Name: three-dimensional culture of murine colonic crypts to study intestinal stem cell function ex vivo
Uploaded: 2022-10-11T14:00:00
Description: Watch this Scientific Journal Video about Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo at JoVE.com

这项研究由NIH DK103166资助。

所有动物实验和程序均由东卡罗来纳大学（ECU）动物护理和使用委员会（IACUC）批准，并按照美国国立卫生研究院和ECU关于实验动物护理和使用指南进行。通过将C57BL6claudin-7-flox转基因小鼠与Villin-CreERT2小鼠杂交产生诱导的肠特异性claudin-7基因敲除小鼠19。本研究使用3个月大的雄性和雌性小鼠。
1. 试剂/设备准备
<ol><li>在开始相关实验之前冷却以...

<ol>
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(80), e50611 (2013).</li><li>Sugimoto, K., 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=Cell+adhesion+signals+regulate+the+nuclear+receptor+activity.">Cell adhesion signals regulate the nuclear receptor activity.</a> Proceedings of the National Academy of Sciences. 116 (49), 24600-24609 (2019).</li><li>Mansour, 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=Connexin+30+expression+and+frewuency+of+connexin+heterogeneity+in+astrocyte+gap+junction+plaques+increase+with+age+in+the+rat+retina.">Connexin 30 expression and frewuency of connexin heterogeneity in astrocyte gap junction plaques increase with age in the rat retina.</a> PLoS One. 8 (3), 57038 (2013).</li><li>Miranda, M., 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=Antioxidants+rescue+photoreceptors+in+rd1+mice:+relationship+with+thiol+metabolism.">Antioxidants rescue photoreceptors in rd1 mice: relationship with thiol metabolism.</a> Free Radical Biology and Medicine. 48 (2), 216-222 (2010).</li><li>Wang, L., 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=Mesenchymal+stromal+cells+ameliorate+oxidative+stress-induced+islet+endothelium+apoptosis+and+functional+impairment+via+Wnt4-&#946;-catenin+signaling.">Mesenchymal stromal cells ameliorate oxidative stress-induced islet endothelium apoptosis and functional impairment via Wnt4-&#946;-catenin signaling.</a> Stem Cell Research and Therapy. 8 (1), 188 (2017).</li><li>Almeqdadi, M., Mana, M., Roper, J., Yilmaz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+organoids:+mini-tissues+in+culture+to+study+intestinal+physiology+and+disease.">Gut organoids: mini-tissues in culture to study intestinal physiology and disease.</a> American Journal of Physiology-Cell Physiology. 317 (3), 405-419 (2019).</li></ol>

类器官培养是研究干细胞功能、肠道生理学、药物发现、人类肠道疾病以及组织再生和修复的优秀模型7，8，9，10，11，26。虽然它有很多优点，但建立起来可能具有挑战性。在整个协议的所有步骤中都必须小心，但最重要的是在电镀阶段。将分离的...

肠道类器官培养是一种三维（3D）离体系统，其中干细胞从原代组织的肠隐窝中分离并接种到凝胶基质中1，2。这些干细胞能够自我更新，自我组织和器官功能2。类器官模拟组织微环境，与二维（2D）体外细胞培养模型更类似于体内模型，尽管可操作性不如细胞3，4。该模型消除了2D模型中遇到的障碍，例如缺乏适当的细胞-细胞粘附，细胞-基质相互作用和同质群体，并且还减少了动物模型的局限性，包括高成本和长时间5。肠道类器官 - 也称为结肠类器官，用于从结肠隐窝来源的干细胞生长的类结肠 - 本质上是包含上皮的微型器官，包括体内存在的所有细胞类型以及腔。该模型允许操纵系统来研究肠道的许多方面，例如干细胞生态位、肠道生理学、病理生理学和肠道形态发生3，5，6。它还为药物发现、研究炎症性肠病 （IBD） 和结直肠癌等人类肠道疾病、患者特异性个性化治疗开发以及研究组织再生4，7，8，9 提供了一个很好的模型。此外，类器官系统还可用于研究细胞通讯、药物代谢、活力、增殖和对刺激的反应7，8。虽然动物模型可用于测试肠道病理状况的潜在治疗方法，但它们非常有限，因为同时研究多种药物是一项挑战。体内混杂变量较多，相关成本和时间分别高而长。另一方面，类器官培养系统允许在较短的时间内一次筛选多种治疗方法，并且还允许通过使用患者来源的类器官培养物进行个性化治疗4，8。结肠类器官模仿组织组织、微环境和功能的能力也使它们成为研究再生和组织修复的优秀模型9。本实验室建立了小肠类器官培养系统，研究claudin-7对小肠干细胞功能的影响10。在这项研究中，建立了一个大型肠道类器官培养系统来研究干细胞在条件claudin-7敲除（cKO）模型中自我更新，分化和增殖的能力或缺乏能力。
Claudin-7是一种非常重要的紧密连接（TJ）蛋白，在肠道中高度表达，对于维持TJ功能和完整性至关重要11。cKO小鼠患有IBD样表型，表现出严重的炎症，溃疡，上皮细胞脱落，腺瘤和细胞因子水平升高11，12。虽然人们普遍认为克劳迪斯对上皮屏障功能至关重要，但克劳迪斯的新角色正在出现;它们参与增殖、迁移、癌症进展和干细胞功能10，12，13，14，15，16，17。目前尚不清楚claudin-7如何影响结肠干细胞的干细胞生态位和功能。由于肠道大约每5-7天快速自我更新一次，因此维持干细胞生态位和活性干细胞的正常功能至关重要18。在这里，建立了一个系统来检查claudin-7对结肠干细胞生态位的潜在调节作用。

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			<td>0.09 cubic feet space-saver vacuum desiccator&#160;</td>
			<td>United States Plastic Corp</td>
			<td>78564</td>
			<td>anesthesia chamber</td>
		</tr>
		<tr>
			<td>0.5 M EDTA pH 8.0</td>
			<td>Invitrogen</td>
			<td>AM9261</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>ThermoFisher</td>
			<td>69715</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Dulbecco&#8217;s Phosphate buffered saline</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methylbutane</td>
			<td>Sigma</td>
			<td>277258</td>
			<td></td>
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		<tr>
			<td>4% paraformaldehyde</td>
			<td>ThermoFisher</td>
			<td>J61899.AK</td>
			<td></td>
		</tr>
		<tr>
			<td>4-hydroxytamoxifen (4OH-TAM)</td>
			<td>Sigma</td>
			<td>579002</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m nylon cell strainer</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
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			<td>96 well culture plate</td>
			<td>Greiner Bio-One</td>
			<td>655180</td>
			<td></td>
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			<td>B-27 Supplement (50x)</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Fisher Scientific</td>
			<td>BP1605-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Claudin-7 anti-murine rabbit antibody</td>
			<td>Immuno-Biological Laboratories&#160;</td>
			<td>18875</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass (24 x 50-1.5)</td>
			<td>Fisher Scientific</td>
			<td>12544E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomolds</td>
			<td>vwr</td>
			<td>25608-916</td>
			<td></td>
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		<tr>
			<td>Cultrex RCF BME, Type 2</td>
			<td>R&#38;D Systems</td>
			<td>3533-005-02</td>
			<td>gel matrix</td>
		</tr>
		<tr>
			<td>Cy3 anti-rabbit antibody</td>
			<td>Jackson Immunoresearch</td>
			<td>111-165-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Dewar Flask</td>
			<td>Thomas Scientific</td>
			<td>1173F61</td>
			<td></td>
		</tr>
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			<td>DMEM High Glucose with L-Glutamine</td>
			<td>ATCC</td>
			<td>30-2002</td>
			<td></td>
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		<tr>
			<td>EVOS FLoid Imaging System</td>
			<td>ThermoFisher</td>
			<td>4477136</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoro-Gel II with DAPI</td>
			<td>Electron Microscopy Sciences</td>
			<td>17985-50</td>
			<td></td>
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		<tr>
			<td>GlutaMAX (100x)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>JT Baker</td>
			<td>4059-02</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M) Buffer Solution</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>ThermoFisher</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>In situ cell death detection kit, TMR Red</td>
			<td>Roche</td>
			<td>12156792910</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pivetal</td>
			<td>07-893-8440</td>
			<td></td>
		</tr>
		<tr>
			<td>L-WRN Media</td>
			<td>Harvard Medical School Gastrointestinal Organoid Derivation and Culture Core</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse surgical kit</td>
			<td>Kent Scientific Corporation</td>
			<td>INSMOUSEKIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine EGF</td>
			<td>PeproTech</td>
			<td>315-09-500UG</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement (100x)</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimum cutting temperature (OCT) compound&#160;</td>
			<td>Agar Scientific</td>
			<td>AGR1180</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequenza Rack</td>
			<td>vwr</td>
			<td>10129-584</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Fisher Scientific</td>
			<td>S-279</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S9378</td>
			<td></td>
		</tr>
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			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum filter (0.22 &#181;m; cellulose acetate)</td>
			<td>Corning</td>
			<td>430769</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris Bioscience</td>
			<td>1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional culture of murine colonic crypts to study intestinal stem cell function ex vivo

肠上皮每5-7天再生一次，并由位于隐窝区域底部的肠上皮干细胞（IESC）群控制。IESC包括活性干细胞，其自我更新并分化为各种上皮细胞类型，以及静止干细胞，在受伤的情况下作为储备干细胞。肠上皮的再生由这些活性IESC的自我更新和分化能力控制。此外，隐窝干细胞群的平衡和干细胞生态位的维持对于肠道再生至关重要。类器官培养是研究调节干细胞存活和功能的蛋白质、信号分子和环境线索的一种重要且有吸引力的方法。该模型比动物模型更便宜，耗时更少，并且更易于操作。类器官还模仿组织微环境，提供 体内 相关性。本协议描述了结肠隐窝的分离，将这些分离的隐窝细胞嵌入到三维凝胶基质系统中并培养隐窝细胞以形成能够自组织，增殖，自我更新和分化的结肠类器官。该模型允许人们操纵环境 - 敲除特定的蛋白质，如claudin-7，激活/停用信号通路等 - 研究这些效应如何影响结肠干细胞的功能。具体而言，研究了紧密连接蛋白claudin-7在结肠干细胞功能中的作用。Claudin-7对于维持肠道稳态和屏障功能和完整性至关重要。在小鼠中敲除claudin-7诱导炎症性肠病样表型，表现出肠道炎症，上皮增生，体重减轻，粘膜溃疡，上皮细胞脱落和腺瘤。此前，据报道，claudin-7是小肠肠上皮干细胞功能所必需的。在该协议中，建立了结肠类器官培养系统以研究claudin-7在大肠中的作用。

为了检查claudin-7对结肠干细胞的调节作用，如上所述从鼠结肠组织中分离结肠隐窝，如图 1A所示。从原代组织中分离出隐窝后，将它们接种在96孔板中的3D基质中以生长11天（图1）。正常健康的隐窝将在第2天关闭管腔并变成球状体，并最终在大约第5天开始出芽并形成各种上皮细胞类型（图1B）。允许结肠类生长到第11天，然后收获...

Watch this Scientific Journal Video about Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo at JoVE.com

