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

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....

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.......

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 fu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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