这项研究得到了ZonMw的资助，作为LSH 2Treat计划（436001003）和荷兰肾脏基金会（14a2d507）的一部分。N.A.K.感谢欧洲研究理事会（851960）的支持。我们感谢荷兰科学研究组织（024.003.013）资助的引力计划"材料驱动再生"。

作者没有什么可透露的。

此处描述的生物反应器允许系统地评估剪切应力和循环拉伸对管状可重新吸附脚手架的炎症和组织再生的个体贡献和综合影响。这种方法还使血管构造能够进行各种各样的分析，在具有代表性的结果部分就说明了这一点。这些结果表明，不同的血液机载系统（即剪切和拉伸的不同组合）对 TEVG 结构的生长和改造都有独特的影响。这些见解通过这个体外平台收集，有助于优化新开发的原地 TEVG 的脚手架设计参数。为了确保适当的实验工作流程，了解本协议的关键步骤和局限性非常重要。
协议中最关键的步骤与向样品应用拉伸有关。对于拉伸应用程序，设置必须无泄漏。系统中有两个弱点:将电喷管连接到压力导管的结以及生物反应器基座和流培养室之间的连接。如第 2.5.2 步和 2.5.4 步所述，多个紧结必须精确定位在雕刻槽处。如果结不够紧或放置在凹槽上方或下方，则可能会有液压油轻微渗入流培养室。此泄漏可检测为液压储层中的压力下降和应变泵的电压稳步增加，以达到设定的压力值。此外，这会导致流动培养室的流动受到干扰，细胞培养受到污染的风险增加，介质稀释。一旦发生这种情况，流培养室必须从实验中取出，液压储层上的螺丝线应用白色 Luer 盖关闭。这个取出完整样本的主要措施，是确保其他七个流文化室的实验得到适当继续所必须的，它强调了准确放置紧结的重要性，正好在雕刻的凹槽中。只有这样，才能确保水库水与流水室介质之间适当分离。为了提高脚手架安装的坚固性，压力导管中的凹槽将在生物反应器的修订版中稍微深一点，从而更轻松、更好的结位，从而确保液压液与介质分离。
另一个潜在的泄漏来源是生物反应器基座的螺丝线和流培养室的白色Luer连接器之间。由于特氟隆材料可能磨损，可以添加额外的硅胶 O 环以防止泄漏 （步骤 5.2.3）。此外，应允许应变泵的铁氟龙波纹管在实验前一天 （第1.4步）稍微扩大O/N。如果发生泄漏，损失的水力液应通过八根螺丝线之一（使用带针头和柔性线的注射器）添加少量超纯水来补偿。将流文化室放回螺丝线和流文化室底部隔间的白色 Luer 连接器之间的一小块准膜。为了在未来的实验中克服这一泄漏问题，生物反应器基座上的特氟隆螺丝线将被下一代生物反应器中的不锈钢螺纹所取代，以防止系统磨损。
拉伸（图3C）的变化大于WSS（图3D）的变化，因为拉伸更难控制。然而，除了防止泄漏的措施外，还有其他措施限制了拉伸的变化:（i） 避免长笛中的气泡（第 1.4 步和 5.2.1 步），（ii）确保不同样品之间（第 2.5.3 步）之间的一致预拉伸，（iii） 确保不同样品之间一致的脚手架属性（第 1.3 步）。
最后，在将电子喷头脚手架安装到硅胶管（第 2.3 步）上时，需要格外小心。具体来说，当脆弱的电喷头脚手架需要拉过拉伸的硅胶管时，重要的是不要施加太大的力来防止电喷管被损坏，尤其是在电喷管内部。如果电勺移植物内部发生损坏，无论是强力拉力还是硅胶管拉伸过弱，则只有在收获和分析结构后，才会可见电勺纤维的损坏程度。在目前的设置中，种子的成功只能在牺牲样品后，通过免疫荧光或免疫组织化学分析来确认。然而，以纤维蛋白作为细胞载体的播种过程是一种成熟的方法67，通常导致具有足够大孔径的脚手架的同质细胞分布。细胞播种最重要的方面之一是确保脚手架在播种前尽可能干燥（第4.4步），以防止纤维蛋白凝胶与细胞通过湿脚手架，导致异质播种。最后，虽然紫外线辐射和浸入30%乙醇的电孢子移植的净化方法不如体内研究准备的电孢子移植物的灭菌严格， 通常由氧化乙烯或伽马辐照消毒，这足以进行体外培养实验，这种实验可以持续长达20天，没有任何污染迹象（例如，见图3、图4、图5、图6以及范哈芬和威辛（2020）44）。此外，这里使用的PCL-BU材料不允许长时间接触高乙醇浓度。根据所使用的材料，可以选择最合适的灭菌方法。
除了以前进行的共同文化研究的结果外，还可以在同一系统下进行更广泛的研究。该系统以前曾被用来执行肌纤维细胞（辅助图1A B）和巨噬细胞（辅助图1C-D）的动态单体培养，以调查血液机载量对单个细胞类型及其寄生虫信号19，43的影响。不同的血液机载系统导致肌纤维细胞（辅助图1A）的明显作用素纤维方向，并在10天后明显不同的胶原蛋白沉积（补充图1B）。THP1 衍生巨噬细胞的细胞因子生产在不同的血液动力学负载 （辅助图 1C）之间有着巨大的差异，在加载时表现出更有利于炎症的轮廓（辅助图 1D）。其他经验证的可能性包括使用额外的泵和流体单元应用振荡流。介质的粘度可以增加对血液粘度的范围（例如，通过添加桑坦口香糖）69。调节中等粘度是扩大适用剪切应力范围的附加变量。最后，尽管上述协议采用了"Ibidi"流量调节设置，但只要可以应用类似的流程系统，其他制造商的设置也可以使用。
使用这种生物反应器系统的主要优点之一是相对较大的构造（约15毫米 x 10.5 毫米），可以按血液学方式加载，从而允许从单个示例中提取各种可能的读出参数。同时，构造大小也可以被视为一种限制，因为此设置需要相对大量的（有时是昂贵的）材料，特别是如果使用原细胞，或者如果培养介质需要昂贵的添加剂。此外，该设置的吞吐量相对较低。因此，目前的设置特别适合假设驱动的研究，其中对数量有限的变量进行全面测试，而不是筛选大量读出量有限的变量。对于未来的实验，目前正在对目前的设置进行小的改进，以便能够选择安装较小的脚手架和缩小中型水库的大小。关于后者，需要中型水库的当前体积，以使足够的体积流量速率达到所需的剪切应力。所需的流量 -并因此，通过增加介质的粘度（例如，通过添加以前确定的69）来减少介质储层的体积。
最后，这种生物反应器允许量化剪切应力和循环拉伸对组织生长的个体和组合影响，并在各种弹性3D生物材料脚手架上进行改造。生物反应器可以在各种装载条件下培养多达8个血管结构。由于其设计，生物反应器特别适合研究血液动力学和原位血管TE过程之间的相互作用。

