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。
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。
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。
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?…
The authors have nothing to disclose.
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”。
advanced Dulbecco’s modified EagleMedium (aDMEM) | Gibco | 12491-015 | cell culture medium for fibroblasts |
Aqua Stabil | Julabo | 8940012 | prevent microorganism growth in bioreactor-hydraulic reservoir |
Bovine fibrinogen | Sigma | F8630 | to prepare fibrinogen gel to seed the cells on the electrospun scaffold |
Bovine thrombin | Sigma | T4648 | to prepare fibrinogen gel to seed the cells on the electrospun scaffold |
Centrifuge | Eppendorf | 5804 | to spin down cells and conditioned medium |
Clamp scissor – "kelly forceps" | Almedic | P-422 | 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) |
CO2 cell culture incubators | Sanyo | MCO-170AIC-PE | for cell culturing |
Compressed air reservoir | Festo | CRVZS-5 | smoothing air pressure fluctuations and create time delays for pressure build-up |
Custom Matlab script to calculate the maximum stretches | Matlab | R2017. The Mathworks, Natick, MA | calculate the minimum and maximum outer diameter of the electrospun scaffold |
Data acquisition board | National Instruments | BNC-2090 | data processing in between amplifier system and computer |
Ethanol | VWR | VWRK4096-9005 | to keep sterile working conditions |
Fetal bovine calf serum (FBS) | Greiner | 758087 | cell culture medium supplement; serum-supplement |
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 | Custom made, Department of Biomedical Engineering, Eindhoven University of Technology | n.a. | flow culture chamber compartments (autoclave at step 1, step 7) |
Glass Pasteur pipet | Assistant | HE40567002 | apply vacuum on electrospun scaffold (autoclave at step 1) |
Glass tubes of the flow culture chamber | Custon made, Equipment & Prototype Center, Eindhoven University of Technology | n.a. | part of the flow culture chamber (clean and store in 70% ethanol, at step 1 and 7) |
GlutaMax | Gibco | 35050061 | cell culture medium amino acid supplement, minimizes ammonia build-up |
High speed camera | MotionScope | M-5 | 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) |
High speed camera lens – Micro-NIKKOR 55mm f/2.8 – lens | Nikon | JAA616AB | 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) |
Hose clip | ibidi GmbH | 10821 | block medium flow (autoclave at step 1, step 7) |
Hydraulic reservoir with 8 screw threads for 8 flow culture chambers | Custom made, Department of Biomedical Engineering, Eindhoven University of Technology | n.a. | 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) |
Ibidi pump system (8x) including ibidi pump, PumpControl software, fluidic unit, perfusion set (medium tubing), air pressure tubing, drying bottles with orange silica beads | ibidi GmbH | 10902 | 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. |
Knives (no.10 sterile blades, individual foil pack) and scalpel handle (stainless steel, individually wrapped) | Swann Morton | 0301; 0933 | to cut the silicone tubing in the correct size for the scaffold and to cut the suture material |
LabVIEW Software | National Instruments | version 2018 | to control the stretch applied to the scaffolds |
Laminar flow biosafety cabinet with UV light | Labconco | 302310001 | to ensure sterile working conditions. The UV is used to decontaminate everything that cannot be autoclaved, or touched after autoclaving |
Large and small petri dishes | Greiner | 664-160 | for sterile working conditions |
L-ascorbic acid 2-phosphate (vitamin C) | Sigma | A8960 | cell culture medium supplement, important for collagen production |
LED light cold source KL2500 | Zeiss | Schott AG | to aid in visualization for the time lapse of the scaffolds during monitoring of the stretch |
Luer (female and male) locks and connectors, white luer caps | ibidi GmbH | various, see (https://ibidi.com/26-flow-accessories) | to close or connect parts of the bioreactor and the ibidi pump (autoclave at step 1, step 7) |
