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

Summary

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Abstract

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Introduction

VCA is an emerging option for patients requiring complex surgical reconstruction. Traumatic injuries or tumor resections result in volumetric tissue loss that can be difficult to reconstruct. VCA offers the transplantation of multiple tissues such as the skin, bone, muscle, nerves, and vessels as a composite graft from a donor to a recipient¹. Despite its promising nature, VCA is limited due to long-term immunosuppressive regimens. Lifelong use of such drugs results in increased risk for opportunistic infections, malignancies, and end-organ toxicity^1,2,3. To help reduce and/or eliminate the need for immunosuppression, tissue-engineered scaffolds using decellularization approaches for VCA show great promise.

Tissue decellularization entails retaining the extracellular matrix structure while removing the cellular and nuclear contents. This decellularized scaffold can be repopulated with patient-specific cells⁴. However, preserving the ECM network of composite tissues is an added challenge. This is due to the presence of multiple tissue types with varying tissue densities, architectures, and anatomic locations within a scaffold. The present protocol offers a surgical technique and a decellularization method for a rat hindlimb. This is a proof-of-concept model for applying this tissue engineering technique to composite tissues. This can also prompt subsequent efforts to regenerate composite tissues through recellularization.

Protocol

Cadaveric male Lewis rats (300-430 g) obtained from the Toronto General Hospital Research Institute were used for all experiments. For all surgical procedures, sterile instruments and supplies were used to maintain aseptic technique (see the Table of Materials). All procedures were performed in compliance with guidelines from the Animal Care Committee at Toronto General Hospital Research Institute, University Health Network (Toronto, ON, Canada). A total of four hindlimbs were decellularized.

1. Presurgical preparation

1. Prepare 50 mL of 5% heparinized saline. From a 50 mL saline bag, take out 2.5 mL of saline solution using a 5 mL syringe and discard. Using a 5 mL syringe, add 2.5 mL of heparin to the saline bag. Invert the saline bag to mix its contents.
2. Place a cadaveric rat in a supine position under a blue pad. Shave the hindlimb and groin area circumferentially using an electric shaver and remove hair.
3. Bring the rat to the surgical station and apply Povidone iodine scrub solution using a gauze to the hindlimb and groin area. Subsequently, apply 70% isopropyl alcohol to wipe off the scrub solution using gauze.
4. Discard the blue pad from step 1.2 and change into new, sterile gloves.

2. Procurement of rat hindlimb

1. Make a skin incision using a #10 surgical blade and a #3 blade runner along the inguinal ligament level, moving from a lateral to a medial direction (Figure 1A). Use Adson forceps to hold the surrounding skin to ensure a smooth incision.
2. When the underlying fat is exposed, use blunt dissection to carefully dissect through the fat. Locate the superior epigastric vessels.
3. Use microscissors to dissect proximally and expose the underlying femoral nerve, artery, and vein at the inguinal ligament level.
4. 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 (Figure 1B).
5. Ligate the femoral artery and vein separately using 6-0 sutures.
6. Conduct circumferential dissection around the remainder of the hindlimb without disrupting the ligated femoral vessels.
7. Transect the femoral bone mid-length using a bone cutter.
8. To fully isolate the hindlimb, transect the ligated femoral vessels below the ligatures using microscissors.
9. Cannulate the femoral artery using a 24 G angiocatheter under the dissection microscope. Use fine forceps to insert the cannula carefully. Flush with heparinized saline until clear outflow is observed from the femoral vein.
10. Secure the cannula by tying one suture around the cannulated vessel and another suture distally around the cannula itself. Ensure the cannula is placed proximally to prevent blocking the bifurcation points.
11. Submerge the procured hindlimbs in phosphate-buffered saline (PBS) until decellularization.

Figure 1: Procurement of rat hindlimb. (A) Marking of skin incision at the inguinal ligament level from lateral to medial. (B) View of the femoral vein and the femoral artery, which have been dissected proximally toward the inguinal ligament, indicated by the dotted line. Abbreviations: L = lateral; M = medial; FV = femoral vein; FA = femoral artery. Please click here to view a larger version of this figure.

