Name: micropatterning and assembly of 3d microvessels
Uploaded: 2016-09-09T15:00:00
Description: Watch this Scientific Journal Video about Micropatterning and Assembly of 3D Microvessels at JoVE.com

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）。

1.图案聚二甲基硅氧烷（PDMS）与网络设计的微细<ol><li> 晶圆制造到创造的网络设计的负模板 <ol><li>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）。 </li><li>生成使用高...

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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...

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（&lt;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。

<table border="0" cellpadding="0" cellspacing="0" width="1363">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Wafer Fabrication</td>
			<td style="width:225px;"></td>
			<td style="width:140px;"></td>
			<td style="width:540px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AutoGlow Plasma System</td>
			<td>AutoGlow</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Headway Spin Coater</td>
			<td>Headway Research, Inc&#160;</td>
			<td colspan="2">PWM32 Spin Coater&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ABM Contact Aligner</td>
			<td>AB-M</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alpha Step Profilometer</td>
			<td>Tencor</td>
			<td>Alpha Step 200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 Developer</td>
			<td>Microchem</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 Resist</td>
			<td>Microchem</td>
			<td>SU-8 2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8&#34; silicon wafer</td>
			<td>Wafer World Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tabletop Micro Pattern Generator</td>
			<td>Heidelberg Instruments</td>
			<td>&#956;PG 101</td>
			<td>For generation of photomask</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hot plate</td>
			<td>VWR</td>
			<td>97042-646</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ispropyl alcohol</td>
			<td>Avantor Performance Materials</td>
			<td>9088</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes (120 x 120 mm, square)</td>
			<td>Sigma-Aldrich</td>
			<td>Z617679</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trichloro(3,3,3-trifluoropropyl)silane</td>
			<td>Sigma-Aldrich</td>
			<td>MKBG3805V</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Polydimethylsiloxane (PDMS) elastomer base and curing agent</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td>Mixed at 10:1 (w/w)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vacuum desiccator</td>
			<td>Sigma-Aldrich</td>
			<td>Z119024-1EA</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Oven</td>
			<td>VWR</td>
			<td>9120976</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Device Fabrication and Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">poly(methyl methacrylate) (PMMA)</td>
			<td>Plexiglas</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corona Treater</td>
			<td>Electro-Technic Products, Inc.</td>
			<td>BD-20</td>
			<td>Handheld device for plasma treatment of PMMA devices and PDMS molds</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Soldering Iron</td>
			<td>Weller&#160;</td>
			<td>WTCPS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stainless Steel Truss Head Slotted Machine Screw</td>
			<td>McMaster-Carr&#160;</td>
			<td>91785A096</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stainless steel dowel pins</td>
			<td>McMaster-Carr&#160;</td>
			<td>93600A060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezers&#160;</td>
			<td>Miltex</td>
			<td>24-572</td>
			<td>Any similar tweezers may be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spatula (Micro Spoon)</td>
			<td>Electron Microscopy Services</td>
			<td>62410-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screw driver</td>
			<td></td>
			<td></td>
			<td>Any flat head screwdriver may be used, autoclaved</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass coverslips (22 x 22 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-542B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bleach</td>
			<td>Clorox</td>
			<td>4460030966</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes (150 X 25mm)</td>
			<td>Corning</td>
			<td>430599</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes (100 X 20 mm)</td>
			<td>Corning</td>
			<td>2909</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cotton, cut into 1 cm x 3 cm pieces</td>
			<td></td>
			<td></td>
			<td>Autoclaved</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethyleneimine (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>P3143</td>
