这项工作得到了上海科学技术委员会 (15411951800, 15410723200) 的资助。作者感谢复旦大学病原微生物系玉梅教授和赵教授的技术支持。

该方法是根据复旦大学华山医院研究伦理学委员会批准的指导方针和规定进行的。每个步骤都遵循相应的标准安全措施。有关示意性演示文稿, 请参阅图 1。
1. 细胞培养和质粒结构
<ol><li>例行维持人脐静脉内皮细胞 (HUVEC) 在 Dulbecco 的改良鹰的培养基 (DMEM) 补充10% 胎牛血清 (血清), 100 单位/毫升青霉素, 100 µg/毫升链霉素在加湿 5% CO2孵化器摄氏37摄氏度。</li>
	<li>摘要 PLKO 1 质粒, ApaI 和 EcoRI 合成 shRNA 片段。确保 VHL shRNA 向量的寡核苷酸序列 CCGGGATCTGGAAGACCACCCAAATCTCGAGA TTTGGGTGGTCTTCCAGATCTTTTTG14。</li>
	<li>然后, 混合20µΜ系统与前5µL 的寡核苷酸, 5 µL 的反向寡聚, 5 µL 10x 的纳布缓冲器和35µL ddH2O 一起 (总容量为50µL)。将混合物加热98摄氏度4分钟, 在几个小时内慢慢冷却到室温。</li>
	<li>结扎退火的寡核苷酸和 PLKO 1 质粒与 T4 连接酶在4°c 过夜。16小时后, 添加5µL 结扎组合成25µL 主管 DH5α细胞。然后, 筛选成功结扎的质粒, 并通过测序验证插入片段。</li>
</ol>2. 慢病毒北疆包装和感染
<ol><li>总计, 混合10µg 的 PLKO 1-shVHL 或 PLKO 1-shScramble 向量, 7.5 µg 的包装质粒 psPAX2, 2.5 µg 的信封质粒 pMD2 500 µL 的 DMEM 培养基没有胎牛血清 (血清) 25 分钟。</li>
	<li>添加 7 mLDMEM, 而不给上述解决方案。</li>
	<li>培养293FT 细胞在 DMEM 没有在10厘米直径组织培养皿中的血清。使用单元格计数器确保293FT 单元格的数量为 1 x 107 。</li>
	<li>将上述溶液的1毫升添加到293FT 细胞中以进行转染。 注意:293FT 细胞系来源于293F 细胞系, 稳定表达 SV40 大 T 抗原。因此, 它是一个适合慢病毒载体生产的主机。</li>
	<li>在6小时后将培养基与含有10% 的 DMEM 的培养基替换。</li>
	<li>转染后用吸管在48小时收集介质。 注意:病毒存在于含有10% 血清的 DMEM 中。死细胞漂浮在培养基上。使用0.45 µm 无菌膜过滤死细胞和杂质。一般而言, 没有必要检查感染的多重性。这是建立一个稳定的细胞线。在最后一步, 所有没有成功感染的活细胞将死于嘌呤霉素。shScramble 组和 HUVEC 细胞为对照组。确保所有 HUVEC 细胞与所使用的嘌呤霉素浓度 72 h 一起死亡。每个井都有相同数量的细胞。</li>
	<li>将慢病毒载体介质中的 HUVEC 细胞培养为72小时。然后, 将嘌呤霉素添加到 HUVEC 细胞的培养基中, 浓度为2µg/毫升, 24 小时. 使用 HUVEC 细胞而不接受任何治疗作为对照组。确保所有控制单元在此时死亡。 注意:活细胞代表 VHL 击倒细胞和 shSramble 细胞的稳定细胞系。在96井板中, 镀层密度为 5000/井。</li>
</ol>3. 内皮细胞球体的生成
<ol><li>血管内皮细胞细胞和并用重悬在 DMEM 培养基中胰蛋白酶处理10% 的血清。在10厘米的培养皿中有适量的细胞, 加入1毫升的 typsin EDTA。</li>
	<li>将3D 圆底96井板中的细胞种子, 在细胞密度为 1 x 103单元格/好。用一个能容纳0.50 µL 球体的井来悬浮球体。按照制造商的说明, 使用单元格计数器系统计数单元格。</li>
	<li>连续孵化37°c 和 5% CO2的细胞为 72 h。在这些条件下, 确保悬浮细胞自动形成细胞球体。36小时后替换培养基中的一半。</li>
</ol>4.体外血管生成试验
<ol><li>解冻的凝胶溶液在4°c 和稀释它与减少的血清培养基在稀释率为1:5。</li>
	<li>用微吸 DMEM 培养基中的球体。洗涤后的球体与减少血清培养基的5毫升, 暂停球体轻轻地和谨慎地在稀释凝胶。确保没有气泡。</li>
	<li>在15井板中嵌入300µL 混合液。孵化后在37°c 和 5% CO2为 1 h, 增加400µL 减少的血清培养基对每个井。在水井周围填充无菌水, 以产生湿润的环境, 以阻止蒸发。</li>
	<li>尔后, 文化板材在37°c 在 5% CO2在100% 湿气为 1 h。</li>
	<li>小心地吸入旧培养基, 用600µL 减少血清培养基, 并将每一个井中的1% 个内皮细胞生长补充剂替换。</li>
	<li>在12小时或24小时以后, 在倒置的光显微镜下拍摄图像 (40 *)。</li>
</ol>5. 数据分析
<ol><li>将图像上传到在线图像分析平台上, 以获取发芽长度数据 (材料表)。</li>
	<li>在网页上, 通过电子邮件创建帐户。</li>
	<li>然后单击 "上载" 按钮上载图像。</li>
	<li>单击 "下载" 按钮下载结果。 注意:该软件将生成处理后的图像和数据。数据包含五个部分: 累计发芽长度、平均发芽长度、芽长的标准偏差、发芽面积和球体面积。在处理过的图像中, 每个参数都用不同的颜色勾勒出来, 以简化处理后的图像中的识别: 红色代表芽的骨架, 黄芽的数量, 蓝色的芽结构, 白色芽端, 和橙色球体。</li>
</ol>