3. Preparation of solutions

1. In a 6 L glass flask, prepare a 5 L detergent reservoir of 0.25% sodium dodecyl sulfate (SDS) by dissolving 12.5 g of SDS powder in 5 L of ultrapure distilled water. Cover the opening of the flask with parafilm to seal it.
2. In a 1 L glass jar, prepare 1 L of 0.25% SDS solution separately by dissolving 2.5 g of SDS in 1 L of ultrapure distilled water. Add a stir bar to mix the solution on a magnetic stirrer until all the SDS is dissolved.
3. Prepare 1 L of 1x PBS wash solution with 1% Antibiotic-Antimycotic (AA) solution. In a 1 L flask, add 990 mL of 1x PBS solution and 10 mL of AA.
4. Separately, in a 500 mL glass jar, prepare 1x PBS + 1% AA solution again using 495 mL of 1x PBS solution and 5 mL of AA.
5. Prepare 200 mL of 1% peracetic acid (PAA)/4% ethanol (EtOH) solution. Prepare this solution under a fume hood.

4. Bioreactor and perfusion circuit construction

NOTE: Refer to Figure 2 for the configuration of the bioreactor and perfusion circuit throughout the listed steps.

1. In a 500 mL chamber (plastic container), drill three holes (1/4 in) at the labeled locations in Figure 2: Port A is the inlet line, Port B is the replenishing line, and Port C is the outlet line. Remove and discard excess plastic. Spray and wipe the chamber with 70% ethanol.
2. Cut three 5 cm long silicone tubes and insert one halfway into each port of the chamber. Connect a male Luer connector on the opening of Port A facing the inside of the chamber and a female Luer connector on the other end of the tube outside of the chamber.
3. Connect a female Luer connector at Port B and Port C on the ends of the tubes facing out of the chamber.
4. In an air-tight lid for the plastic container, drill one hole on the surface of the lid.
5. Cut a 3 cm silicone tube and insert it into the hole of the lid. Ensure approximately 2 cm of the tube is located out of the lid, as shown in Figure 2.
6. Sterilize both the chamber and the lid with the tubing.
7. Cut two 30 cm silicone tubes using scissors. Connect a 1/16 in to a 1/8 in connector on one end of each tube.
8. Separately, cut two 12 cm silicone tubes using scissors. Connect a 1/16 in to a 1/8 in connector on one end of both tubes. Connect a male Luer connector on the other end of one tube and a female Luer connector to the other tube.
9. Sterilize all the tubing material from step 4.7 and step 4.8. Include two 3-stop pump tubings (1.85 mm) in the sterilization.
10. Prepare three 3-way stopcocks, one syringe filter, two 1 mL serological pipettes, and one 10 mL syringe for the decellularization setup. Remove the filter from the 1 mL serological pipettes.

Figure 2: Preparation of bioreactor and perfusion circuit construction. Apparatus shown of the perfusion circuit including (A) peristaltic pump and (B) corresponding cassettes for both inlet and outlet lines. (C, D) Silicone tubings of 12 cm and 30 cm are also shown with respective connectors. (E) Tubing for peristaltic pump (1.85 mm). Bioreactor chamber with labeled ports for (F) inflow, (G) replenishing port, and (H) outflow. (I) Bioreactor lid shown with ventilation port. Please click here to view a larger version of this figure.

