我们感谢Christopher Hughes博士实验室的成员对所述程序的宝贵投入，以及Abraham Lee博士实验室的合作者在平台设计和制造方面的帮助。这项工作得到了以下资助：UG3/UH3 TR002137、R61/R33 HL154307、1R01CA244571、1R01 HL149748、U54 CA217378 （CCWH） 和 TL1 TR001415 和 W81XWH2110393 （SJH）。

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

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CCWH拥有Aracari Biosciences， Inc.的股权，该公司正在将本文所述技术的一个版本商业化。该安排的条款已由加州大学欧文分校根据其利益冲突政策进行审查和批准。没有其他利益冲突。

身体中几乎每个组织都通过脉管系统获得营养和氧气，使其成为现实疾病建模和 体外药物筛选的关键组成部分。此外，几种恶性肿瘤和疾病状态由血管内皮功能障碍和高通透性定义 3。值得注意的是，在癌症中，肿瘤相关的脉管系统通常灌注不良、破坏和渗漏，从而成为治疗和免疫细胞递送至肿瘤的障碍。此外，脉管系统可作为癌细胞转移以播种远处组织的管道，并促进?...

癌症仍然是全球主要的健康问题，也是美国的第二大死因。仅在 2023 年，美国国家卫生统计中心就预计美国将有超过 190 万例新发癌症病例和超过 600,000 例癌症死亡1，这凸显了对有效治疗方法的迫切需要。然而，目前，只有5.1%的进入临床试验的抗癌疗法最终获得了FDA的批准。有希望的候选药物未能成功通过临床试验可部分归因于在临床前药物开发过程中使用了非生理模型系统，例如 2D 和球状培养物 2。这些经典癌症模型缺乏肿瘤微环境的基本组成部分，例如基质生态位、相关免疫细胞和灌注脉管系统，这些都是治疗耐药性和疾病进展的关键决定因素。因此，为了改善临床前发现的临床转化，需要一种更好地模拟人类 体内 肿瘤微环境的新模型系统。
组织工程领域正在迅速发展，为在实验室环境中研究人类疾病提供了改进的方法。一个重要的发展是微生理系统（MPS）的出现，也称为器官芯片或组织芯片，它们是功能性的小型化人体器官，能够复制健康或疾病条件3,4,5。在此背景下，肿瘤芯片是基于三维微流控的体外人类肿瘤模型，已被开发用于肿瘤学研究2,3,4,5,6,7,8,9,10,11,12,13 .这些先进的模型在动态肿瘤微环境中结合了生物化学和生物物理线索，使研究人员能够在更相关的生理环境中研究肿瘤行为和对治疗的反应。然而，尽管有这些进步，但很少有小组成功地整合了活的、功能性的脉管系统，特别是响应生理流动的自我模式的脉管系统3,4,5,6。功能性血管网络的加入至关重要，因为它允许对影响药物或细胞递送的物理屏障、细胞归巢到不同微环境以及肿瘤、基质和免疫细胞的跨内皮迁移进行建模。通过包含这一特征，肿瘤芯片可以更好地表示在体内肿瘤微环境中观察到的复杂性。
为了满足这一未满足的需求，我们开发了一种新型药物筛选平台，使微流控装置8,9,10,11,12,13,14,15,16形成微血管网络。这种基础器官芯片平台被称为血管化微器官 （VMO），几乎可以适应任何器官系统，以复制原始组织生理学，用于疾病建模、药物筛选和个性化医疗应用。VMO 是通过共培养内皮集落形成细胞来源的内皮细胞 （ECFC-EC）、HUVEC 或 iPSC-EC（以下简称 EC）和腔室中的多个基质细胞（包括重塑基质的正常人肺成纤维细胞 （NHLF）和包裹和稳定血管的周细胞）来建立的。VMO也可以通过将肿瘤细胞与相关基质共培养来建立为癌症模型系统，以创建血管化微肿瘤（VMT）8,9,10,11,12,13或肿瘤芯片模型。通过在动态流动环境中共培养多种细胞类型，灌注的微血管网络在装置的组织室中从头形成，其中血管生成受到间质流速的密切调节14,15。培养基由静水压力头驱动通过设备的微流体通道，静水压力头仅通过微血管为组织室的周围细胞提供营养，渗透系数为 1.2 x 10-7 cm/s，类似于体内毛细血管的可见值 8。
将自组织微血管纳入VMT模型代表了一项重大突破，因为它：1）模拟体内血管化肿瘤肿块的结构和功能;2）可以模拟转移的关键步骤，包括肿瘤-内皮细胞和基质细胞相互作用;3）建立营养和药物递送的生理选择性屏障，改善药物筛选;4）允许直接评估具有抗血管生成和抗转移能力的药物。通过在复杂的 3D 微环境中复制营养物质、药物和免疫细胞的体内递送，VMO/VMT 平台是一种生理相关模型，可用于进行药物筛选和研究癌症、血管或器官特异性生物学。重要的是，VMT 支持各种类型肿瘤的生长，包括结肠癌、黑色素瘤、乳腺癌、胶质母细胞瘤、肺癌、腹膜癌、卵巢癌和胰腺癌 8,9,10,11,12,13。除了成本低、易于建立和排列用于高通量实验外，微流控平台还完全光学兼容，可用于肿瘤-基质相互作用和对刺激或治疗的反应的实时图像分析。系统中的每种细胞类型都用不同的荧光标记物标记，以便在整个实验过程中直接可视化和跟踪细胞行为，从而为动态肿瘤微环境创造一个窗口。我们之前已经表明，与标准培养模式相比，VMT 更忠实地模拟体内肿瘤生长、结构、异质性、基因表达特征和药物反应10。重要的是，VMT支持患者来源细胞（包括癌细胞）的生长和研究，与标准球状体培养物相比，它更好地模拟了母体肿瘤的病理学，并进一步推进了个性化医疗工作11。这份手稿概述了建立VMT的方法，展示了其在研究人类癌症方面的效用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fabrication</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(3-Mercaptopropyl)trimethoxysilane, 95%&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>175617-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Greiner Bio-One&#160;&#956;Clear Bottom 96-well Polystyrene Microplates</td>
			<td>Greiner Bio-One</td>
			<td>655096</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol &#8805;99.8% ACS</td>
			<td>VWR Chemicals BDH</td>
			<td>BDH1135-1LP</td>
			<td></td>
		</tr>
		<tr>
			<td>MILTEX Sterile Disposable Biopsy Punch with Plunger, 1mm diameter,</td>
			<td>Integra Miltex</td>
			<td>33-31AA-P/25</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS membrane</td>
			<td>PAX Industries</td>
			<td>HT-6240</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner PDC-001</td>
			<td>Harrick Plasma</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Smooth-Cast 385</td>
