Name: stepwise cell seeding on tessellated scaffolds to study sprouting blood vessels
Uploaded: 2021-01-14T15:00:00
Description: Watch this Scientific Journal Video about Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels at JoVE.com

这项研究得到了密歇根大学-以色列研究伙伴关系的资助。作者要感谢乌里·默德勒、利奥·黛比和加利亚·本·大卫给予的巨大帮助和支持，感谢密歇根大学卢里纳米制剂设施博士王纳丁博士和皮拉尔·埃雷拉-菲罗博士，以及路易斯·索洛里奥博士对光刻技术的启发性讨论。

1. 泰瑟尔脚手架制造
注：光刻技术是一种广泛的技术，需要专门设备，通常位于纳米制泡设施/实验室内。本议定书中规定的方法尽可能为观众所采用：但是，根据读者可获得的设备，可能需要对程序进行轻微更改。我们建议在纳米制剂设施的洁净室中执行这些程序，以确保最高的工艺质量。在开始之前，请访问面膜对齐器（或一些紫外线曝光设置）、旋转涂层、热板、溶剂洗...

<ol>
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在工程组织内需要丰富的血管，这对构建生存和适当的功能至关重要。虽然工程血管系统一直是大量研究的重点，但还有许多问题有需要研究和理解。特别是，在重建特定组织时，微血管应相应地表现和组织。微维斯生成最常见的方法是在适合细胞附着、增殖和血管形成的合适 3D 环境中共同播种内皮细胞和支持细胞。这种方...

组织工程领域正在迅速向制造工程结构，以取代缺失或损坏的器官和组织1。然而，由于为组织营养而生成操作血管网络仍然是一个突出的挑战，但尚未实现全功能结构。如果没有适当的血管化，工程组织仅限于氧气和营养物质的被动扩散传输，将最大可行的组织厚度限制在扩散极限，约200μm2。这种厚度不适合修复大型组织缺陷或全器官制造，这使得功能血管网络的存在成为功能和植入组织3的必修特征。
血管系统由多种血管组成，具有不同的大小、表型和组织，与宿主组织密切相关。了解发育和发芽的血管的行为、反应和迁移决定可以指导它们融入工程组织4。目前，创建体外血管网络的最常见方法是将内皮细胞 （ECs） 与支持细胞 （SCs） 相结合，从而能够分化成壁画细胞），在三维微环境中播种。这种环境提供化学和物理线索，使细胞附着，增殖和自我组装到容器网络2，5，6，7，8。当共同培养时，SCs 分泌细胞外基质 （ECM） 蛋白质，同时为形成管状结构的 EC 提供机械支持。此外，两种细胞类型之间的交叉相互作用促进管状形成、血管发芽和迁移，此外，SCs 成熟和分化成α光滑的肌肉作用表达 （αSMA） 壁画细胞4。船舶网络开发最常在使用水凝胶、多孔聚合物脚手架或其组合创建的 3D 环境中进行研究。后一种选择同样为细胞和 ECM9提供了细胞友好的环境和所需的机械支持。
研究血管发育的工作很多，包括共同培养水凝胶10上的细胞，水凝胶-脚手架组合11，12，2D平台，和微流体装置13。然而，水凝胶很容易被细胞施加的力14变形，而2D和微流体系统无法重建一个更接近自然的环境，以获得更外推的反应15，16。了解成型容器如何对其周围环境做出反应，可以提供关键见解，使工程环境能够以可预测的方式指导船舶发展。了解血管形成现象对于跟上亚微米到微米尺度制造技术的迅速出现尤为关键，如立体成形、数字投影光刻、连续液体接口生产、3D熔融电喷射写、基于溶液的3D电喷射写写，以及新兴的生物打印技术17、18、19、20、21等。将这些微制造技术的控制与加深对血管生物学的理解相协调，是为目标组织创建适当的工程血管学的关键。
在这里，我们提出了一个3D系统，以研究新的成形和发芽的容器对周围脚手架几何的反应，观察它们的发芽起源和随后的迁移22。通过利用带分壳隔间几何形状的 3D 脚手架和两步播种技术，我们成功地以清晰易分析的方式创建了高度有序的血管网络。分壳几何形状提供高吞吐量系统，每个单元包含对当地环境有反应的船只。使用五颜六色的ECs，我们跟踪芽的形成起源和随后的迁移模式，与隔间几何和SCs位置22相关。
虽然拟议的协议已准备分析几何线索对血管化行为的影响，但这种方法可以扩展到各种新应用。特有脚手架和易于成像的网络允许直接分析不同的 UC 和 SCs 相互作用、增加特定器官细胞及其与血管网络的相互作用、药物对血管网络的影响等等。我们建议的系统结果非常多才多艺，简单制造和处理。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Angiotool freeware</td>
			<td>NIH-CCR</td>
			<td></td>
			<td>Free download at https://ccrod.cancer.gov/confluence/display/ROB2/Home</td>
		</tr>
		<tr>
			<td>Bovine albumin serum Probumin</td>
			<td>Millipore</td>
			<td>82-045-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental pulp stem cells</td>
			<td>Lonza</td>
			<td>PT-5025</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM media + bullet kit</td>
			<td>Sciencell</td>
			<td>#1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Gadot-Group</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Evicel fibrin sealant</td>
			<td>Johnson&#38;Johnson</td>
			<td>EVB05IL</td>
			<td>Provides both thrombin and fibrinogen (BAC2) solutions</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse Cy3 antibody</td>
			<td>Jackson</td>
			<td>115-166-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa-Fluor 488</td>
			<td>Thermo- Fisher Scientific</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Human adipose microvascular cells</td>
			<td>Sciencell</td>
			<td>#7200</td>
			<td></td>
		</tr>
		<tr>
			<td>Human fibronectin</td>
			<td>Sigma</td>
			<td>F0895-5MG</td>
			<td>Stock concentration: 1 mg/mL</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>Free download at https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Gadot-Group</td>
			<td>67-63-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Lift-off reagent</td>
			<td>Kayaku Advanced Materials, Inc</td>
			<td>G112850</td>
			<td>Commercial name Omnicoat</td>
		</tr>
		<tr>
			<td>Low-glucose DMEM</td>
			<td>Biological Industries</td>
			<td>01-050-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-SMA antibody</td>
			<td>Dako</td>
			<td>M0851</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Gibco</td>
			<td>11140068</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4% in PBS</td>
			<td>ChemCruz</td>
			<td>SC-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Nystatin Solution</td>
			<td>Biological Industries</td>
			<td>03-032-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-vWF antibody</td>
			<td>Abcam</td>
			<td>ab9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>Silicon Valley Microelectronics (SVM)</td>
			<td></td>
			<td>Wafers 4&#34;, Type N-1-10, 500-550 microns thick</td>
		</tr>
		<tr>
			<td>SU-8 2050 photoresist</td>
			<td>Kayaku Advanced Materials, Inc</td>
			<td>Y11058</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer</td>
			<td>Kayaku Advanced Materials, Inc</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryton-X 100</td>
			<td>BioLab LTD</td>
			<td>57836</td>
		</tr>
	</tbody>
</table>