心血管组织工程（TE）正在作为替代治疗选择，目前使用的永久心血管假肢（如血管移植，心脏瓣膜置换），这是不理想的大组患者1，2，3，4。许多抢手的应用包括组织工程血管移植 （TEVG）5，6和心脏瓣膜 （TEHV）7，8.最常见的是，心血管TE方法利用可重新吸收的生物材料（无论是天然的还是合成的），作为新组织形成的有启发性的支架。新组织的形成可以完全在体外设计，通过用细胞播种脚手架，并在植入前在生物反应器中培养（体外TE）9、10、11或直接就地植入，在原位植入合成脚手架时无需预先培养，以诱导体内（原位TE）12、13、14等新组织的形成。对于体外和原位心血管TE方法，成功的功能再生主要取决于宿主对植入结构的免疫反应和适当的生物力学负荷。
生物力学负荷对心血管TE的重要性是众所周知的。在心血管植入物中，填充脚手架的细胞暴露于循环拉伸和剪切压力，这些压力是血友病环境引起的。许多研究已经报告了（循环）拉伸对基质成分形成的刺激作用，如胶原蛋白16，17，18，19，糖氨酸（GAGs）20，和弹性蛋白21，22，由各种细胞类型。例如，黄等人证明，双轴拉伸利用血管生物反应器23，提高了胶原蛋白和弹性蛋白在体外特威格斯中的沉积和组织。虽然重点通常在于拉伸作为主要负载，但这些研究通常使用流驱动的生物反应器，其中样本也暴露在剪切流中。虽然对剪切应力对3D组织形成和炎症的孤立影响知之甚少，但提供了一些数据。例如，Hinderer等人和Eoh等人证明，剪切流除了3D脚手架微结构外，对于人类血管平滑肌肉细胞在体外模型系统24、25中形成成熟的弹性蛋白非常重要。总之，这些发现说明了环伸和剪切应激对心血管TE的相关性。
TE植入物成败的另一个重要决定因素是宿主对植入移植物26的免疫反应。这对于物质驱动的原位TE策略尤其重要，它实际上依赖于对脚手架的急性炎症反应来启动细胞流入和内源性组织形成和重塑的后续过程。巨噬菌体是功能组织再生的关键启动器，28、29、30等多项研究表明。类似于伤口愈合，组织的再生是由巨噬细胞和组织产生细胞之间的寄生虫信号，如成纤维细胞和肌纤维细胞31，32，33。除了协调新的组织沉积外，巨噬细胞还参与外来脚手架材料34、35的主动吸收。因此，体外对生物材料的宏年反应已被确定为植入物36、37、38体内成功的预测参数。
植入式脚手架的宏观反应取决于脚手架的设计特征，如材料组成和微观结构35，39，40。除了脚手架属性外，对脚手架及其与肌纤维细胞的相声的宏法响应也受到血液动力载荷的影响。例如，环状拉伸被证明是巨噬细胞表型41，42，43，44和细胞因子43，44，45，46在3D电子喷支架分泌物的重要调节剂。Battiston等人利用巨噬细胞和血管平滑肌肉细胞的共培养系统证明，巨噬细胞的存在导致弹性蛋白和GAG水平增加，中度循环拉伸水平（1.07-1.10）刺激胶原蛋白I和弹性蛋白47的沉积。在以往的著作中，我们已经证明剪切应力是单核细胞招募到3D电喷支架48，49，剪切应力和循环拉伸影响人类单核细胞和间质频闪细胞50之间的寄生虫信号的重要决定因素。Fahy等人证明，剪切流增加了人类单核细胞51的亲炎细胞因子的分泌。
综合起来，上述证据表明，对血液机载荷的充分理解和控制对心血管TE至关重要，因此必须考虑炎症反应来实现这一点。许多生物反应器以前曾被描述为体外52，53，54，55，56，57，58或前体内59，60，61心血管组织培养。然而，所有这些系统都旨在尽可能模仿生理血液机载量条件。虽然这在体外创建心血管组织或维持前体内培养物方面具有很高的价值，但此类系统不允许系统地研究个体线索的个体影响。这是因为在这些生物反应器中应用循环拉伸和剪切应力是由相同的加压流驱动的，而加压流与它们有着内在的联系。虽然用于精确多提示机械操作的微系统已用于 2D 基材62或 3D 水凝胶设置63，64，但此类设置不允许纳入弹性 3D 生物材料脚手架。
在这里，我们介绍了管状生物反应器系统的应用，该系统独特地实现了剪切应力和循环拉伸的脱钩，并有助于机械地研究其个体和组合效应。该系统允许测试各种组织工程血管移植物（例如，合成或自然起源，不同的微结构，各种孔隙）。为了有效地将剪切应力和拉伸的应用脱钩，生物反应器使用的关键概念是（1） 使用独特的泵系统分离剪切应力和拉伸控制，（2） 以计算驱动尺寸的"从内到外"方式刺激脚手架。通过使用流泵将流量应用于管状脚手架的外表面，而脚手架的周长则通过使用单独的应变泵来扩展安装脚手架的硅胶管。硅胶管和包含构造的玻璃管的尺寸是使用计算流体动力学模拟仔细选择和验证的，以确保脚手架上的剪切应力（由于流量）和环状拉伸（由于管膨胀）不会显著影响彼此。这种从内到外的设计有几个实用的理由。如果拉伸由发光流体压力（类似于生理负荷）施加，则固有要求样品设计无泄漏。此外，拉伸样品所需的压力将完全由样品刚度决定，样本刚度可能因样品而异，并且随着时间的推移，很难控制样品的拉伸。这种生物反应器将组织工程移植物安装在硅胶管周围，并允许在移植物的外壁上应用壁切变应力 （WSS），并从内部给移植物加压。这样，样品之间和样品内部的同等装载条件可以保证，而且，样品允许泄漏，就像多孔血管支架19一样。这种从内到外的生物反应器是专门用来系统研究剪切和/或拉伸的影响，而不是在体外进行原生血管的工程，而传统的血管生物反应器设置更适合于此。有关生物反应器设计图纸 的图 1A-B 及其相应的 表 1， 了解生物反应器主要组件背后的功能描述和原理。
生物反应器的使用是根据我们小组最近的一系列研究证明的，其中我们调查了剪切应力和循环拉伸对可再吸附电喷支架中炎症和组织形成的个体和组合影响，用于原位心血管组织19、43、44。为此，我们使用单体或共培养中的人类巨噬细胞和肌纤维细胞来模拟原位再生级联的各个阶段。我们已经证明，人类巨噬细胞的细胞因子分泌明显受到循环拉伸和剪切应力的影响，通过parcrine信号和直接接触19、43、44，通过这些脚手架中的人类肌纤维细胞影响基质沉积和组织。值得注意的是，这些研究表明，在剪切应力和拉伸的结合应用的情况下，对组织形成和炎症的影响要么由两个负载之一主导，要么有两个负载的协同效应。这些发现说明了将两个负载脱钩以更好地了解机械环境对 TE 过程的贡献的相关性。这一理解可用于系统地优化相关血液动力负荷系统中的脚手架设计参数。此外，来自这些控制良好的环境的机械数据可以作为正在开发的数字模型的输入，以预测原位组织重塑的过程，正如最近为TEVG65或TEHV66报告的那样，以进一步提高预测能力。

<table><tbody><tr><td>advanced Dulbecco&amp;rsquo;s modified EagleMedium (aDMEM)</td><td>Gibco</td><td>12491-015</td><td>cell culture medium for fibroblasts</td></tr><tr><td>Aqua Stabil</td><td>Julabo</td><td>8940012</td><td>prevent microorganism growth in bioreactor-hydraulic reservoir</td></tr><tr><td>Bovine fibrinogen</td><td>Sigma</td><td>F8630</td><td>to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td></tr><tr><td>Bovine thrombin</td><td>Sigma</td><td>T4648</td><td>to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804</td><td>to spin down cells and conditioned medium</td></tr><tr><td>Clamp scissor - &quot;kelly forceps&quot;</td><td>Almedic</td><td>P-422</td><td>clamp the silicone tubing and apply pre-stretch to the scaffold so the scaffold can be sutured into the engraved groove (autoclave at step 1, step 7)</td></tr><tr><td>CO2 cell culture incubators</td><td>Sanyo</td><td>MCO-170AIC-PE</td><td>for cell culturing</td></tr><tr><td>Compressed air reservoir</td><td>Festo</td><td>CRVZS-5</td><td>smoothing air pressure fluctuations and create time delays for pressure build-up</td></tr><tr><td>Custom Matlab script to calculate the maximum stretches</td><td>Matlab</td><td>R2017. The Mathworks, Natick, MA</td><td>calculate the minimum and maximum outer diameter of the electrospun scaffold</td></tr><tr><td>Data acquisition board</td><td>National Instruments</td><td>BNC-2090</td><td>data processing in between amplifier system and computer</td></tr><tr><td>Ethanol</td><td>VWR</td><td>VWRK4096-9005</td><td>to keep sterile working conditions</td></tr><tr><td>Fetal bovine calf serum (FBS)</td><td>Greiner</td><td>758087</td><td>cell culture medium supplement; serum-supplement</td></tr><tr><td>Flow culture chamber compartments, consisting of a pressure conduit with engraved grooves and small holes to apply pressure on silicone tubing, a screw thread, nose cone, top compartment with flow inlet and bottom compartment flow outlet, adapter bushing</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>flow culture chamber compartments (autoclave at step 1, step 7)</td></tr><tr><td>Glass Pasteur pipet</td><td>Assistant</td><td>HE40567002</td><td>apply vacuum on electrospun scaffold (autoclave at step 1)</td></tr><tr><td>Glass tubes of the flow culture chamber</td><td>Custon made, Equipment &amp;amp; Prototype Center, Eindhoven University of Technology</td><td>n.a.</td><td>part of the flow culture chamber (clean and store in 70% ethanol, at step 1 and 7)</td></tr><tr><td>GlutaMax</td><td>Gibco</td><td>35050061</td><td>cell culture medium amino acid supplement, minimizes ammonia build-up</td></tr><tr><td>High speed camera</td><td>MotionScope</td><td>M-5</td><td>to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td></tr><tr><td>High speed camera lens - Micro-NIKKOR 55mm f/2.8 - lens</td><td>Nikon</td><td>JAA616AB</td><td>to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td></tr><tr><td>Hose clip</td><td>ibidi GmbH</td><td>10821</td><td>block medium flow (autoclave at step 1, step 7)</td></tr><tr><td>Hydraulic reservoir with 8 screw threads for 8 flow culture chambers</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to apply pressure to the silicone mounted constructs (clean outside with a paper tissue with 70% ethanol, rinse reservoir with 70% ethanol followed by demi water, at step 1 and 7)</td></tr><tr><td>Ibidi pump system (8x) including ibidi pump, PumpControl software, fluidic unit, perfusion set (medium tubing), air pressure tubing, drying bottles with orange silica beads</td><td>ibidi GmbH</td><td>10902</td><td>set up used to control the flow in the flow culture chambers. Note 1: the ibidi pumps were modified by the manufacturer to enable 200 mbar capacity. Note 2: can be replaced by pump system of other manufacturer, as long as same flow regimes can be applied.</td></tr><tr><td>Knives (no.10 sterile blades, individual foil pack) and scalpel handle (stainless steel, individually wrapped)</td><td>Swann Morton</td><td>0301; 0933</td><td>to cut the silicone tubing in the correct size for the scaffold and to cut the suture material</td></tr><tr><td>LabVIEW Software</td><td>National Instruments</td><td>version 2018</td><td>to control the stretch applied to the scaffolds</td></tr><tr><td>Laminar flow biosafety cabinet with UV light</td><td>Labconco</td><td>302310001</td><td>to ensure sterile working conditions. The UV is used to decontaminate everything that cannot be autoclaved, or touched after autoclaving</td></tr><tr><td>Large and small petri dishes</td><td>Greiner</td><td>664-160</td><td>for sterile working conditions</td></tr><tr><td>L-ascorbic acid 2-phosphate (vitamin C)</td><td>Sigma</td><td>A8960</td><td>cell culture medium supplement, important for collagen production</td></tr><tr><td>LED light cold source KL2500</td><td>Zeiss</td><td>Schott AG</td><td>to aid in visualization for the time lapse of the scaffolds during monitoring of the stretch</td></tr><tr><td>Luer (female and male) locks and connectors, white luer caps</td><td>ibidi GmbH</td><td>various, see (https://ibidi.com/26-flow-accessories)</td><td>to close or connect parts of the bioreactor and the ibidi pump (autoclave at step 1, step 7)</td></tr><tr><td>Measuring amplifier (PICAS)</td><td>PEEKEL instruments B.V.</td><td>n.a.