离体 在3D打印生物反应器中对大血管进行灌注培养
Summary
该协议介绍了新开发的3D打印生物反应器的设置和操作，用于灌注中血管 的离体 培养。该系统旨在易于被其他用户采用，实用，价格合理，并适应不同的实验应用，如基础生物学和药理学研究。
Abstract
血管疾病是大多数心血管疾病（CVD）的基础，心血管疾病仍然是全球死亡和发病的主要原因。迫切需要有效的手术和药物干预来预防和治疗血管疾病。在某种程度上，翻译模型的缺乏限制了对血管疾病中涉及的细胞和分子过程的理解。 体 外灌流培养生物反应器为在受控动态环境中研究大型动物血管（包括人类）提供了一个理想的平台，结合了 体外 培养的易用性和活组织的复杂性。然而，大多数生物反应器都是定制制造的，因此难以采用，从而限制了结果的可重复性。本文介绍了一种3D打印系统，该系统可以在任何生物实验室中轻松生产和应用，并为其设置提供了详细的协议，使用户能够进行操作。这种创新且可重复的 ex vivo 灌注培养系统可在生理条件下培养血管长达 7 天。我们预计采用标准化的灌注生物反应器将有助于更好地了解大型动物血管中的生理和病理过程，并加速新疗法的发现。
Introduction
血管壁以反应性稳定状态存在，这确保了对外部刺激（即压力变化、血管收缩剂）和防止血液凝固和炎症细胞浸润的一致非活化表面的作用1。为了应对衰老和生活方式依赖性刺激以及直接损伤，血管壁会激活重塑过程，例如再狭窄和动脉粥样硬化，这些过程是常见心血管疾病 （CVD） 的已知因素，例如缺血性中风和心肌梗塞2。虽然经皮血运重建和支架置入术等介入方法可用于解决血管疾病的晚期表现，但已知这些方法会引起进一步的血管损伤，通常会导致复发。此外，只有有限的预防和早期解决方案可用。了解维持血管壁稳态并驱动其功能障碍的机制是开发新疗法的核心3.
尽管分子生物学和组织工程不断发展和进步，但动物研究仍然是血管生物学研究的重要组成部分。体内动物研究为血管稳态和病理学的机制提供了丰富的见解;然而，这些程序成本高昂，吞吐量相对较低，并存在重大的伦理问题。此外，小动物对人类血管生理学的代表性很差，而较大的动物实验要昂贵得多，并会产生进一步的伦理考虑4,5。随着快速老龄化人口对制药和医疗解决方案的需求不断增长，动物使用的缺点被放大，影响了结果的可重复性、可靠性和可转移性，用于患者护理6.
体外 系统为研究基本机制提供了一个简化的平台，但未能概括整个组织的复杂性、细胞与细胞外基质之间的相互作用以及机械力，这些都是血管疾病发展的关键决定因素7.
对在人工控制环境中维持的整个组织进行的离体研究模拟了体内复杂性，同时实现了相对高通量的研究8.鉴于能够密切控制培养条件和环境，离体模型允许进行广泛的复杂研究，并提供合适的替代方案来减少血管生物学中动物程序的使用。静态血管环培养提供了有趣的见解，但未能纳入关键的血流动力学元素9。事实上，对离体血管系统的研究提出了与应用于血管壁内细胞的许多动态力相关的特定挑战。管腔流动、湍流、剪切应力、压力和壁变形等刺激会显着影响组织病理生理学10,11,12。
灌注生物反应器对于研究血管稳态和响应损伤或血流动力学变化的重塑至关重要13.此外，灌注培养可用于改善组织工程血管 （TEBV） 的成熟度和耐久性，为血管移植物提供合适的替代方案14。
市售灌注生物反应器在灵活性和适应性方面受到限制，而且成本高昂。许多现有的内部开发的生物反应器很难在其他实验室中复制，因为描述有限且无法获得特制组件7,8,9,10,11,12。为了克服这些局限性，我们最近开发了一种新的生物反应器（EasyFlow），该反应器生产经济，可容纳各种组织，并能够进行相对简单的修改以适应不同的研究需求13。该插件是 3D 打印的，可以安装在标准 50 mL 离心管的盖子上。其模块化设计和 3D 打印制造使其可在不同的实验室中访问和复制，并且易于修改以适应不同的科学需求。该协议描述了生物反应器系统在动脉灌注环境中的组装和基本操作。
Protocol
该协议描述了由两个EasyFlow（生物反应器）插入物组成的系统的组装和使用：一个代表反应室（C），包含灌注的动脉样本，一个用作培养基储液器（R）（图1 和 图2A）。颈动脉采集于英国皮尔布赖特研究所的4-6周龄雄性和雌性仔猪（6-12公斤）。动物程序是根据内政部动物（科学程序）法案（1986年）（ASPA）进行的，并由皮尔布赖特研究所的动物福利和?…
Representative Results
这项研究建立了一个多功能且经济实惠的灌注系统（EasyFlow）13。该系统的 3D 打印设计有助于其他实验室采用该系统，从而鼓励可重复性。 制造的灌注插入物被安置在 50 mL 离心管中，创造了一个隔离的环境。使用两个灌注插件，可以建立一个灌注回路，其中包含一个储液器和一个反应室，生物样品在其中孵育。然后将灌注系统连接到蠕动泵和可选的采集系?…
Discussion
离体 血管灌注系统构成了一个独特的平台，用于在受控条件下研究血管细胞在其天然组织内的功能和行为，从而能够解剖复杂过程，例如损伤后血管重塑22。然而，大多数报道的生物反应器都是基于定制组件的内部制造系统，通常难以被其他人复制23.存在替代的商业解决方案，但在设计上缺乏灵活性，并且可能相对昂贵24.
<p class="jove…Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢萨里大学兽医学院的兽医病理学中心提供的组织学服务。我们还要感谢皮尔布赖特研究所（英国皮尔布赖特）的 L. Dixon、A. Reis 和 M. Henstock 博士在采购动物组织方面的支持，以及萨里大学生化科学系，尤其是技术团队的持续支持。RSM得到了博士学院学生奖（萨里大学）的支持，DM和PC得到了国家动物替代，改进和减少研究中心的支持（资助号：NC / R001006 / 1和NC / T001216 / 1）。
Materials
| EasyFlow | – | – | 3D printed by MultiJet Fusion by Protolabs |
| PA12 – 3D printing | Protolabs | – | – |
| Peristaltic pump | Heidolph | PD5201 | |
| Culture media components: | |||
| Amphotericin B solution, 250 mug/mL in deionized water | Sigma-Aldrich | A2942-20ML | |
| Dextran from Leuconostoc spp. | Sigma-Aldrich | D8802-25ML | |
| Dulbecco's Modified Eagle's Medium – high glucose, w/ 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate | Sigma-Aldrich | D6429-6X500ML | |
| Fetal Bovine Serum | Sigma-Aldrich | F9665 | |
| Penicillin-Streptomycin | Sigma-Aldrich | P4333-100ML | |
| Immunostaining materials: | |||
| Cryostat | LEICA | CM3050 S | |
| DAPI | Sigma-Aldrich | D9542-10MG | |
| Goat serum | Sigma-Aldrich | G9023-10ML | |
| Goat α-Rabbit Alexa Fluor 488 | Thermo Fisher Scientific | A11008 | |
| Invitrogen eBioscience Fluoromount G | Thermo Fisher Scientific | 50-187-88 | |
| MX35 Premier + Microtome Blade | Thermo Scientific | 3052835 | |
| Optimal Cooling Tempearure Compound – OCT | Agar Scientific | AGR1180 | |
| Rabbit α-CD31 antibody | Abcam | ab28364 | |
| Sudan Black B | Santa Cruz Biotechnology | SC-203760 | |
| X72 SuperFrost Plus Adhesion slide, 25x75x1mm, White, 90° Ground Edges, Frosted Area 20mm, 72/box | Fisher Scientific | J1800AMNZ | |
| α-Smooth Muscle Actin (SMA) Alexa Fluor® 647-conjugated antibody | R&D Systems | IC1420R | |
| Material for laser cutting of components: | |||
| Clear Plastic Sheet, 1250 mm x 610 mm x 1 mm (for laser cutting of washers) | RS Components | 258-6590 | |
| RS PRO Translucent Rubber Sponge Sheet, 600 mm x 600 mm x 1.5 mm (for laser cutting of silicone seals) | RS Components | 840-5541 | |
| Optional pressure monitors: | |||
| Pressure sensor | Parker Hannifin | 080-699PSX-3P-5 | |
| SciPres Pressure Monitor | Parker Hannifin | 206-200-M | |
| Pre-sterilized single use plasticware: | |||
| 0.2 um filter | Sarstedt | 70.1114.210 | |
| 20 mL Sterile syringe | IMS Euro | 40004 | |
| 50 mL Centrifuge Tube | Thermo Fisher Scientific | Sarstedt – 62.547.254 | |
| Small components: | |||
| Cable ties | – | – | |
| Masterflex Adapter Fittings, Female Luer to Hose Barb | Cole-Parmer | WZ-30800-10 | Barb Adaptor |
| Masterflex Polycarbonate Luer Fittings | Cole-Parmer | AU-45504-84 | |
| Nylon Miniature Check Valve | Cole-Parmer | 98553-00 | |
| RS PRO Translucent Rubber Sponge Sheet, 600 mm x 600 mm x 1.5 mm (for laser cutting of silicone seals) | RS Components | 840-5541 | |
| Stainless Steel M2 Hex Nuts | RS Components | 527-218 | |
| Stainless Steel M2 x 6 mm Screws | RS Components | 418-7426 | |
| Stainless Steel M5 Hex Nuts | RS Components | 189-585 | |
| Surgical vessel loop | Vascular Silicone Ties,International Medical Supplies | 10-1003 | |
| Three-way valves | IMS Euro | 91000 | |
| Surgical Equipment | |||
| Anatomical Forceps, GRAEFE, Curved, 10 cm SKU: BD-07 | International Medical Supplies | SKU: BD-07 | |
| Micro Forceps, Angled, 0.3 mm, 11 cm | International Medical Supplies | SKU: BD-361 | |
| Micro Scissors Noyes, Curved, 12 cm | International Medical Supplies | SKU: FD-12 | |
| Troge Surgical Scalpels – Size 23 – Box of 100 | International Medical Supplies | 63114 | |
| Tubing: | |||
| Eppendorf silicone tubing (I.D.1.6 mm, O.D.4.7 mm) | Eppendorf | M0740-2396 | System tubing |
| Masterflex PharMed BPT 3-Stop Tubing | ISMATEC | 95714-48 | Soft wall tubing (for clamp) |
| RS PRO Transparent Hose Pipe, 0.8 mm ID, Silicone | RS Components | 667-8432 | Resistance tubing (small inner diameter) |
| Tygon for food (I.D. 4.8 mm, W.T. 1.6 mm) | Heidolph | 525-30027-00-0 | One way valve tube |
| Verderflex Yellow Hose Pipe, 6.4 mm ID, Verderprene | RS Components | 125-4042 | Pump Tubing |
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Cite This Article
Matos, R. S., Jawad, A. J., Maselli, D., McVey, J. H., Heiss, C., Campagnolo, P. Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor. J. Vis. Exp. (197), e65465, doi:10.3791/65465 (2023).