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毫摩尔/ L的NaCl，1.6毫摩尔/ L的KCl，43.4毫摩尔/ L蔗糖， 54.9 mmol / L的D-山梨糖醇，0.5毫摩尔/ L的DL-二硫苏糖醇在蒸馏水中）在冰上60分钟。 </li><li>螯合作用后，正常的肠上皮细胞脱落，而肿瘤细胞的间充质细胞将保持附着。螯合缓冲吸含有正常的上皮细胞洗净残余肿瘤碎片一个更多的时间与5毫升冷螯合缓冲。 </li><li>吸螯合缓冲区，用5毫升冷1X PBS洗涤肿瘤碎片。 </li><li>吸出的1倍PBS，孵育肿瘤片段消化缓冲液中（2.5％胎牛血清，1单位/ ml青霉素，1微克/毫升链霉素，和2.5毫微克/毫升的两性霉素B，200 U / ml的Ⅳ型胶原酶， 125微克/毫升分散酶II型的Dulbecco改良的Eagle培养基）2小时，在37℃下</li><li>允许肿瘤片段来解决正常重力下1分钟，收集上清液在15毫升猎鹰管。颗粒的单细胞瘤悬浮液上清，通过离心分离，在200×g离心3分钟，并以200×g离心3分钟，用5ml PBS洗一次。 </li></ol> 2。肠道肿瘤文化<ol><li>用500μlPBS重悬肿瘤细胞沉淀，孤立的单个肿瘤细胞使用血球计数。 </li><li>颗粒肿瘤细胞占rifuging 3分钟后，以200×g，并重新悬浮在5 mg / ml的基底膜上的冰和板在24孔培养板中，每孔15000个细胞每50μl的基底膜。 </li><li>让基底膜在37℃下聚合反应15分钟，并加入500微升/孔的基础培养基（1单位/ ml青霉素，1微克/毫升链霉素，和2.5毫微克/毫升两性霉素B，10 mmol / L的HEPES N2补充2mm GLUTAMAX的，1个，1个B27的补充，1 mmol / L的N-乙酰半胱氨酸高级贝科的修改鹰Medium/F12）含有50毫微克/毫升鼠表皮生长因子。 </li></ol> 3。维护既定的组织体<ol><li>改变基底含有表皮生长因子的培养基中每星期一次，每2天及通过类器官1:5。 </li><li>传代，更换新鲜的基础培养基培养基。机械破坏的类器官和基底膜使用P1000的吸管切断与技巧，并转移到15毫升猎鹰管。进一步的机械分离是通过使用火抛光的巴斯德管TTE。 </li><li>洗离解类器官的基础培养基中，并在200×g离心2分钟离心，用5毫升。 </li><li>弃去上清液，将沉淀重悬于与基底膜和如上所述的培养基中添加。 </li></ol> 4。存储和恢复既定的组织体<ol><li>对于长期储存，冻结液体N 2类器官的稳定，至少2年。对于冻结类器官，扰乱使用P1000的吸管与技巧切断，并转移到15毫升猎鹰管。 </li><li>洗离解类器官的基础培养基中，并在200×g离心2分钟离心，用5毫升。 </li><li>弃去上清液，将沉淀重悬于含20％胎牛血清（FBS）和10％二甲亚砜（DMSO）的Dulbecco改进的Eagle培养基（DMEM）。 </li><li>传输到1.5毫升冻存管细胞，然后把管一个的Nalgene雾先生冷冻集装箱和储存在-80℃的冰箱中实现了冷却速率以-1℃/分钟。过夜培养后，转成液态N 2管。 </li><li>对于恢复，迅速解冻冷冻类器官在37°C的热水浴，用基础培养基，降速，重悬在基底膜和文化与上述条件。 </li></ol> 5。提取RNA，蛋白质的提取及免疫组化<ol><li> RNA的提取:收集细胞，按上述通路。 RNA分离与PicoPure TM RNA提取试剂盒，根据制造商的指示。 </li><li>蛋白质提取方法:如上所述，并溶解在100μl放射免疫沉淀法测定缓冲液（RIPA，收集细胞沉淀，50 mmol / L的pH为7.5的Tris-HCl，150毫摩尔/ L的NaCl，2毫摩尔/ L的EDTA，1％NP-40，0.1 ％SDS）中与1倍的蛋白酶抑制剂。 </li><li>免疫组化:吸细胞培养液中，P1000移液器转移肿瘤组织体切断成低温模具的提示，并立即冻结那朵用干冰冷冻介质（OCT）提起诉讼。在7μm切片，切片冷冻染色按如前所述，26。 </li></ol>

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我们什么都没有透露。

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?...

Watch this Scientific Journal Video about In vitro Organoid Culture of Primary Mouse Colon Tumors at JoVE.com

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 ...

in-vitro-organoid-culture-of-primary-mouse-colon-tumors

Research

JoVE Journal

Medicine

In vitro Organoid Culture of Primary Mouse Colon Tumors

34.9K 次观看.  University of Michigan. 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。

视频: In vitro Organoid Culture of Primary Mouse Colon Tumors

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

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

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

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

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

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

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

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

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

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

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

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

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

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

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

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

Here we present a protocol to detect miRNA expression in breast cancer patient samples 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

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

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

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

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

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

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

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized &#945;4&#946;1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

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

We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized &#945;4&#946;1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points.

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 ...

在体外化培养基层小鼠结肠肿瘤

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