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作者没有什么可透露的。

近年来, 血管内皮细胞15的研究刺激了多领域的维管束生物学研究。本文以内皮球发芽技术为实验模型, 研究了 VHL 基因在操作内皮细胞中的功能丧失对血管形成的作用, 以确定新的候选分子血管生成叶。根据我们的知识, 这是第一个检查内生改良内皮细胞血管生成效应的报告。
组织 (包括肿瘤组织) 新生血管是一个复杂的过程, 发生在发育过程中的通过血管生成和血管机制, 以及在生理和病理条件下12,15,16. 球状发芽检测的特点是复杂的事件的层叠,17,18, 包括嵌入在3D 细胞基质培养系统中的内皮细胞的自聚集, 从而导致发芽和复制毛细血管的3D 网络19。该3D 凝胶嵌入球形模型已成为组织工程中广泛应用的一种研究血管形成及其机制的系统, 因为它能够在合适的基质中提供较好的仿环境。然而, 这一生物过程最关键的步骤是激活静态内皮细胞17,20,21,22。在这个过程中, 内皮细胞被激活的内生沉默的 VHL 基因, 调节生长基因 neovessels23,24。不同于传统的内皮启动通过基质的外源诱导和相应的胞外环境 (包括外源性血管生长因子), 这种修正方法可以扩展到许多用于解开内生遗传机制的应用。其次, 为了减少外源矩阵的影响, 通过稀释凝胶对该协议进行了优化, 这是实验中的一个简单步骤。此外, 它只花了几分钟的时间上传图像到平台, 并获得分析结果。该平台提供了一种图像分析服务, 使实验者能够对在线发芽检测图像进行客观、可比和自动的图像分析。因此, 该协议克服了人为因素造成的测量和计算误差。
对新生血管级联个体步骤的机械理解为目前正在核可的肿瘤临床应用的抗血管治疗的发展提供了合理的依据。本文建立了一种简单、健壮的球形检测模型, 研究了由操作性内皮细胞产生的血管形成, 并利用VHL基因丧失功能来识别新生血管的新候选分子。级联, 可用于许多血管生成和血管发生相关的应用。作者推测, 这种体外模型可能提供额外的信息, 以帮助评估血管生成在某些实体肿瘤中的作用 (如肿瘤血管模仿), 并研究其他可能的祖细胞的潜在作用。产生肿瘤 neovessels在体内, 特别是在肿瘤患者, 作为一个群体, 是没有反应的治疗与抗血管生成剂只。
球形发芽法已成为研究血管生成和相关机制的一种广泛应用的方法。通过提供比经典的2D 文化更好的模拟环境, 该检测具有重要的优势。最近, 建立了3D 的共培养模型, 以研究模拟肿瘤微环境中血管生成的异质性和复杂性的肿瘤血管生成25。在这个模型中, 内皮细胞直接培养癌细胞, 以及在3D 环境中的基质成分。此外, 3D体外培养系统已被证明比常规细胞培养系统更准确地反映了体内对治疗剂的反应。此外, 该方法用于胚胎干细胞的分化、组织生长、创面愈合、缺血和炎症。与经典方法相比, 我们的方法是有效的, 易于实现。我们相信它是一个有用的工具, 研究血管生成在体外, 它开辟了一个新的方法来更好地了解这个过程。

