我们真诚地感谢她的帮助与MATLAB程序和癌症研究所的新泽西病理学设施和显微镜设施文锦陈博士。我们也感谢他们的意见和建议的评论。这项研究是由雷蒙德和富康赛克勒基金会的支持。

1。制备1％琼脂糖涂层的24孔板<ol><li> 1 G琼脂糖（RPI的，A20090-500）加入去离子水100毫升，在250-500毫升瓶和高压消毒的琼脂糖（121℃，15磅，15分钟高压灭菌机）。转入70℃水浴瓶消毒琼脂糖。琼脂糖是随时可以倒，当它冷却到70°C，这需要一个小时左右。确保琼脂糖无菌时刻保持。 </li><li>获得24平底板（猎鹰，353047），任何一种24孔平底板，将工作，如果该板块可以根据相衬显微镜成像。 </li><li>在生物安全柜和漩涡周围以及琼脂糖使其覆盖的良好均匀的Eppendorf中继吸管，每孔200μL琼脂糖免除。 200μL琼脂糖是适当的，以形成凹面，使球体能形成一致的球形。不得小于200μl琼脂糖以及完全覆盖，但超过200μl琼脂糖可能不会形成凹面和它生长的球体，可能无法形成球形的形状。 </li><li>琼脂糖涂层的24孔板在使用约10分钟准备。 （可选）介质平衡琼脂糖前种子细胞培养基中加入100μL。 </li><li>此外，琼脂糖完全凝固后，加入100μL培养基中，每孔和密封无菌密封胶带（NUNC，236366）板块。长达一个星期，这些板块可以储存在4°C。 </li></ol> 2。单细胞悬浮液的制备<ol><li>所有的实验都使用了标准的无菌技术在生物安全柜和所有的文化都生长在37℃培养箱孵化，5％CO 2在空气中，除另有指明。 </li><li> NET BON-1人胰腺癌细胞维持在含10％胎牛血清（FBS，Invitrogen公司的DMEM/F12（Invitrogen公司，10565），16000-044）在100毫米的中等×20毫米无菌组织培养皿（康宁，430167）。 </li><li>当单层BON-1细胞达到70-80％汇合，中被删除。细胞用PBS洗两次，从盘脱落增加5分钟孵育1毫升TrypLE（Invitrogen公司，12604）在37°C TrypLE类似胰蛋白酶，但稳定在15 - 30°C以及在4°C。显微镜下检查，以确保所有的细胞都从盘脱节。 </li><li> 9毫升培养基中加入胰酶消化细胞，用吸管吸取细胞上下几次，以确保即使是单细胞悬液。 </li><li>使用70微米的细胞过滤器（费舍尔，22363548）筛选细胞。 </li><li>收集细胞，进行细胞计数，用番石榴PCA-96相应的系统（Millipore公司）。 </li><li>准备苯酚的DMEM/F12 10％FBS的培养基中的每毫升5000细胞的悬浮液。 </li></ol> 3。球体文化<ol><li>覆盖200μL单细胞悬浮液的琼脂糖涂层的24孔板和24孔板密封无菌的密封胶带，以防止蒸发。 </li></ol>注： - 镀细胞的数量应根据经验来确定的基础上，在6天的球体的大小; 300-400微米直径的球体将开始药物治疗的理想规模1000 BON-1和; H727细胞接种。 - 我们不建议开始药物治疗后第6天，因为三维细胞的活力开始下降，约10天（数据未显示，在准备稿件）。 <ol start="2"><li>将上述琼脂糖涂层的24孔板上的轨道摇床苯教-1细胞在5％CO 2在空气中过夜，在37℃培养箱孵化低于40 RPM非常缓慢的搅拌。然后将这些板块一个稳定的平台，以保持不断增长的文化。 </li><li>加入200μl的生长介质以及上述板块的每个的每3天。请勿打扰的球体。通过触摸枪头一侧以及让中等入井流了下来，尝试通过每口井的墙壁添加介质。 </li><li>在显微镜下，在24孔板监察球体形成。 </li></ol> 4。药物治疗<ol><li>当球体达到约300-400微米，直径在6天，3D球体药物治疗。这是记为0天的药物治疗。 </li><li>在第6天，每板以及包含大约400μL培养基。为了确保1X终浓度为每口井，每个治疗剂量的3倍系列稀释的药物治疗，从药物库存在5毫升15毫升锥形管，这是8足够的复制生长介质。 </li><li>在24孔板的每一行的球体被归类为汽车，剂量1，2，3，4及5。有4个重复治疗的剂量为每一个24孔板（见<stroNG&gt;表1）。我们通常把3D球体，至少在2月24孔板，这使得至少有8个，每个治疗剂量复制。 </li><li>降速为5分钟800转的24孔板，在4°C至确保所有封箱胶带凝聚入井。由于浮动的3D球体的功能，在每口井的介质不会被删除，以避免干扰的球体。 </li><li>仔细地添加到适当的井每口井的墙沿200μL上述各剂量国产治疗。 </li><li>封箱胶带密封24孔板和培育的文化，在37°C间湿润的孵化器与空气中的5％CO 2。 </li><li>如果有必要，撤退在各自的剂量与治疗后72-96小时，每口井的球体。 </li></ol> 5。显示器3D球体文化<ol><li>在选择药物治疗后，每个时间点（0，24，48，72小时），球体在每个井的T他24孔板成像与蔡司Axiovert 200/M-based的相衬显微镜使用5倍的目标。 </li></ol>注：图片可以采取任何光学显微镜校准与微米和像素之间的尺度。 5倍的目标是理想的，因为球体迅速长大成像领域与10倍的目标。 <ol start="2"><li>球体的分析，可以进行手动蔡司AxioVision 4.8.2软件使用原始图像。 </li><li>此外，定制开发的MATLAB程序用于估计球体的大小，自动/半自动自动。自动程序实现了主动轮廓算法准确定位在一个给定的图像球体的轮廓，并测量其主要（L）和未成年人（宽）轴自动。一个相同的算法的半自动版本，允许用户启动程序时，强大的神器存在。所有的图像必须为TIFF或JPEG文件格式导出程序读取原始图像。球体的体积估计为0.5 x长x宽2。 </li></ol> 6。 NET 3D球体HistoGel嵌入<ol><li> 10-24球体收集从24孔板，PBS洗涤两次，在2小时10％福尔马林固定，洗净，在50％和70％乙醇15分钟两次，分别是准备HistoGel嵌入。此外，他们可以保持在4°C，70％的乙醇在几个月。 </li><li>管冷HistoGel（Thermo Scientific的，HG-4000-012）在装满水的烧杯中。在1分钟微波加热烧杯直到凝胶完全液化，然后保留在任何时候都在同一个装满水的烧杯。 </li><li>转入活检的cryomold（组织TEK，4565樱花Finetek），用牙签在70％以上的乙醇的球体。确保所有的球体被转移。让球体坐在一分钟下沉至底部的生物PSY cryomold。尝试得到摆脱活检cryomold的液体与Kimwipes同时，非常轻轻推球体中心使用一次性塑料巴斯德吸管在的活检cryomold的底部。要在解剖显微镜下进行，这一步可能会更容易。 </li><li>一层薄薄一个温暖HistoGel的添加到活检cryomold来稳定在活检cryomold底部的球体;同时，牙签是用来保持在球体中心在活检cryomold底部非常轻柔的。不添加太多HistoGel，但刚好够稳定在活检cryomold底部的球体。 </li><li>让球体/ HistoGel的坐在在室温或，直到HistoGel凝固1-2分钟。然后填写与温暖HistoGel的活检cryomold休息。 </li><li>允许它在室温下固化，约10分钟。 </li><li>只是前弹出球体/ HistoGel块活检cryomold，LEA在4°C，1分钟，这有助于完整球体/ HistoGel的块弹出了VE的。球体/ HistoGel块，然后包裹在一块的生物套（Surgipath，01090）和组织的投入和活检卡带（Fisher Scientific则15-182-701D）。组织活检磁带存储在用70％乙醇的容器。然后常规石蜡包埋组织处理程序。 </li><li>石蜡块切片到3微米厚层幻灯片。幻灯片可以与苏木精伊红染色，以确定病理改变，也可用于免疫组化染色。 </li></ol> 7。代表结果人体胰腺神经内分泌肿瘤细胞株BON-1（由瑞典乌普萨拉大学的谢尔·奥伯格，Verto研究所提供），QGP物质-1（日本健康科学基金会，大阪，日本），人肺神经内分泌肿瘤细胞系H727（ATCC）种植1琼脂糖E-涂层的24孔板。在图1可以看出，这三个细胞株显示出不同的形态。 BON-1和H727细胞形成三维的球体。在显微镜下，H727细胞显示比BON-1细胞的球体更紧密和更紧凑的。我们推测，H727细胞增加细胞与细胞粘附能力，并因此形成更紧密的路口。 QGP物质-1细胞显示大葡萄状多孔的群众，这是不作为球体。由于胰腺神经内分泌肿瘤研究近年来的兴趣上升，BON-1细胞用于其余的数字。雍慧阁-1细胞在琼脂糖涂层的24孔板播种后，形成多细胞球体，通常会出现3 路或4 天的第 6天的球体成为全才。由于养分和氧气的限制，在致密的球体核心的细胞开始死亡，约10天，17日，球体开始瓦解，由于大尺寸或在如此我的情况下弹射坏死核心。通常的球体可以长大到直径1000微米。 图2显示BON-1三维球体的形成，生长和解体的部分。 药物治疗开始时，球体保留约300-400微米和细胞存活率高（准备稿）。 6日3D球体被选中作为药物治疗的起点。据报道，组蛋白去乙酰酶抑制剂表明对NET细胞增殖和凋亡的影响。6，我们选择了一个（TSA）作为一个例子来说明药物治疗的三维网状球体的效果。 图3A显示了组蛋白去乙酰化酶抑制剂曲古抑菌治疗后，TSA的。 图3B，3D球体的形态显示TSA（Sigma公司，T8552）治疗后的3D球体的体积。