			<td>Dilute to 1% in cell culture grade water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G6257</td>
			<td>Dilute to 0.1% in cell culture grade water</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sterile H2O</td>
			<td></td>
			<td></td>
			<td>Autoclaved DI H2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Type I collagen, dissolved in 0.1% acetic acid</td>
			<td></td>
			<td></td>
			<td>Isolated from rat tails as described in Rajan et. al. 2006 (ref #37)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 mL syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 mL syringe</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL conical tubes</td>
			<td>Corning</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">30 mL conical tubes</td>
			<td>Corning</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199 10X Media&#160;</td>
			<td>Life Technologies</td>
			<td>11825-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1N NaOH (sterile)</td>
			<td>Sigma-Aldrich</td>
			<td>415413</td>
			<td>Dilute to 1N in cell culture grade water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HUVECs&#160;</td>
			<td>Lonza</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Endothelial growth media</td>
			<td>Lonza</td>
			<td>CC-3124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum (FBS)</td>
			<td>Thermofisher Scientific</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran from Leuconostoc spp. (70kDa)</td>
			<td>Sigma-Aldrich</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemocytometer</td>
			<td>Hausser Scientific Co.</td>
			<td>3200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel loading tips</td>
			<td>VWR</td>
			<td>37001-152</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18G Blunt Fill Needle</td>
			<td>BD&#160;</td>
			<td>305180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20G Stainless Steel Dispensing Needle</td>
			<td>McMaster-Carr</td>
			<td>75165A123</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tygon 1/32&#8221; ID, 3/32&#34; OD Silicon Tubing</td>
			<td>Cole-Parmer</td>
			<td>EW-95702-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1/16&#34; Tube-to-tube Coupling</td>
			<td>McMaster-Carr</td>
			<td>5116K165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">90&#176; Elbow Connectors, Tube-to-Tube</td>
			<td>McMaster-Carr</td>
			<td>5121K901</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luer Lock Coupling (Female, 1/16&#34; ID)</td>
			<td>McMaster-Carr</td>
			<td>51525K211</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic Forceps, with Jaw Grips</td>
			<td>Electron Microscopy Services</td>
			<td>72971</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dual Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL Polystyrene Round-bottom tube</td>
			<td>Fisher Scientific</td>
			<td>14-959-2A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Device Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A8806-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T-9284</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti-hCD31</td>
			<td>Abcam</td>
			<td>ab32457</td>
			<td>1:25 working dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FITC conjugated anti-von Willebrand Factor antibody</td>
			<td>Abcam</td>
			<td>ab8822</td>
			<td>1:100 working dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rabbit 568 secondary antibody</td>
			<td>Thermofisher Scientific</td>
			<td>A-11011</td>
			<td>1:100 working dilution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoescht</td>
			<td>Thermofisher Scientific</td>
			<td>H1399</td>
			<td>Resuspended in DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium cacodylate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C0250</td>
			<td>To make 0.2M cacodylate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>VWR International</td>
			<td>BDH1164-4LP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40kDa FITC-conjugated Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD40S&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Additional Culture Reagents&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHIR-99021</td>
			<td>Selleck Chem</td>
			<td>S2924</td>
			<td>Small molecule GSK-3 inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human recombinant VEGF</td>
			<td>Peprotech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human recombinant bFGF</td>
			<td>Peprotech</td>
			<td>AF-100-18B</td>
			<td></td>
		</tr>
	</tbody>
</table>