stepwise cell seeding on tessellated scaffolds to study sprouting blood vessels

心血管系统是人体生理学的关键角色，为人体大多数组织提供营养：容器存在不同的大小，结构，表型和性能取决于每个特定的香水组织。组织工程领域，旨在修复或替换受损或缺失的身体组织，依靠受控血管生成在工程组织内创建适当的血管化。如果没有血管系统，厚重的工程结构就无法充分滋养，这可能导致细胞死亡、不良的灌输，并最终失败。因此，了解和控制工程血管的行为是该领域的一个突出挑战。这项工作提出了一个高通量系统，允许创建有组织和可重复的船舶网络，用于研究船舶行为在3D脚手架环境中。此两步播种协议显示系统内的容器对脚手架地形做出反应，根据船舶居住的隔间几何形状呈现独特的发芽行为。可应用这一高吞吐量系统获得的结果和理解，以便更好地实现 3D 生物打印脚手架构造设计，其中使用 3D 打印作为蜂窝化生物环境的基础时，无法快速评估各种 3D 几何形状的制造。此外，从这种高吞吐量系统的理解可以用来改进快速药物筛选，快速发展共培养模式，研究机械刺激血管形成，加深血管系统的认识。

所提出的协议，使用立体摄影技术，允许制造由SU-8光分辨率器制成的特星脚手架。获得具有不同隔间几何形状（正方形、六边形和圆圈）的脚手架，以及高度准确和可重复的特征（图 1）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61995/61995fig01.jpg"> 图1：代表扫描电子显?...

Watch this Scientific Journal Video about Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels at JoVE.com

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

工程组织严重依赖适当的血管网络来提供重要的营养和气体，并清除代谢废物。在这项工作中，内皮细胞和支持细胞的分步播种协议在高通量平台中创建高度有序的血管网络，用于研究在受控的 3D 环境中开发血管行为。