5. Decellularization of rat hindlimbs

1. Place the sterilized chamber and screw on three single-use 3-way stopcocks at the inlet, outlet, and replenishing lines. Ensure the stopcock for the replenishing port is capped at its remaining two ports to prevent leakage.
2. Attach the tubes made in step 4.8 to the stopcocks at the inlet and outlet lines.
3. Connect peristaltic tubing to the tubes in the step above. Secure the cassette on the peristaltic tubing and place it on the peristaltic pump. Do not secure the cassettes with tubing in place yet.
4. Connect one tube from step 4.7 to the end of the peristaltic tubing of the outlet line from the step above. Connect a 1 mL serological pipette on the other end. Suspend the end attached with a serological pipette in the waste reservoir flask.
5. Repeat step 4.7 for the inlet line. Suspend the end of the tube attached to the serological pipette into the detergent reservoir. Seal the opening of the detergent reservoir flask with parafilm immediately. See Figure 3A,B for an overview of the decellularization circuit.
6. Add 0.25% SDS from the 1 L glass jar (step 3.2) into the bioreactor chamber at the halfway level.
7. Take the procured hindlimb using Adson forceps and suspend it into the bioreactor chamber carefully.
8. Use two pairs of Adson forceps to 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. Ensure the cannula is not pulled or twisted to prevent decannulation.
9. Once secured, add more 0.25% SDS from the 1 L glass jar to fully submerge the limb, as needed. Ensure the outlet port is also submerged in the bioreactor reservoir to ensure that consistent outflow is maintained (Figure 3C).
10. Attach a single-use syringe filter to the ventilation port on the lid of the bioreactor chamber, referring to step 4.4 and step 4.5.
11. Secure the lid on the bioreactor and ensure the chamber is sealed from all sides (Figure 3B).
12. To remove air from tubing and prime the perfusion circuit, use a new, single-use 10 mL syringe to draw detergent from the detergent reservoir using the 3-way stopcock at the inlet line.
13. Once drawn, use the same fluid to insert into the 3-way stopcock at the outlet line. Ensure that there is detergent present throughout the tubing at both the inlet and outlet lines.
14. Press down and secure the cassettes with the tubing into the peristaltic pump. Turn the peristaltic pump on using the power button.
    1. On the peristaltic pump screen, proceed to the second tab using the arrow key to set the perfusion rate for the first channel. Input flow rate as the mode of delivery and set the perfusion rate at 1 mL/min. Ensure the direction of flow is correct according to apparatus set-up. Repeat for the second channel.
    2. Calibrate the peristaltic pump to ensure the amount of fluid delivered through the inlet line and/or taken from the outlet line is flowing at a consistent rate between the two. Ensure that the tubing ID is set to 1.85 mm.
15. Begin decellularization via machine perfusion at 1 mL/min for both the inlet and outlet lines by pressing the power button on the keypad. Monitor and ensure that the flow is consistent and ongoing at both the inlet and outlet lines.
16. Continue decellularization and monitor daily. Use the 1 L of 0.25% SDS (step 3.2) to replenish the bioreactor reservoir through the replenishing port, as needed. Look for a white, translucent appearance of the tissue, which will appear by day 5, indicating decellularization of the rat hindlimbs.

Figure 3: Overview of perfusion decellularization bioreactor circuit of rat hindlimb. (A) Schematic representation of bioreactor perfusion circuit. Blue arrows indicate the direction of detergent and waste flow. (B) Overview of the decellularization circuit with bioreactor containing rat hindlimb. The SDS reservoir (left flask) leads into the peristaltic pump and into the inlet tubing of the bioreactor. The outflow is connected to the waste reservoir (right flask) through the peristaltic pump. (C) (I) Bioreactor containing rat hindlimb with inlet tubing connected to the cannulated femoral artery. (II) Replenishing port located in the corner for perfusing detergent. (III) Outflow tubing suspended in suspension reservoir. Abbreviation: SDS = sodium dodecyl sulfate. Please click here to view a larger version of this figure.

6. Post-decellularization washing and sterilization

1. Following the confirmation of decellularization, replace the detergent reservoir with the 1x PBS + 1% AA reservoir. Seal the opening of the flask with parafilm. Begin 1x PBS + 1% AA perfusion at 1 mL/min and continue for 2 days.
2. Replace the 1x PBS + 1% AA reservoir with 200 mL of 0.1% PAA/4% EtOH reservoir and begin perfusion at 1 mL/min for 2 h.
3. 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. Ensure that the cannula is not pulled to prevent decannulation.
4. Store the limb in a 500 mL glass jar containing 1x PBS + 1% AA at 4 °C until further use.

Representative Results

The procurement protocol was successful in isolating and cannulating the common femoral arteries for subsequent perfusion steps. The representative dissection images in Figure 1A,B show the incision location and exposure of the femoral vessels with sufficient distance from the bifurcation points. Figure 2 shows the apparatus required for preparing the bioreactor and perfusion circuit. The endpoint of decellularization was determined by observing a white, translucent-like appearance of the tissue. The ex vivo machine perfusion system was successful in the perfusion decellularization of the rat hindlimb. A single-pass, closed-system circuit was maintained (Figure 3). The gross morphology of the native hindlimb changed into a white, pale appearance after 5 days of 0.25% SDS perfusion (Figure 4).

