Name: real time analysis of metabolic profile in ex vivo mouse intestinal crypt organoid cultures
Uploaded: 2014-11-03T15:00:00
Description: Watch this Scientific Journal Video about Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures at JoVE.com

这项研究是由赠款RO1 CA 135561，R01 CA151494，R01 CA174432和P3013330来自美国国立卫生研究院的支持。 我们要感谢米歇尔·休斯顿，艾琳娜Dhima和安娜Velcich博士开发的地下室隔离协议提出宝贵的意见。 我们也感谢糖尿病的培训和医学用NIH P60DK20541支持的阿尔伯特爱因斯坦医学院的研究中心，以及迈克尔·布朗利博士和博士学梁笃，直接和运营海马工厂，分别是谁。

按照指南中的美国国立卫生研究院实验动物的护理和使用的建议进行研究。该方案经委员会医学爱因斯坦医学院动物实验的伦理。 1.地穴的分离与培养<ol><li>隔离小肠隐窝： <ol><li>从任何感兴趣的小鼠模型中分离出肠腺。安乐死的小鼠用CO 2，随后通过颈脱位。 </li><li>打开腹部纵向和补小肠（SI）的用冰冷的磷酸盐缓冲盐水，PBS（-Ca 2+ -Mg 2+）用2×抗?...

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	<li>Jemal, A., 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=.">.</a> CA Cancer J Clin. 58, 71-96 (2008).</li><li>Slattery, M. L., Boucher, K. M., Caan, B. J., Potter, J. D., Ma, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eating+patterns+and+risk+of+colon+cancer.">Eating patterns and risk of colon cancer.</a> Am J Epidemiol. 148, 4-16 (1998).</li><li>Warburg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+cancer+cells.">On the origin of cancer cells.</a> Science. 123, 309-314 (1956).</li><li>Coles, N. W., Johnstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamine+metabolism+in+Ehrlich+ascites-carcinoma+cells.">Glutamine metabolism in Ehrlich ascites-carcinoma cells.</a> Biochem J. 83, 284-291 (1962).</li><li>Medina, M. A., Castro I, N. u. n. e. z. d. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutaminolysis+and+glycolysis+interactions+in+proliferant+cells.">Glutaminolysis and glycolysis interactions in proliferant cells.</a> Int J Biochem. 22, 681-683 (1990).</li><li>Anso, E., 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=Metabolic+changes+in+cancer+cells+upon+suppression+of+MYC.">Metabolic changes in cancer cells upon suppression of MYC.</a> Cancer Metab. 1, 7 (2013).</li><li>Malmgren, S., 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=Coordinate+changes+in+histone+modifications,+mRNA+levels,+and+metabolite+profiles+in+clonal+INS-1+832/13+&#946;-cells+accompany+functional+adaptations+to+lipotoxicity.">Coordinate changes in histone modifications, mRNA levels, and metabolite profiles in clonal INS-1 832/13 &#946;-cells accompany functional adaptations to lipotoxicity.</a> J Biol Chem. 288 (17), 11973-11987 (2013).</li><li>Whitehead, R. 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=Establishment+of+conditionally+immortalized+epithelial+cell+lines+from+both+colon+and+small+intestine+of+adult+H-2Kb-tsA58+transgenic+mice.">Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice.</a> Proc Natl Acad Sci U S A. 90 (2), 587-591 (1993).</li><li>Roig, A. I., 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=Immortalized+epithelial+cells+derived+from+human+colon+biopsies+express+stem+cell+markers+and+differentiate+in+vitro.">Immortalized epithelial cells derived from human colon biopsies express stem cell markers and differentiate in vitro.</a> Gastroenterology. 138 (3), 1012-1021 (2010).</li><li>Sato, T., 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=Single+LGR5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single LGR5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459, 262-265 (2009).</li><li>Sato, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+self-organizing+mini-guts+from+a+single+intestinal+stem+cell:+mechanism+and+applications.">Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications.</a> Science. 340, 1190-1194 (2013).</li><li>Yilmaz, O. 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=mTORC1+in+the+Paneth+cell+niche+couples+intestinal+stem-cell+function+to+calorie+intake.">mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake.</a> Nature. 486, 490-495 (2012).</li></ol>

我们测试的耗氧率（OCR）和隐窝中分离出8个月大的小鼠并生长成组织体在体外的细胞外酸化速率（ECAR）的基础量的测定之后，隐窝代谢物通过增加寡霉素，羰基氰化物评价-p-trifluoromethoxyphenylhydrazone（FCCP）和鱼藤酮，按顺序。 基底OCR和基底ECAR记录从0 - 29分钟（ 图2A和2B）。在第 29 届分钟，寡霉素（来自端口A）注射到每...