估计每一个人球体的大小自动通过CUS汤姆开发的MATLAB计算机程序。增加了4手工绘图步骤，以帮助收购的球体，这是在给定的图像边缘。在每个时间点至少有8个球体被测定，每剂量的TSA处理。每个3D球体的体积为0.5 x长x宽2计算。在图3A和3B，相对车辆，所有的数字显示TSA的剂量表现出某种程度的生长抑制BON-1三维球体。其中，125纳米和250纳米TSA的浓度强烈抑制3D球体的生长，导致细胞的死亡在96小时的时间点。 理解NET 3D球体的组织学，，H＆E和免疫组化（IHC）染色进行三维球体，已培养了17天。 图4A显示了一个方法来处理为HistoGel嵌入3D球体，这是关键三维球状石蜡包埋的一步<html> OIDS。 图4B H＆E染色，裂解的caspase-3（细胞凋亡的标志）和Ki-67免疫组织化学染色（增殖细胞标记）3D球体上的抗体，这已培养了17天。内核心的3D球体的细胞大部分坏死/凋亡和外层细胞积极增殖。 <img src="/files/ftp_upload/4218/4218fig1.jpg" alt="图1" /> 图1。 NET 3D球体形态。从三个人类神经内分泌肿瘤细胞株的3D球体上形成琼脂糖涂层的24孔板。 1000 BON-1，H727和QGP的-1细胞接种到24孔板琼脂糖涂层，在正常生理条件下生长，在协议中详细。这些都是QGP的1 BON-1和H727或细胞聚集，培养10天的3D球体的图像。 18/4218fig2.jpg“ALT =”图2“/&gt; 图2。 BON-1三维球体的生长跟踪苯教-1三维球体的生长追踪：三维球体的形成，生长和解体。在详细的协议，1000 BON-1细胞接种到24孔板琼脂糖涂层。细胞生长在一个标准的DMEM/F12培养基，10％胎牛血清对轨道振动筛在生理条件下过夜，然后搬到一个稳定的平台上，并在同等条件下种植。电镀后的前5小时，每隔一小时由成像细胞的三维细胞生长跟踪随访，然后每隔1-3天用蔡司Axiovert 200/M-based相衬显微镜使用5倍的目标。首先把群众聚集的细胞，然后形成多细胞的集合体，小的球体，球体大，最终球体瓦解。 <img src="/files/ftp_upload/4218/4218fig3.jpg" alt="图3" /> 图3。 BON-1 TSA处理3D球体。（一）3D球体TSA处理后的图像。治疗组：五 - 汽车（0.0025％二甲基亚砜），D1-1（15.​​625纳米TSA）的剂量，D2-2剂量（31.25 NM TSA）的，D3：3（62.5 NM TSA）的剂量，D4：4剂量（125纳米技术协议）; D5：剂量（250纳米，TSA）5。治疗时间：0小 - 时间点6天3D球体开始治疗24小时，48小时，72 h和96 h时间点，治疗开始后。 3D球体影像像以前那样在每个时间点。 （二）TSA处理后的3D球体的生长。主要（L）和小型（宽）在（A）的3D球体的轴，估计Matlab程序自动在每次治疗的时间点（0，24，48，72和96小时）。三维球体的体积估计为0.5 x长x宽2。在图球体的体积平均8复制和计算错误栏8复制基地。 <img alt="表1" src="/files/ftp_upload/4218/4218table1.jpg" /> 表1。布局24孔板上的药物治疗<img src="/files/ftp_upload/4218/4218fig4A.jpg" alt="图4a" /> 图4A。插图的方法处理为HistoGel嵌入的3D球体。3D球体（一）3D球体处理为HistoGel嵌入方法概述HistoGel嵌入和免疫组化染色的方法来处理3D球体。 3D球体，封装在HistoGel中，在相同的在活检cryomold的水平，然后HistoGel /球体块被包裹在一个蓝色的生物套转移到活检石蜡包埋黄色录像带。 <img src="/files/ftp_upload/4218/4218fig4B.jpg" alt="图4B" /> 图4B。 H＆E染色和免疫组化染色17日3D球体（B）H＆E染色，免疫组化染色，Ki-67和劈开的为期17天的3D球体Caspase-3的。在Paraffin嵌入式切片幻灯片脱蜡和抗原检索使用CCI（Verana细胞空调解决方案，医疗系统，＃711-065-152）。多克隆Ki-67的抗体（Abcam.＃Ab833）和cleaved caspase 3的抗体（细胞信号技术，＃9661）在1:75和1:200的浓度，分别在室温下为1小时。应用抗兔二抗（杰克逊Immunolab，＃711-065-152）在1:500为1小时，在室温下，发色检测试剂盒DABMap（Ventana公司医疗系统，＃760-124）。幻灯片与苏木和脱水counterstained和前盖片从二甲苯清除。

