Name: measuring the confluence of ipscs using an automated imaging system
Uploaded: 2020-06-10T13:30:00
Description: Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

该研究得到了Bambino Gesù基金会和Ricerca Corrente（意大利卫生部）对C.C.的资助。 我们要感谢Enrico Bertini博士（神经科学系，神经肌肉和神经退行性疾病部门，分子医学实验室，Bambino Gesù儿童研究医院），Stefania Petrini博士（共聚焦显微镜核心设施，研究实验室，Bambino Gesù儿童研究医院），Giulia Pericoli（肿瘤血液学，基因和细胞治疗科，儿童研究医院Bambino Gesù） Roberta Ferretti（Bambino Gesù儿童研究医院肿瘤血液学、基因和细胞治疗科）进行科学讨论和技术帮助。玛丽亚·芬奇是“英国癌症儿童奖学金”的获得者。

1. 涂覆 96 孔板
注意：在同一板中测试不同的涂层，但不同的孔（见 补充文件）。
<ol><li>在DMEM中以1：100稀释基质胶。向96孔板中每孔加入100μL，并在室温下孵育1小时。之后，除去溶液并用 100 μL DMEM 洗涤孔两次。</li>
	<li>在PBS（含钙和镁）中稀释层粘连蛋白（20μg/ mL，LN-521）。向孔中加入100μL并在4°C下孵育过夜。第二天在接种细胞之前用DMEM进行两次洗涤。</li>
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使用iPSCs进行疾病建模和未来的药物筛选以及它们在精准医学中的可能应用使其成为一项具有重要意义的技术，因此我们认为有必要清楚地了解更类似于胚胎干细胞生理状况的体外培养条件。在这种情况下，我们使用野生型iPSCs测试了不同的ECM涂层，以了解允许细胞保持健康和未分化状态的条件。除此之外，一个关键点是在MEF和基质胶的异种成分中培养iPSC，这可能解释了一式三份之间的实验变异?...

诱导多能干细胞（iPSC）是从体细胞中获得的，可以分化成不同的细胞类型。它们通常被用作模拟疾病发病机制或进行药物筛选的系统，并且还提供了用于个性化医疗的潜力。由于iPSCs具有巨大的潜力，因此充分表征它们以用作可靠的模型系统非常重要。我们之前展示了在缺氧环境中生长iPSCs的重要性，因为这些细胞依赖于糖酵解，而有氧环境会导致氧化还原失衡1。iPSCs也容易受到其他培养条件的影响，特别是细胞外环境。优化培养条件是保持其健康和增殖的关键问题。健康的iPSC培养将产生健康的分化细胞，这些分化细胞通常是用于了解特定人类疾病或细胞过程的分子，细胞和功能特征的模型的终点。
在这项研究中，已经使用一个简单的方案来测试在不同孔中使用不同包衣条件的iPSCs的汇合度。iPSCs需要鼠胚胎成纤维细胞（MEF）的饲养层才能正确附着，但是iPSCs和MEF的共存使得难以进行RNA或蛋白质提取等分析，因为存在两个细胞群。为了避免饲养层，属于细胞外基质（ECM）的不同蛋白质已被用于重建天然细胞生态位并具有无饲养层iPSC培养物。特别是，Matrigel是从Engelbreth-Holm-Swarm（EHS）小鼠肉瘤中提取的可溶基底膜制剂，其富含细胞外基质蛋白（即层粘连蛋白，胶原蛋白IV，硫酸乙酰肝素蛋白聚糖，entactin/nidogen和生长因子）2，3。其他使用的包被条件是纯化的蛋白质，这些蛋白质在构建ECM中具有已知的相关性：已知层粘连蛋白-521由胚胎内部细胞团中的人多能干细胞（hPSCs）分泌，它是出生后体内最常见的层粘连蛋白之一4，5，6，7，8，9， 10，11;玻连蛋白是一种无异种细胞培养基质，已知可支持hPSC12，13，14，15，16的生长和分化;纤连蛋白是一种ECM蛋白，对脊椎动物发育以及胚胎干细胞在多能状态下的附着和维持很重要17，18，19，20，21，22，23，24，25。由于存在不同的包被条件，我们根据它们对iPSC汇合的影响来比较它们。

