成像引导的生物反应器，用于生成生物工程气道组织
Summary
该协议描述了一种支持成像的生物反应器，该反应器允许从大鼠气管中选择性地去除内源性上皮，并在管腔表面均匀分布外源细胞，然后对细胞组织构建体进行长期 体外 培养。
Abstract
气道组织反复损伤可损害肺功能并引起慢性肺病，如慢性阻塞性肺病。再生医学和生物反应器技术的进步为生产实验室培养的功能性组织和器官结构体提供了机会，这些结构和结构体可用于筛选药物，模拟疾病和工程组织替代物。在这里，描述了一种小型化生物反应器与成像方式相结合，该成像方式允许在体外组织操作和培养期间原位可视化外植大鼠气管的内腔。使用这种生物反应器，该方案展示了成像引导的内源性细胞成分的选择性去除，同时保留了气道组织基质的内在生化特征和超微结构。此外，还显示了外源性细胞在去细胞化气道腔上的递送，均匀分布和随后的延长培养，并进行光学原位监测。结果强调，成像引导的生物反应器可能用于促进功能性体外气道组织的产生。
Introduction
呼吸道的腔内表面衬有一层上皮，主要由多纤毛，球杆，高脚杯和基底干细胞1，2组成。上皮层是肺的主要防御机制，充当生物物理屏障，保护下面的气道组织免受吸入病原体，颗粒物或化学气体的侵害。它 通过 多种机制保护气道组织，包括细胞间紧密连接形成，粘膜纤毛清除以及抗菌和抗氧化剂分泌3，4。气道上皮缺陷与破坏性呼吸系统疾病相关，例如慢性阻塞性肺疾病 （COPD）5、原发性睫状肌运动障碍 （PCD）6 和囊性纤维化 （CF）7。
肺芯片（LOC）技术的进步代表了研究人类肺部发育，模拟各种肺部疾病以及在严格监管 的体外 环境中开发新治疗材料的机会。例如，气道上皮和内皮可以在薄的多孔膜的相对侧培养，以模拟交换肺组织的气体，从而允许忠实的疾病建模和药物测试8。同样，已经创建了 体外 疾病模型来模拟 体外气道疾病，例如COPD9 和囊性纤维化10。然而，LOC装置的一个主要挑战是重述肺组织的复杂三维（3D）结构和动态细胞 – 组织基质 相互作用在体外11。
最近，已经开发了创新的组织工程方法，允许操作 离体 肺组织12。使用这些方法， 可以通过化学， 物理和机械处理从肺组织中去除内源性细胞来制备脱落的同种异体或异种组织移植物13。此外，去细胞化肺支架中保存的天然组织细胞外基质（ECM）为植入细胞附着，增殖和分化提供了物理模拟结构，生化和生物力学线索14，15。
在这里，报道了通过结合LOC和组织工程技术创建的成像引导的生物反应器系统，以允许 体外 组织操作和外植大鼠气管组织的培养。使用这种气道组织生物反应器，该方案证明选择性地去除内源性上皮细胞，而不会破坏气道组织的潜在上皮下细胞和生化成分。接下来，我们通过滴注细胞加载的胶原I预凝胶溶液，展示新接种的外源细胞（如间充质干细胞（MSCs））在剥脱的气道腔上的均匀分布和瞬时沉积。此外，通过使用集成到生物反应器中的微光学成像装置，还可以在上皮去除和内源性细胞递送过程中实现气管腔的可视化。此外，还表明气管和新植入的细胞可以在生物反应器中培养4天而不会出现明显的细胞死亡和组织降解。我们设想，本研究中使用的成像生物反应器平台，基于薄膜的去上皮化技术以及细胞递送方法可用于生成用于 体外 疾病建模和药物筛选的气道组织。
生物反应器包括一个矩形腔室，连接到可编程注射泵，灌注泵和呼吸机，用于培养分离的大鼠气管。生物反应器具有连接到气管或组织培养室的入口和出口，以分别向气管的内部和外部空间供应试剂（例如培养基）（图1）。定制的成像系统可用于在细胞水平上可视化 体外培养的大鼠气管内部（图2）。 通过 滴注基于洗涤剂的去细胞化溶液，然后进行振动辅助气道清洗，去除气管的内源性上皮（图3）。水凝胶溶液，如I型胶原蛋白，被用作在剥脱的气管腔内均匀和瞬时接种外源细胞的递送载体（图4）。用于构建生物反应器和进行实验的所有材料均在 材料表中提供。
Protocol
下面的动物组织方案已获得史蒂文斯理工学院动物护理和使用委员会研究所（IACUC）的动物福利指南和法规的批准，并且符合美国国立卫生研究院（NIH）关于使用实验动物的指南。 1. 影像引导大鼠气管生物反应器的设计与搭建 大鼠气管生物反应器的设计与制造 使用 CAD 生成器软件创建具有相关设计（如入口、出口和组织培养室）的生物反应器腔室的…
Representative Results
基于GRIN晶状体 的原位 成像方式可以原 位 显示气管内腔（图5A）。使用这种成像方法，可以获得天然和去上皮化气管的明场和荧光图像（图5B，C）。在CFSE标记之前，没有从天然气管中观察到荧光信号（图5Bii）。然而，当用CFSE染料标记气管上皮时，在整个上皮中观察到均匀的荧光信号（绿色）（<strong cl…
Discussion
在这项工作中，我们创建了一种成像引导的生物反应器，可以允许（i）在细胞去除和外源性细胞递送后 原位 监测气管腔，以及（ii）细胞接种气管组织的长期 体外 培养。使用这种定制的生物反应器，我们证明了（i）使用洗涤剂和振动辅助气道洗涤从气管腔中选择性地去除内源性上皮细胞，以及（ii）使用细胞负载的胶原I预凝胶将外源性细胞均匀分布到脱毛气管腔表面上。此外，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了美国胸科学会基金会研究计划，新泽西州健康基金会和国家科学基金会（CAREER奖2143620）的部分支持。和美国国立卫生研究院（P41 EB027062）至G.V.N.
