Name: 3d culturing of organoids from the intestinal villi epithelium undergoing dedifferentiation
Uploaded: 2021-04-01T13:00:00
Description: Watch this Scientific Journal Video about 3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation at JoVE.com

本出版物得到了国家卫生研究院国家癌症研究所颁发的K22号CA218462-03奖的支持。表达R-斯庞丁1的 HEK293-T细胞是迈克尔·韦尔齐博士的慷慨礼物。

所有进行的小鼠实验，包括使用塔莫西芬和子宫颈错位安乐死，都得到了史蒂文斯生态学研究所的机构动物护理和使用委员会的批准。
1. 老鼠
注： 斯马德4f/f的生成：卡顿布洛克斯 （前 3）/*维林-克雷ERT2 小鼠此前曾被描述过3只。成年雌性小鼠在8至12周之间使用。
<ol><li>诱导斯马德</su...

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O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SMAD4+Suppresses+WNT-Driven+Dedifferentiation+and+Oncogenesis+in+the+Differentiated+Gut+Epithelium.">SMAD4 Suppresses WNT-Driven Dedifferentiation and Oncogenesis in the Differentiated Gut Epithelium.</a> Cancer Research. 78 (17), 4878-4890 (2018).</li><li>Roche, J. 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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dll1++secretory+progenitor+cells+revert+to+stem+cells+upon+crypt+damage.">Dll1+ secretory progenitor cells revert to stem cells upon crypt damage.</a> Nature Cell Biology. 14 (10), 1099-1104 (2012).</li><li>Tetteh, P. 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这种方法可以确认在体内获得干细胞标记的去分化上皮的自我更新能力。正常的肠道上皮可以产生器官从墓穴，但不是维利隔间，当培养在3D，因为干细胞的存在在墓穴1。因此，从3D培养的去差异化的维利上皮培养的器官形成确认干细胞形成从细胞命运逆转。关于肠道上皮因突变和/或损伤引起的细胞命运逆转的报告正在增加2，3，9，10，11，12。<s...

肠道隐士，但不是维利，形成器官时，培养在马特里格尔或BME-R1矩阵。这些器官是自组织结构，通常被称为"迷你肠"，因为存在各种差异化的血统，祖细胞和干细胞存在于肠道上皮在体内。从墓穴中形成器官的可能性归因于干细胞1的存在。另一方面，肠道病毒只由分化细胞组成，因此不能形成器官。然而，突变2或条件，允许去差异的维卢斯上皮可能导致干细胞在维利2，3。这种命运的变化导致在维利上皮的茎可以通过电镀在3D基质中分化的维卢斯上皮来确定其器官形成潜力，作为在维卢斯上皮的去诺沃茎的指标来证实。因此，此过程的关键方面是确保没有墓穴污染。
Smad4KO：β-卡特宁GOF 有条件突变导致肠道上皮的分化，其特征是病毒中增殖和干细胞标记的表达， 最终在被称为异位性墓穴的细胞中形成类似墓穴的结构 这些分离的维利的干细胞的存在是由异位性墓穴（体内）中干细胞标记的表达和突变的维利形成器官的能力决定的镀马特里格尔3。上述程序详细说明了用于确认 Smad4KO：β-卡特宁GOF 突变小鼠中脱位肠道上皮的茎的方法。这种隔离病毒的方法的一个关键特点是使用刮肠流明，而不是EDTA溷合方法4。与 EDTA 溷合方法不同，通过刮擦来隔离维利保留了大部分底层，并允许调整刮擦压力，从而在没有系绳的墓穴的情况下产生 villi。刮擦的压力是主观的操作员，因此，最佳压力，以产生维利没有密码系绳必须由操作员经验决定。此过程的关键方面是通过对 BME-R1 矩阵中电镀前后的 villi 进行微观检查，确保没有墓穴污染。
肠道病毒被刮掉肠道流明与玻璃滑梯，并放置在70μm过滤器和洗涤与PBS摆脱松散的细胞或墓穴，如果有的话，在BME-R1矩阵电镀之前。该方法强调以下标准，以避免墓穴污染：a） 限制 villi 收获的十二指肠的近半部分，其中 villi 是最长的，b） 尽量减少葡萄球产的刮伤数量，c） 通过六井盘中的一系列 PBS 清洗含有 villi 的过滤器，d） 通过在 BME-R1 矩阵中电镀之前和之后的显微检查确认没有墓穴污染。Villi 隔离通过刮擦，而不是通过 EDTA 溷合，防止潜在的介质的完全丧失，可能提供利基信号5，6，7，8，如果需要的话， 从虚列上皮启动器官.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM F-12 media</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3-diaminobenzidine</td>
			<td>Vector Labs</td>
			<td>SK-4105</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well U-bottom plate</td>
			<td>Fisher Scientific</td>
			<td>FB012932</td>
			<td></td>
		</tr>
		<tr>
			<td>ABC kit</td>
			<td>Vector Labs</td>
			<td>&#160;PK4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled scissor</td>
			<td>Fisher Scientific</td>
			<td>11-999</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), minus vitamin A</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA) Protease-free Powder</td>
			<td>Fisher Scientific</td>
			<td>BP9703100</td>
			<td></td>
		</tr>
		<tr>
			<td>CD44 antibody</td>
			<td>BioLegend</td>
			<td>1030001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdx2 antibody</td>
			<td>Cell Signaling</td>
			<td>12306</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn oil</td>
			<td>Sigma-Aldrich</td>
			<td>C8267-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 70-micron cell strainer</td>
			<td>Life Sciences</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Reduced Growth Factor Basement Membrane Extract, Type R1</td>
			<td>R&#38;D</td>
			<td>3433-005-R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Fisher Scientific</td>
			<td>22-079-747</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>17-456-209</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax (100X)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK 293-T cells expressing RSPO-1</td>
			<td>Gift from Dr. Michael Verzi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Histogel</td>
			<td>Thermoscientific</td>
			<td>HG-4000-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh filter</td>
			<td>Fisher Scientific</td>
			<td>07-201-431</td>
			<td></td>
		</tr>
		<tr>
			<td>Micrscope glass slide</td>
			<td>VWR</td>
			<td>89218-844</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>p200 Blunt tips</td>
			<td>VWR</td>
			<td>46620-642</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin (50mg/mL)</td>
			<td>Invivogen</td>
			<td>ant-pm-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Quality Biological Inc PBS (10X)</td>
			<td>Fisher Scientific</td>
			<td>50-146-770</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Signal diluent</td>
			<td>Cell Signaling</td>
			<td>8112L</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>T5648-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Fisher Scientific</td>
			<td>50-146-770</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d culturing of organoids from the intestinal villi epithelium undergoing dedifferentiation