</td><td>to amplify the signal from the pressure sensor and feedback to LabView</td></tr><tr><td>Medium reservoir (large syringes 60 mL) and reservoir holders</td><td>ibidi GmbH</td><td>10974</td><td>medium reservoir (autoclave at step 1, step 7)</td></tr><tr><td>Medium tubing with 4.25 mm outer diameter and 1 mm inner diameter</td><td>Rubber BV</td><td>1805</td><td>to allow for a larger flow rate, the ibidi medium tubing with larger diameter is used. Note: the part of medium tubing guided through the fluidic unit valves are the same as the default ibidi medium tubing</td></tr><tr><td>Motion Studio Software</td><td>Idtvision</td><td>2.15.00</td><td>to make the high speed time lapse images for stretch monitoring</td></tr><tr><td>Needle (19G)</td><td>BD Microlance</td><td>301700</td><td>together with thin flexible tubing used to fill the hydraulic reservoir with ultrapure water without adding air bubbles</td></tr><tr><td>Needle driver</td><td>Adson</td><td>2429218</td><td>to handle the needle of the nylon suture through the silicone tube (autoclave at step 1, step 7)</td></tr><tr><td>Paper tissues</td><td>Kleenex</td><td>38044001</td><td>for cleaning of the equipment with 70% ethanol</td></tr><tr><td>Parafilm</td><td>Sigma</td><td>P7793-1EA</td><td>quick fix if leakage occurs</td></tr><tr><td>Penicillin/streptomycin (P/S)</td><td>Lonza</td><td>DE17-602E</td><td>cell culture medium supplement; prevent bacterial contamination</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma</td><td>P4417-100TAB</td><td>for storage and washing steps (autoclave at step 1)</td></tr><tr><td>Plastic containers (60 mL) with red screw caps</td><td>Greiner</td><td>206202</td><td>to prepare the fibrinogen solution</td></tr><tr><td>Pneumatic cylinder</td><td>Festo</td><td>AEVC-20-10-I-P</td><td>to actuate the Teflon bellow (clean with a paper tissue with 70% ethanol at step 1 and 7)</td></tr><tr><td>Polycaprolactone bisurea (PCL-BU) tubular scaffolds (3 mm inner diameter, 200 &amp;micro;m wall thickness, 20 mm length)</td><td>SyMO-Chem, Eindhoven, The Netherlands</td><td>n.a.</td><td>produced using electrospinning from 15% (w/w) chloroform (Sigma; 372978) polymer solutions. See Van Haaften et al Tissue Engineering Part C (2018) for more details</td></tr><tr><td>Pressure conduit without holes (for static control)</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to mount electrospun tubes on silicon tubing (autoclave at step 1, step 7)</td></tr><tr><td>Pressure sensor and transducer</td><td>BD</td><td>TC-XX and P 10 EZ</td><td>the air pressure going to the pneumatic actuated pump is raised until it reaches the set pressure</td></tr><tr><td>Proportional air pressure control valve and pressure sensor</td><td>Festo</td><td>MPPES-3-1/8-2-010, 159596</td><td>provides compressed air to the pneumatic actuated pump</td></tr><tr><td>Roswell Park Memorial Institute 1640 (RPMI-1640)</td><td>Gibco</td><td>A1049101</td><td>cell culture medium for monocyte/macrophage</td></tr><tr><td>Safe lock Eppendorf tubes (1.5 mL)</td><td>Eppendorf</td><td>30120086</td><td>multiple applications (autoclave at step 1)</td></tr><tr><td>Sodium dodecyl sulfate solution 20%</td><td>Sigma</td><td>5030</td><td>Used to clean materials, at a concentration of 0.1%.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Silicone O-rings</td><td>Technirub</td><td>1250S</td><td>to prevent leakage (autoclave at step 1, step 7)</td></tr><tr><td>Silicone tubing (2.8 mm outer diameter, 400 um wall thickness)</td><td>Rubber BV</td><td>1805</td><td>to mount the electrospun tubes on the pressure conduits (autoclave at step 1)</td></tr><tr><td>Sterile tube (15 mL)</td><td>Falcon</td><td>352095</td><td>multiple applications</td></tr><tr><td>Suture, 5-0 prolene with pre-attached taper point needle</td><td>Ethicon, Johnson&amp;amp;Johnson</td><td>EH7404H</td><td>Prolene suture wire 5-0 (75cm length, TF taper point needle, 1/2 circle, 13 mm needle length)</td></tr><tr><td>Syringe (24 mL)</td><td>B. Braun Melsungen AG</td><td>2057932</td><td>to add the ultrapure water or medium to the hydraulic reservoir or flow culture chamber</td></tr><tr><td>Syringe filter (0.2 &amp;micro;m)</td><td>Satorius</td><td>17597-K</td><td>to filter the fibrinogen solution</td></tr><tr><td>T150 cell culture flask with filter cap</td><td>Nunc</td><td>178983</td><td>to degas culture medium</td></tr><tr><td>T75 Cell culture flask with filter cap</td><td>Nunc</td><td>156499</td><td>to culture static control samples</td></tr><tr><td>Teflon bellow</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to load the hydraulic reservoir (clean outside with a paper tissue with 70% ethanol at step 1 and 7)</td></tr><tr><td>Tray (stainless steel)</td><td>PolarWare</td><td>15-248</td><td>for easy transport of the fluidic culture chambers and the bioreactor from incubator to laminar flow cabinet and back (clean with a paper tissue with 70% ethanol before and after use)</td></tr><tr><td>Tweezers</td><td>Wironit</td><td>4910</td><td>sterile handling of individual parts (autoclave at step 1 and 7)</td></tr><tr><td>Ultrapure water</td><td>Stakpure</td><td>Omniapure UV 18200002</td><td>to correct for medium evaporation, mixed with aqua stabil mixed and used as hydraulic fluid. (autoclave ultrapure water at step 1)</td></tr><tr><td>UV light</td><td>Philips</td><td>TUV 30W/G30 T8</td><td>for decontamination of grafts and bioreactor parts before seeding</td></tr></tbody></table>