			<td>Smooth-On</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>SP Bel-Art Lab Companion Clear Polycarbonate Cabinet Style Vacuum Desiccator</td>
			<td>Bel-Art</td>
			<td>F42400-4031</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Lids with Condensation Rings, 96-well plate</td>
			<td>VWR</td>
			<td>82050-827</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD 184 Silicone Elastomer Kit (PDMS)</td>
			<td>Dow</td>
			<td>4019862</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture/Loading</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Lionheart FX Automated Microscope</td>
			<td>Agilent&#160;</td>
			<td>CYT5MFAW</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLvo Human Endothelial Progenitor Cells</td>
			<td>StemBioSys</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I, rat tail</td>
			<td>Enzo Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum (type 4)</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Hank&#8217;s Balanced Salt Solution, 1X without calcium and magnesium</td>
			<td>Corning</td>
			<td>21-021-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate</td>
			<td>Corning</td>
			<td>10013CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 Endothelial Cell Growth Medium-2 BulletKit</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen from bovine plasma</td>
			<td>Neta Scientific</td>
			<td>SIAL-341573</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin human plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F0895</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;dextran (70kDa)</td>
			<td>Sigma-Aldrich</td>
			<td>FD70S-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase from sheep testes (type 4)</td>
			<td>Sigma-Aldrich</td>
			<td>H6254</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein</td>
			<td>Gibco</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP8</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>ATCC</td>
			<td>HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>NHLF &#8211; Normal Human Lung Fibroblasts</td>
			<td>Lonza</td>
			<td>CC-2512</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 4% in 0.1M Phosphate BufferSaline, pH 7.4</td>
			<td>Electron Microscopy Sciences&#160;</td>
			<td>15735-90-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>PBMCs - Peripheral blood mononuclear cells</td>
			<td>Lonza</td>
			<td>CC-2702</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Grade Fetal Bovine Serum (FBS), Heat Inactivated</td>
			<td>Avantor Seradigm</td>
			<td>97068-091</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick-RNA Microprep Kit</td>
			<td>Zymo Research</td>
			<td>R1051</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin from bovine plasma</td>
			<td>Sigma-Aldrich</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 (Electrophoresis),</td>
			<td>Fisher BioReagents</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1X), phenol red</td>
			<td>Gibco</td>
			<td>12605028</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>Vasculife</td>
			<td>Lifeline Cell Technology</td>
			<td>LL-0003</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing a physiologic human vascularized micro-tumor model for cancer research