micropatterning and assembly of 3d microvessels

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。

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...

Watch this Scientific Journal Video about Micropatterning and Assembly of 3D Microvessels at JoVE.com

Micropatterning and Assembly of 3D Microvessels

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。

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

The overall goal of using engineered micro-vessels is to replicate the structure and function of vessels in the body to study vascular physiology in normal and diseased states. This method can help answer key questions in the field of vascular biology such as how different anathema cells respond to flow, how they influence tissue function, and how vascular diseases initiate and progress over time. The main advantage of this technique is it can be easily modified to mimic different organ systems and disease states.This can be done by changing the cell types, the matrix and media composition, and other variables. Generally individuals new to this method could struggle before they become aware of common mistakes that can lead to poor vessel quality. Visual demonstration of this method is critical because there are several tricks to successful fabrication during the injection molding and assembly steps.For this procedure, have the flat and micro-patterned PDMS molds and the PMMA housing pieces prepared in advance. A description of how to make these parts is provided in the text protocol. Autoclave the PDMS molds along with the screws, pins, forceps, and a spatula.Do not autoclave the PMMA pieces. They must be sterilized in bleach under a hood. After allowing the PMMA pieces to soak in bleach for at least an hour, rinse off the bleach with autoclaved water two times.Then, place the pieces in sterile dishes. Aspirate excess water and let them air dry. With everything cleaned, start the protocol by treating the inner wells of both the top and bottom PMMA pieces using a handheld plasma treater for approximately one minute.Turn the light off to help visualize the coverage of the plasma treatment. Next, add 1%PEI onto the inner wells. If the PEI doesn't spread easily, increase the plasma treatment time.After letting the PEI react for 10 minutes, remove the solution and then rinse the devices off with sterile water and let them air dry. Once dried, apply 0.1%glutaraldehyde onto the treated surfaces and allow the glutaraldehyde to react for half an hour. Then, rinse the pieces with sterile water twice and allow them to air dry.Now, prepare fresh 0.75%neutralized collagen for vessel fabrication by combining the pre allocated stock collagen volume with the neutralization solution of medium and sodium hydroxide. Stir slowly and carefully with a spatula until the solution is homogeneous and is orange in color. Then, if needed, add cells at the desired concentration and continue to stir the solution until the cells are uniformly distributed.In preparation for the assembly process, place the micropatterned PDMS mold into a 100 by 20 millimeter dish and treat the mold with plasma for one minute. Next, align the top PMMA piece onto the PDMS so the square reservoirs line up with the inlet and outlet reservoirs. Then, put stainless steel dowel pins into the inlet and outlet reservoir holes so they will remain open after collagen injection.Now, load the collagen cell mixture into a one milliliter syringe without introducing air bubbles. Then using forceps, gently press down on the top PMMA piece to secure it to the PDMS mold and slowly inject about one half milliliter of collagen cell mixture into the injection port. The collagen must fill the 20 by 20 millimeter area over the pattern and not leak out.Then, carefully close the dish without jostling the dowel pins. Next, into the bottom PMMA piece place a 22 square millimeter glass coverslip in the inner well. Then, dispense about 0.25 milliliters of collagen evenly onto the glass.Complete the bottom half by gently lowering the flat PDMS into the collagen so it is flat against the PMMA with no air bubbles in between. Now, carefully place both halves of the device into a 37 degree Celsius incubator and allow the collagen solution to gel for 30 minutes before proceeding. After gelation, add enough PBS to surround the PDMS on the bottom piece.Then, use forceps to remove any excess collagen around the edges and slowly peel the flat PDMS piece off while keeping the collagen hydrated with PBS. To prepare the top piece, use forceps to lift the PMMA and PDMS assembly and flip it over. Then add a few drops of PBS to the inner face and remove the PDMS mold from the top PMMA piece, peeling quickly and firmly.Next, with a second pair of forceps, gently remove the pins from the other side of the PMMA housing. Ensure the inlet and outlet are clear of excess collagen by moving the pins up and down before their removal. If the pins fall out on their own, simply use another sterile dowel pin to ensure that the inlet and outlet are clear.Then, drop more PBS onto the micro patterned collagen, flip the piece over, and place screws into the four corner holes. Now, gently lay the top piece on the bottom piece aligning the screws into their holes. Do