a multi-cue bioreactor to evaluate the inflammatory and regenerative capacity of biomaterials under flow and stretch

从转化的角度来看，使用可再吸附生物材料直接在体内诱导再生是一种有吸引力的策略。这种材料在植入时会引起炎症反应，这是随后材料吸收和新组织再生的驱动因素。这一战略，也被称为原位组织工程，旨在获得心血管置换，如组织工程血管移植。炎症和再生过程都由脚手架上的局部生物力学线索（即拉伸和剪切应力）决定。在这里，我们详细描述了定制开发的生物反应器的使用，这种反应器能够使管状脚手架上的拉伸和剪切应力脱钩。这允许系统和标准化地评估管状脚手架在控制良好的机械负载的影响下的炎症和再生能力，我们利用人类巨噬细胞和肌纤维细胞进行动态共培养实验来证明这一点。详细讨论了该方法的关键实际步骤:生物反应器的建造和建立、脚手架和细胞播种的制备、拉伸和剪切流的应用和维护以及样品采集分析。

该生物反应器的开发是为了研究剪切应激和循环拉伸对血管组织生长和3D生物材料脚手架改造的个体和组合效应。生物反应器的设计允许在各种装载条件下培养多达8个血管结构（图1A）。血管结构位于流动培养室（图1B），其中周长伸展和WSS都可以独立控制。流文化室的顶部隔间持有一个流直发器，以相对较短的沉降长度（图1C）稳定流。直流直发器的下游，鼻锥通过环形通道均匀地分配流量（图 1D）。当协议的所有步骤都以正确的方式执行时，流培养室中的血管支架可以通过脚手架和玻璃墙之间的环形通道连续单向流动，并由气动泵绕行（图 1E F）。在安装脚手架之前，电喷管应切成25毫米管（图1G），并可用SEM进行检查，以分析微结构（图1H-I）。请务必注意，本示例中的 PCL-BU 移植物可以由任何其他弹性组织工程移植（自然或合成产地、不同的微结构或孔隙性）所取代。从内到外的设计允许测试高孔隙移植物，因为它不一定非必须无泄漏。流文化室的示意图图显示了对方程中用于描述WSS（方程1）、压力梯度（方程2）和周长拉伸（方程3）（图1J）中所用参数的物理解释。
对协议关键步骤执行不当可能导致一些情况。例如，由于安装错误结，液压储层可能会发生泄漏，导致液压液从压力管道泄漏，孔通过硅胶管并进入流培养室（图2A）。当硅胶环位置不好或在实验前一天不允许特氟隆波纹管稍微膨胀 O/N 时，螺纹管和生物反应器基座连接的液压油也会泄漏（图 2B）。此外，当介质不脱气，或者如果中型储层中的中等水平不均衡，一个中型储层干涸，导致空气被吸入系统，则流培养室（图2C）中可能会出现气泡，从而干扰WSS拍子，损害细胞的生存能力和随后的组织生长。最后，当底部隔间中的硅胶 O 环放置不正确时，可在流培养室（图 2D）下方观察到中等溢出。
由于这种生物反应器设置允许应用单个和组合的血液动力载荷，多个血液机加载实验组可以包括在一个实验（图3A）。以前，不同的血液动力负荷（即两个剪切应力系统和两个拉伸系统）通过应用各种可能的系统设置（图 3B）得到验证。当拉伸 （图 3C）和 WSS （图 3D）在长期培养期间进行监测时，可以验证这些时间可以保持在相对恒定的水平，持续长达 20 天。
生物反应器特别适合研究血液同位素加载对生长和改造的影响，在原位血管TE上下文。原位TE的阶段被假定为反映自然伤口愈合反应的阶段（图4A）。如这里所述，单细胞衍生巨噬细胞和从人类下垂静脉中提取的肌纤维细胞的共同培养物被确定为体外模拟增殖阶段。播种三天后，免疫荧光染色显示两种细胞类型在整个脚手架上均匀分布（图4B）。经过20天的共培养，仅循环拉伸就产生了更多更厚的胶原蛋白I型纤维的沉积，而在具有混合血态负荷的组中，循环拉伸的这种效应被剪切应力所压倒，导致胶原蛋白I型沉积不那么明显，免疫荧光染色（图4C）就是例证。要成功地进行原位组织再生，需要在组织生产和脚手架吸收之间取得紧密的平衡。除了组织形成，生物反应器还允许细胞驱动的脚手架吸收感应。例如，在电喷变移植物上培养8天的巨噬细胞单一培养时，在所有血液机载荷系统中观察到纤维侵蚀和纤维裂口，静态组的吸收最明显，剪应力组的吸收最少（图4D）。这些结果表明，不同的血液动力载重系统对生长和改造的影响。这些见解有助于优化新开发的原地 TEVG 的设计参数。
组织再生过程的另一个重要决定因素是亲炎和抗炎细胞因子的存在。由于生物反应器是一个闭环系统，系统中的细胞将不断暴露在分泌因子的寄生虫刺激下。早期和后期对人类外周血单核细胞（PBMC）衍生巨噬细胞和人类肌纤维细胞的动态加载共生介质中的细胞因子分泌剖面进行了分析（图5A）。这些具有代表性的结果说明了循环拉伸和剪切应力对共同培养中细胞因子分泌特征的影响。有趣的是，两个负载的组合效应显示了两个负载之一的主导地位（例如，白细胞介素-6（IL-6）和单细胞化疗性蛋白质-1（MCP-1）的循环拉伸）或两个负载的协同效应（例如 IL-10）（图 5B）。这些见解通过这个体外测试平台收集，为基于大噬菌体驱动的原位组织再生原理的原位 TEVG 的发展提供了宝贵的信息。
巨噬细胞和肌纤维细胞的共同培养实验表明，机械环境和由此产生的依赖负荷的炎症环境调节了肌纤维细胞的表型。经过20天的血液动力学加载，肌纤维细胞标记的基因表达在加载的个体和组合影响上以及巨噬细胞对肌纤维细胞的直接和寄生虫信号（图6A）上表现出明显的差异。此外，收缩标记α平滑肌肉作用素的基因表达模式与蛋白质合成相关（图6B）。此外，循环拉伸刺激胶原蛋白和弹性基质基因表达和衰减矩阵金属蛋白酶1/组织抑制剂基质金属蛋白酶1介导胶原蛋白重塑，而剪切应激的稳定效果观察到共培养（图6C）。这些长期共培养实验表明，有可能研究后期组织重塑阶段（图4A）在组织工程血管移植与这种生物反应器的各种血液同位素加载系统。由于 TEVG 安装在硅胶管上"从内到外"，因此圆形拉伸和 WSS 可以应用于更长的文化周期。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61824/61824fig01.jpg"> 图1:生物反应器的设计和概述。