Name: a three-dimensional model of spheroids to study colon cancer stem cells
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Description: Watch this Scientific Journal Video about A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells at JoVE.com

我们确认成像和阿尼帕德重新切切组织学平台 （CRCL， CLB）.我们感谢里昂·贝拉德中心（CLB）医院的药房，感谢福尔福克斯和福尔菲里赠送的实物礼物。我们还感谢布里吉特·曼希普对手稿的批判性阅读。这项工作得到了FRM（2018年FRM，DEQ20181039598）和印加（PLBIO19-289）的支持。MVG 和 LC 得到了 FRM 的支持，CF 得到了 ARC 基金会和莱昂·贝拉德中心的支持。

注：所有试剂和材料的详细信息都列在 材料表中。
1. 球形形成
<ol><li>球形文化媒体<ol>
<li>准备基础介质，包括杜尔贝科的改良鹰介质 （DMEM）， 辅以 4 mM L-阿拉尼尔- L - 谷氨酰二肽.</li>
		<li>从步骤 1.1.1 开始，在基础介质中准备包含 10% 胎儿牛血清 （FBS） 和 1% 青霉素链霉素 （笔/链球菌） 的 DMEM 完整介质。</li>
		<li>从步骤 1.1.1 开始，在基础介质中准备包含 2.5%...

<ol>
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体外3D模型克服了2D癌细胞培养的主要实验缺陷，因为它们在回顾典型的肿瘤特征（包括微环境和细胞异质性）方面似乎更可靠。常用的球体 3D 模型是无脚手架（在低附件条件下培养）或基于脚手架（使用生物材料培养细胞）。这些方法由于取决于使用的脚手架的性质或产生结构和大小变化的球体而呈现不同的缺点。
本协议报告了使用无脚手架方法从结肠腺癌细胞系 Caco2 中?...

