作者感谢洛桑大学医院的M.菲切提供的隆乳照片，M.维尔特和瑞士研究所实验癌症研究能力的研究分子肿瘤学国家中心，生命科学学院，巴黎高等联邦理工学院的A. Ayyanan英国曼彻斯特大学的洛桑技术援助和R·克拉克的批评。这项研究导致这些结果根据赠款协议已获得支持SNF3100A0-112090，Oncosuisse 531817，以及创新药物计划联合执行体没有。 115188，资源，都是由欧盟第七框架计划（FP7 / 2007-2013）和制药工业协会的实物出资公司的欧洲联盟财政贡献。创新药物行动的网址是<a href="http://www.imi.europa.eu/" target="_blank">http://www.imi.europa.eu/</a> 。&lt;/ P&gt;

一般考虑:提前，成立了一个道德的协议，准备调查问卷的患者，并请培训临床人员。使用的材料从乳房缩小成形术手术前，采购授权，并确保病人的同意。组织可以是感染性和需要进行相应的处理。该协议被批准ISREC的伦理委员会 - 瑞士研究所的实验癌症研究。 1.组织回收，加工和生物银行以确保用于生物银行最佳样品的质量，一些步骤进行，在运送到实验室现有的医院。 医院: <ol><li>获得人乳房组织从乳房缩小成形术的手术和血液样本在无菌条件下，如果需要的话。组织需要由病理学家谁将会品尝以排除恶性病变的存在进行审查。 </li><li>将乳房吨发出引擎盖下的无菌板，并准备剪刀，手术刀，哪些是已经无菌蒸汽灭菌器钳。使在不同地点的乳腺组织深的伤口用手术刀将其打开。在人类的乳腺组织外观为白色股由导管和小叶，这是嵌在黄色的脂肪组织。 注:白色实质到黄色脂肪组织的比例不同的妇女;一般年轻的女性有更多的上皮组织。 </li><li>挑选白料件并把它们用钳子，然后用剪刀脂肪组织轻轻的将它们分开。避免服用血管。 </li><li>在磷酸盐缓冲盐水（PBS）pH 7.2的漂洗件的组织去除血液。转移组织到250毫升塑料瓶含有F12培养基（Dulbecco氏改良的Eagle氏培养基F12）无酚红，用2％青霉素/链霉素（包括5000单位/ ml青霉素和5000克/ ml链霉素）和1％的tibiotic /两性霉素B（包括10,000单位/ ml青霉素，万微克/毫升链霉素和两性霉素B的25微克/毫升）。商店组织中运输到实验室的媒介。 </li><li>准备3件 - 直径5mm用于RNA和蛋白质提取和闪光灯用冷（干冰）的异戊烷冻结它们在冷冻管。将它们转移到干冰盒运输到实验室。 </li><li>放的3个 - 5毫米入模具中并用一层最佳切割温度（OCT）覆盖它，然后将模具上的盒子充满冷的异戊烷的平坦底部。完成冷冻后的模具转移到干冰框。 </li><li>固定件5 3 - 直径5mm的多聚甲醛（PFA）的4％和另5个中的甲醛（FA）的在PBS中的4％在室温下的组织学分析以后。两种不同的固定剂被推荐为一些抗体与工作的一个或另一个的2的更好。 </li></ol>在实验室中: <ol start="8"><li>办cument关于乳房缩小成形术的所有信息，手术是否样品日期来源于左与右乳房。 </li><li>将冷冻的样品在-80℃和2小时内固定后转移固定组织成组织学盒中。然后把它们无论是在PBS或70％的乙醇，对O / N储存在4℃。第二天，转移组织学盒到组织学设施对石蜡包埋。 </li></ol> 2.组织消化和激素刺激<ol><li>传送含有组织样品的瓶层流柜中的P2的细胞培养室。将组织块在无菌砧板（29×19厘米）。握住钳材料刮去剩余的脂肪和船只远离乳腺实质（白色部分）与手术刀。将白色材料PBS在10cm的培养皿中。 </li><li>根据安全规则的所有作品去除脂肪物质和处置后，他们实验室S，把含有上皮组织放回案板的白色部分。使用相对的手术刀切割组织并将其砍至高达2毫米直径的碎片。 </li><li>将切碎的材料进足够的规模管:最多1个，2.5和10毫升到5，15或50ml试管，分别。通常有5毫升管产量足够的材料为石蜡包埋和激素刺激，和一个15毫升管产量足够的材料用于荧光激活细胞分选（FACS）。较大的管用于冷冻等分试样。 </li><li>准备消化主结构由介质（DMEM / F12无酚红）与1％青霉素/链霉素/两性霉素B和胶原酶A（梭状芽胞杆菌A以上的溶组织梭菌 ）。根据消化倍剂量胶原酶。 <ol><li>为12，24和36小时培养中，使用1.5毫克/毫升，1.0毫克/毫升和0.5毫克胶原酶的终浓度/毫升分别。加入消解预混高达4倍将消化的材料的体积，以确保有足够的搅拌和通气。 注意:根据实验设计和激素刺激的持续时间，期间或酶消化步骤和显微恢复后添加激素。 </li></ol></li><li>添加激素（s）的期望的浓度，以测试样品和所述车辆到控制。