结直肠癌（CRC）是癌症相关死亡在美国的第三大原因。散发性大肠癌- 即后来的生活产生（&gt; 50岁），并没有明确的遗传易感因素-占〜80％ ​​的情况下，随着发病率的长期膳食模式1,2的强烈影响。这些肿瘤中表现出对依赖性代谢移对氧化糖酵解，被称为Warburg效应，这可能部分地使较高浓度的蜂窝构件和能量可用（通过谷氨酰胺），以允许和也许驱动的肿瘤细胞的增殖3-5率高。结肠癌的研究以及其他胃肠道癌症，包括小肠肿瘤提供重要的洞察肿瘤形成的原因。调查胃肠器官系统，可协助DET正常的，有利于肿瘤发生和肿瘤发生的国家之间的代谢差异发芽对肿瘤发展的相对风险，以及早期发现肿瘤的。此外，了解生物能量代谢涉及到线粒体呼吸和糖酵解提供基本的深入了解细胞生理学，老化和疾病状态扰乱肠道内环境稳定。生物能学分析技术，细胞外流量分析，可以利用在细胞生长在培养实时6,7同时评估线粒体呼吸和糖酵解的速度。 直到最近， 在体外研究小肠被限制在由良性或恶性肿瘤8,9的细胞系，并没有代表正常的肠道上皮细胞的生理和他们所在的微环境的影响。在2009年，Sato 等人 10导入离体培养体系生长的三维（3D）的小鼠的肠上皮组织体，或epithelial“微型胆”，适用于实验，诊断和治疗的研究10,11。此外，隐窝从calorically限制小鼠中分离维持其改变的生长特性类器官在这样的文化12。相比于转化的细胞系，隐窝类器官培养物可以被用于产生生理相关数据呈现出好得多的模型，以了解在体内的状态。 我们调整生物能量学分析技术测定肠隐窝组织体的能量代谢。小鼠小肠隐窝类器官培养体外发展提出的墓穴类器官的能量代谢研究。耗氧率（OCR）和隐窝组织体的细胞外酸化速率（ECAR）测定在不存在和两种不同的代谢抑制剂（寡霉素，鱼藤酮），和离子载体（羰基氰 - 对 - trifluoromethoxyphenylhydrazone）存在下进行。地穴奥尔加这些化学物质NOID代谢反应，通过改变ECAR和OCR值成功体现。 细胞生物能的研究将阐明代谢状态和疾病的风险和表型之间的相互交流的癌症，肥胖症，糖尿病，代谢紊乱和线粒体疾病，并有助于与转化医学直接影响预先筛选方法。在这里，我们描述了一个详细的协议来隔离小肠隐窝和文化隐窝类器官。此外，我们引入了一种新的方法来使用地穴类器官培养的代谢试验。