肠道上皮器官的克隆性归因于其中干细胞的存在。小鼠小肠上皮被分割成墓穴和卵石：茎和增殖细胞被限制在墓穴中，而上皮只包含分化细胞。因此，正常的肠道隐士，但不是病毒，可以产生器官在3D文化。此处描述的程序仅适用于导致茎的脱位的维卢斯上皮。所述方法使用 Smad4 功能丧失：β-卡特宁功能增益 （Smad4 KO：β-卡特宁GOF） 有条件突变鼠标。突变导致肠道病毒分化，并在病毒中产生干细胞。接受脱位的肠道病毒使用玻璃滑梯刮掉肠道，放置在 70 μm 滤网中，并清洗多次，在 BME-R1 矩阵中电镀之前过滤掉任何松散的细胞或凹痕，以确定其器官形成潜力。两个主要标准用于确保产生的器官是从分化的紫罗兰隔间而不是从墓穴中发育出来的：1） 对孤立的 villi 进行微观评估，以确保在 3D 矩阵中电镀之前和之后没有任何系绳的切除器，2） 从 villi 监测器官形成的时间过程。从病毒启动的器官在电镀后仅发生两到五天，并且看起来形状不规则，而来自同一肠道上皮的隐性器官在电镀后的十六小时内就明显出现球形。然而，该方法的局限性是，形成的器官数量和从病毒中启动器官所需的时间因分化程度而异。因此，根据突变的特异性或导致分化的侮辱，必须从经验上确定可以收获病毒以检测其器官形成潜力的最佳阶段。

该过程成功的决定因素是防止密码污染。通过确认四个主要标准来确保从 villi（而不是从任何污染的墓穴）中进行有机物发育：1） 通过在 BME-R1 中电镀 villi 前后进行微观检查，确保收获的葡萄球体的纯度：2） 每口井电镀有限数量的 villi，以便单独可视化所有镀膜的 villi：3） 每天对器官发育进行监控：时间过程的图像显示从维利（图3）和4）开始的器官的动力学和形态外?...

Watch this Scientific Journal Video about 3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation at JoVE.com