Materials
| 1× PBS | Gibco, Thermo Fisher Scientific | 10-010-031 | |
| 3-port connector | World Precision Instruments | 14048-20 | |
| 4-port connector | World Precision Instruments | 14047-10 | |
| Accelerometer | STMicroelectronics | IIS3DWBTR | |
| Achromatic doublet | Thorlabs | AC254-150-A-ML | |
| Aluminum pin stub | TED PELLA | 16111 | |
| Antibiotic-antimycotic | Thermo Fisher Scientific | 15240062 | |
| Assembly rod | Thorlabs | ER1 | |
| Button head screws | McMaster-Carr | 91255A274 | |
| Cage cube | Thorlabs | C4W | |
| Carbon double-sided conductive tape | TED PELLA | 16073 | |
| CFSE labelling kit | Abcam | ab113853 | |
| Citrisolv (clearing agent) | Decon | 1061 | |
| C-mount adapter | Thorlabs | SM1A9 | |
| Collagen I | Advanced BioMatrix | 5153 | |
| Conductive liquid silver paint | TED PELLA | 16034 | |
| Dichroic mirror | Semrock | DI03-R488 | Reflected laser wavelengths: 473.0 +- 2 nm 488.0 +3/-2 nm |
| Dulbecco's modified Eagle’s medium | Gibco, Thermo Fisher Scientific | 11965118 | |
| Female luer bulkhead to hose barb adapter | Cole-Parmer | EW-45501-30 | |
| Female luer to tubing barb | Cole-Parmer | EW-45508-03 | |
| Female to male luer connector | Cole-Parmer | ZY-45508-80 | |
| Fetal bovine serum | Gibco, Thermo Fisher Scientific | 10082147 | |
| Filter lens | Chroma Technology Corp | ET535/50m | |
| Fluorescent microscope | Nikon | Eclipse E1000 – D | |
| Fusion 360 | Autodesk | ||
| Hex nut | McMaster-Carr | 91813A160 | |
| Hexamethyldisilazane (HMDS) | Fisher Scientifc | AC120585000 | |
| Imaging fiber | SELFOC, NSG group | GRIN lens | |
| Laser | Opto Engine | MDL-D-488-150mW | |
| Lens tubes | Thorlabs | SM1L40 | |
| LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen) | Thermo Fisher Scientific | L3224 | |
| MACH 3 CNC Control Software | Newfangled Solutions | ||
| Objective lens | Olympus | UCPLFLN20X | |
| Peristaltic Pump | Cole Parmer | L/S standard digital pump system | |
| Recombinant human FGF-basic | PeproTech | 100-18B | |
| Retaining ring | Thorlabs | SM1RR | |
| Scientific CMOS camera | PCO Panda | PCO Panda 4.2 | |
| Sodium dodecyl sulfate | VWR | 97064-472 | |
| Solidworks (2019) | Dassault Systèmes | ||
| Stackable lens tube | Thorlabs | SM1L10 | |
| Subwoofer plate amplifier | Dayton Audio | SPA250DSP | |
| Subwoofer speaker | Dayton Audio | RSS21OHO-4 | Diaphragm diameter: 21 cm |
| Syringe Pump | World Precision Instruments | AL-4000 | |
| Threaded cage plate | Thorlabs | CP33 | |
| Threaded luer adapter | Cole-Parmer | EW-45513-81 | |
| Tube lens | Thorlabs | AC254-150-A-ML | |
| Tygon Tubing | Cole-Parmer | 13-200-110 | |
| XY Translator | Thorlabs | CXY1 |
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Cite This Article
Mir, S. M., Chen, J., Pinezich, M. R., O’Neill, J. D., Guenthart, B. A., Vunjak-Novakovic, G., Kim, J. Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue. J. Vis. Exp. (182), e63544, doi:10.3791/63544 (2022).