结肠直肠癌（CRC）仍然是世界第二大癌症相关死亡原因。CRC的发展是基因突变和/或表观遗传改变2，3逐步获取和积累的结果，包括肿瘤基因的激活和肿瘤抑制基因3，4的灭活。此外，非遗传因素（如微环境）可以促进和促进原基因转化，从而参与CRC5的演变。重要的是，CRC由不同的细胞群组成，包括未分化的CSC和显示一些分化特征的散装肿瘤细胞，它们构成了一个层次结构，让人联想到正常结肠密码6、7中的上皮组织。
CSC被认为是负责肿瘤外观8，其维持和生长，转移能力，和耐常规疗法6，7。在肿瘤中，癌细胞，包括CSC，在它们独特的突变和表观遗传特征、形态和表型差异、基因表达、新陈代谢、增殖率和转移潜力方面表现出高度的异质性和复杂性。因此，为了更好地了解癌症生物学，肿瘤进展，并获得抗药性治疗及其转化为有效的治疗，人类前科模型捕捉这种癌症的异质性和层次性是重要的10，11。
体外2D癌细胞系已被长期使用，为肿瘤的发展和治疗分子疗效的机制提供了宝贵的见解。然而，由于原始肿瘤中缺乏表型和遗传异质性，它们的限制现在已得到广泛认可。此外，营养物质、氧气、pH梯度和肿瘤微环境不复发生，微环境对于维持包括CSC11、12在内的不同细胞类型的特别重要。为了克服这些主要缺点，已经开发了几种3D模型，以实验性地解决和再现癌症的复杂性和异质性。实际上，这些模型可回顾肿瘤细胞异质性、细胞相互作用和空间结构，类似于体内12、13、14中观察到的模型。从新鲜肿瘤以及细胞线衍生球体中建立的原发性肿瘤器官主要采用15、16。
球体可以以无脚手架或脚手架的方式培养，以迫使细胞形成细胞并在细胞聚合体中生长。无脚手架的方法基于非粘附条件下的细胞培养（例如，悬垂法或超低附着板），而基于脚手架的模型则依赖于自然、合成或混合生物材料来培养细胞12、13、14。基于脚手架的球体呈现不同的缺点，因为最终的球体形成将取决于所用（生物）材料的性质和组成。虽然脚手架无球体方法，到目前为止不依赖于基板的性质，他们产生球体，在结构和大小17，18变化。
这项工作旨在设计一个强大和可重复的球体3D培养系统，其大小是同质的，由Caco2结肠腺癌细胞组成，以研究CSC生物学。Caco2细胞特别感兴趣，因为它们能够随着时间的推移分化19，20，强烈地暗示了干细胞般的潜力。因此，球体的长期培养揭示了不同CSC人群对化疗有不同的反应。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>37 &#181;m Reversible Strainer, Large&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>27250</td>
			<td>To be used with 50 mL conical tubes</td>
		</tr>
		<tr>
			<td>5-Fluorouracil</td>
			<td>Gift from Pharmacy of the Centre Leon Berard (CLB)</td>
			<td>-</td>
			<td>stock solution, 5 mg/100 mL; final concentration, 50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Aggrewell 400 24-well plates</td>
			<td>STEMCELL Technologies</td>
			<td>34411</td>
			<td>1,200 microwells per well for spheroid formation and growth</td>
		</tr>
		<tr>
			<td>Anti Caspase3 - Rabbit</td>
			<td>Cell Signaling</td>
			<td>9661</td>
			<td>dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti Musashi-1 (14H1) - Rat</td>
			<td>eBioscience/Thermo Fisher</td>
			<td>14-9896-82</td>
			<td>dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution x 100 mL</td>
			<td>STEMCELL Technologies</td>
			<td>07010</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD133 (13A4) - Rat</td>
			<td>Invitrogen</td>
			<td>14-133-82</td>
			<td>dilution 1:100</td>
		</tr>
		<tr>
			<td>Anti-CD44 -Rabbit</td>
			<td>Abcam</td>
			<td>ab157107</td>
			<td>dilution 1:2000</td>
		</tr>
		<tr>
			<td>Anti-PCNA - Mouse</td>
			<td>Dako</td>
			<td>M0879</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Anti-&#946;-catenin - Mouse</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-7963</td>
			<td>dilution 1:50</td>
		</tr>
		<tr>
			<td>Black multiwell plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>237108</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid Monohydrate</td>
			<td>Sigma</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>CLARIOstar apparatus&#160;</td>
			<td>BMG Labtech</td>
			<td></td>
			<td>microplate reader</td>
		</tr>
		<tr>
			<td>Dako pen</td>
			<td></td>
			<td></td>
			<td>marker pen to mark circles on slides for creating barriers for liquids</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21202</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10037</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21206</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10042</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) Glutamax (L-alanyl-L-glutamine dipeptide)</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorogel mounting medium with DAPI</td>
			<td>Interchim</td>
			<td>FP-DT094B</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11077</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td></td>
			<td></td>
			<td>Spheroid image analysis</td>
		</tr>
		<tr>
			<td>Irinotecan&#160;</td>
			<td>Gift from Pharmacy CLB</td>
			<td>-</td>
			<td>stock solution, 20 mg/mL; final concentration, 100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>iScript reverse transcriptase&#160;</td>
			<td>Bio-Rad</td>
			<td>1708891</td>
			<td></td>
		</tr>
		<tr>
			<td>Leucovorin</td>
			<td>Gift from Pharmacy CLB</td>
			<td>-</td>
			<td>stock solution, 50 mg/mL; final concentration, 25 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>Nucleospin RNA XS Kit</td>
			<td>Macherey-Nagel</td>
			<td>740902 .250</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxaliplatin</td>
			<td>Gift from Pharmacy CLB</td>
			<td>-</td>
			<td>stock solution, 100 mg/20 mL;final concentration, 10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140130</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR qPCR Premix Ex Taq II (Tli RNaseH Plus)</td>
			<td>Takara</td>
			<td>RR420B</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX- Green</td>
			<td>Thermo Fisher Scientific</td>
			<td>S7020</td>
			<td>nucleic acid stain; dilution 1:5000</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05&#160;%)</td>
			<td>Gibco</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss-Axiovert microscope</td>
			<td></td>
			<td></td>
			<td>inverted microscope for acquiring images of spheroids</td>
		</tr>
	</tbody>
</table>

a three-dimensional model of spheroids to study colon cancer stem cells

结肠直肠癌的特点是异质性和分层组织，由负责肿瘤开发、维持和抗药性的癌症干细胞（CSC）群体组成。因此，更好地了解CSC特性对于其特定目标来说是有效治疗的先决条件。然而，缺乏适合深入调查的可适用的前科模型。虽然体外二维（2D）癌细胞系为肿瘤生物学提供了有价值的见解，但它们不会复制表型和遗传肿瘤异质性。相比之下，三维（3D）模型处理和再现近生理癌症的复杂性和细胞异质性。这项工作的目的是设计一个强大和可重复的3D培养系统来研究CSC生物学。目前的方法描述了从Caco2结肠腺癌细胞中产生3D球体的条件的发展和优化，这种模型可用于长期培养。重要的是，在球体中，围绕流明状结构组织的细胞的特点是微分细胞增殖模式，以及CSC的存在，这些细胞表示一组标记物。这些结果为研究细胞异质性和CSC生物学（包括对化疗的反应）的这种3D方法的恰当性提供了第一个概念证明。

由于球体大小缺乏同质性是目前可用的3D球形培养系统13的主要缺点之一，这项工作的目的是建立一个可靠和可重复的协议，以获得同质球体。首先，为了建立理想的工作条件，使用专用板（表1）测试了不同数量的Caco2细胞，每个微孔/球体的细胞从50到2000个不等。实际上，这些板块中的每口井都含有1，200个微孔，使每口井形成相同数量的球?...

Watch this Scientific Journal Video about A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells at JoVE.com

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

该协议提出了一个新型的，强大的，可重复的文化系统，以产生和生长三维球体从Caco2结肠腺癌细胞。研究结果为研究癌症干细胞生物学（包括化疗反应）的方法的恰当性提供了第一个概念证明。

研究结肠癌干细胞的球体三维模型

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

这种球形培养的新方法可用于研究肿瘤原性，分析不同人群的癌症干细胞的存在，也可以很好地应用于通过高放屏的新药。这是一个强大和重复的培养系统，用于生成大量均匀大小的球体。此外，这些球体可用于研究癌干细胞。这种方法是为了研究慢性癌症生物学而建立的，但通过改变3D培养条件，可以很容易地建立起来研究其他癌症。一定要注意介质中的气泡，在球体形成过程中不要移动盘子太多，在将球体收集到滤网时要小心。首先对每口井500微升防粘性冲洗液的24口井板进行预处理。几秒钟后，将板在摆动桶转子中离心，用2毫升温暖的玄武岩介质冲洗每口油井。观察显微镜下的板，以确保气泡已完全从微井中去除。如果没有观察到气泡，请再次冲洗油井，然后再向每口井添加一毫升温暖的DMEM地下室膜基质介质。要诱导球体形成，请参阅 DMEM 中 10 厘米的盘中 10 厘米盘中 2D 单层中 Caco-2 细胞的预期浓度，并辅以 10% FBS 和 1% 抗生素。在37摄氏度和5%的潮湿大气中培养。当达到80%的汇合度时，用五毫升PBS清洗细胞，然后用两毫升的Trypsin-EDTA在37摄氏度和5%的二氧化碳下处理培养物两到五分钟。当细胞分离中和特里普辛与四毫升完整的DMEM。计数后，通过离心收集细胞，并以适当体积的 DMEM 基层基质基质介质重新分配调色板，以达到每口井一毫升中等细胞的预期浓度。在预付费 24 油井板的每口井中加入一毫升细胞，然后向每口井添加一毫升 DMEM 基膜基质介质。播种后，立即离心板，并使用光显微镜，以验证细胞已均匀分布在微井。然后将盘子放在细胞培养孵化器中48小时，而不会干扰细胞。要收获球体，在孵化结束时，使用血清学移液器和每口井中一半的超自然物轻轻地将球体从其微井中分离。当所有球体都分离后，使用移液器将 3D 培养物从每口井小心地转移到 15 毫升圆锥管顶部的 37 微米可逆滤应变器。每口井洗三次，每次洗一毫升预热的Basal介质，以收集任何剩余的球体，并将这些额外的球体添加到过滤器中。最后一次洗涤后，检查显微镜下的板，以确认所有球体已从微井中去除。然后将滤网倒置到新的 15 毫升管上，用新鲜的 DMEM 底层膜基质介质清洗滤网底部，将球体收集到管中。球体从500个细胞中上升，表明随着时间的推移，其大小会增加最均匀。球体外围有500和600个细胞，确定是实现良好生长和低变异性的最佳细胞数量。地下室膜基质的添加可增强球体生长和均匀性。在长期培养中，细胞在第三天以密集的多层身份出现。虽然在单层中排列的扁平细胞在第 10 天清晰可见。有趣的是，从第五天开始，球体中就出现了流明。此外，在第5天和第7天观察到增殖细胞核抗原阳性细胞明显增加，到第10天下降。令人惊讶的是，激活的Caspace3只在第三天和第五天在极少数细胞中观察到。虽然每个时间点都观察到高水平的β-卡泰宁表达。球体分化成前体的潜力从其碱性磷酸盐和溶解载体家族2A5 mRNA表达中得到了证明。此外，这种球形表示典型的癌症干细胞标记，并响应常规给结肠直肠癌患者施用的联合化疗治疗。因此，球体由不同的细胞群组成。然后，它们可以很好地用于分析细胞对刺激（如耐火器）的特异性反应，并最终用于药物反应。

Research

JoVE Journal

Cancer Research

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

研究病毒载体在三维肝模型重新填充与人肝癌细胞株HepG2

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

科研

癌症研究

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

结肠-26癌荷瘤小鼠为癌症恶病质的研究模型

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

一种确定环境富集对小鼠结肠肿瘤模型结肠菌群生物多样性影响的方法

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

三维肿瘤球体的纵向形态和生理监测

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

在结肠直肠癌细胞的三维球体培养物中使用无细胞益生菌超常剂评估细胞死亡

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...