Name: a brain tumor/organotypic slice co-culture system for studying tumor microenvironment and targeted drug therapies
Uploaded: 2015-11-07T14:00:00.000+00:00
Duration: 10 min 13 s
Description: Watch this Scientific Journal Video about A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies at JoVE.com

This work is supported by grants from the NIH (P01CA142536 to RAS, T32CA009361 to DPY) and the Pediatric Low Grade Astrocytoma foundation.

伦理声明:以下步骤涉及动物受试者按照卫生准则的国家机构完成并批准Dana-Farber癌症机构动物护理和使用委员会。所有的人类受试者的工作得到了机构审查委员会委员会的布莱根妇女医院和Dana-Farber癌症研究所，以及斯坦福大学的适当使用，所有受试者在需要时获得了知情同意书，并适当减免同意要求审查为最低风险的研究获得。 时间线切片文化协议: <img alt="图1" src="/files/ftp_upload/53304/53304fig1.jpg" /> 图1.时间表脑肿瘤/器官切片共培养协议 。此图描绘了切片培养过程的时间表涵盖所有8天Øf显示实验并且过程的主要步骤。时间轴是相对于0天当细胞以突出电镀细胞前开始的程序天的重要性镀到切片。 <a href="https://www.jove.com/files/ftp_upload/53304/53304fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 1.解剖缓冲区<ol><li>制备15mM的HEPES（1μM），6.5mg / ml的葡萄糖，1.3毫硫酸镁（1μM），20mM的氯化钾（2 M）和1％青霉素/链霉素在1×HBSS中。 </li><li>调节pH至7.4，用NaOH。 </li><li>过滤并储存在-20℃。 </li></ol> 2.切片文化传媒<ol><li>制备2％的B-27，1％N2补充，1％青霉素/链霉素，1％Glutamax的，和1.5mg / ml的葡萄糖中的Neurobasal-A培养基减去苯酚红。 </li><li>过滤并储存在-20℃。根据需要解冻，和1周后丢弃在4℃。 注意:下面的过程可以做到上Ë日开始解剖前。 </li><li> 3％低熔点琼脂糖 注意:下面的过程可以做高达一天开始解剖前。 </li><li>琼脂糖混合0.9克低熔点成〜31毫升夹层缓冲液，微波25秒，瓶盖松动。微波，直到融化并确保混合物不溢出。 </li><li>保持在55-65°C，直到需要。 注意:下面的过程可以做高达一天开始解剖前。 </li></ol> 4.大衣切片文化插入与层粘连蛋白注意:下面的过程可以做高达一天开始解剖前。 <ol><li>在组织培养罩，用无菌镊子，将一个切片培养插管在一个6孔板的每个孔（符合打算获得的片数，大约为12可以从一个过程回收。下面的过程用于书面6个片层）。填写一个15毫升的锥形牛逼<html> UBE用1×PBS（800微升/插入），并添加层粘连蛋白（10微克/毫升）。 </li><li>添加800微升层粘连蛋白混合物与每个切片培养插管的顶部。直到需要在37℃。 </li></ol> 5.准备解剖<ol><li>填充6孔板的每个孔与1200微升切片培养介质。填充任何未使用的井1200微升PBS和填充板的中心空间用5毫升1X PBS。存放该板在37℃。 </li><li>消毒在70％乙醇清扫工具。