（A） 生物反应器基座的施工图纸和（B） 流体培养室的爆炸视图，所有部分都指示（步骤 2.4/2.8）。流文化室的顶舱有一个流直发器。（C） 球形钝化鼻锥位于流直器后，通过环形通道（以粉红色表示的流向）分配流。（D） 这些组件共同控制和引导流动方向。有关上述各个部分的功能描述，请参阅表 1。（E） 完整的生物反应器设置（第5.2步）和（F）流培养室和气动气缸的特写视图（第5.6.1步）。（G） 播种前电喷PP伦-BU脚手架的毛外观（标尺滴答声1毫米）。扫描管状电子弹头PCL-BU脚手架的电子微观图像，内径为3毫米，平均纤维直径为5微米，放大倍数（H）100微米，（I）10微米（步骤1.3）。（J） 文化室的示意图图像，由管状电喷支架组成，外半径（r1）以内半径玻璃管（r2）为中心。流（Q） 入口和插座连接到环形环，通道高度h用于应用壁切应力（t）。压力/拉伸（P）入口连接到硅胶安装的脚手架，用于从内部（第 6.1 步）向安装的电子弹脚手架施加环状拉伸（+（t）。缩写:多丙酮双磺酸（PCL-BU）。C组、D组和G-J组改编自范·哈夫滕等人19。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61824/61824fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>生物反应器部件</td>
			<td>功能描述</td>
		</tr>
<tr>
<td colspan="2">拉伸应用程序</td>
		</tr>
<tr>
<td>气缸</td>
			<td>激活特氟隆波纹管。</td>
		</tr>
<tr>
<td>特氟隆波纹管</td>
			<td>装载水力储层。</td>
		</tr>
<tr>
<td>水力储层</td>
			<td>可连接多达8个流文化室，充满德米水，对硅胶安装结构施加压力。</td>
		</tr>
<tr>
<td>螺纹</td>
			<td>流培养室与水库之间的连接。硅胶安装结构的压力入口。</td>
		</tr>
<tr>
<td>白色吕尔连接器</td>
			<td>用于拧紧流培养室，拧紧到液压储层/生物反应器基座上的八根螺丝线之一。</td>
		</tr>
<tr>
<td>带小孔的压力导管</td>
			<td>与水库水直接相连。当液压储层加压时，压力管用水填充硅胶管（安装在压力管上）之间的空间，将硅胶管向外推，导致硅胶安装移植物从内部绕行。</td>
		</tr>
<tr>
<td>硅胶管</td>
			<td>将电喷管移植到压力导管上。硅胶管从内部呈环状拉伸。</td>
		</tr>
<tr>
<td colspan="2">流应用</td>
		</tr>
<tr>
<td>流泵系统</td>
			<td>用于控制流培养室中的流。（以我们为例:使用了宜必迪泵系统。</td>
		</tr>
<tr>
<td>顶部和底部带流口和出口的隔间</td>
			<td>将流培养室连接到流环中。</td>
		</tr>
<tr>
<td>玻璃管</td>
			<td>在中心包含加压脚手架，并允许灌注脚手架。</td>
		</tr>
<tr>
<td>流直发器</td>
			<td>在相对较短的沉降长度内稳定流量。</td>
		</tr>
<tr>
<td>鼻锥</td>
			<td>均匀地分配流量。</td>
		</tr>
<tr>
<td>适配器衬里</td>
			<td>修理玻璃管。</td>
		</tr>
<tr>
<td>硅胶 O 环</td>
			<td>防止介质泄漏。</td>
		</tr>
</tbody></table>表1:生物反应器主要特征的功能描述，与图1A-D中的指示部分相对应。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61824/61824fig02.jpg"> 图2:协议中关键步骤执行不准确的结果。在生物反应器设置中，如果关键步骤没有以正确的方式执行时，可以观察到某些事件的照片。（A） 如果结不够紧或未完全放置在刻有凹槽（箭头表示），则可能发生液压液向流文化室的轻微泄漏（第 2.5 步）。（B） 如果在实验前一天或硅胶环位置不好时，不允许特氟隆波纹管膨胀O/N，则螺丝线与生物反应器基座连接的液压液泄漏可能发生（箭头表示）（步骤1.4和5.2.3）。（C） 流动培养室中的气泡（箭头表示）会导致剪切应力模式紊乱。始终淡化培养介质，确保中型水库的中层保持平衡，防止一个水库干涸，空气被吸入流量室系统（步骤 1.2 和 5.5.4）。（D） 当底部隔间中的硅胶环放置不当时，可以观察到介质的溢出（箭头表示）（步骤 2.4.2）。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61824/61824fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61824/61824fig03.jpg"> 图3:控制剪切应力和伸展。由于生物反应器允许独立和组合地应用拉伸和剪切，（A）多个实验组可以包含在一个实验中。（B） 在四个不同的系统设置（由颜色表示）下测试的特定时间点的最大拉伸和剪切压力变化示例。黑色矩形表示每个设置的测量值的平均±标准偏差。虚线被计算为拉伸（水平线）和剪切（垂直线）的均值，以指示四个不同的装载条件。（C） 循环环状伸展在循环伸展和组合组在20天的实验过程中，基于外径测量的脚手架结构监测与高速相机（步骤6.2）的延时。（D） 根据注射器中介质水平的变化（第6.1步），在实验过程中监测剪切应力和组合组。A、C和D小组改编自范哈芬和威辛等人44:B面板改编自范哈夫滕等人19。