Name: in vitro organoid culture of primary mouse colon tumors
Uploaded: 2013-05-17T12:00:00
Description: Watch this Scientific Journal Video about In vitro Organoid Culture of Primary Mouse Colon Tumors at JoVE.com

这项研究得到补助YMS从美国国家卫生研究院（CA148828），密歇根胃肠肽中心大学，杰弗里A.科尔比结肠癌研究的美国密歇根大学综合癌症中心的汤姆刘纪念基金。

1。结肠癌的分离和细胞分离<ol><li>肠道肿瘤或治疗引起的任何零星结肠癌模型可以隔绝。应的小鼠安乐死与CO 2。冒号然后收集，用冷的磷酸盐缓冲盐水（PBS）冲洗，并沿纵向打开。识别区，其中包含肿瘤，利用体视显微镜，解剖用一把剪刀，用冷PBS洗。 </li><li>孵育肠片段：含有肿瘤EDTA螯合缓冲液（2 mM的EDTA，5.6毫摩尔/ L的Na 2 HPO 4，8.0毫摩尔/ L KH 2 PO 4，96.2<...

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在这个协议中描述的实验程序将允许分离培养初级小鼠结肠肿瘤。该协议是改编自做开创性的工作由博士Clevers组1,24,27。我们优化的消化时间和胶原酶浓度的肿瘤组织体，以获得更好的收益率。关键步骤包括肿瘤细胞消化成单个细胞，基底膜再悬浮，选择性培养。对于肿瘤细胞的消化中，为了获得有效的结肠肿瘤的离解，并维持其生存能力，胶原酶的消化和浓度应根据经验得出和优化。例...

肠上皮细胞增殖，并以惊人的速度，超过了所有其他组织在脊椎动物体内2,3。分裂的细胞，包括肠干细胞（ISC）和中转放大细胞分化（高脚杯，潘氏和肠内分泌）分为分泌细胞或肠3。国际学习中心坐落在该基地的地穴。潘氏细胞向下移动到隐窝底部，寿命长，而其他谱系向上迁移的绒毛3,4。在这里，细胞暴露于肠道内容包括微生物群，并通过的脱落凋亡诱导的细胞凋亡的机制从绒毛提示脱落。虽然结肠没有绒毛和帕内特细胞，能维持体内平衡机制，是相似图4。 有牵连的Wnt信号通路发挥了至关重要的作用在肠道增殖和ISC维护4。删除THê转录因子TCF4，Wnt信号通路的下游效应，导致肠道干细胞的损失和随后的崩溃组织5。同样，转基因的表达，Wnt信号抑制剂DKK1时间上皮细胞增殖和耗尽分泌细胞谱系6。相反，过度的Wnt受体激动剂R-脊椎蛋白1诱导强有力的和快速的肠隐窝细胞增殖的7。 鉴于Wnt信号肠道动态平衡的重要性，Wnt信号通路的突变常常可以观察到在结肠癌8。结肠癌是第三大死因，癌症在美国9。红肉和酒精，过量饮食摄入量与体力活动减少，并继承和体细胞突变被认为是结肠癌的危险因素10,11。腺瘤性息肉病（APC）基因，一个关键Wnt信号因子，在大多数PA突变患者家族性的，零星的，和结肠炎相关性结肠癌12,13。其他因素参与Wnt信号通路包括AXIN2和β-连环蛋白的突变也观察到在结肠癌14,15。但是，确切的机制和有效的治疗结肠癌仍然缺乏。为方便调查为结肠癌的分子机制已经建立，代表不同的癌的进展阶段，从良性积极的细胞类型的人类结肠癌细胞株16-18。小鼠结肠癌细胞株具有不同转移特性也19,20。然而，原代细胞或优于类器官文化转化的细胞系，因为它们紧密地模仿体内状态，并产生更多的生理相关数据21。最受欢迎的结肠癌衍生细胞系生长附着板的单分子层，或作为细胞悬浮液，缺位克顶端基底取向和细胞间紧密连接。此外，正常和肿瘤的肠上皮细胞在体内经过一个自发的形式，被称为细胞凋亡脱落凋亡，分化的细胞到达绒毛的提示和脱落22。这些功能是困难的，扼要重述在细胞系，但结肠癌23的发展过程中是很重要的。这些初级类器官的功能得到维护。此外，肿瘤类器官培养提供了一个有效的系统，以评估肿瘤的自主功能的基因相比， 在体内研究。遗传操纵体内的肠道是一个耗时的过程，主要是通过建立转基因和/或基因敲除小鼠肠道特定的驱动程序。然而，肿瘤类器官易于病毒介导的遗传操作和一个伟大的工具，用于评估精确的分子机制。原发性小肠肿瘤类器官培养甲肝已经证明是一个可行的和强大的技术。原发性肠细胞培养可以建立肠道功能的类器官体外隐窝绒毛结构从单一的成人LGR5 +干细胞24。这些类器官可移植和嫁接到受损的结肠组织再生25。进一步适应的文化条件作出了类似的上皮组织体小鼠结肠和人类小肠和结肠可行1。主正常结肠上皮细胞培养，基础培养基以及生长因子EGF，Noggin与中，R-脊椎和Wnt3a的是必不可少的，而基础培养基和EGF是足够的增长的主要小鼠结肠肿瘤类器官1。在这里，我们描述了一个详细的协议，分离，培养，并产生结肠肿瘤类器官。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>LS004188</td>
			<td>375 U/mg</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>1.8 U/mg</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF), Murine, Natural</td>
			<td>Invitrogen</td>
			<td>53003-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>100 x</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50 x</td>
		</tr>
		<tr>
			<td>Glutamax-I</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle Medium</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PicoPureTM RNA Isolation Kit</td>
			<td>Invitrogen</td>
			<td>KIT0204</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vitro organoid culture of primary mouse colon tumors