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1123">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">10 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td style="width:281px;">Corning</td>
			<td style="width:77px;">4488</td>
			<td style="width:176px;">Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL high-clarity polypropylene (PP) conical centrifuge tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>Tool</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (With Ca2+; Mg2+)</td>
			<td>Thermofisher</td>
			<td>14040133</td>
			<td>Medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (without Ca2+; Mg2+)</td>
			<td>Euroclone</td>
			<td>ECB4004L</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td>Corning</td>
			<td>4487</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture microplate, 96 WELL, PS, F-Bottom</td>
			<td>Greiner Bio One</td>
			<td>655090</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture plate, 6 well</td>
			<td>Costar</td>
			<td>3516</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM (Dulbecco&#39;s Modified Eagle&#39;s Medium- high glucose)</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>ED4SS-500g</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epi Episomal iPSC Reprogramming Kit</td>
			<td>Invitrogen</td>
			<td>A15960</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FAST - READ 102</td>
			<td>Biosigma</td>
			<td>BVS100</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Merck</td>
			<td>FC010</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>Reagent</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">H2O</td>
			<td></td>
			<td>MILLIQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst</td>
			<td>Thermofisher</td>
			<td>33342</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminin 521</td>
			<td>Stem Cell Technologies</td>
			<td>77003</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>LS25030081</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>Corning Matrigel hESC-Qualified Matrix</td>
			<td>354277</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">Mouse embryonic fibroblasts (MEF)</td>
			<td>Life Technologies</td>
			<td>A24903</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Medium</td>
			<td>Stem Cell Technologies</td>
			<td>85851</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Supplement</td>
			<td>Stem Cell Technologies</td>
			<td>85852</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vitronectin</td>
			<td>Stem Cell Technologies</td>
			<td>7180</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>Reagent</td>
		</tr>
	</tbody>
</table>

measuring the confluence of ipscs using an automated imaging system

本研究的重点是了解在不同ECM包衣底物上生长iPSC如何影响细胞汇合。已经建立了实时评估iPSC汇合的协议，而无需对单细胞悬液中的细胞进行计数，以避免任何生长扰动。使用高内涵图像分析系统以自动方式评估 4 种不同 ECM 上的 iPCS 汇合度。使用不同的分析设置来评估贴壁iPSC的细胞汇合度，并且仅观察到使用60%、80%或100%面膜的微小差异（在层粘连蛋白24和48小时）。我们还表明，与基质胶，玻连蛋白和纤连蛋白相比，层粘连蛋白导致最佳汇合。

在这项研究中，我们研究了在不同包衣条件下生长时的iPSCs汇合。使用细胞仪，我们能够在5天内获得一式三份的简单信息结果。由于iPSCs几乎不附着在塑料容器上，并且需要涂层来支持其增殖，因此我们决定监测人iPSC的汇合，因为它表明细胞培养物的健康状况，并可能反映其分化潜力。体外扩增后，我们将iPSCs接种在不同的ECM底物上，并通过观察在明场中获得的样品图像并使用鬼笔环肽染色（用?...

Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

Measuring the Confluence of iPSCs Using an Automated Imaging System

该方案的目标是比较不同的细胞外基质（ECM）包被条件，以评估差异涂层如何影响诱导多能干细胞（iPSC）的生长速率。特别是，我们的目标是创造条件以获得iPSC培养物的最佳生长。

使用自动成像系统测量 iPSC 的汇合度。

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

Research

JoVE Journal

Biology

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

被截肢者的智能植入种植体设计系统的生物电分析

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

科研

生物学

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

测量细菌细胞的抗弯刚度，使用光学陷阱

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

研究蛋白质的质量控制体系<em&gt;Ð。菌</em&gt;使用温控活细胞成像

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)