为刺激17-β雌二醇，和promegestone（R5020）的使用为20nM的最终浓度。 注:R5020是合成孕酮受体激动剂，这比在介质天然孕酮更稳定。 <ol><li>地方管上的培养箱内的滚筒混合器，在37℃和40rpm下O / N。在放置样品的调音台，其中重悬很好地确保所有材料沿离心管分配。 </li></ol></li><li>作为消化至少需要12小时（O / N），对于小于12小时激素刺激倍，微结构的恢复后添加激素（德下步3尾）。添加激素，你悬浮组织和放置在超低附着板暂停。组织的微观结构，可以保持在超低附着板长达7天的生长因子的培养基中保持约70％的存活率。 </li><li>用于RNA和蛋白质分析，收集组织显微离心在400×g离心5分钟，并快速冷冻在离心管中。 </li></ol> 3，恢复组织微结构<ol><li>离心反应管（多个），在400×g离心5分钟。从离心管的顶部吸出脂肪和传输剩余的含水上清液，其富含成纤维细胞的上清液，向一个新的离心管中并离心，在1250×g离心5分钟。 注意:作为乳房组织的微结构是非常粘性的，对于所有后续步骤通过轻轻搅拌该管重悬沉淀。移液不因丢失的材料的危险，建议。 </l我&gt; <li>悬浮富集的微观结构和在在PBS，2％胎牛血清（FCS）的衍生自水性上清液的成纤维细胞的小球。接着旋两个管在1250×g离心5分钟，然后弃去上清液。 </li><li>悬浮颗粒在3 - 5毫升红血细胞裂解液和他们转移到清洗管。孵育他们5分钟，在室温。 </li><li>添加PBS，2％FCS中的2倍量和离心它们在1250×g离心5分钟。在1250×g离心洗用PBS，2％FCS和离心机的粒料5分钟。重复此步骤两次。 </li></ol> 4.样品的流式细胞仪<ol><li>加入1 - 2 ml胰蛋白酶0.25％至沉淀并通过吹吸用1000微升的Pipetman以解离细胞混合它和向下轻轻2分钟。 </li><li>通过加入9毫升的PBS，2％FCS中抑制胰蛋白酶活性。离心机在450×g离心5分钟，重悬沉淀在10ml的PBS，2％FCS中。 </li><li>细胞悬液转移到40微米的细胞滤网置于吨一50ml离心管中的运算以制备单细胞。如果细胞悬液几微保留在过滤器的顶部上，帮助他们通过过滤器从过滤器的另一侧吸移它们与一个吸管，并将它们添加到经过滤的微结构。 </li><li>分离免疫细胞，从微结构与抗CD45的鸡尾酒成纤维细胞和内皮细胞，抗FAP和抗CD31抗体，然后对其进行排序用FACS基于泛上皮标记的EpCAM和CD10的分离腔和基底细胞分别为10 。 </li></ol> 5.样品的组织学<ol><li>洗涤上述的机械和酶促消化材料之后，从固定1.5毫升初始材料粒料在1ml的4％PFA的PBS中在RT下30分钟。 </li><li>离心机在1250×g离心5分钟，并用1洗净 - 2毫升的PBS两次。 </li><li>离心在16000×g离心10分钟。在此期间，制备1％的多用途琼脂糖在Tris-醋酸盐，乙二胺四乙酸（TAE）缓冲液中并加热，在微波炉的溶液，直到该溶液来煮沸。 <ol><li>除去从微波炉的溶液，摇动烧瓶轻轻以混合溶液重悬任何剩余的琼脂糖颗粒。凉爽琼脂糖溶液至50℃，并倒在石蜡基模1毫升尺寸31×23×13.5毫米。 </li></ol></li><li>弃去上清液，然后将粒料在固化层的顶部 - 琼脂糖使用刮刀具有平面端部（1 3毫米）。覆盖所有琼脂糖的附加层（40°C）。 </li><li>琼脂糖凝固后，通常在5分钟内，将琼脂糖块放入纸盒组织学石蜡包埋。随着琼脂糖样品放入一个白色的办公用纸与有关乳房缩小成形术数，实验条件和日期写在铅笔小写字母的所有信息的磁带;写这篇文章承受所有后续处理步骤。 </li><li>把磁带中PBS O / N，或70％的乙醇储存在上周末，在4℃。我们有没有注意到这两个存储解决方案之间的任何差别。然后，组织学磁带转移到组织学设施石蜡包埋。 </li></ol> 6.冷冻微观结构（未来培养） <ol><li>重悬组织微结构在冷冻介质组成的FCS，用10％二甲基亚砜（DMSO），并冻结1ml等分在冷冻管。所得冷冻管的数量取决于从乳房缩小成形术获得的主材料的量。 </li><li>冻结含使用一个受控速率冷冻器与以下程序组织微结构的冷冻管:5分钟栖居在4℃时，温度为6℃/分速率降低到-7℃，降低温度至-12℃，用率的6℃/分钟，10分钟的住宅中，温度为6℃/分速率降低到-40℃，在温度下降到-80℃下用率的6℃/分钟。然后将离心管转移到-80℃的冰箱中。 </li><li>第二天，从-80℃转移至冷冻管液氮罐长期贮存。 </li></ol>

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作者什么都没有透露。

此处所描述的体外培养提供了含有完整导管和小叶，连同在人类女性乳房通常存在的其它细胞类型的乳房组织的微观结构。在通常从降低mammoplasties获得的人类乳房组织的处理中，去除脂肪组织，基质基质的机械和酶消化，和血红细胞裂解的丰富的牛奶导管片段和终端导管小叶单位。轻柔酶促消化在合适的浓度，为右持续时间是至关重要，以确保组织的微结构保持不变，保留多种细胞类型，...

A correction was made to <a href="/video/52436">An Ex Vivo Model to Study Hormone Action in the Human Breast</a>. There was an error with the author, Marie Shamsheddin&#39;s, name. The author&#39;s name has been corrected to:
Marie Shamseddin
instead of:
Marie Shamsheddin

A correction to the author list has been made for the article <a href="/video/52436">An Ex Vivo Model to Study Hormone Action in the Human Breast</a>.

Erratum: An Ex Vivo Model to Study Hormone Action in the Human Breast

关于乳腺癌的突变的景观信息正以迅猛的速度。更少的受到人们的重视，以影响乳腺癌的发展全身因素。暴露于生殖激素对疾病进展1-3产生重大影响。然而，通过该生殖激素撞击在人乳腺癌的机制知之甚少。遗传工程小鼠模型的工作表明，它们涉及到一些下游效应4细胞的内在和旁分泌信号。 有关激素作用于人体乳房的知识有限，主要是由于缺乏足够的模型。大多数工作对雌激素受体（ER）和孕酮受体（PR）信令的机制已被执行激素受体阳性的乳腺癌细胞系，如MCF-7和T47D。这些来源于胸腔积液患者晚期乳腺癌谁曾alreaDY接受多次治疗5。在人乳腺癌的这种简单的体外模型的发现的生物相关性是在这些体外模型确定可疑和靶基因是不同于那些在动物模型6鉴定靶基因。当主人乳腺上皮细胞在体外培养它们往往会失去激素受体 ​​表达，因此激素反应7,8。这个问题可以通过使用基质胶尖端的3D方法来circumventd。以这种方式，克拉克和同事成功地建立了该保持激素受体 ​​的表达，并显示出对孕酮刺激9增殖反应乳腺上皮细胞。然而，2 体内孕激素受体 ​​的靶基因重要的是， 的Wnt-4和RANKL，均未在孕酮刺激诱 ​​导在该系统9。这种方法是最近采取进一步在体外 </EM&gt;激素预处理和RANKL诱导达到10。一个警告仍然是基质胶具有是批次依赖性的活动，是昂贵的，并且要求的实验设计，易于仅用于小细胞数。 基于这一发现， 在体内 ER和PR信号在很大程度上是由旁分泌相互作用11介导的，我们认为，细胞间的相互作用需要维护。即失去作为组织的另一个重要因素是解离为单细胞的体外培养是上皮细胞与细胞外基质之间的相互作用;但这些都是上皮细胞分化的关键和他们的破坏是肿瘤发生的重要12。考虑到这一点，我们建立了一个方法，从新鲜手术丢弃材料13分离乳腺组织的微观结构。乳房实质，由两层上皮内管腔和外myoepitheli人的细胞中，从脂肪组织解剖距离，并进行机械和酶解。洗涤和离心后，乳管的片段保留与许多基质细胞密切相互作用获得的。这些组织的微观结构保持激素反应。该模型在临床标本13进行了验证。这样，本程序可以帮助研究激素作用在乳房中的生物和临床相关的上下文。