几个人类和小鼠结肠癌细胞系已经建立，结肠肿瘤，如多细胞层，基底顶端极性，分化能力和失巢凋亡的生理完整性不保持在结肠癌细胞系。目前的研究表明原代培养小鼠结肠肿瘤类器官改编自佐藤T 等人 ，保留了重要的生理功能，结肠肿瘤的方法。这种方法包括小鼠结肠肿瘤组织收集，相邻的正常结肠上皮解离，结肠肿瘤细胞消化成单个细胞，基底膜嵌入结肠肿瘤细胞，选择性培养的原则基础上，肿瘤细胞保持增长限制比较正常营养条件上皮细胞。 原发肿瘤类器官，如果从转基因小鼠中分离，提供了非常有用的系统，来评估肿瘤自治区此功能特定基因的离子。此外，肿瘤类器官适合病毒预谋基因传递的遗传操作，也可以被广泛的研究，因此在结肠肿瘤发生的信号通路参与过表达或击倒。原发肿瘤类器官培养提供了有关的生理和可行的手段来研究结肠肿瘤的发生机制和治疗方式。

从3个月大的APC 分钟/ +鼠标如图1所示的结肠肿瘤类器官形成的时间过程。单细胞在第0天，可以观察到几个小时后镀（ 图1A）。在第1天，存活结肠癌上皮细胞难治核可以观察到。在第3天，细胞的大小增加一倍。在第6天，类器官的大小扩大超过10倍，表现出细胞凋亡在中间。在第14天，的orgnoids会生长成不规则的车厢（ 图1B）。在这里，我们不得?...

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In vitro Organoid Culture of Primary Mouse Colon Tumors

一个简单的方法来建立初级小鼠结肠肿瘤类器官。这种方法利用的功能，结肠肿瘤细胞含有生长因子有限的媒体生存和发展成类器官，而正常结肠上皮不。

在体外化培养基层小鼠结肠肿瘤

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

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Medicine

In vitro Organoid Culture of Primary Mouse Colon Tumors

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

推导人类纤维原细胞的皮肤穿刺活检植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Primary Culture of Human Vestibular Schwannomas

人体前庭神经鞘瘤的原代培养

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

原发性肿瘤和/或转移相关的临床前和治疗的临床开发的离体处理响应

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

在小鼠大肠传入结尾的体外功能研究

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

利用miRNA的miRNA表达的临床病理分析乳腺癌组织<em&gt;原位</em&gt;杂交

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

分离培养犬从动脉和静脉主血管内皮细胞

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

毛喉素诱导在肠组织体溶胀：一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

小鼠尿的解剖和外植体培养对囊附着的作用分析

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...