Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

This method describes the surgical procurement of three vascularized porcine flaps and their subsequent perfusion decellularization. This method can aid efforts to regenerate porcine flaps using a tissue decellularization and recellularization approach. The main advantage of this technique is its versatility to achieve profusion decellularization across a variety of vascularized porcine flaps in a simple to assemble bioreactor setup.

The technique described here can be broadly applied to other vascularized flaps as a potential tissue engineering solution using porcine flaps in reconstructive surgery. To begin, make an inflow and outflow tubing by measuring approximately 50 centimeters of LS16 platinum-coated silicone tubing. Connect one end to a yellow 3-stop pump tubing and the other to a sterile 2 milliliter serological pipette to carry perfusate from the detergent reservoirs into the bioreactor chamber.

Autoclave the chamber and tubings in individually wrapped sterile packaging. For omental flap procurement, place a 12-week old anesthetized Yorkshire pig in the supine position, and then prepare the abdomen in the usual sterile fashion. Perform a xipho-umbilical laparotomy to enter the abdomen and mobilize the stomach cephalad, and place the omentum into the operative field.

Starting at the left aspect of the greater curvature, where the left gastroepiploic artery joins the splenic artery, skeletonize the left gastroepiploic artery and vein using straight Stevens tenotomy scissors. Then ligate and divide both separately using surgical ties or clips. Proceed along the greater curvature of the stomach from left to right to ligate and divide the short vascular branches arising from the omentum, supplying the greater curvature of the stomach.

Continue the mobilization of the greater omentum until the junction of the right gastroepiploic vessels and gastroduodenal artery is encountered. Then ligate and divide the right gastroepiploic artery and vein to free the omental flap and allow for flap cannulation later. For tensor fascia lata flap procurement, place the pig in the lateral decubitus position.

Mark the interior flap boundary from the anterior superior iliac spine, or ASIS, towards the lateral patella, and a posterior boundary approximately 8 to 10 centimeters caudally. Use a scalpel to make a sharp incision at the distal margin at the patella. Deepen the incision through the subcutaneous tissues to reach the fascia overlying the rectus femoris, and continue the incisions towards the ASIS along the marked boundaries.

To isolate the fascial flap alone, remove the overlying skin component from the underlying fascia layer. Then ligate or cauterize small perforator vessels to the skin. Return to the distal border of the flap, and incise the deep fascia.

Mobilize the fascia off the underlying vastus lateralis muscle towards the ASIS. After tracing the pedicle toward the deep femoral vessels, skeletonize the lateral circumflex femoral artery and vein supplying the fascial flap. Then ligate and divide flap vessels proximally, where they join the deep femoral vessels and allow for later cannulation.

For radial forearm flap procurement, mark a 3 by 3 centimeter square on the radial aspect of an extended pig forearm. Draw a line between the midpoint of the proximal flap margin and the antecubital fossa to denote the approximate course of the radial artery pedicle. Confirm the arterial course with palpation.

Next, incise the skin using a scalpel at the distal aspect with a depth up to the antebrachial fascia. Blunt dissect with tenotomy scissors until the distal radial artery and its vena comitantes are encountered. Then ligate and divide the artery and veins with surgical ties or clips.

Continue the skin incisions on the radial and ulnar margins of the marked skin square. Then mobilize the flap off the underlying radial bone with a surgical blade or cautery, proceeding from an ulnar to radial direction while raising the skin flap proximally toward the antecubital fossa. Using tenotomy scissors, skeletonize the radial artery and vena comitantes proximally at the antecubital fossa, where they join the brachial artery and veins, respectively.

Dissect the vessels from the surrounding fibro-fatty tissues to isolate the vascular pedicle. Then ligate and divide the artery and vein separately to free the radial forearm flap. Next, cannulate the pedicle vessels with 20 to 24-gauge angiocatheter secured with 3-0 silk ties.

Flush heparinized saline into the flap arterial cannula until a clear venous outflow is observed. While working in a laminar flow Biosafety cabinet, place flaps in the bioreactor tissue chamber. Prime inflow tubing with sterile heparinized saline to remove residual air and arrange flaps to allow connection between the flap arterial cannula and the male luer connector.

Then use sterile hemostats to screw the arterial cannula onto the connector. Then flush the heparinized saline through the stopcock to check that the luer connection is without leaks, and that the flops can be perfused with evidence of venous outflow. After closing the tissue chamber lid, connect the inflow tubing with a yellow 3-stop pump tubing to the inflow stopcock of the tissue chamber.

Also, attach an inline pressure sensor transducer to the inflow 3-way stopcock for perfusion pressure monitoring. Next, turn on the inflow peristaltic pump and on the initial screen, move to the third tab using the arrow to set the tubing ID to 1.85 millimeters. Then set the perfusion rate from the second tab.

The input rate as the mode of delivery is set to 2 milliliters per minute. Ensure the flow direction is correct as displayed on the screen. Load the 3-stop pump tubing to the peristaltic pump with a compatible cassette.

Set the outflow pump setting to a rate at 4 milliliters per minute. Connect the outflow tubing with a yellow 3-stop pump tubing to the outflow stopcock of the tissue chamber. Then load the 3-stop pump tubing to the peristaltic pump, and start the flow for both pumps by pressing the power button on each pump.

Begin the perfusion decellularization of flaps with heparinized saline in the tissue chamber for 15 minutes to remove any retained blood clots. Upon completion, replace the residual saline with 500 milliliters of sodium dodecyl sulfate, or SDS, decellularization solution to submerge the flap. Next, transfer the inflow tubing to the SDS solution and profuse the tissue.

After SDS decellularization, aspirate the remaining SDS before perfusing the flap with PBS for one day. After SDS perfusion decellularization, sequentially perfuse the flaps in DNA solution for two hours, and then in PBS for one day, followed by transferring the inflow tubing into peracetic acid and ethanol in distilled water to sterilize the flaps by perfusing for three hours. Once done, remove the flaps aseptically and immerse them into two washes of PBS containing 1%antibiotics antimycotics for 15 minutes each.

Store flaps at 4 degrees Celsius until ready for recellularization. Use a 3 to 10 millimeter punch biopsy tool to obtain tissue samples for evaluation of decellularized flaps. The isolated decellularized flaps flushed under manual control demonstrated evidence of outflow from the freely draining venous cannula of omentum, tensor fascia lata, and radial forearm flap.

The gross morphology of the native omentum, tensor fascia lata, and radial forearm flaps appeared pink colored immediately after the procurement. In comparison, decellularized tissues were characteristically white or opaque in appearance. Histological examination of native tissues with hematoxylin and eosin showed the presence of blue nuclei.

In decellularized flaps, histological staining revealed loss of cellular material without blue nuclear staining, indicating an acellular tissue scaffold. Additional quantification of DNA content showed a significant decrease in DNA in acellular scaffolds compared to native tissues. The most important consideration is a surgical technique used during procurement, taking care to avoid damage to the flap pedicle in order to permit subsequent perfusion decellularization.

Following perfusion decellularization, the resulting flaps may be recellularized with tissue specific cell populations in order to regenerate the native tissue compartments.