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

该程序描述了从小鼠肠道上皮分离，进行分化以确定其器官形成潜力。

肠道维利表皮的器官3D培养进行分化

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

这种方法可以确认在体内获得干细胞标记的去分化上皮的器官形成能力。villi 的收集是通过刮擦而不是 EDTA 溷合方法进行的，方法可防止潜在的介质完全丧失，如果器官启动需要，这些夹层可能会提供利基信号。这种方法适用于上皮组织，其中增殖和分化隔间被物理划定，去分化细胞在体内表达干细胞标记。本协议中的某些因素需要经验确定，包括收获葡萄糖的最佳阶段和刮葡萄糖的最佳压力，这需要时间和实践。连续四天通过腹内注射塔莫西芬和玉米油，诱导突变小鼠的Smad4敲除和β-卡特宁功能突变增益。对小鼠实施安乐死后，用70%乙醇喷洒腹部，以防止老鼠毛皮进入腹腔。用解剖剪刀打开腹腔，露出肠道，用剪刀和钳子隔离肠道。分解十二指肠的近半部分，然后用5毫升冰冷PBS在10毫升注射器中冲洗十二指肠，以清除亮性含量。用斜剪刀纵向打开十二指肠，将十二指肠平放在冰上15厘米的培养皿上，十二指肠的流明朝操作者。在开始刮擦之前，在六井组织培养板的一口井中放置一个70微米的网状过滤器。用四毫升1X PBS填充所有油井，并将板放在冰上。用两个微小的玻璃滑梯刮擦玻璃片，一个用来压住十二指肠，另一个用来刮。刮十二指肠的发光侧表面两次，以消除粘液不删除维利，然后刮十二指肠两次，以收集幻灯片上的葡萄球，而不系绳的墓穴。使用含有 PBS 的一毫升转移移液器将 villi 转移到六井盘中的 70 微米网状过滤器中。每次刮伤后收集维利。为了去除松散的地窖，通过将过滤器通过六井盘中的一系列井（每口油井含有四毫升冰冷 PBS）中的一系列井，用 70 微米滤网清洗收集的 villi。使用 P1000 移液器，将大约 3 毫升 PBS 中的 villi 悬浮液从 70 微米滤网转移到冰上新的 15 毫升管。使用 0.1% BSA 涂层的钝端 P200 移液器尖端将 50 微升的 villi 悬架体积转移到玻璃滑梯上。计算 4 倍放大倍数下 50 微升液滴中的维利数，以确定 PBS 悬架中维利的浓度，并确认没有系绳的暗箱。要平板 villi，请使用 0.1% BSA 涂层的 P200 钝端移液器尖端将 villi 转移到微中心管中，在 BME-R1 矩阵的 12.5 微升中将每口井的电镀密度为 6 维利。在摄氏4度200度时旋转两分钟。取出超高纳特并重复离心以去除任何残留的 PBS。在层压流罩中，将维利颗粒轻轻地重新沉积在冰上解冻所需的冷 BME-R1 量中。使用P20移液器，在BME-R1基质中，每口96井U底板的villi板12.5微升预热至37摄氏度。在37摄氏度的组织培养培养器中孵化板15分钟，使BME-R1基质凝固。在每口井中，添加 125 微升预热 ENR 介质，并补充文本手稿中描述的。在37摄氏度的组织培养孵化器中用5%的二氧化碳孵育镀的维利，每隔一天更换一次介质。丢弃任何在两天前出现器官的井。从墓穴中浮现的器官与同一小鼠肠中去分化的上皮在外观上是有区别的。加密衍生的器官在一夜之间以具有明确边界的球形结构出现。检查了从维利开始的器官的动力学和形态外观，这些器官最初看起来形状不规则，需要两到五天才能在显微镜下看到。从两个不同的维利的有机启动显示。具有器官形成潜力的病毒似乎密集，可能是由于基础粘膜的保留。从别墅的有机启动是明显的第二天从维利之一。而在其他别墅，风琴出现在第四天。在收获病毒和种植由此产生的器官时，采取适当步骤避免墓穴污染至关重要。在此过程之后，观察了从同一突变体的切口中产生的器官和病毒之间的表型差异，因此可以进行进一步的实验，以寻找两者之间的分子差异。本协议可用于研究内源性密码产生的器官与非分化产生的异位性密码之间的差异，从而可以解决去分化的影响。

Research

JoVE Journal

Biology

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

一个培养人关节软骨细胞的三维系统在滑液

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

科研

生物学

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

小鼠视网膜色素上皮细胞的原代培养

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

使用活性氧敏感荧光探针分析小鼠肠道类器官中的氧化应激

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

用于类器官培养和 3D 成像的高通量平台

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

Establishing 3D Endometrial Organoids from the Mouse Uterus

从小鼠子宫建立3D子宫内膜类器官

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

冷冻保存的上皮隐窝仔猪肠道3D类器官的培养和细胞单层的建立

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

3D Culture of Organoids from Murine Intestinal Crypts and Single Stem Cell

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research (Video) | JoVE 

小鼠肠隐窝和单个干细胞的类器官的3D培养用于类器官研究

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

Generation and Manipulation of Rat Intestinal Organoids (Video) | JoVE 

大鼠肠道类器官的产生和操作

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

Construction of a 3D Intestinal Mucosa Model with Paraffin Embedding

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models (Video) | JoVE

具有免疫成分的基本三维 （3D） 肠道模型系统

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...