Measuring amplifier (PICAS) | PEEKEL instruments B.V. | n.a. | to amplify the signal from the pressure sensor and feedback to LabView |
Medium reservoir (large syringes 60 mL) and reservoir holders | ibidi GmbH | 10974 | medium reservoir (autoclave at step 1, step 7) |
Medium tubing with 4.25 mm outer diameter and 1 mm inner diameter | Rubber BV | 1805 | 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 |
Motion Studio Software | Idtvision | 2.15.00 | to make the high speed time lapse images for stretch monitoring |
Needle (19G) | BD Microlance | 301700 | together with thin flexible tubing used to fill the hydraulic reservoir with ultrapure water without adding air bubbles |
Needle driver | Adson | 2429218 | to handle the needle of the nylon suture through the silicone tube (autoclave at step 1, step 7) |
Paper tissues | Kleenex | 38044001 | for cleaning of the equipment with 70% ethanol |
Parafilm | Sigma | P7793-1EA | quick fix if leakage occurs |
Penicillin/streptomycin (P/S) | Lonza | DE17-602E | cell culture medium supplement; prevent bacterial contamination |
Phosphate Buffered Saline (PBS) | Sigma | P4417-100TAB | for storage and washing steps (autoclave at step 1) |
Plastic containers (60 mL) with red screw caps | Greiner | 206202 | to prepare the fibrinogen solution |
Pneumatic cylinder | Festo | AEVC-20-10-I-P | to actuate the Teflon bellow (clean with a paper tissue with 70% ethanol at step 1 and 7) |
Polycaprolactone bisurea (PCL-BU) tubular scaffolds (3 mm inner diameter, 200 µm wall thickness, 20 mm length) | SyMO-Chem, Eindhoven, The Netherlands | n.a. | 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 |
Pressure conduit without holes (for static control) | Custom made, Department of Biomedical Engineering, Eindhoven University of Technology | n.a. | to mount electrospun tubes on silicon tubing (autoclave at step 1, step 7) |
Pressure sensor and transducer | BD | TC-XX and P 10 EZ | the air pressure going to the pneumatic actuated pump is raised until it reaches the set pressure |
Proportional air pressure control valve and pressure sensor | Festo | MPPES-3-1/8-2-010, 159596 | provides compressed air to the pneumatic actuated pump |
Roswell Park Memorial Institute 1640 (RPMI-1640) | Gibco | A1049101 | cell culture medium for monocyte/macrophage |
Safe lock Eppendorf tubes (1.5 mL) | Eppendorf | 30120086 | multiple applications (autoclave at step 1) |
Sodium dodecyl sulfate solution 20% | Sigma | 5030 | Used to clean materials, at a concentration of 0.1%. |
Silicone O-rings | Technirub | 1250S | to prevent leakage (autoclave at step 1, step 7) |
Silicone tubing (2.8 mm outer diameter, 400 um wall thickness) | Rubber BV | 1805 | to mount the electrospun tubes on the pressure conduits (autoclave at step 1) |
Sterile tube (15 mL) | Falcon | 352095 | multiple applications |
Suture, 5-0 prolene with pre-attached taper point needle | Ethicon, Johnson&Johnson | EH7404H | Prolene suture wire 5-0 (75cm length, TF taper point needle, 1/2 circle, 13 mm needle length) |
Syringe (24 mL) | B. Braun Melsungen AG | 2057932 | to add the ultrapure water or medium to the hydraulic reservoir or flow culture chamber |
Syringe filter (0.2 µm) | Satorius | 17597-K | to filter the fibrinogen solution |
T150 cell culture flask with filter cap | Nunc | 178983 | to degas culture medium |
T75 Cell culture flask with filter cap | Nunc | 156499 | to culture static control samples |
Teflon bellow | Custom made, Department of Biomedical Engineering, Eindhoven University of Technology | n.a. | to load the hydraulic reservoir (clean outside with a paper tissue with 70% ethanol at step 1 and 7) |
Tray (stainless steel) | PolarWare | 15-248 | 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) |
Tweezers | Wironit | 4910 | sterile handling of individual parts (autoclave at step 1 and 7) |
Ultrapure water | Stakpure | Omniapure UV 18200002 | to correct for medium evaporation, mixed with aqua stabil mixed and used as hydraulic fluid. (autoclave ultrapure water at step 1) |
UV light | Philips | TUV 30W/G30 T8 | for decontamination of grafts and bioreactor parts before seeding |