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCF-7+cells+as+a+three-dimensional+model+for+the+study+of+human+breast+cancer.">MCF-7 cells as a three-dimensional model for the study of human breast cancer.</a> Tissue Eng. Part C Methods. 17, 1097-1107 (2011).</li><li>Hirschhaeuser, F., Menne, H., Dittfeld, C., West, J., Mueller-Klieser, W., Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroids:+An+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: An underestimated tool is catching up again.</a> Journal of Biotechnology. 148, 3-15 (2010).</li></ol>

没有利益冲突的声明。

我们已经证明了一个简单，可靠和可重复性的方法，形成多细胞球体三维人体神经内分泌肿瘤细胞，用琼脂糖凝胶涂层的24孔板。这项技术的成功，是能够获得一个单一的球体均匀大小和均匀6天的形状固定数量的细胞开始。关键的一步是板与NET的第16-24小时后电镀细胞缓慢搅拌。这种方法也适用于普通的人类乳腺癌细胞系MCF-7，人结肠腺癌细胞系HT-29，人非小细胞肺癌细胞系H1299和人类胚胎肾脏细胞株的HEK-293。他们都可以形成均匀，致密的三维球体治疗药物筛选（数据未显示）。几种方法虽然是3D球体上产生报告，7-10的方法不需要专门的板或试剂，因此简单和成本效益。未发表的数据我们正在示威é琼脂糖增长NET 3D球体，模仿体内 NET移植瘤的几何形状和分子时，比单层二维细胞。这是已经从其他人类癌症细胞株的3D球体报道相似。11,12三维细胞培养模型的限制是，它不能完全取代测试体内的任何药物或治疗方法的疗效，因为3D球体股骨头缺血性肿瘤一样，这是从体内肿瘤在这方面的不同。然而，这种方法将有助于弥合的差距和评估NET治疗药物在体内体外的某些方面。 多细胞球体的免疫荧光染色是非常难以执行，因为浮动的3D球体的大尺寸和几何形状和共聚焦显微镜的限制。在这里，我们说明了一个方法来处理HistoGel石蜡包埋嵌入，允许3D球体的序列切片幻灯片的免疫组化染色。此外，我们的定制开发的MATLAB程序，有助于准确和重复性估计球体的大小，有效监测药物疗效，并可以在净患者的药物开发的高通量筛选中。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>的DMEM/F12媒体</td><td> Gibco公司</td><td> 10565-018 </td><td></td></tr><tr><td> TrypLE </td><td> Gibco公司</td><td> 12604 </td><td></td></tr><tr><td> 24孔板</td><td>鹘</td><td> 353047 </td><td></td></tr><tr><td> 70毫米的细胞过滤器</td><td> Fisher Scientific则</td><td> 22363548 </td><td></td></tr><tr><td>无菌密封胶带</td><td> NUNC </td><td> 236366 </td><td></td></tr><tr><td>曲古菌素A </td><td>西格玛</td><td> T8552 </td><td></td></tr><tr><td>轨道摇床</td><td> Fisher Scientific则</td><td> 11-402-12 </td><td></td></tr></tbody></table>

human neuroendocrine tumor cell lines as a three-dimensional model for the study of human neuroendocrine tumor therapy