not let the two pieces slide against each other.Then, gently screw the pieces together using a spatula and a gentle touch so they are not overtightened. It is critical to be gentle and precise during assembly. By applying an ample amount of PBS, the friction between the inner face is reduced.While securing the screws, proceed slowly and stop as soon as any resistance is met. Next, aspirate the PBS surrounding the PMMA surfaces and place a small piece of cotton under one edge of the device. Then, aspirate any PBS from the reservoirs.Now, refill the reservoirs with cell culture medium and incubate the device for at least an hour for gelation. Once the device is assembled, use an endothelial cell suspension at 10 million cells per milliliter to seed the device. Remove the medium in the inlet and outlet reservoirs and using a 20 microleter pipette with gel loading tips, add 10 microliters of the cell suspension into the center of the inlet reservoir.The cells will immediately flow into and coat the network. Once the cells have filled the network and the flow has equilibrated, add 150 microliters of medium into both reservoirs. Then incubate the device for an hour or more so the cells can spread and attach.For long term culture, remove the medium every 12 hours and add back 150 microliters of fresh medium to the inlet reservoir and 50 microliters to the outlet reservoir. After 24 hours of culturing, a syringe pump can be used to establish continuous flow conditions. After bringing the materials to the bio hood, remove the tubing from the autoclave bags and place it in a dish.Then, prepare a sterile flow dish by melting side holes and then threading the tubing through. Prepare a 10 milliliter syringe with culture medium containing 3.5%Dextran. Next, attach the filled syringe to the lure lock at the end of the inlet tubing.Then, secure the syringe to the pump and profuse medium until any trapped air bubbles have exited the tubing. Once the tubing is filled with medium and is clear of bubbles, transfer the device to a new dish and insert the inlet connector joint into the housing inlet. Then, set the syringe pump to the desired flow rate and begin profusion.Prepare the outlet by filling the tubing with cell culture media using an 18 gauge needle and a one milliliter syringe. Clamping it and removing the needle. Then, melt a hole in the cap of the outlet collection tube using a soldering iron.Afterwards, add media to the collection tube, insert the tubing, and attach the connector joint into the housing outlet. Throughout this process, a clamp is used to retain the medium in the tubing. Once completed, transfer the entire setup into an incubator.Depending on the experimental conditions, the syringe may need to be refilled after 24 to 36 hours. During syringe replacement, ensure bubbles are not introduced into the tubing. Human umbilical vein and ethelial cells were profused through the collagen embedded micro fluidic network to form a patent lumen and confluent endothelium.Several vessel geometries were used to establish different flow conditions. Continuous flow culture conditions were used to apply constant shear stress on the endothelium. Devices cultured under gravity driven flow conditions also maintained long term viability and patency.A critical feature of these micro-vessels is their ability to respond to inflammatory stimuli. This was investigated by profusing the micro-vessels with whole blood. Non-stimulated micro-vessels contained non-activated endothelium and remained quiescent.Stimulated micro-vessels became activated and their response initiated thrombus formation. Another interesting application for the system is to study phenotypic changes in vasculature formed from different endothelial cell populations. For example, micro-vessels formed from unique populations of stem cells exhibited different angiogenic sprouting capabilities.After watching this video you should have a good understanding of how to fabricate micro-vessels and use them to study vascular biology and to build complex model organ systems. While attempting this procedure it is important to remember that practice will improve with the rate of successful vessel formation and allow for more consistent results. Once mastered, several devices can be made in three to four hours.Following this procedure, other methods like the profusion of whole blood, Dextran, or pharmaceutical treatments can be performed to assess endothelial quiescence, permeability, drug response, and the general morphology of cells and vessels.

Research

JoVE Journal

Bioengineering

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

科研

生物工程

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

温度控制组件和液滴接口比莱耶的特征

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

通过红外激光辅助微模式控制细胞几何

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

A Micropatterning Assay for Measuring Cell Chirality

一种用于测量细胞手性的微图案化检测

Chirality is an intrinsic cellular property, which depicts the asymmetry in terms of polarization along the left-right axis of the cell. As this unique ...

Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

明胶甲基丙烯酰基颗粒水凝胶支架：高通量微凝胶制造、冻干、化学组装和 3D 生物打印

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation ...