Name: a robust method for the large-scale production of spheroids for high-content screening and analysis applications
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作者承认爱尔兰科学基金会（SFI）（16 / RI / 3745）对JCS的基础设施研究资助的支持。ucd细胞筛选实验室的工作由ucd科学学院支持。ASC由爱尔兰研究委员会（IRC）爱尔兰政府研究生奖学金（GOIPG / 2019 / 68）资助。作者还感谢实验室所有成员的投入和有益的讨论。图 1 中的图稿是在 BioRender 中生成的。

1. 细胞培养
<ol><li>准备介质<ol>
<li>根据细胞系的类型制备特定的细胞培养基。确保所有用于细胞维护的培养基都含有10%的胎儿牛血清（FBS）。 注意：不同的细胞系使用不同的培养基。HT-29结肠癌细胞（ATCC HTB-38）在McCoys 5A + 10%FBS中生长。HepG2肝细胞癌细胞（ATCC HB-8065）在最低必需培养基+ 1%L-谷氨酰胺+ 10%FBS中生长。H358支气管肺泡癌细胞（ATCC CRL-5807）在RPMI 1640培养基+ 1%L-谷氨酰胺+ 10...

<ol>
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这里描述的方法详细介绍了一个平台，该平台以适合HCS和HCA的方式每孔产生数百个球体。与其他流行的方法相比，例如使用平底和圆底ULA板，这些板允许每个孔仅形成一个旋转椭球体18，19，这种方法为以筛选格式从大量球体中提取高分辨率信息提供了机会。值得注意的是，这种方法已经在3种不同的细胞系中得到证明，代表了各种肿瘤类型，突出了它对...

传统上，基于细胞的测定是在固体底物上生长的单层中进行的，这实际上可以被认为是二维（2D）环境。然而，人们越来越认识到，2D细胞培养模型在某些情况下缺乏生理相关性，无法复制细胞之间发生的许多复杂相互作用1。三维（3D）细胞培养方法在研究人员中迅速流行，3D细胞模型显示出更好地模拟细胞在组织环境中遇到的生理条件的巨大潜力2。已经使用了几种不同类型的3D细胞组件，但最常见的两种类型是球体和类器官。球状体可以从许多不同的细胞系中生长，它们可以根据所使用的细胞类型及其组装方法采用各种形状和大小3。此外，当球状体从癌细胞系生长时，它们也可以被称为多细胞肿瘤球体（MCTS），这些模型已经发现特别适用于临床前 体外 药物递送和毒性研究4，5。另一方面，类器官旨在更好地模仿我们体内的组织和器官，并且可以采用更复杂的形态安排。类器官的产生涉及使用成体干细胞或多能干细胞，它们可以重新编程到适当的细胞中以类似于感兴趣的组织或器官。它们主要用于研究器官的发育，并对疾病和宿主-病原体相互作用进行建模6。
有一系列不同的方法可用于生成 3D 单元组件。基于支架的方法提供了一种底物或支撑物，细胞可以附着或生长在其内。这些脚手架可以有各种形状，可以由各种不同的材料制成。最常见的是细胞外基质（ECM）成分和水凝胶，它们被设计成类似于细胞的天然细胞外环境，从而促进生理相互作用4，7。ECM基底材料已从Engelbreth-Holm-Swarm小鼠肉瘤肿瘤中提取，并显示含有丰富的ECM成分混合物，包括层粘连蛋白，IV型胶原蛋白和perlecan8。然而，尽管其成分有利，但其使用存在两个主要挑战，即其批次间变异性，以及它在10°C8，9以下和上方具有两种不同的聚集状态。相比之下，水凝胶在组件和刚度方面具有柔韧性的优势，并且可以定制以适应所需的特定3D细胞组件7，10。基于支架的方法对于类器官生长至关重要，但也广泛用于球体。无支架方法通过防止细胞附着在它们生长的表面上起作用，通常只与球体组装兼容。例子包括超低附着（ULA）板，具有平底或U形底，允许细胞聚集成球状体，或使用旋转瓶中细胞的连续搅拌10。
使用3D细胞组件来研究各种生物事件正在迅速普及;然而，为他们的养殖选择的方法必须适当，并且与下游分析计划兼容。例如，使用ULA板可产生高稠度的球体;然而，这种方法仅限于生产每孔的单个球体，从而限制了通量。在计划对3D结构进行荧光成像时，需要特别考虑。生长组件的基板或板需要具有光学相容性，并且必须注意将可能使用的任何支架引起的光散射的影响降至最低11。随着显微镜物镜的数值孔径增加，这一特殊问题变得更加尖锐。
可以说，选择使用3D细胞模型的主要原因之一是提取不仅关于整个组件的体积成像数据，还提取其中单个细胞的体积成像数据。特别是MCTS模型，对于加深我们对治疗药物如何从外部传递到中心细胞（就像它们在肿瘤中需要的那样）的理解方面开始变得非常强大12，因此从不同层的单个细胞中获得知识至关重要。从单个细胞中提取定量信息的成像技术被称为高内涵分析（HCA），是筛选环境中的一种强大方法13。迄今为止，HCA几乎只应用于单层培养物，但人们越来越意识到这种方法具有应用于3D培养物的能力，从而能够研究广泛的细胞功能和过程14。它具有明显的优势，可以分析大量的3D组件，从而可能从每个结构中提供单元级数据。然而，需要克服与潜在厚细胞组件的成像以及生成的大型数据集相关的挑战。
本文介绍了一种基于支架的稳健方法，用于大规模生产96孔格式的MCTS。该方法有助于在每个孔中生产数百个3D单元组件。显示了三种不同细胞类型的示例，代表肝脏，肺和结肠的实体瘤模型。形成的球体可以是各种大小，因此HCA用于选择特定大小和/或形态的结构。这一特征提供了额外的优势，即观察到的任何表型都可以在不同大小的球体之间进行比较，但在同一孔中以相同的方式处理。这种方法与高分辨率成像兼容，重要的是提供来自相同细胞组件的细胞水平和亚细胞水平的定量数据。与每孔产生单个球体的方法相比，这种球体生产方法具有额外的优势，即每个孔中产生的大量球体可能为其他下游分析（如转录组和蛋白质组分析）提供足够的生物量。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA (1x), phenol red</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A6003</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>10050070</td>
			<td></td>
		</tr>
		<tr>
			<td>CellCarrier-96 Ultra Microplates, tissue culture treated, black, 96-well with lid</td>
			<td>Perkin Elmer</td>
			<td>6055302</td>
			<td>These plates have been renamed as Phenoplates</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum (FBS), qualified, EU approved, South America origin, heat inactivated</td>
			<td>Gibco</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP381-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine solution, 200 mM</td>
			<td>Gibco</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>14533</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher Scientific</td>
			<td>10647032</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix, Phenol Red-free, LDEV-free, 10 mL</td>
			<td>Corning</td>
			<td>356237</td>
			<td>This Matrigel formulation can be also found with the same catalogue number at BD Biosciences</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced Matrigel</td>
			<td>BD Biosciences</td>
			<td>356231</td>
			<td>This Matrigel formulation can be also found with the same catalogue number at Corning</td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5A medium</td>
			<td>Gibco</td>
			<td>26600023</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5A medium with L glutamine and sodium bicarbonate, without phenol red</td>
			<td>Hyclone</td>
			<td>10358633</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM)</td>
			<td>Gibco</td>
			<td>21090022</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM), without glutamine, without phenol red</td>
			<td>Gibco</td>
			<td>51200046</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-LAMP1 antibody (concentrate)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>H4A3-a</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Hirschmann</td>
			<td>8100203</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunclon tissue culture dish with lid, polystyrene, 92 mm x 17 mm</td>
			<td>ThermoFisher Scientific</td>
			<td>150350</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera Phenix HCS System and Harmony HCA software</td>
			<td>Perkin Elmer</td>
			<td>HCSHH14000000</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) tablets</td>
			<td>Sigma Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>Sigma Aldrich</td>
			<td>P5927</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium, GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>61870010</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium, without glutamine, without phenol red</td>
			<td>Gibco</td>
			<td>11835063</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup sterile vacuum filter units</td>
			<td>Millipore</td>
			<td>SCGVU05RE</td>
			<td></td>
		</tr>
	</tbody>
</table>

a robust method for the large-scale production of spheroids for high-content screening and analysis applications

高内涵筛选（HCS）和高内涵分析（HCA）是使研究人员能够从细胞中提取大规模定量表型测量的技术。这种方法已被证明对于加深我们对细胞生物学中广泛的基本和应用事件的理解是强大的。迄今为止，该技术的大多数应用都依赖于使用单层生长的细胞，尽管人们越来越意识到这些模型并不能概括组织中发生的许多相互作用和过程。因此，在开发和使用3维（3D）细胞组件（例如球体和类器官）方面出现了。尽管这些3D模型在癌症生物学和药物递送研究的背景下特别强大，但它们以适用于HCS和HCA的可重复方式进行生产和分析，带来了许多挑战。这里详述的方案描述了一种产生多细胞肿瘤球体（MCTS）的方法，并证明它可以以与HCS和HCA兼容的方式应用于三种不同的细胞系。该方法有助于每孔产生数百个球体，提供了特定的优点，即当用于筛选制度时，可以从每孔数百个结构中获得数据，所有这些都以相同的方式处理。还提供了示例，其中详细介绍了如何处理球体以进行高分辨率荧光成像，以及HCA如何在球体水平以及从每个球体内的单个细胞中提取定量特征。该协议可以很容易地应用于回答细胞生物学中的各种重要问题。

在该协议中，详细介绍了一种以球体形式产生3D细胞培养组件的可靠方法，使用不同的细胞类型来表示各种肿瘤组织。该方法允许每孔产生数百个球体，这使得基于细胞的测定能够以高内涵的方式进行（图1）。这种方法以前已被用于研究HT-29球体16 中的纳米颗粒摄取和HepG2球体中纳米颗粒诱导的毒性17。该方法依赖于在光学质量板的孔底部?...

Watch this Scientific Journal Video about A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications at JoVE.com

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

该协议详细介绍了生产三种不同类型的球体的方法，使其适用于大规模高内涵筛选和分析。此外，还提供了示例，展示了如何在球体和单个细胞水平上分析它们。

用于高内涵筛选和分析应用的大规模生产球体的稳健方法

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale ...

该协议提供了一种方法，可以在适用于自动显微镜的多孔板的每个孔中产生数百个多细胞肿瘤球体。产生的球体可以分为不同的大小类别，并且可以从每个细胞中提取亚细胞信息。该技术产生足够的血清，可用于直接筛选和RNA干扰研究。使用保持在零下20摄氏度的预冷尖端将ECM基底材料稀释在冷酚红血清游离培养基中。我打赌15微升的ECM基底材料和培养基溶液进入96孔成像板。将板在4摄氏度下以91倍G离心20分钟。将板在37摄氏度下孵育不超过30分钟。将行程和 EDTA 添加到单元格中。在孵育结束时将细胞在37摄氏度下孵育，我们将细胞悬浮在含有10%FBS的完整培养基中。将10微升细胞悬浮液移液，放入血细胞计数器计数室中。计算腔室四个象限中的细胞数量。Centra在室温下以135倍G观察细胞四分钟。一种是沉淀该细胞，吸出上清液并将细胞重悬于无酚红的完整培养基中，以获得每毫升10至6个细胞的浓度。在不含酚红的完全培养基中稀释细胞。看到细胞以圆周运动以每孔35微升的总体积下降。将细胞在37摄氏度下在具有5%二氧化碳的加湿培养箱中孵育一小时。每孔1.2微升ECM基底材料至23.8微升无酚红完整培养基，最终体积为每孔60微升。将细胞在37摄氏度下在具有5%二氧化碳的加湿培养箱中孵育。第二天，用100微升新鲜酚红游离完全培养基替换培养基。将含有球体的96孔板插入共聚焦高内涵筛选显微镜中。选择合适的激光器来激发所使用的地板。按顺序获取通道以避免串扰，使用适当的物镜对椭球体进行成像。为了分析球体的形态，根据荧光共轭的FLoid和染色对球体进行分割。根据六角三，四，二染色来分割细胞核。通过细胞质中存在的残留的三，三，四，二，染色剂分割每个细胞的细胞质。计算旋转椭球体的不同形态特性，包括体积、表面积、球形度和每个球体的原子核数量。罗斯有进一步的图像来去除小于75立方米和大于900， 000立方米的球体。要分析球体中的单个细胞，请根据荧光共轭的球体染色来分割球体。根据六角三，四，二染色来分割细胞核。通过细胞质中存在的残留的六角形三，三，三，四，二染色剂来分割每个细胞的细胞质。根据二抗染色对溶酶体进行分段。计算每个球体内细胞的不同形态学性质，包括每个细胞的溶酶体数量，细胞体积和细胞表面积。该图很好地显示了三种不同类型的球体的概述，包括不同共聚焦平面上的图像。还显示了整个旋转椭球体的体积视图。该图显示了pH分割过程，面板A显示了体积视图中的整个球体。图B显示单个球体的所有细胞内的细胞质，细胞核和溶酶体的集合分割。细胞质和细胞核根据六角三，四，二染色进行分段。溶酶体根据抗实验室一抗体染色进行分段。面板 C 显示的分段与面板 B 相同，但以三个普通视图显示。可以在椭球体水平上进行各种形态测量。这些测量包括计算每个球体的原子核数量，以及这些费利西度，体积和pH的表面积。三种球体类型中每个细胞的典型体积。以及每个细胞的溶酶体数量显示在此图中。在 SP 生成开始时优化初始单元座密度是关键步骤。该图显示，添加过多的细胞仍然允许球体形成。但是，如果球体，这可能导致聚变。以这种方式产生的球状体可以进行转录组学和蛋白质组学分析。该技术用于定量分析药物递送这种纳米颗粒的摄取，并评估它们对球状体中单个细胞的毒性。

Research

JoVE Journal

Biology

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Infinium Assay for Large-scale SNP Genotyping Applications

Infinium检测的大型SNP基因分型应用

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

在大规模的基因敲除<em&gt; C。线虫</em&gt;使用双链RNA料库产生失去功能的鲁棒表型

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

敏感，大规模定量代谢组学的策略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

高内涵筛选分析法评价有害和潜在有害成分的毒性效应（HPHC）

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

开源高内涵分析利用自动荧光寿命成像显微镜

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

ODELAY：用于酵母生长多参数定量的大规模方法

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

用于大规模表观遗传学研究的半自动ChIP-Seq程序

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...