Name: procurement and decellularization of rat hindlimbs using an ex vivo perfusion-based bioreactor for vascularized composite allotransplantation
Uploaded: 2022-06-09T15:00:00
Description: Watch this Scientific Journal Video about Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation at JoVE.com

Figure 3A was created in BioRender.com.

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.
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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 fu...

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 toxicity1,2,3. 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.

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			<td>0.9% Sodium Chloride Injection USP 50 mL</td>
			<td>Baxter Corporation</td>
			<td>JB1308M</td>
			<td></td>
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		<tr>
			<td>1 mL Disposable Serological Pipets</td>
			<td>VWR</td>
			<td>75816-102</td>
			<td></td>
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			<td>10 cc Disposable Syringes</td>
			<td></td>
			<td></td>
			<td>Obtained from Research Institution</td>
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			<td>3-way Stopcock</td>
			<td></td>
			<td></td>
			<td>Obtained from Research Institution</td>
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			<td>5cc Disposable Syringes</td>
			<td></td>
			<td></td>
			<td>Obtained from Research Institution</td>
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			<td>70% Isopropyl Alcohol</td>
			<td></td>
			<td></td>
			<td>Obtained from Research Institution</td>
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			<td>Acrodisc Syringe Filter 0.2 &#181;m</td>
			<td>VWR</td>
			<td>CA28143-310</td>
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			<td>Adson Forceps, Straight</td>
			<td>Fine Science Tools</td>
			<td>11006-12</td>
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			<td>Angiocatheter 24 G 19 mm (&#190;&#8221;)&#160;</td>
			<td>VWR</td>
			<td>38112</td>
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			<td>Antibiotic-Antimycotic Solution (100x) 100 mL</td>
			<td>Multicell</td>
			<td>450-115-EL</td>
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			<td>Bone Cutter</td>
			<td>Fine Science Tools</td>
			<td>12029-12</td>
			<td></td>
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			<td>Connectors for&#160; 1/16&#34; to 1/8&#34; Tubes</td>
			<td>McMasterCarr</td>
			<td>5117K52</td>
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			<td>Female Luer to barbed adapter (PVDF) - 1/8&#34; ID</td>
			<td>McMasterCarr</td>
			<td>51525K328</td>
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			<td>Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
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			<td>Fine Forceps with Micro-Blunted Tips</td>
			<td>Fine Science Tools</td>
			<td>11253-20</td>
			<td></td>
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			<td>Heparin Sodium Injection 10,000 IU/10 mL</td>
			<td>LEO Pharma Inc.</td>
			<td>006174-09</td>
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			<td>Male Luer to barbed adapter (PVDF) - 1/8&#34; ID</td>
			<td>McMasterCarr</td>
			<td>51525K322</td>
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			<td>Micro Needle Holder</td>
			<td>WLorenz</td>
			<td>04-4125</td>
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			<td>Microscissors</td>
			<td>WLorenz</td>
			<td>SP-4506</td>
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			<td>Peracetic Acid</td>
			<td>Sigma Aldrich</td>
			<td>269336-100ML</td>
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			<td>Peristaltic Pump, 3-Channel</td>
			<td>Cole Parmer</td>
			<td>RK-78001-68</td>
			<td></td>
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			<td>Phosphate Buffered Saline 1x 500 mL</td>
			<td>Wisent</td>
			<td>311-425-CL</td>
			<td></td>
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		<tr>
			<td>Povidone Surgical Scrub Solution</td>
			<td></td>
			<td></td>
			<td>Obtained from Research Institution</td>
		</tr>
		<tr>
			<td>Pump Tubing, 3-Stop, Tygon E-LFL</td>
			<td>Cole Parmer</td>
			<td>RK-96450-40</td>
			<td></td>
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			<td>Pump Tubing, Platinum-Cured Silicone</td>
			<td>Cole Parmer</td>
			<td>RK-96410-16</td>
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			<td>Scalpel Blade - #10</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
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			<td>Scalpel Handle - #3&#160;</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
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			<td>Sodium Dodecyl Sulfate Reagent Grade: Purity: &#62;99%, 1 kg</td>
			<td>Bioshop</td>
			<td>SDS003.1</td>
			<td></td>
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			<td>Surgical Suture #6-0</td>
			<td>Covidien</td>
			<td>VS889</td>
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procurement and decellularization of rat hindlimbs using an ex vivo perfusion-based bioreactor for vascularized composite allotransplantation

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.

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.&#160;The endpoint of decellularization was determined by obse...

Watch this Scientific Journal Video about Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation at JoVE.com

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

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.

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

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

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