小脑 (哈佛商学院) 是良性血管肿瘤, 是完全在人类中枢神经系统 (CNS) 发现。他们发展在患者与冯冯·希佩尔·林道-林道 (VHL) 疾病或零星的损伤。VHL-哈佛商学院是难以治愈的外科治疗, 由于频繁复发和多病变, 造成这种遗传紊乱1。尽管 VHL 抑癌基因的失活已被认为是 VHL-哈佛商学院肿瘤发生的根本原因, 但哈佛商学院的细胞学起源 (包括新生血管) 和进化过程仍然很有争议2。因此, 更好地了解 HB-新生血管的生物机制, 可以为 VHL-哈佛商学院最有前途的抗血管策略提供有益的见解。
最近的研究表明, HB-新生血管类似于胚胎血管发生 3, 4, 5.典型的血管内皮生长因子 (VEGF) 介导的血管生成是由血管内皮引起的, 由 VHL 功能丧失所驱动, 导致增殖和新生血管形成, 这已被挑战6。在 1965年, Cancilla 和齐默尔曼使用电子显微镜发现, 哈佛商学院起源于内皮细胞7。后来发现基质细胞来源于 vasoformative 元素8。在 1982年, Jurco et 等发现基质细胞是内皮源9。因此, 我们假设, 人血管内皮细胞是血红蛋白的原细胞-新生血管的10。虽然最好使用从 VHL 患者手术中提取的 hb 细胞中的主要培养物, 但我们以前的研究表明, 来自 hb 的主要培养基不稳定, 细胞系无法建立3。此外, 3D 环境中的主要文化无法识别乙肝新生血管的细胞学来源, 因为它们包括 hb-血管成分的祖细胞10,11。因此, 作为一种原始和经典的内皮细胞模型, 人血管内皮细胞 (HUVEC) 可以作为哈佛商学院的替代细胞模型。
球形发芽试验是组织工程中的一个新模型12,13。本文开发了一种基于3D 胶原的共培养系统体外, 其目的是评价哈佛商学院是否存在经典的肿瘤血管生成, 以及它在哈佛商学院的作用。

<table><tbody><tr><td>human umbilical vein endothelial cell</td><td>Fudan IBS Cell Center</td><td>FDCC-HXN180</td><td></td></tr><tr><td>dulbecco&amp;rsquo;s modified eagle&amp;rsquo;s medium&amp;nbsp;</td><td>Gibco</td><td>11995040</td><td></td></tr><tr><td>fetal bovine serum</td><td>Gibco</td><td>&amp;nbsp;26400044</td><td></td></tr><tr><td>PLKO.1-puro vector</td><td>Addgene</td><td>#8453</td><td></td></tr><tr><td>packing plasmid psPAX2&amp;nbsp;</td><td>Addgene</td><td>#12260</td><td></td></tr><tr><td>envelope plasmid pMD2.G</td><td>Addgene</td><td>#12259</td><td></td></tr><tr><td>3D round-bottom 96-well plates</td><td>S-Bio</td><td>MS-9096M</td><td></td></tr><tr><td>matrigel</td><td>BD Biosciences</td><td>354234</td><td></td></tr><tr><td>Opti-MEM medium</td><td>Gibco</td><td>31985-070</td><td>reduced serum medium&amp;nbsp;</td></tr><tr><td>15-well plate</td><td>Ibidi</td><td>81501</td><td>Air bubbles in the gel can be reduced by equilibrating the &amp;mu;&amp;ndash;Slide angiogenesis before usage inside the incubator overnight</td></tr><tr><td>endothelial cell growth supplements</td><td>Sciencell</td><td>#1052</td><td></td></tr><tr><td>10-cm culture dish</td><td>Corning</td><td>Scipu000813</td><td></td></tr><tr><td>Puromycin</td><td>Gibco</td><td>A1113802</td><td></td></tr><tr><td>typsin-EDTA</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>Automated Cell Counter System &amp;nbsp;</td><td>BioTech</td><td></td><td></td></tr><tr><td>Image Analysis software&amp;nbsp;</td><td>Winmasis</td><td></td><td>http://mywim.wimasis.com&amp;nbsp;</td></tr></tbody></table>

a comprehensive procedure to evaluate the in vitro performance of the putative hemangioblastoma neovascularization using the spheroid sprouting assay

