在定制灌注生物反应器中获取和灌注-脱细胞猪血管化皮瓣
1Latner Thoracic Surgery Research Laboratories,Toronto General Hospital Research Institute, University Health Network, 2Institute of Laboratory Medicine and Pathobiology,University of Toronto, 3Division of Thoracic Surgery, Department of Surgery,University of Toronto, 4Division of Plastic and Reconstructive Surgery, Department of Surgery,University of Toronto
Summary
该协议描述了通过在定制的灌注生物反应器中通过皮瓣脉管系统灌注十二烷基硫酸钠洗涤剂来手术获取和随后的血管化猪皮瓣的脱细胞。
Abstract
大量软组织缺损会导致功能缺陷，并会极大地影响患者的生活质量。虽然手术重建可以使用自体游离皮瓣移植或血管化复合同种异体移植（VCA）进行，但这种方法也有缺点。供体部位发病率和组织可用性等问题限制了自体游离皮瓣移植，而免疫抑制是 VCA 的重要限制。使用去细胞化/再细胞化方法的重建手术中的工程组织代表了一种可能的解决方案。使用去除天然细胞物质同时保留底层细胞外基质（ECM）微结构的方法生成去细胞化组织。然后，这些无细胞支架随后可以用受体特异性细胞重新细胞化。
该协议详细介绍了用于在猪模型中实现无细胞支架的获取和脱细胞方法。此外，它还提供了灌注生物反应器设计和设置的描述。皮瓣包括猪网膜、阔筋膜张肌和桡骨前臂。通过低浓度十二烷基硫酸钠（SDS）洗涤剂的 离体 灌注进行脱细胞，然后在定制的灌注生物反应器中进行DNase酶处理和过氧乙酸灭菌。
成功的组织脱细胞的特征是肉眼下皮瓣呈白色不透明外观。无细胞瓣显示组织学染色时没有细胞核，DNA 含量显着降低。该协议可以有效地用于生成具有保留ECM和血管微结构的脱细胞软组织支架。这种支架可用于随后的细胞再生研究，并具有在重建手术中的临床转化潜力。
Introduction
创伤性损伤和肿瘤切除可导致大而复杂的软组织缺损。这些缺陷会损害患者的生活质量，导致功能丧失，并导致永久性残疾。虽然自体组织皮瓣移植等技术已被普遍采用，但皮瓣可用性和供体部位发病率问题是主要限制1，2，3。血管化复合同种异体移植（VCA）是一种有前途的替代方案，可将复合组织（例如肌肉，皮肤，脉管系统）作为一个单元转移给受体。然而，VCA需要长期免疫抑制，这会导致药物毒性、机会性感染和恶性肿瘤4，5，6。
组织工程无细胞支架是这些限制的潜在解决方案7。无细胞组织支架可以使用去细胞化方法获得，该方法从天然组织中去除细胞物质，同时保留底层细胞外基质（ECM）微结构。与在组织工程中使用合成材料相比，使用生物衍生支架提供了一种仿生ECM底物，可实现生物相容性和临床翻译的潜力8。脱细胞后，随后用受体特异性细胞对支架进行再细胞化，然后可以产生几乎没有免疫原性的功能性血管化组织9，10，11。通过开发使用灌注脱细胞技术获得无细胞组织的有效方案，可以设计多种组织类型。反过来，基于该技术允许应用于更复杂的组织。迄今为止，已经使用简单的血管化组织（例如啮齿动物 12、猪 13 和人类模型14 的全层筋膜皮瓣）以及猪腹直肌骨骼肌15 研究了血管化软组织的灌注脱细胞化。此外，复杂的血管化组织也已灌注脱细胞，如猪和人耳16，17模型和人全脸移植模型18所示。
在这里，该协议描述了使用生物衍生的ECM支架的血管化游离瓣的去细胞化。我们介绍了三个临床相关皮瓣的去细胞化：1）网膜，2）阔筋膜张量和3）桡骨前臂，所有这些都代表了重建手术中常规使用的主力皮瓣，并且以前从未在组织脱细胞背景下的动物研究中进行过检查。这些生物工程皮瓣提供了一个多功能且易于获得的平台，具有临床应用的潜力，可用于大型软组织缺损修复和重建领域。
Protocol
所有涉及动物受试者的程序均已获得大学健康网络机构动物护理和使用委员会（IACUC）的批准，并按照大学健康网络动物资源中心的协议和程序以及加拿大动物护理指南委员会执行。所有实验均使用五头约克郡猪（35-50公斤;约12周龄）。 1. 灌注生物反应器制造 有关灌注生物反应器中使用的所有组件，请参见 图1 。对于组织室的制造，请使用市售的聚…
Representative Results
这种使血管化猪瓣脱细胞的方案依赖于离子基洗涤剂SDS通过定制灌注生物反应器中的皮瓣脉管系统的灌注。在脱细胞之前，根据猪模型的主要供应血管采购和插管三个血管化皮瓣。采购后立即冲洗瓣，以保持专利，可灌注脉管系统，以便成功脱细胞。使用密封的卡扣盖容器，设计了一种定制的生物反应器，以允许在封闭环境中进行瓣灌注。使用连接到组织室的两个蠕动泵以单程方式实现生物反应?…
Discussion
拟议的方案使用低浓度SDS的灌注来使一系列猪源性皮瓣脱细胞。通过此程序，可以使用有利于低浓度 SDS 的方案成功脱细胞，无细胞网膜、阔筋膜张肌和桡骨前臂皮瓣。初步优化实验已确定，当使用组织学技术分析时，2天至5天之间低浓度（0.05%）的SDS能够去除网膜，阔筋膜张肌和桡骨前臂瓣的细胞物质（补充图1，补充图2，补充图3）。这种方法提供了一种简单的方法，可以在短时间?…
Declarações
The authors have nothing to disclose.
