缩微和3D微血管大会
Summary
该原稿提出了一种注射模制方法来设计该概括内皮生理特性微血管。基于微流体过程创建具有可调整的条件，如流量，细胞组成，几何形状和生化梯度专利3D血管网络。制造过程和潜在的应用例子中描述。
Abstract
体外平台研究血管内皮细胞和血管生物学很大程度上限于2D内皮细胞培养物，与聚合物或基于玻璃基板，和基于水凝胶的管形成测定流动腔室。这些测定，而信息量大，不概括腔的几何形状，正确的细胞外基质，和多细胞的接近，这在调节血管功能中发挥关键作用。该原稿描述了一种注射成型方法，以产生直径为100μm量级的工程化血管。微血管被嵌入的本地I型胶原凝胶内的微流体通道播种内皮细胞制造。通过通道形成之前胶原基质内掺入实质细胞，组织特异性微环境进行建模和分析。流体力学性质和介质组合物的附加的调制允许对所需的微环境内复杂的血管功能的控制。这个平台允许血管周围细胞募集，血内皮细胞相互作用，流动响应，并组织微血管相互作用的研究。工程改造微血管提供来隔离从血管利基的单个组件的影响的能力，并精确地控制其化学，机械和生物性质，研究血管生物学两个健康和疾病。
Introduction
各器官微血管帮助定义组织微环境，保持组织的平衡和调节炎症反应，透气性，血栓形成，纤溶1,2。的微血管内皮，尤其是血流和周围组织并因此之间的界面起着响应于刺激如流体动力和循环细胞因子和激素3调制血管和器官功能起关键作用– 5。理解内皮，血液之间的详细的相互作用，与周围组织的微环境是血管生物学和疾病进展的研究很重要。然而，在研究这些相互作用的进展已经阻碍通过体外工具不体内微血管结构概括和功能6,7-限制。其结果是，外地和治疗的进步主要依赖昂贵和耗时消费的动物模型，往往不能转化为人类的8成功– 10。而在体内模型中的疾病机制和血管功能的研究非常宝贵的，它们是复杂的，且往往缺乏个体细胞，生化的精确控制，和生物物理提示。
血管遍布全身拥有与扩张毛细血管床结合了成熟的分层结构，提供优化的灌注和养分运输同时11。最初，血管的形式作为早期发育过程中12,13整理到一个层次支网络原始丛。尽管许多参与这些过程的信号的充分理解14 – 16日 ，它的血管形成图案是如何这样确定的15仍然难以捉摸。反过来， 在体外扼要这个过程中为工程师举办的血管网络具有蜂Ñ 困难的。许多现有体外平台建模脉管，如二维内皮细胞培养物，缺乏如多细胞接近度，三维管腔几何形状，流量，和细胞外基质的重要特征。在三维水凝胶管形成测定（胶原或纤维蛋白）17 – 19或入侵检测20,21已被用于研究在3D和它们之间的相互作用内皮功能与其他血管17,22或组织的细胞类型23。然而，组装在这些测定流明缺乏互连性，血流动力学流动，以及适当的灌注。此外，对于在这些管形成测定24血管退化的倾向防止长期培养和成熟这限制了可以进行功能性研究的程度。因此，存在一个新兴需要工程师体外微血管网络，可以适当地进行建模连接的平台内皮特性，并能够长期培养的。
各种血管的工程技术已经出现多年来为医疗应用，以取代或患有血管疾病旁路血管的影响。如聚乙烯对苯二甲酸酯（PET），和聚四氟乙烯（ePTFE）由合成材料制成的大直径容器已经与长期通畅（平均95％的通畅超过5年）25相当的治疗成功。虽然小直径合成移植物（<6毫米）通常面临的并发症，如内膜增生和血栓形成26 – 28日 ，组织工程与生物材料制成的小直径移植取得了显著的进展29,30。尽管有这样的进步，在微尺度工程船仍然是一个挑战。到微血管充分模拟，有必要产生具有萨夫复杂网络模式够的机械强度，以保持通畅，并用基质组合物，其允许为实质细胞和细胞重塑既养分渗透。
该协议提出了一种新的人工血管perfusable网络模拟体内的天然具有可调可控的微环境设置31 – 34。所描述的方法生成的直径为100微米量级上的工程微血管。工程微血管由通过嵌入软I型胶原蛋白凝胶中的微流体通道灌流内皮细胞制造。该系统具有生成具有开放的管腔结构图案网络，复制的多细胞相互作用，调节细胞外基质成分，并应用生理学相关的血流动力学部队的能力。
Protocol
1.图案聚二甲基硅氧烷（PDMS）与网络设计的微细 晶圆制造到创造的网络设计的负模板 创建使用任何计算机辅助设计（CAD）软件的网络格局。确保入口和出口之间的对角线尺寸匹配在未来的步骤（见2.1.1）的壳体装置的入口和出口储槽之间的距离。 注意：本身是定义这取决于用户的具体的研究目标的图案的设计（ 见图1，例如图案）。 生成使用高…
Representative Results
该工程船平台创建嵌入式天然I型胶原基质内的微血管功能，并允许进行体外细胞，生物物理和生物化学环境的严格控制。为了制造工程改造的微血管，人脐静脉内皮细胞（HUVECs）通过胶原包埋微流体网络，其中它们连接，以形成一个专利腔和汇合内皮灌注。如在图1A-C中所示，容器的几何形状可以被专门设计用于回答有关的流速，扭曲的问题，和分支角…
Discussion
改造的微血管是一个体外模型，其中诸如鲁米几何形状，流体动力，和多细胞相互作用生理特性是存在和可调。这种类型的平台的是，它提供模拟和在各种情况下，其中在体外培养条件可以匹配，有关的微环境的研究内皮行为的能力强大。例如，驾驶内皮过程，如血管生成的机制，已知在不同器官和在不同的病理状态，例如健康和疾病44不同发生。出于这个原因，平面内皮细…
Disclosures
The authors have nothing to disclose.
Acknowledgements