冯·希佩尔·林道-林道 (VHL) 抑癌基因的失活在人类中枢神经系统 (CNS) 的小脑 (HBs) 的发展中起着至关重要的作用。然而, 哈佛商学院 (包括新生血管) 的细胞学起源和进化过程仍然存在争议, VHL-哈佛商学院的抗血管生成是基于典型的 HB 血管生成, 在临床试验中产生了令人失望的结果。抗血管治疗成功的临床翻译的一个主要障碍是对血管肿瘤的新生血管缺乏透彻的了解。在本文中, 我们提出了一个综合的程序来评价在体外是否存在经典的肿瘤血管生成在哈佛商学院, 以及它在哈佛商学院的作用。通过这一程序, 研究人员可以准确地了解乙肝新生血管的复杂性, 并确定其在哈佛商学院的这种常见的血管生成功能。这些协议可用于评估最有前景的肿瘤抗血管治疗, 它具有很高的翻译潜力, 无论是肿瘤治疗或协助优化抗血管生成治疗的哈佛商学院在未来的翻译。结果突出了血红蛋白新生血管的复杂性, 认为这种共同的形态血管生成只是血红蛋白新生血管的补充机制。

原图像采用倒置光镜。控件组和 VHL 组的典型图像显示在图 2A1和图 2A2中。对照组的发芽长度短于 VHL 组。
上传图像后, 在线平台直接提供分析结果。输出由两部分组成: 处理后的图像和相应的分析结果。控制组和 VHL 基因静默组的处理图像标记为不同的颜色 (图 2A3和图 2A4)。对照组和 VHL 沉默组的平均发芽长度分别为65µm 和125µm, 对照组和µm 沉默组分别为680µm 和 1250 VHL, 表明发芽长度由与控制组 (图 2B) 相比, VHL 静默组中大约有两个褶皱。这些结果表明, VHL 缺乏促进血管形成能力的人内皮细胞。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57183/57183fig1.jpg"> 图 1.球体发芽试验的图式.步骤1显示 HUVEC 细胞培养皿。在步骤2中, HUVEC 单元格被移动到3D 圆底96井板上72小时. 步骤3显示 HUVEC 细胞形成内皮细胞球体自动在3D 板。在步骤4中, 将细胞球体添加到稀释的凝胶中。在步骤5中, 将上面准备好的混合物添加到15井板上。步骤6显示了球体在15井板块发芽。步骤7显示了在倒置光显微镜下拍摄的发芽图像。步骤8显示上载的图像和步骤9是从平台 (条形 = 100 µm) 的输出图像。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57183/57183fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57183/57183fig2.jpg"> 图 2.VHL 基因沉默对球形发芽试验 HUVEC 细胞血管生成能力的影响。(A) 12 小时控制 (A1) 和 VHL 静音 HUVEC 球体 (A2) (酒吧 = 100 µm) 后, 有代表性的喷口图像。图 A3 和 A4 分别显示了图 A1 和 A2 平台所分析的图像。红色代表芽的骨架, 黄色的芽数, 蓝色的芽结构, 白色芽端, 和橙色球体。(B)长度的芽。运用非配对学生的 t 检验进行统计学分析。* 代表 P < 0.05。控制; 骗局HUVEC, 人脐静脉内皮细胞。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57183/57183fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay at JoVE.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

用球形发芽法评价 Hemangioblastoma 新生血管的体外性能的综合程序

本文提出了一个综合的方法来评价在体外是否存在经典的肿瘤血管生成在小脑 (哈佛商学院) 及其在哈佛商学院的作用。研究结果突出了血红蛋白-新生血管的复杂性, 认为这种共同的血管生成形式仅仅是血红蛋白-新生血管的补充机制。

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

a-comprehensive-procedure-to-evaluate-vitro-performance-putative

Nanyang Technological University

Research

JoVE Journal

Cancer Research

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

7.9K 次观看.  Fudan University. 本文提出了一个综合的方法来评价在体外是否存在经典的肿瘤血管生成在小脑 (哈佛商学院) 及其在哈佛商学院的作用。研究结果突出了血红蛋白-新生血管的复杂性, 认为这种共同的血管生成形式仅仅是血红蛋白-新生血管的补充机制。

视频: A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

3D球形模型作为癌症相关纤维细胞分化和入侵体内研究的更多生理系统

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

科研

癌症研究

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

胶质母细胞瘤的3D球形模型

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

将毛细血管纳入内皮细胞珠发芽试验

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

发育生物学

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are&#160;modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,&#160;a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

医学

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

生物工程

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

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

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 ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

眼部微血管血管生成前维沃胆球状芽分析

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

生物学

使用划痕伤口迁移和球状体发芽测定法研究血管生成

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

作者聚焦：通过检测开发中的挑战和创新研究血管生成

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

利用划痕伤口迁移测定和球状体发芽测定在功能和分子水平上研究血管生成

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...