神经内分泌肿瘤（网）是一种罕见的肿瘤，发病率两个100％，000个人每年，他们占人类所有恶性肿瘤的0.5％。除少数局部疾病患者的手术，有全身治疗的很少或根本没有生存的利益。因此，有很大的需要更好地了解以英语为母语的英语教师的生物学定义，特别是不能切除或转移性神经内分泌肿瘤患者的新的治疗靶点。三维细胞培养已成为流行的方法为药物筛选，由于其在模拟体内肿瘤组织的组织和微环境的相关。2,3三维多细胞球体，可以提供有价值的信息更及时，更便宜的方式比直接从2D出发细胞培养实验动物（小鼠）模型。 为方便拍拍网新疗法的发现ients，我们已经开发出体外三维多细胞球体使用人类NET细胞株模型。 NET细胞接种在非胶粘涂层琼脂糖24孔板和生理条件下，一个非常缓慢的搅拌电镀后的16-24小时（5％CO 2，37℃）孵育。细胞形成多细胞球体上的3 路或4 天开始。药物治疗，在这一点发起的第 6天，变得更加球形的球体。监测的基础上的形态，形状和大小的球体与相衬光学显微镜上的NET球体的药物治疗的疗效。估计球体的大小自动采用定制开发的基于主动轮廓算法的MATLAB程序。此外，我们演示了一个简单的方法来处理这​​些3D球体HistoGel嵌入，允许使用标准的组织学和免疫组化技术。 <p=“的jove_content”&gt;这是使用NET细胞株，研究治疗药物的效果的3D球体上产生的第一次报告。这些3D球体上，我们还进行组织学，并显示单一药物的效果NET的球体的生长和扩散的一个例子。我们的研究结果支持的净球体NET生物学和药物开发的进一步研究的宝贵。

Watch this Scientific Journal Video about Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy at JoVE.com

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

我们提出一个简单的琼脂糖凝胶覆盖的平台，成长的3D使用神经内分泌肿瘤细胞株的多细胞球体。这种方法提供了一个非常方便的方法来研究神经内分泌肿瘤细胞的治疗药物的效果。它也可以帮助我们建立人类癌症治疗神经内分泌肿瘤球体。

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 ...

human-neuroendocrine-tumor-cell-lines-as-three-dimensional-model-for

Research

JoVE Journal

Medicine

28.5K 次观看.  Raymond and Beverly Sackler Foundation. 我们提出一个简单的琼脂糖凝胶覆盖的平台，成长的3D使用神经内分泌肿瘤细胞株的多细胞球体。这种方法提供了一个非常方便的方法来研究神经内分泌肿瘤细胞的治疗药物的效果。它也可以帮助我们建立人类癌症治疗神经内分泌肿瘤球体。

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

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

科研

医学

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable risks for normal tissue injury and the hazards of physical tumor miss. A first-of-its-kind SBRT platform achieves high-precision ablative radiation treatment through a combination of versatile real-time imaging solutions and sophisticated tumor tracking capabilities. It uses dual-diagnostic kV x-ray units for stereoscopic open-loop feedback of cancer target intrafraction movement occurring as a consequence of respiratory motions and heartbeat. Image-guided feedback drives a gimbaled radiation accelerator (maximum 15 x 15 cm field size) capable of real-time &#177;4 cm pan-and-tilt action. Robot-driven &#177;60&#176; pivots of an integrated &#177;185&#176; rotational gantry allow for coplanar and non-coplanar accelerator beam set-up angles, ultimately permitting unique treatment degrees of freedom. State-of-the-art software aids real-time six dimensional positioning, ensuring irradiation of cancer targets with sub-millimeter accuracy (0.4 mm at isocenter). Use of these features enables treating physicians to steer radiation dose to cancer tumor targets while simultaneously reducing radiation dose to normal tissues. By adding respiration correlated computed tomography (CT) and 2-[18F] fluoro-2-deoxy-&#7429;-glucose (18F-FDG) positron emission tomography (PET) images into the planning system for enhanced tumor target contouring, the likelihood of physical tumor miss becomes substantially less1. In this article, we describe new radiation plans for the treatment of moving lung tumors.

Stereotactic ablative body radiation therapy (SBRT) involves the precise delivery of high-dose radiation to cancer tumor targets. A novel SBRT platform offers a first-of-its-kind gimbaled radiation accelerator mounted within a pivoting O-ring gantry capable of dynamic image-guided tumor tracking. Here, we describe dynamic tumor tracking for lung targets.

动态肺肿瘤跟踪烧蚀立体定向放射身体治疗

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

人类成胶质细胞瘤器官型切片培养模型，用于研究肿瘤细胞迁移和抗创伤药物的患者特异性作用

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...