prepare a decellularized rat kidney, first, obtain a freshly harvested rat kidney along with its major arterial and venous vasculature - the abdominal aorta and inferior vena cava. Tie off the ureter, minor arterial, and venous bifurcations to prevent liquid leakage during the subsequent perfusion process.
Place the kidney in a suitable buffer to ensure organ hydration. Insert appropriately sized catheters into the major vasculatures and secure them. Connect the catheter to a peristaltic pump. Perfuse the kidney with an anticoagulant-containing buffer at an appropriate flow rate and pressure for the desired duration.
This step helps to efficiently remove blood from the organ through the venous outflow. Change the perfusate to a non-ionic surfactant to permeabilize the cellular membranes. Thereafter, perfuse the organ with an anionic surfactant to effectively solubilize the cellular and nuclear membranes, resulting in cell lysis and the release of intracellular contents.
The liberated cellular components and debris flow out of the kidney, gradually rendering it translucent. Inject the decellularized kidney with a suitable buffer containing antibiotics and anticoagulants to remove residual surfactant and any remaining debris from the organ. Antibiotics prevent the likelihood of bacterial growth within the decellularized organ.
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 5 rpm and 37 degrees Celsius. To decellularize the kidney, perfuse it with 1% Triton X-100 for 3 hours and then with 0.75% SDS solution for 6 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.
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