Acknowledgements
没有
Materials
| 0.2 µm pore Acrodisk Filter | VWR | CA28143-310 | |
| 0.9 % Sodium Chloride Solution (Normal Saline) | Baxter | JF7123 | |
| 20 L Polypropylene Carboy | Cole-Parmer | RK-62507-20 | |
| 3-0 Sofsilk Nonabsorbable Surgical Tie | Covidien | LS639 | |
| 3-way Stopcock | Cole-Parmer | UZ-30600-04 | |
| Adson Forceps | Fine Science Tools | 11027-12 | |
| Antibiotic-Antimycotic Solution, 100X | Wisent | 450-115-EL | |
| Atropine Sulphate 15 mg/30ml | Rafter 8 Products | 238481 | |
| BD Angiocath 20-Gauge | VWR | BD381134 | |
| BD Angiocath 22-Gauge | VWR | BD381123 | |
| BD Angiocath 24-Gauge | VWR | BD381112 | |
| Calcium Chloride | Sigma-Aldrich | C4901 | DNAse Co-factor |
| DNase I from bovine pancreas | Sigma-Aldrich | DN25 | |
| DNA assay (Quant-iT PicoGreen dsDNA Assay Kit) | Invitrogen | P7589 | |
| DPBS, 10X | Wisent | 311-415-CL | without Ca++/Mg++ |
| Halsted-Mosquito Hemostat | Fine Science Tools | 13008-12 | |
| Heparin, 1000 I.U./mL | Leo Pharma A/S | 453811 | |
| Ketamine Hydrochloride 5000 mg/50 ml | Bimeda-MTC Animal Health Inc. | 612316 | |
| Ismatec Pump Tygon 3-Stop Tubing | Cole-Parmer | RK-96450-40 | Internal Diameter: 1.85 mm |
| Ismatec REGLO 4-Channel Pump | Cole-Parmer | 78001-78 | |
| Ismatec Tubing Cassettes | Cole-Parmer | RK-78016-98 | |
| Isoflurane 99.9%, 250 ml | Pharmaceutical Partners of Canada Inc. | 2231929 | |
| LB Agar Lennox | Bioshop Canada | LBL406.500 | Sterility testing agar plates |
| Magnesium Sulfate | Sigma-Aldrich | M7506 | DNAse Co-factor |
| Masterflex L/S 16 Tubing | Cole-Parmer | RK-96410-16 | |
| Midazolam 50 mg/10 ml | Pharmaceutical Partners of Canada Inc. | 2242905 | |
| Monopolar Cautery Pencil | Valleylab | E2100 | |
| Normal Buffered Formalin, 10% | Sigma-Aldrich | HT501128 | |
| N°11 scalpel blade | Swann Morton | 303 | |
| Papain from papaya latex | Sigma-Aldrich | P3125 | |
| Peracetic Acid | Sigma-Aldrich | 269336 | |
| Plastic Barbed Connector for 1/4" to 1/8" Tube ID | McMaster-Carr | 5117K61 | |
| Plastic Barbed Tube 90° Elbow Connectors | McMaster-Carr | 5117K76 | |
| Plastic Quick-Turn Tube Plugs | McMaster-Carr | 51525K143 | Male Luer |
| Plastic Quick-Turn Tube Sockets | McMaster-Carr | 51525K293 | Female Luer |
| Punch Biopsy Tool | Integra Miltex | 3332 | |
| Potassium Chloride 40 mEq/20 ml | Hospira Healthcare Corporation | 37869 | |
| Povidone-Iodine, 10% | Rougier | 833133 | |
| Serological Pipet, 2mL | Fisher Science | 13-678-27D | |
| Snap Lid Airtight Containers | SnapLock | 142-3941-4 | |
| Sodium Dodecyl Sulfate Powder | Sigma-Aldrich | L4509 | |
| Surgical Metal Ligation Clips, Small | Teleflex | 001200 | |
| Stevens Tenotomy Scissors, 115 mm, straight | B. Braun | BC004R | |
| TruWave Pressure Monitoring Set | Edwards Lifesciences | PX260 |
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Citar este artigo
Xu, M. S., Karoubi, G., Waddell, T. K., Haykal, S. Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor. J. Vis. Exp. (186), e64068, doi:10.3791/64068 (2022).