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61824/61824fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61824/61824fig04.jpg"> 图4:原位血管组织工程、细胞分布、组织生产和脚手架退化的概念。（A） 一个示意图，描绘了主机功能部位脚手架驱动组织再生的假设阶段。所展示的结果来自专注于增殖阶段的实验，其中巨噬细胞和产生组织的细胞已经殖民了脚手架材料。（B） 来自人类肺静脉（红色）和PBMC衍生巨噬细胞（绿色）的肌纤维细胞在第3天在电子喷射脚手架（灰色）的外侧分布。比例杆 200 μm.（C）共同培养构造的横截面在第 20 天染色胶原蛋白类型 I （绿色）， 胶原蛋白类型 III （红色） 和 DAPI （白色） 。缩放杆 100 μm，*表示结构的外侧，对应流侧。（D） 8天大噬细胞单一栽培的去细胞化移植的SEM图像，显示巨噬细胞退化:比例尺杆 20 μm。缩写:人类下静脉细胞（HVSC）:外周血单核细胞（PBMC）:4+，6-迪亚米迪诺-2-苯林多勒（DAPI）:扫描电子显微镜 （SEM）。A小组改编自威辛和博尼托等人27:B组和C组改编自范哈夫滕和威辛等人44:和面板D改编自威辛等人43。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61824/61824fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61824/61824fig05.jpg"> 图5:3天20天共文化构造的血液负荷炎症环境。人类PBMC衍生巨噬细胞和人类肌纤维细胞从下垂静脉的共同培养。（A） 通过多路复用 ELISA 在超纳坦中测量的细胞因子总分泌的热图。（B） 第20天选择细胞因子的盒图，标准化为总DNA含量。P值是使用克鲁斯卡尔-瓦利斯测试与邓恩的多重比较测试计算的;* p < 0.05， ** p < 0.01， * 缩写:金属蛋白酶（TIMP）、基质金属蛋白酶（MMP）、白细胞间蛋白（IL）、单细胞化疗提取蛋白1（MCP-1）、转化生长因子β1（T）的组织抑制剂 GF-β1）、结缔组织生长因子（CTGF）、肿瘤坏死因子α（TNF-α）、血小板衍生生长因子（PDGF）、静态（ST）、循环拉伸（CS）、剪切应激（SS）。A组和B组由范·哈芬和威辛等人改编而成。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61824/61824fig05large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61824/61824fig06.jpg"> 图6:肌纤维细胞表型的变化和矩阵生长和改造标记的变化，以响应血管结构中的血液同位素负荷在第20天。（A） 肌纤维细胞特异性表型标记的相对基因表达。（B） 交叉部分染色为 αSMA （绿色） 和 DAPI （蓝色）， 比例栏 100 μm.（C） 与胶原体基质、弹性基质、蛋白糖和重塑基因相关的基因的相对表达。P值是使用克鲁斯卡尔-瓦利斯测试与邓恩的多重比较测试计算的;* p < 0.05， ** p < 0.01， *** p < 0.001。缩写: S100 钙结合蛋白 A4 （S100A4），α 平滑肌肉作用素 （+SMA 或 ACTA2）， 钙素 1 （CNN1）， 平滑素 （SMTN）， 维他汀 （VIM）， 胶原蛋白 I （COL1A1）， 弹性蛋白 （ELN）， 维西坎 （VCAN）， 基质金属蛋白酶 （MMP）， 金属蛋白酶 （TIMP） 组织抑制剂， 静态 （ST）， 环状拉伸 （CS）， 剪切应力 （SS）， 4+，6-二苯丙酸酯 （DAPI）.# 在检测极限或低于检测限值时测量。A、B、C小组由范哈芬和威辛等人改编而成。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61824/61824fig06large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
补充图S1:蛋白质表达肌纤维细胞-和巨噬细胞-单一栽培受个人和组合血液动力负荷。（A） 肌纤维细胞代表共聚焦图像，用actin纤维（绿色）、核（红色）和脚手架（蓝色）培养了10天，与无法观察到优先作用纤维方向的静态样品相比，在装载的样品中显示出清晰的ACTIN纤维方向。比例杆 50 μm.（B） 焦化图像相同的肌纤维细胞单一栽培染色胶原蛋白 （绿色） 和核/脚手架 （白色） 。比例尺 50 μm。（C） 静态和动态培养THP1衍生巨噬细胞的蛋白质分泌图的盒状图，根据IL-6、TNF-α、MCP-1（抗炎）和IL-10、IL-13、MMP-9（抗炎）的细胞因子分泌水平计算M1/M2比率。MCP-1 图中的点表示统计离群值。（D） ELISA 数据与支持炎症、抗炎、生长和重塑蛋白质的平均分泌相比，在第 8 天静态和动态培养巨噬细胞的相对蛋白质分泌水平（蛋白质水平针对每组平均 DNA 含量进行了更正）。点和阴影区域分别表示第 50 和第25个百分位。缩写:单细胞化疗提取蛋白1（MCP-1）、白细胞间蛋白（IL）、转化生长因子β（TGF-β）、基质金属蛋白酶（MMP）、血小板衍生生长 因子 （PDGF）， 结缔组织生长因子 （CTGF）， 肿瘤坏死因子α （TNF-α）， 酶链接免疫吸附剂检测 （ELISA.* p < 0.05;** p < 0.01， *** p < 0.001。A组和B组改编自范·哈夫滕等人19。C组和D组是根据威辛等人43号改编的。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61824/61824supfig01.jpg" target="_blank">请点击这里下载此图。</a>

Watch this Scientific Journal Video about A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch at JoVE.com

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

本协议的目标是在管状电喷脚手架中执行人类巨噬细胞和肌纤维细胞的动态共生，以研究物质驱动的组织再生，使用生物反应器，使剪切应力和循环拉伸脱钩。

评估流和拉伸下生物材料的炎症和再生能力的多奎生物反应器

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

a-multi-cue-bioreactor-to-evaluate-inflammatory-regenerative-capacity

Nanyang Technological University

Research

JoVE Journal

Engineering

5.5K 次观看.  Eindhoven University of Technology. 本协议的目标是在管状电喷脚手架中执行人类巨噬细胞和肌纤维细胞的动态共生，以研究物质驱动的组织再生，使用生物反应器，使剪切应力和循环拉伸脱钩。

视频: A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

基于生物工程，人造血管使用脉动生物反应器

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

主动脉环共培养法: 一种方便的工具, 可以评估骨髓间质细胞的血管生成潜能体外

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

发育生物学

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

多器官芯片 - 一种微流体平台的长期多组织共培养

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

骨再生生物材料的生物相容性曲线

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

设计生物反应器以改善工程心脏组织的数据采集和模型通量

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

用于灌注血管离体培养的新型 3D 打印生物反应器

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

作者聚焦:EasyFlow - 一种适用于大型血管培养的经济且适应性强的灌流生物反应器 

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

离体 在3D打印生物反应器中对大血管进行灌注培养

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...

评估流和拉伸下生物材料的炎症和再生能力的多奎生物反应器

摘要

探索更多视频

此视频中的章节

基于生物工程，人造血管使用脉动生物反应器

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

静电纤维组织工程的后期处理

组织工程生物反应器的双轴机械装载设计

主动脉环共培养法: 一种方便的工具, 可以评估骨髓间质细胞的血管生成潜能体外

多器官芯片 - 一种微流体平台的长期多组织共培养

工程三维细胞化胶原凝胶的血管组织再生

骨再生生物材料的生物相容性曲线

设计生物反应器以改善工程心脏组织的数据采集和模型通量

离体在3D打印生物反应器中对大血管进行灌注培养

评估流和拉伸下生物材料的炎症和再生能力的多奎生物反应器

摘要

探索更多视频

此视频中的章节

基于生物工程，人造血管使用脉动生物反应器

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

静电纤维组织工程的后期处理

组织工程生物反应器的双轴机械装载设计

主动脉环共培养法: 一种方便的工具, 可以评估骨髓间质细胞的血管生成潜能体外

多器官芯片 - 一种微流体平台的长期多组织共培养

工程三维细胞化胶原凝胶的血管组织再生

骨再生生物材料的生物相容性曲线

设计生物反应器以改善工程心脏组织的数据采集和模型通量

离体 在3D打印生物反应器中对大血管进行灌注培养

离体在3D打印生物反应器中对大血管进行灌注培养