笔者想承认Lynn和迈克·加维成像实验室的研究所干细胞与再生医学，以及在华盛顿大学华盛顿纳米加工设施。他们也承认美国国立卫生研究院的资助授予DP2DK102258（YZ到），以及培训补助T32EB001650（SSK来和MAR）和T32HL007312（以MAR）。
Materials
| Wafer Fabrication | |||
| AutoGlow Plasma System | AutoGlow | ||
| Headway Spin Coater | Headway Research, Inc | PWM32 Spin Coater | |
| ABM Contact Aligner | AB-M | ||
| Alpha Step Profilometer | Tencor | Alpha Step 200 | |
| SU-8 Developer | Microchem | Y020100 | |
| SU-8 Resist | Microchem | SU-8 2000 | |
| 8" silicon wafer | Wafer World Inc. | ||
| Tabletop Micro Pattern Generator | Heidelberg Instruments | μPG 101 | For generation of photomask |
| Hot plate | VWR | 97042-646 | |
| Ispropyl alcohol | Avantor Performance Materials | 9088 | |
| Petri dishes (120 x 120 mm, square) | Sigma-Aldrich | Z617679 | |
| Trichloro(3,3,3-trifluoropropyl)silane | Sigma-Aldrich | MKBG3805V | |
| Polydimethylsiloxane (PDMS) elastomer base and curing agent | Dow Corning | Sylgard 184 | Mixed at 10:1 (w/w) |
| Vacuum desiccator | Sigma-Aldrich | Z119024-1EA | |
| Oven | VWR | 9120976 | |
| Device Fabrication and Culture | |||
| poly(methyl methacrylate) (PMMA) | Plexiglas | ||
| Corona Treater | Electro-Technic Products, Inc. | BD-20 | Handheld device for plasma treatment of PMMA devices and PDMS molds |
| Soldering Iron | Weller | WTCPS | |
| Stainless Steel Truss Head Slotted Machine Screw | McMaster-Carr | 91785A096 | |
| Stainless steel dowel pins | McMaster-Carr | 93600A060 | |
| Tweezers | Miltex | 24-572 | Any similar tweezers may be used |
| Spatula (Micro Spoon) | Electron Microscopy Services | 62410-01 | |
| Screw driver | Any flat head screwdriver may be used, autoclaved | ||
| Glass coverslips (22 x 22 mm) | Fisher Scientific | 12-542B | |
| Bleach | Clorox | 4460030966 | |
| Petri dishes (150 X 25mm) | Corning | 430599 | |
| Petri dishes (100 X 20 mm) | Corning | 2909 | |
| Cotton, cut into 1 cm x 3 cm pieces | Autoclaved | ||
| Polyethyleneimine (PEI) | Sigma-Aldrich | P3143 | Dilute to 1% in cell culture grade water |
| Glutaraldehyde | Sigma-Aldrich | G6257 | Dilute to 0.1% in cell culture grade water |
| Sterile H2O | Autoclaved DI H2O | ||
| Type I collagen, dissolved in 0.1% acetic acid | Isolated from rat tails as described in Rajan et. al. 2006 (ref #37) | ||
| 1 mL syringe | BD | 309659 | |
| 10 mL syringe | BD | 309604 | |