逐步在泰瑟尔脚手架上播种，研究发芽血管

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

我们的协议允许研究人员以清晰易行的方式研究血管网络形成过程，为新的研究打开大门，并揭示血管行为。该技术提供了创建高度组织和可重复的血管网络，以响应周围环境的可能性。使用该系统可以检索血管疾病患者的内皮细胞，从而有可能重现血管疾病并找到适当的治疗方法。建议的方法可以扩展为研究血管网络行为时，与不同的细胞类型配对，允许观察发展血管与特定组织类型的相互作用。首先将脚手架浸入70%乙醇中至少15分钟，然后用PBS清洗两次。将 1.5 微升纤维素库存溶液与每脚手架 28.5 微升 PBS 混合以进行播种。准备一个纤维素稀释，用于所有脚手架，以避免管道错误。将脚手架稀疏地放在疏水表面的顶部，用 30 微升纤维素稀释覆盖每个脚手架。更换板盖，将纤维素覆盖的脚手架放入一个孵化器中，温度为 37 摄氏度，湿度为 100%，最少一小时。孵化后，轻轻冲洗PBS中的脚手架，去除纤维素残留物。脚手架可在 PBS 中保存在摄氏四度，最长达一周。通过将基底介质与相应的中等套件组件混合，包括制造商指出的抗生素溶液、FBS 和内皮细胞生长补充剂，准备内皮细胞或 EC 介质。使人体脂肪微血管内皮细胞悬浮在EC介质中，每毫升浓度为400万细胞。使用钳子，将每口井一个纤维素涂层脚手架放在非TC 24井板中。用 25 个微滑滴覆盖每个脚手架，确保不要让悬架从脚手架上流出。将盖子盖在盘子上，放在37摄氏度、5%二氧化碳和100%湿度的孵化器中，60至90分钟。孵化后，每口井中填充700微升EC介质。孵化内皮脚手架，直到使用荧光显微镜或三天观察EC汇合。每隔一天更换一次媒体。通过混合 500 毫升低葡萄糖 DMEM、57.5 毫升 FBS、5.75 毫升非必需氨基酸、5.75 毫升麸质MAX 和 5.75 毫升青霉素-链霉素-尼他汀溶液来准备 DPSC 介质。将内皮脚手架转移到新的非TC 24井板中。使用移液器或吸尘器从当前板中丢弃所有介质，注意不要将真空直接涂抹在脚手架上。将一个脚手架放入每个油井的中心，然后用光真空干燥周围区域。与PBS一起稀释血栓和纤维素库存解决方案，最终浓度分别为每毫升5个单位和每毫升15毫克。在血栓稀释中准备每毫升悬架 800 万 DPSC，并将悬架的 12.5 微升分配到每个脚手架的单个微管中播种。将 5 到 50 微升移液器设置为 12.5 微升，并填充纤维蛋白原溶液。无需取出尖端，将移液器设置为 25 微升。尖端的材料应上升并留下空体积。慢慢按下柱塞按钮，直到液体到达尖端开口，但不会泄漏出去。将柱塞放在这个位置，并将尖端放入含有血栓悬架中细胞的微中性管中，确保尖端与液体接触。轻轻释放柱塞按钮，并将单元格悬架拉入尖端。彻底混合两种材料，避免气泡形成。快速将混合材料分配到内皮脚手架上。重复每个脚手架以前的步骤，确保在使用之间更改提示，以避免尖端内意外的纤维蛋白凝胶形成。更换板盖，在37摄氏度、5%二氧化碳和100%湿度下孵化脚手架30分钟。孵化后，在每口井中填充一毫升一对一的 DPSC 和 EC 介质。文化一周，每隔一天改变一次媒体。在文化时期的不同时间点，从井中取出介质，并用共聚焦显微镜对构造进行成像，以研究血管发育或任何其他感兴趣的参数。该协议允许制造由SU-8光还器制成的特塞尔脚手架。获得具有不同隔间几何形状和高度准确和可重复特征的脚手架。传统的内皮细胞和支持细胞同时播种，细胞均匀地分布在脚手架上，导致不可预知和混乱的血管网络。相反，分步细胞播种导致高度有序的血管网络。使用荧光内皮细胞时，可以实时对血管进行成像。表达内皮细胞的红色荧光蛋白在六边形脚手架上培养并成像。支持单元在第一天被添加，血管网络每隔一天被成像，以量化血管发育。每个时间点都拍摄了整个脚手架的宽图像。船舶增长按船舶总长度和面积进行量化。进行了共焦成像延时，允许使用多色内皮细胞进行单个血管跟踪。船只路径是从延时生成的，因此可以观察船只的迁移。容器成熟是由光滑的肌肉作用和支撑细胞的存在观察到的。对于圆形、六边形和方形隔间，细胞会繁殖并组织成结构。到第七天，所有形状都显示出一个丰富而复杂的血管网络。更高的放大图像显示，更密集的平滑肌肉作用和支撑细胞的存在与形成的血管共同本地化，证明支持细胞招募和血管结构周围的分化。该技术可用于研究三维血管网络在暴露于不同条件下的行为，如特定的细胞抑制剂或在存在其他细胞类型时的行为。

Research

JoVE Journal

Bioengineering

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

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

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 ...

科研

生物工程

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

凝血因子VIII的脂质纳米管螺旋组织

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

同心凝胶系统来研究基质微环境的三维细胞迁移的生物物理作用

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Imaging Cell Viability on Non-transparent Scaffolds ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Â Using the Example of a Novel Knitted Titanium Implant

非透明的支架成像细胞活性 - 使用新型针织钛种植体为例

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

Melt Electrospinning Writing of Three-dimensional PolyÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Âµ-caprolactone Scaffolds with Controllable Morphologies for Tissue Engineering Applications

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

标准化和可扩展的测定，以研究iPSC衍生内皮细胞在Vitro的渗透3D血管生成发芽

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative

用心脏灌注和二固定仪对脑和垂体血管进行标记

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one ...