使用 离体 灌注生物反应器进行大鼠后肢的获取和去细胞化血管化复合同种异体移植
1Latner Thoracic Research Surgical Laboratories,University Health Network, Toronto General Hospital, 2Institute of Medical Science, Temerty Faculty of Medicine,University of Toronto, 3Institute of Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine,University of Toronto, 4Division of Plastic & Reconstructive Surgery, Department of Surgery,University of Toronto
Summary
我们描述了复合大鼠后肢的手术技术和去细胞化过程。使用低浓度十二烷基硫酸钠通过 离体 机灌注系统进行脱细胞。
Abstract
严重创伤和组织丢失的患者需要复杂的手术重建。血管化复合同种异体移植（VCA）是一种不断发展的重建途径，用于将多个组织作为复合亚单位转移。尽管VCA具有前景，但由于恶性肿瘤、终末器官毒性和机会性感染的风险增加，长期免疫抑制需求是一个重大限制。无细胞复合支架的组织工程是减少免疫抑制需求的潜在替代方案。本文描述了大鼠后肢的获取及其随后使用十二烷基硫酸钠（SDS）的去细胞化。所提出的采购策略基于股总动脉。构建了基于机器灌注的生物反应器系统，用于后肢 离体 脱细胞。成功进行了灌注脱细胞，导致后肢呈白色半透明状外观。观察到整个后肢的完整、可灌注的血管网络。组织学分析显示，所有组织隔室的核内容物去除和组织结构的保存。
Introduction
VCA是需要复杂手术重建的患者的新兴选择。创伤性损伤或肿瘤切除会导致难以重建的体积组织丢失。VCA 提供多种组织的移植，例如皮肤、骨骼、肌肉、神经和血管，作为从供体到受体的复合移植物1.尽管VCA具有希望的性质，但由于长期免疫抑制方案而受到限制。终身使用此类药物会增加机会性感染、恶性肿瘤和终末器官毒性的风险1，2，3。为了帮助减少和/或消除对免疫抑制的需求，使用VCA脱细胞方法的组织工程支架显示出巨大的前景。
组织脱细胞需要保留细胞外基质结构，同时去除细胞和核内容物。这种脱细胞支架可以用患者特异性细胞重新填充4。然而，保存复合组织的ECM网络是一个额外的挑战。这是由于支架内存在多种组织类型，具有不同的组织密度、结构和解剖位置。本协议提供了一种用于大鼠后肢的手术技术和去细胞化方法。这是将这种组织工程技术应用于复合组织的概念验证模型。这也可以促使随后的努力通过细胞再生再生复合组织。
Protocol
从多伦多总医院研究所获得的尸体雄性刘易斯大鼠（300-430g）用于所有实验。对于所有外科手术，使用无菌器械和用品来维持无菌技术（见 材料表）。所有程序均按照多伦多总医院研究所动物护理委员会的指导方针进行，大学健康网络（加拿大安大略省多伦多市）。共有4个后肢被去细胞化。 1.术前准备 准备 50 mL 的 5% 肝素化盐水。从 50 mL 盐?…
Representative Results
采购方案成功地分离和插管股总动脉以进行后续灌注步骤。 图1A，B 中的代表性解剖图像显示了距分叉点足够距离的股血管的切口位置和暴露。 图2 示出了制备生物反应器和灌注回路所需的装置。通过观察组织的白色半透明外观来确定去细胞化的终点。 离体 机灌注系统成功地实现了大鼠后肢的灌注去细胞化。维持单通道闭合系统电?…
Discussion
大鼠后肢可用作VCA5中的实验模型。无细胞支架的组织工程是解决与VCA相关的长期免疫抑制方案缺点的第一步。鉴于存在多种组织，每种组织都具有独特的功能、免疫原性和结构特性，复合移植物的使用带来了额外的挑战。本协议显示了获得无细胞复合大鼠后肢的成功方法。这些支架可以进一步重新细胞化，并代表VCA的概念验证模型。
为了确保成功的采购和?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
图 3A 是在 BioRender.com 中创建的。
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 |
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Cite This Article
Adil, A., Karoubi, G., Haykal, S. Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation. J. Vis. Exp. (184), e64069, doi:10.3791/64069 (2022).