| 15 mL conical tubes | Corning | 352097 | |
| 30 mL conical tubes | Corning | 352098 | |
| M199 10X Media | Life Technologies | 11825-015 | |
| 1N NaOH (sterile) | Sigma-Aldrich | 415413 | Dilute to 1N in cell culture grade water |
| HUVECs | Lonza | ||
| Endothelial growth media | Lonza | CC-3124 | |
| Trypsin | Corning | 25-052-CI | |
| Fetal bovine serum (FBS) | Thermofisher Scientific | 10082147 | |
| Dextran from Leuconostoc spp. (70kDa) | Sigma-Aldrich | 31390 | |
| Phosphate Buffered Saline (PBS) | Corning | 21-031-CV | |
| Hemocytometer | Hausser Scientific Co. | 3200 | |
| Gel loading tips | VWR | 37001-152 | |
| 18G Blunt Fill Needle | BD | 305180 | |
| 20G Stainless Steel Dispensing Needle | McMaster-Carr | 75165A123 | |
| Tygon 1/32” ID, 3/32" OD Silicon Tubing | Cole-Parmer | EW-95702-00 | |
| 1/16" Tube-to-tube Coupling | McMaster-Carr | 5116K165 | |
| 90° Elbow Connectors, Tube-to-Tube | McMaster-Carr | 5121K901 | |
| Luer Lock Coupling (Female, 1/16" ID) | McMaster-Carr | 51525K211 | |
| Plastic Forceps, with Jaw Grips | Electron Microscopy Services | 72971 | |
| Dual Syringe Pump | Harvard Apparatus | 70-4505 | |
| 5 mL Polystyrene Round-bottom tube | Fisher Scientific | 14-959-2A | |
| Device Analysis | |||
| Formaldehyde | Sigma-Aldrich | F8775 | |
| Bovine Serum Albumin | Sigma-Aldrich | A8806-5G | |
| Triton X-100 | Sigma-Aldrich | T-9284 | |
| Rabbit anti-hCD31 | Abcam | ab32457 | 1:25 working dilution |
| FITC conjugated anti-von Willebrand Factor antibody | Abcam | ab8822 | 1:100 working dilution |
| Goat anti-rabbit 568 secondary antibody | Thermofisher Scientific | A-11011 | 1:100 working dilution |
| Hoescht | Thermofisher Scientific | H1399 | Resuspended in DMSO |
| Sodium cacodylate | Sigma-Aldrich | C0250 | To make 0.2M cacodylate buffer |
| Ethanol | VWR International | BDH1164-4LP | |
| 40kDa FITC-conjugated Dextran | Sigma-Aldrich | FD40S | |
| Additional Culture Reagents | |||
| CHIR-99021 | Selleck Chem | S2924 | Small molecule GSK-3 inhibitor |
| Human recombinant VEGF | Peprotech | 100-20 | |
| Human recombinant bFGF | Peprotech | AF-100-18B | |
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Cite This Article
Roberts, M. A., Kotha, S. S., Phong, K. T., Zheng, Y. Micropatterning and Assembly of 3D Microvessels. J. Vis. Exp. (115), e54457, doi:10.3791/54457 (2016).