<table border="0" cellpadding="0" cellspacing="0" width="1633">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">BD Matrigel Basement Membrane Matrix, GFR, Phenol Red-free, LDEV-free</td>
			<td style="width:229px;">BD Biosciences</td>
			<td style="width:135px;">356231</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">PBS (phosphate buffered saline), no magnesium, no calcium, pH 7.2</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">20012-027</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Advanced DMEM/F-12 (1X)</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">12634-028</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Dulbecco&#8242;s Modified Eagle&#8242;s Medium without glucose, L-glutamine, Phenol Red, sodium pyruvate and sodium bicarbonate</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">D5030</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Phenol Red sodium salt&#160;</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">P4758</td>
			<td style="width:456px;">Final Concentration 15 mg / L in DMEM (D5030) - step 2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Antibiotic-Antimycotic, 100X, 100ml</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15240-062&#160;</td>
			<td style="width:456px;">Final Concentration 1 X or 2 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Penicilin-Streptomycin, liquid</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15140-122</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; GlutaMAX&#8482; supplement</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">35050061</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid), 1 M</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15630-080</td>
			<td style="width:456px;">Final Concentration 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">N-Acetyl-L-Cysteine, 25g</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">A9165-25G</td>
			<td style="width:456px;">Final Concentration 1 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">100X N-2 supplement, liquid</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:135px;">17502-048</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">50X B-27&#174; supplement minus Vitamin A, liquid</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:135px;">12587-010</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Mouse R-Spondin 1, CF, 50ug</td>
			<td style="width:229px;">R&#38;D Systems</td>
			<td style="width:135px;">3474-RS-050&#160;</td>
			<td style="width:456px;">Final Concentration 500 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Murine EGF, 100ug&#160;</td>
			<td style="width:229px;">Peprotech</td>
			<td style="width:135px;">315-09&#160;</td>
			<td style="width:456px;">Final Concentration 50 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Murine Noggin, 20ug&#160;</td>
			<td style="width:229px;">Peprotech</td>
			<td style="width:135px;">250-38</td>
			<td style="width:456px;">Final Concentration 100 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; L-glutamine, 200 mM</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">25030-081</td>
			<td style="width:456px;">Final Concentration 2 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; Glucose powder</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15023-021</td>
			<td style="width:456px;">Final Concentration 5 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Ambion&#174; 0.5 M EDTA (Ethylenediaminetetraacetic acid), pH 8.0</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">AM9260G</td>
			<td style="width:456px;">Final Concentration 3 mM for step 1.1.5; 2 mM for step 1.1.8&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">DTT (Dithiothreitol), 1M</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">P2325</td>
			<td style="width:456px;">Final Concentration 3 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Albumin from bovine serum (BSA)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">A2058</td>
			<td style="width:456px;">0.1 % in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">16000-044</td>
			<td style="width:456px;">1 % in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recovery&#8482; Cell Culture Freezing Medium</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">12648-010</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">ROCK inhibitor (Y-27632)&#160;</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">Y0503</td>
			<td style="width:456px;">Final Concentration 10 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Oligomycin</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">O4876</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Carbonyl cyanide-p-trifluoro-methoxy-phenyl-hydrazone (FCCP)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">C2920</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Rotenone</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">R8875</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Sodium hydroxide</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">221465</td>
			<td style="width:456px;">Final Concentration 0.1 N in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">XF24 Extracellular Flux Analyzer (XF Analyzer)</td>
			<td style="width:229px;">Seahorse Bioscience</td>
			<td style="width:135px;"></td>
			<td style="width:456px;"></td>
		</tr>
	</tbody>
</table>

real time analysis of metabolic profile in ex vivo mouse intestinal crypt organoid cultures

小肠粘膜表现出组织成两个基本结构的重复架构：绒毛，伸入肠腔和成熟肠细胞，杯状细胞和肠内分泌细胞组成;和隐窝，驻留近端粘膜下层和肌层，窝藏成体干细胞和祖细胞和成熟的帕内特细胞，以及基质和隐窝微环境的免疫细胞。直到最近几年， 在小肠体外研究仅限于从良性或恶性的肿瘤衍生的细胞系，并没有表示的正常肠上皮细胞的生理机能和在它们所在的微环境的影响。在这里，我们展示了从培养C57BL / 6小鼠的原代小鼠小肠隐窝类器官改编自佐藤等人的方法。（2009）。此外，我们目前使用的crypt类器官培养法检测进行实时测量地穴新陈代谢换货基础耗氧量，糖酵解速率，ATP的产生和呼吸的能力。类器官维持他们的源代码中定义的属性，并保留其代谢改编耗氧量和胞外酸化率反映的各个方面。在这个地穴化培养系统实时代谢研究是一个强大的工具来研究隐窝类器官的能量代谢，以及如何通过营养和药物因素的调节。

地穴类器官是8月龄C57BL / 6小鼠饲喂建立纯化营养76A（AIN76A）的啮齿动物饲料美国学院。肠隐窝组织体可以生长在培养物从单个隐窝（ 图1A，单红色箭头）长时间。组织体生长出隐窝状结构在18-20天后在培养物（ 图1B，红色箭头）。隐窝传代，每3周和组织体有效回收下列各通道。 海马生物能量的仪器可以在细胞生长在培养的实时同步​​评估线粒体呼...

Watch this Scientific Journal Video about Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures at JoVE.com

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

小肠隐窝类器官培养离体提供的组织培养系统，该系统概括隐窝依赖于干细胞和它们的生态位的增长。我们建立了一个方法，以测定在实时在原代小​​鼠隐窝类器官的代谢分布图。我们发现组织体保持了其源定义的生理特性。

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JoVE Journal

Biology

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

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Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Metabolic Profile Analysis of Zebrafish Embryos

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

逆转录病毒感染的原发性肠化培养的视频协议

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

小鼠小肠的混合原代培养拟肠激素分泌的研究和活细胞成像的肠内分泌细胞的供

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

实时在体记录了拟南芥钙信号在使用荧光传感器的昆虫饲喂时

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

眼部微血管血管生成前维沃胆球状芽分析

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

类器官来源的上皮单层：临床相关的肠道屏障功能体外模型

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

使用实时细胞代谢通量分析仪监测成骨细胞生物能量学

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...