Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We have established a small animal model for decellularization of a limb. This is useful for facilitating proof of concept research on de and recellularization of vascularized composite tissues. Immunosuppression regimens are one of the main limitations in vascularized composite allotransplantation.

Tissue immunogenicity can be reduced using decellularization. To begin, make a skin incision using a number 10 surgical blade along the inguinal ligament, moving from a lateral to a medial direction, while holding the surrounding skin with Adson Forceps. When the underlying fat is exposed, use blunt dissection to carefully dissect through the fat, and locate the superior epigastric vessels.

Using micro scissors, dissect proximally, and expose the underlying femoral nerve, artery, and vein at the inguinal ligament level. Under a dissection microscope, identify the femoral vessels, and dissect the artery and vein proximally using fine forceps to obtain sufficient length from the bifurcation points of the arterial network. Then, ligate the femoral vein and artery separately using 6-0 sutures.

Next, conduct circumferential dissection around the remainder of the hindlimb without disrupting the ligated femoral vessels, and transect the femoral bone mid-length using a bone cutter. To fully isolate the hindlimb, transect the ligated femoral vessels distal to the ligatures using micro scissors, and cannulate the femoral artery using a 24 gauge angiocatheter under the dissection microscope. Then, flush with heparinized saline until clear outflow is observed from the femoral vein.

Secure the cannula by tying one suture around the cannulated vessel in another suture distally around the cannula itself, ensuring that the cannula is placed proximally to prevent blocking the bifurcation points. Afterward, submerge the procured hindlimbs and PBS until decellularization. To construct the bioreactor, place the sterilized chamber, and screw on three single use three-way stopcocks at the inlet, outlet, and replenishing lines, ensuring that the stopcock for the replenishing port is capped at its remaining two ports to prevent leakage.

Attach the previously made silicone tubes to the stopcocks at the inlet and outlet lines. Then, connect peristaltic tubing to the silicone tubes. After securing the cassette on the peristaltic tubing, place it on the peristaltic pump.

Next, connect one of the silicone tubes to the end of the peristaltic tubing of the outlet line from the step above. On the other end, connect a one milliliter serological pipette, and resuspend the end attached with a serological pipette in the waste reservoir flask. Next, suspend the end of the tube attached to the serological pipette into the detergent reservoir, and immediately sealed the detergent reservoir flask's opening with parafilm.

Add 0.25%SDS from the one liter glass jar into the bioreactor chamber at the halfway level. Then, take the procured hindlimb using Adson Forceps, and suspend it into the bioreactor chamber carefully. Using two pairs of Adson Forceps, guide the cannulated portion of the hindlimb to the inlet line while holding the cannula with one pair of forceps.

Use the other pair of forceps to twist and secure the inlet line to the cannula. Once secured, add more 0.25%SDS from the one liter glass jar to fully submerge the limb as needed, ensuring that the outlet port is also submerged in the bioreactor reservoir, and consistent outflow is maintained. Then, attach a single-use syringe filter to the ventilation port on the lid of the bioreactor chamber, and secure the lid on the bioreactor, ensuring that the chamber is sealed from all sides.

To remove air from the tubing and prime the perfusion circuit, use a new single use 10 milliliter syringe to draw detergent from the detergent reservoir using a three-way stopcock at the inlet line. Once drawn, use the same fluid to insert into the three-way stopcock at the outlet line. Next, press down and secure the cassettes with the tubing into the peristaltic pump.

Then, turn the peristaltic pump on using the power button. On the peristaltic pump screen, proceed to the second tab using the arrow key to set the perfusion rate for the first channel with the input flow rate as the mode of delivery, and set the perfusion rate at one milliliter per minute, ensuring the direction of flow is correct according to apparatus setup. Calibrate the peristaltic pump to ensure the amount of fluid delivered through the inlet line, and/or taken from the outlet line flows consistently between the two, ensuring that the tubing ID is set to 1.85 millimeters.

Begin decellularization via machine perfusion at one milliliter per minute for both the inlet and outlet lines by pressing the power button on the keypad. Monitor and ensure that the flow is and ongoing at both the inlet and outlet lines. Using 1 liter of 0.25%STS, replenish the bioreactor reservoir through the replenishing port as needed.

Then, look for a white translucent appearance of the tissue, which will appear by day five, indicating decellularization of the rat hindlimbs. Following the confirmation of decellularization, replace the detergent reservoir with the PBS and 1%Antibiotic-Antimycotic reservoir, and seal the opening of the flask with parafilm. Begin PBS and 1%Antibiotic-Antimycotic perfusion at one milliliter per minute, and continue for two days.

Replace the PBS and 1%Antibiotic-Antimycotic reservoir with 200 milliliters of 0.1%parasitic acid, 4%ethanol reservoir, and begin perfusion at one milliliter per minute for two hours. Under a biosafety cabinet, disconnect the hindlimb from the inlet line using two pairs of sterile Adson Forceps with one pair of forceps to twist the inlet line, and the other holding the cannula, ensuring that the cannula is not pulled to prevent decannulation. Store the limb in a 500 milliliter glass jar containing PBS, and 1%Antibiotic-Antimycotic at 4 degree Celsius until further use.

Both decellularized femoral artery and vein showed loss of nuclear content across all layers and surrounding connective tissue, given the lack of blue stained nuclei otherwise present in the native vessels. The tunica intima media and adventitia of both the femoral vein and arteries were maintained in the decellularized vessels. The femoral nerve showed preservation of tissue structure, including the endoneurium.

The bone showed overall structural retention with loss of stained nuclei of osteocytes, and from surrounding endosteum and periosteum layers post-decellularization. The skin showed a loss of cells from the epidermis and dermis. The dermis showed retained collagen fibers similar to native skin tissue.

Lastly, the transverse view of skeletal muscle showed loss of nuclei otherwise located in the peripheries of the endomysium. The myofibril content remained retained within respective fascicles post-decellularization. DNA quantification using PicoGreen was also performed, where DNA content was significantly reduced across the femoral vessels, nerve, skin, muscle, and bone.

Following this protocol, the decellularized limb can be further characterized. This involves examining extracellular matrix using both histological and biochemical methods, and by assessing vascular in using imaging. The decellularized stem can also be recellularized using tissue-specific cells.