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

本协议描述了建立鼠结肠类器官系统以研究claudin-7敲除模型中结肠干细胞的活性和功能。

小鼠结肠隐窝三维培养离体研究肠道干细胞功能

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

该协议提供了一种在可操纵环境中研究结肠干细胞功能的方法，其时间成本低于动物模型。类器官培养允许在无免疫环境中研究干细胞功能。这允许精确定位特定原因的影响，例如敲除claudin-7。了解干细胞功能的机制可能会揭示炎症性肠病和结直肠癌等衰弱疾病的新治疗靶点。要开始从安乐死小鼠中分离结肠隐窝，请在小鼠中线下方切开约两英寸的切口。然后，将皮肤向后固定以露出腹部。从近端切开结肠正下方的结肠，从远端切开直肠上方的结肠，以隔离结肠。接下来，使用镊子去除附着在结肠上的脂肪组织。用镊子的平端从分离的结肠中推出粪便后，纵向切开组织。用冷PBS清洗纸巾10至15次，方法是在用镊子洗涤之间在PBS中旋转纸巾。然后用一把干净、锋利的剪刀，将组织切成大约三到五毫米的小块。从另一只安乐死小鼠中分离结肠后，将所有组织碎片合并到含有冷上皮解离培养基的50毫升离心管中，并将结肠组织碎片在上皮解离培养基中孵育90分钟在4摄氏度下轻轻摇动。孵育后，让组织碎片沉入管底部。一旦沉淀，丢弃上皮解离介质而不破坏组织碎片。用冷PBS清洗纸巾10至15次时重复该过程。在最后一次洗涤期间尽可能多地丢弃PBS。接下来，将冷隐窝解离介质添加到50毫升管中洗涤的结肠组织碎片中，并用手摇动5至10分钟。在细胞培养罩下，用70微米尼龙细胞过滤器将含有组织的培养基过滤到新鲜的50毫升离心管中。过滤后，在室温下以200G离心管10分钟，然后丢弃上清液而不干扰颗粒状的隐窝。将沉淀重悬于三到四毫升冷PBS中。在显微镜载玻片上沿线移取10微升隐窝悬浮液。使用显微镜，计算完整、长地穴的数量以估计隐窝浓度，并计算在 96 孔板中每微升接种 10 个隐窝所需的适当隐窝体积。在 1.5 毫升微量离心管中以 200 G 在 4 摄氏度下离心适量分离的隐窝五分钟。然后，使用1， 000微升移液管除去上清液，而不会破坏颗粒状的隐窝。然后，向颗粒状隐窝中加入100微升凝胶基质，而不会引入气泡。等待一到两分钟，直到凝胶基质部分凝固。然后，将10微升凝胶基质与预热的96孔培养板的每个孔中的隐窝混合，以形成圆顶形状。通过将板置于37摄氏度和5%二氧化碳的培养箱中，等待10至20分钟以使凝胶基质完全凝固。最后，向每个孔中加入100微升补充有抗生素的L-WRN培养基的新鲜制备的工作溶液，并将板在37摄氏度下在5%二氧化碳下孵育24小时。要收获类器官，请通过真空抽吸从孔中去除旧培养基，并在室温下用4%多聚甲醛固定类器官一小时。然后通过真空抽吸从孔中除去4%的多聚甲醛，并在4摄氏度下每孔用100微升30%蔗糖处理类器官。24小时后，通过真空抽吸从孔中除去30%的蔗糖，然后向每个孔中加入10微升PBS。使用移液器吸头轻轻刮擦孔底部，以解离含有圆顶的类器官。接下来，用移液管从孔中取出含有解离类器官的PBS，并将其转移到填充90%最佳切割温度或OCT化合物的标记塑料模具中。继续该过程，直到从所有孔中取出所有类器官。将2-甲基丁烷加入含有干冰颗粒的不锈钢杜瓦瓶中，足以覆盖颗粒。快速冷冻含有OCT块的类器官，将其稳定地保持在2-甲基丁烷上方。最后，将含有类器官的OCT块储存在零下80摄氏度，直到准备好切片。代表性图像突出显示了含有claudin-7的正常隐窝中结肠类化合物的成功生长。含有claudin-7的地穴在第二天开始形成球状体，在第五天开始萌芽，并继续生长和萌芽，直到第九天收获。相比之下，缺乏claudin-7的隐窝未能形成适当的结肠。用4-羟基他莫昔芬治疗两到三天后，claudin-7敲除隐窝显示为圆形细胞团块。与对照组不同，隐窝没有长成健康的球体。收获的对照中claudin-7的免疫荧光染色和条件敲除类器官证实了培养物中claudin-7的成功敲除。对照结肠在第九天表现出很少的凋亡信号。然而，claudin-7敲除结肠同时表现出高凋亡性。确保电镀前部分凝固。部分凝固前电镀会导致凝胶基质扩散，不会形成圆顶，影响隐窝的存活和生长。该协议可用于药物发现，研究细胞通讯，药物代谢，活力，增殖以及开发患者特异性个性化治疗。类器官培养的建立彻底改变了疾病研究和个性化医学。

Research

JoVE Journal

Cancer Research

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

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

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

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. ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

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 ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

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 ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

木尿造血干细胞和祖细胞和MLL-AF9驱动白血病线粒体反应氧物种的流细胞学分析

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

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 ...

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

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 ...