The removal of cellular content was observed when stained with hematoxylin and eosin (H&E) in the femoral vessels, skin, nerve, bone, and muscle where no nuclei were found. The structures of each tissue structure were analyzed relative to native tissue. 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 (Figure 5A,B and Figure 5D,E). The tunica intima, media, and adventitia of both the femoral vein and arteries were maintained in the decellularized vessels (Figure 5D,E). The femoral nerve showed preservation of tissue structure, including the endoneurium (Figure 5C and Figure 5F). The bone retained its overall tissue structure post-decellularization, with an observable loss of stained nuclei of osteocytes from the bone and from surrounding endosteum and periosteum layers (Figure 5G and Figure 5J). The skin showed a loss of cells from the epidermis and dermis. The dermis showed retained collagen fibers, similar to native skin tissue (Figure 5H and Figure 5K). Lastly, the transverse view of skeletal muscle showed loss of nuclei otherwise located in the peripheries of the endomysium. The myofiber content remained retained within respective fascicles post-decellularization (Figure 5I and Figure 5L). DNA quantification using Picogreen was also performed, where DNA content was significantly reduced across the femoral vessels, nerve, skin, muscle, and bone (Figure 6).

Figure 4: Gross morphology of native and decellularized rat hindlimbs. (A) Femoral artery of native hindlimb cannulated with a 24 G angiocatheter following procurement. (B) White, translucent appearance of hindlimb after 5 days of decellularization with 0.25% SDS. Please click here to view a larger version of this figure.

Figure 5: Histological staining of rat hindlimb tissues using hematoxylin and eosin. H&E-stained native (top panel) and decellularized (bottom panel) (A, D) femoral vein and (B, E) artery, (C, F) nerve, (G, J) bone, (H, K) skin, and (I, L) muscle. Loss of nuclei and cellular content visible in all decellularized samples. Scale bars = 200 µm (vessels, nerve, bone) and 300 µm (skin, muscle). Please click here to view a larger version of this figure.

Figure 6: DNA quantification of native and decellularized rat hindlimb tissues. DNA content is reduced across native and decellularized vessels, nerve, muscle, skin, and bone, expressed in ng/mg dry weight. Tissues were dried and digested in papain overnight at 65 °C. DNA was fluorescently detected using PicoGreen. Multiple unpaired t-tests were performed. Data presented as mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001. Abbreviations: N = native; D = decellularized. Please click here to view a larger version of this figure.

Discussion

Rat hindlimbs are useful as experimental models in VCA⁵. Tissue engineering of acellular scaffolds represents the first step in addressing the shortcomings of long-term immunosuppression regimens associated with VCA. The use of composite grafts poses an added challenge given the presence of multiple tissues, each having unique functional, immunogenic, and structural properties. The present protocol shows a successful method for obtaining acellular composite rat hindlimbs. These scaffolds can be further recellularized and represent a proof-of-concept model for VCA.

To ensure successful procurement and decellularization, there are various critical steps in the surgical and decellularization phases. To ensure systemic distribution of the detergent and sterilant solution throughout the native vasculature, the critical steps include ligation of the epigastric vessels, as well as obtaining sufficient distance from the bifurcation points of the arteriovenous network prior to the ligation of the femoral vessels. The described sterilization procedure helps to decrease the bioburden for recellularization experiments where a sterile environment is required for cell attachment, survival, and growth⁶.

We also present a perfusion bioreactor system that was designed to implement decellularization. Critical components include the capability of continuous perfusion using a peristaltic pump and allowing single-pass perfusion of the detergent and sterilant through the cannulated artery. The peristaltic pump was also set to a pulsatile flow, similar to physiologic conditions. Lastly, a replenishing port was included for replenishing the suspension reservoir in the bioreactor without exposing the hindlimb to the external environment. This bioreactor system is, therefore, advantageous as it can decellularize and sterilize a rat hindlimb ex vivo in a closed-system, single-pass fashion. The components of the circuit are autoclavable and can be sterilized prior to each decellularization cycle. Given the density and relatively large presence of muscle in the hindlimb, both detergent perfusion and submersion methods were incorporated in the design of this ex vivo circuit to help access and decellularize the muscle.