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an ex vivo model to study hormone action in the human breast

的激素作用在人乳房的研究已经阻碍了缺乏适当的模型系统。经体外培养，初级乳腺上皮细胞往往会失去激素受体 ​​的表达。广泛使用的激素受体 ​​阳性的乳腺癌细胞系的体内情况相关性有限。在这里，我们描述了一个体外模型来研究激素作用于人的乳房。新鲜的人乳腺组织标本从外科废弃材料如还原mammoplasties或mammectomies被机械和酶促消化以获得包含导管和小叶和多种基质细胞类型的组织碎片。保持在基础培养基不含生长因子，这些组织的微结构保持它们的细胞间接触的组织架构，并保持激素反应数天。它们可容易地处理用于RNA和蛋白质的提取，组织学分析或存储在冷冻介质。荧光激活细胞分选（FACS）可以用来富集特定细胞群。该协议提供了具有高度复杂，多样的人体标本翻译研究一个简单的，标准的方法。

研究雌激素和孕激素的作用，更好地了解人类乳腺癌的分子功能，我们收集获得他们的知情同意后进行还原mammoplasties（ 图1）患者的新鲜人类乳腺组织标本。我们还获得了患者的医疗和生育史，以及血液样品，以确定血清孕酮水平在手术的时间。从新鲜还原mammoplasties组织被机械地和酶促解离。所得的组织微结构具有导管和小叶的原点（ 图2A）的乳腺组织的特性。琼脂糖?...

Watch this Scientific Journal Video about An Ex vivo Model to Study Hormone Action in the Human Breast at JoVE.com

An Ex vivo Model to Study Hormone Action in the Human Breast

我们已经开发出一种新的体外模型来研究激素作用在人的乳房。它是基于组织的微结构从外科乳腺组织标本其中保存组织结构，细胞间的相互作用，以及旁分泌信号隔离。

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

an-ex-vivo-model-to-study-hormone-action-in-the-human-breast

Research

JoVE Journal

Medicine

An Ex vivo Model to Study Hormone Action in the Human Breast

11.3K 次观看.  Ecole polytechnique fédérale de Lausanne. 我们已经开发出一种新的体外模型来研究激素作用在人的乳房。它是基于组织的微结构从外科乳腺组织标本其中保存组织结构，细胞间的相互作用，以及旁分泌信号隔离。 

视频: An Ex vivo Model to Study Hormone Action in the Human Breast

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

科研

医学

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

一个人力<em&gt;前体内</em&gt;动脉粥样硬化斑块模型研究病变的生物学

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

人掌腱膜挛缩的<em&gt;离体</em&gt;文化为肌成纤维细胞的研究和细胞外基质相互作用

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

母乳促进 Enteroids 的生长: 一个前体细胞增殖模型

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...