缺乏经过验证的癌症模型来概括 体外 实体癌的肿瘤微环境仍然是临床前癌症研究和治疗开发的重大瓶颈。为了克服这个问题，我们开发了血管化微肿瘤（VMT）或肿瘤芯片，这是一种微生理系统，可以真实地模拟复杂的人类肿瘤微环境。VMT通过在动态生理流动条件下共培养多种人类细胞类型，在微流控平台 内从头 形成。这种组织工程微肿瘤结构包含一个活的灌注血管网络，该网络支持不断增长的肿瘤块，就像新形成的血管 在体内一样。重要的是，药物和免疫细胞必须穿过内皮层才能到达肿瘤， 从而 模拟体内治疗递送和疗效的生理障碍。由于VMT平台是光学透明的，因此可以通过直接可视化组织内荧光标记的细胞来实现免疫细胞外渗和转移等动态过程的高分辨率成像。此外，VMT保留了 体内 肿瘤异质性、基因表达特征和药物反应。几乎任何肿瘤类型都可以适应该平台，来自新鲜手术组织的原代细胞在VMT中生长并对药物治疗做出反应，为真正的个性化医疗铺平了道路。本文概述了建立VMT并将其用于肿瘤学研究的方法。这种创新方法为研究肿瘤和药物反应开辟了新的可能性，为研究人员提供了推进癌症研究的有力工具。

Cancer remains a major health concern worldwide and is the second leading cause of death in the United States. For the year 2023 alone, the National Center for Health Statistics anticipates more than 1.9 million new cancer cases and over 600,000 cancer deaths occurring in the US1, highlighting the urgent need for effective treatment approaches. However, currently, only 5.1% of anti-cancer therapeutics entering clinical trials ultimately gain FDA approval. Failure of promising candidates to successfully progress through clinical trials can be partially attributed to the use of non-physiological model systems, such as 2D and spheroid cultures, during preclinical drug development2. These classical cancer models lack essential components of the tumor microenvironment, such as a stromal niche, associated immune cells, and perfused vasculature, which are key determinants of therapeutic resistance and disease progression. Thus, a new model system that better mimics the human in vivo tumor microenvironment is necessary to improve the clinical translation of preclinical findings.
The field of tissue engineering is rapidly advancing, providing improved methods for studying human diseases in laboratory settings. One significant development is the emergence of microphysiological systems (MPS), also known as organ chips or tissue chips, which are functional, miniaturized human organs capable of replicating healthy or diseased conditions3,4,5. Within this context, tumor chips, which are three-dimensional microfluidic-based in vitro human tumor models, have been developed for oncology research2,3,4,5,6,7,8,9,10,11,12,13. These advanced models incorporate biochemical and biophysical cues within a dynamic tumor microenvironment, enabling researchers to study tumor behavior and responses to treatments in a more physiologically relevant context. However, despite these advancements, few groups have successfully incorporated a living, functional vasculature, particularly one that self-patterns in response to physiologic flow3,4,5,6. The inclusion of a functional vascular network is crucial as it allows for modeling physical barriers that affect drug or cell delivery, cell homing to distinct microenvironments, and transendothelial migration of tumor, stromal, and immune cells. By including this feature, the tumor chip can better represent the complexities observed in the in vivo tumor microenvironment.
To address this unmet need, we have developed a novel drug-screening platform that enables micro-vessel networks to form within a microfluidic device8,9,10,11,12,13,14,15,16. This base organ chip platform, termed the vascularized micro-organ (VMO), can be adapted to virtually any organ system to replicate original tissue physiology for disease modeling, drug screening, and personalized medicine applications. VMOs are established by co-culturing endothelial colony-forming cell-derived endothelial cells (ECFC-EC), HUVEC or iPSC-EC (hereafter EC), and multiple stromal cells in the chamber, including normal human lung fibroblasts (NHLF), which remodel the matrix, and pericytes that wrap and stabilize the vessels. The VMO can also be established as a cancer model system by co-culturing tumor cells with the associated stroma to create a vascularized micro-tumor (VMT)8,9,10,11,12,13, or tumor chip, model. Through the co-culture of multiple cell types in a dynamic flow environment, perfused microvascular networks form de novo in the tissue chambers of the device, where vasculogenesis is closely regulated by interstitial flow rates14,15. Medium is driven through the microfluidic channels of the device by a hydrostatic pressure head that supplies the surrounding cells of the tissue chamber with nutrients exclusively through the micro-vessels, with a permeability coefficient of 1.2 x 10-7 cm/s, similar to what is seen for capillaries in vivo8.
The incorporation of self-organizing micro-vessels into the VMT model represents a significant breakthrough because it: 1) mimics the structure and function of vascularized tumor masses in vivo; 2) can model key steps of metastasis, including tumor-endothelial and stromal cell interactions; 3) establishes physiologically selective barriers for nutrient and drug delivery, improving pharmaceutical screening; and 4) allows direct assessment of drugs with anti-angiogenic and anti-metastatic capabilities. By replicating the in vivo delivery of nutrients, drugs, and immune cells in a complex 3D microenvironment, the VMO/VMT platform is a physiologically relevant model that can be used to perform drug screening and study cancer, vascular or organ-specific biology. Importantly, the VMT supports the growth of various types of tumors, including colon cancer, melanoma, breast cancer, glioblastoma, lung cancer, peritoneal carcinomatosis, ovarian cancer, and pancreatic cancer8,9,10,11,12,13. In addition to being low-cost, easily established, and arrayed for high throughput experiments, the microfluidic platform is fully optically compatible for real-time image analysis of tumor-stromal interactions and response to stimuli or therapeutics. Each cell type in the system is labeled with a different fluorescent marker to allow direct visualization and tracking of cell behavior throughout the entire experiment, creating a window into the dynamic tumor microenvironment. We have previously shown that the VMT more faithfully models in vivo tumor growth, architecture, heterogeneity, gene expression signatures, and drug responses than standard culture modalities10. Importantly, the VMT supports the growth and study of patient-derived cells, including cancer cells, which better models the pathology of the parent tumors than standard spheroid cultures and further advances personalized medicine efforts11. This manuscript outlines the methods for establishing the VMT, showcasing its utility for studying human cancers.

Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

建立用于癌症研究的生理性人血管化微肿瘤模型

按照此处概述的方案，使用商业购买的 EC、NHLF 以及用于 VMT 的三阴性乳腺癌细胞系 MDA-MB-231 建立 VMO 和 VMT。已建立的 VMO 也灌注癌细胞以模拟转移。在每个模型中，在共培养的第 5 天，血管网络响应重力驱动的流经组织室而自组装，作为 体内 营养物质、治疗药物、癌症或免疫细胞输送到基质生态位的管道（图 4）。首先通过将 mCherry 标记的 EC 引入组织室来建立 VMO，?...

Watch this Scientific Journal Video about Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research at JoVE.com

该协议提出了一种生理学相关的肿瘤芯片模型，用于执行高通量基础和转化人类癌症研究，推进药物筛选、疾病建模和个性化医疗方法，并描述了加载、维持和评估程序。

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

我们的肿瘤芯片平台旨在对人类血管化微肿瘤的生长进行生理建模，并使我们能够回答基础和转化研究问题。问题侧重于基础肿瘤生物学、药物疗效、肿瘤反应性、潜在的免疫学过程、潜在的免疫治疗干预和个性化治疗方法。有效地将临床前发现转化为有效的癌症治疗方法取决于忠实地复制疾病状态和模型系统。该协议概述了血管化微肿瘤（VMT）的建立，以模拟体外肿瘤微环境。VMT忠实地再现了人类肿瘤的关键方面，产生了临床相关的发现。我们的模型使我们能够进行与生理相关的转化人类癌症研究。具体来说，与其他模型相比，我们的方案结合了动态流动条件，实现了免疫细胞和免疫疗法的整合，并进一步允许我们纳入个性化医疗方法。我们正在采用VMT模型来推动临床前和临床领域的个性化医疗计划。该模型加深了我们对细胞间相互作用中微环境因素的理解，影响了治疗耐药性和癌症进展。此外，VMT在发现定制的患者特异性治疗方法以增强临床护理方面显示出希望。

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Unit 1

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

Pharmacokinetics: Drug Biotransformation

SCIENTISTS IN ACTION

1.3K 次观看.  University of California, Irvine. 我们的肿瘤芯片平台旨在对人类血管化微肿瘤的生长进行生理建模，并使我们能够回答基础和转化研究问题。问题侧重于基础肿瘤生物学、药物疗效、肿瘤反应性、潜在的免疫学过程、潜在的免疫治疗干预和个性化治疗方法。有效地将临床前发现转化为有效的癌症治疗方法取决于忠实地复制疾病状态和模型系统。该协议概述了血管化微肿瘤（VMT）的建立，以模拟体外肿?...

视频: Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.