Although the bioreactor chamber and the decellularization circuit were carefully designed and tailored to the rat hindlimb, it has a reproducible design that can be modified when adapting this protocol for other tissues. Additional modifications may include incorporating a bubble trap in the perfusion circuit to ensure the flow rate is not disrupted due to air bubbles^7,8. Further, we did not incorporate a pressure monitoring system to monitor the perfusion pressure throughout the duration of decellularization. It is possible for perfusion pressures to fluctuate due to intraluminal cellular debris. Recently, Cohen et al. reported temporary fluctuations in perfusion pressure during SDS perfusion in an approach to generate vascular chimerism in the rat hindlimb, using a similar target flow rate of 1-2 mL/min. Perfusion pressure was stabilized following treatment for potential clogs⁹. For future perfusion system design modifications for the current protocol, the incorporation of a pressure monitor can be informative of any intraluminal occlusions and indicate the need for treatment to help prevent damage to the vasculature.

In this model, the outflow was observed during and after decellularization. To ensure viability and functionality of the scaffolds, vascular patency is required for the delivery of oxygen and nutrients to different tissues in a composite graft¹⁰. With the potential of this decellularization protocol being extended into further recellularization studies, the observation of outflow is critical so that the decellularized vascular tree can be repopulated and refunctionalized during recellularization. Vascular imaging can be used post-decellularization to confirm vascular patency.

To date, few studies have been conducted on decellularization of the rat hindlimb, with limited results on the success across each of the tissue compartments^9,11. The representative results in the present study show the impact of decellularization across all tissue compartments present in the hindlimb. The surgical method also maintains large amounts of muscle and skin that can be serially biopsied and used for further analyses. Additionally, this protocol suggests a less toxic decellularization approach by employing a lower SDS concentration than is typically used in decellularization protocols for composite and isolated tissues¹². The proposed ex vivo bioreactor system can also be adapted for other tissues and models.

In conclusion, the proposed protocol offers a reliable and reproducible surgical technique and decellularization method for rat hindlimbs using an ex vivo machine perfusion system. Future applications include repopulating this scaffold with tissue-specific cells and examining avenues for regenerating functional capacity in tissues such as bone, muscle, and nerve. Future studies may also characterize the extracellular matrix, the retention of the native vasculature, and biochemical properties.

Materials

0.9% Sodium Chloride Injection USP 50 mL	Baxter Corporation	JB1308M
1 mL Disposable Serological Pipets	VWR	75816-102
10 cc Disposable Syringes			Obtained from Research Institution
3-way Stopcock			Obtained from Research Institution
5cc Disposable Syringes			Obtained from Research Institution
70% Isopropyl Alcohol			Obtained from Research Institution
Acrodisc Syringe Filter 0.2 µm	VWR	CA28143-310
Adson Forceps, Straight	Fine Science Tools	11006-12
Angiocatheter 24 G 19 mm (¾”)	VWR	38112
Antibiotic-Antimycotic Solution (100x) 100 mL	Multicell	450-115-EL
Bone Cutter	Fine Science Tools	12029-12
Connectors for  1/16" to 1/8" Tubes	McMasterCarr	5117K52
Female Luer to barbed adapter (PVDF) – 1/8" ID	McMasterCarr	51525K328
Fine Forceps	Fine Science Tools	11254-20
Fine Forceps with Micro-Blunted Tips	Fine Science Tools	11253-20
Heparin Sodium Injection 10,000 IU/10 mL	LEO Pharma Inc.	006174-09
Male Luer to barbed adapter (PVDF) – 1/8" ID	McMasterCarr	51525K322
Micro Needle Holder	WLorenz	04-4125
Microscissors	WLorenz	SP-4506
Peracetic Acid	Sigma Aldrich	269336-100ML
Peristaltic Pump, 3-Channel	Cole Parmer	RK-78001-68
Phosphate Buffered Saline 1x 500 mL	Wisent	311-425-CL
Povidone Surgical Scrub Solution			Obtained from Research Institution
Pump Tubing, 3-Stop, Tygon E-LFL	Cole Parmer	RK-96450-40
Pump Tubing, Platinum-Cured Silicone	Cole Parmer	RK-96410-16
Scalpel Blade – #10	Fine Science Tools	10010-00
Scalpel Handle – #3	Fine Science Tools	10003-12
Sodium Dodecyl Sulfate Reagent Grade: Purity: >99%, 1 kg	Bioshop	SDS003.1
Surgical Suture #6-0	Covidien	VS889