擦拭vibratome刀片用丙酮去除油污，使用前消毒。 </li><li>将三个35毫米2菜肴和五个10cm 2的组织培养皿中的油烟机。填写1个10 平方厘米的菜在冰上解剖缓冲和地点。 注意:进程幼仔一次一个。 </li></ol> 6.解剖注意:进程幼仔一次一个。 ES / ftp_upload / 53304 / 53304fig2.jpg"/&gt; 图2.解剖削减。这些图像显示了必要的清扫削减从P6小鼠的大脑取出的头骨。切口是如虚线所示。（A）的剪切1被示出。削减1和2被从脑干/后部两侧连接到眼窝每侧..（B）的剪切3和4示出制成。切3从一个眼窝向其他削减连接1和2.切4开始在切割3的中线，并继续朝着鼻子划分嗅球之间的头骨（比例尺= 4.4Hz毫米）。尖<a href="https://www.jove.com/files/ftp_upload/53304/53304fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>麻醉用异氟醚曝光（100％异氟醚）的P6鼠标。大约5分钟呼吸变慢或出现呼吸的迹象时，迅速斩首鼠标用锋利的剪刀。喷雾头，用70％的乙醇，并装在一个组织培养皿。 </li><li>采用大钝钳捏住鼻子头抱稳。使用中型清扫剪刀，去除多余的皮肤露出头骨。 </li><li>使削减1和2切割颅骨两侧，从上向两侧前方的颅骨后，连接切到眼窝。保持尽可能靠近颅骨尽可能避免组织损伤的剪刀的尖端。 </li><li>对于切3，用小弹簧剪刀连接削减1和2。然后用小钝钳轻轻剥离头骨。 </li><li>使用小弹簧剪刀，轻轻沿中线切开剩余颅骨上顶，这样剩余颅骨变为2半部（ 图2中 ，切＃4）。使用镊子剥离颅骨以显示两个嗅球。 </li><li>将平面铲（沾夹层缓冲，所以组织会不会坚持下去）的B之间脑和颅底ottom，轻轻取出大脑并放入冰夹层缓冲的菜。 </li><li>重复步骤6，以获得第二脑切片</li></ol> 7.嵌入在琼脂糖大脑<ol><li>放置两个第35毫米2碗碟上的冰开放和擦拭的一对大钝钳用70％的乙醇。 </li><li>倾3％低熔点琼脂糖（在步骤3中制备）进入两个菜直到在顶部的圆顶形式。 3分钟设置计时器。在3分钟（试验通过触摸琼脂糖填充圆顶的边缘，如果能够观察到琼脂糖的聚合，它已经准备好）拿起1脑钝钳和地方变成琼脂糖填充35mm培养皿的一侧，然后移动它的菜心（这去除多余的夹层缓冲大脑周围所以它挂载更好）。 </li><li>重复的第二大脑。启动一个定时器10分钟。 </li><li> 10分钟后，将平铲入之间的空间琼脂糖和菜弹出包含P6大脑聚合琼脂糖。 </li><li>使用平刀片修剪琼脂糖到周围的脑立方体，确保其边缘尽量伸直。 </li><li>将强力胶的vibratome板两条。然后，拿起琼脂糖安装的大脑，轻轻地往下砸过胶，定位于矢状切片。用同样的方法在其他脑。确保两个大脑一字排开彼此。 </li><li>让胶水干在室温下5-8分钟。在这段时间里，填充用碎冰和水的vibratome的冰保持器区域。 </li></ol> 8.与Vibratome切片<ol><li>将切割藏进了冰架区，移动的标签来锁定到位的权利。 </li><li>使用圆形头螺丝刀，拿起圆vibratome板粘在它的大脑，并适应它进入水库。取出螺丝刀和固定板的地方有个Ë六角螺丝刀。确保板和切割水库坚决到位。 </li><li>使用镊子，皮卡vibratome刀片，将它列入切割头，锁到位内六角螺丝刀。然后，固定切削头与附接的叶片上使用轮螺丝vibratome。 </li><li>调出剃刀到尽可能靠近琼脂糖包埋脑尽可能的位置，同时确保剃刀是在相同的高度（或正上方）的大脑。填充室具有足够的冷解剖缓冲器以覆盖刀片。 </li><li>按↕按钮（你应该看到它闪烁，该按钮定义了边界时将刀片来回走），然后按住"前进"手动剪切嵌入的大脑，释放到剃刀清除琼脂糖块。立即再次按下↕按钮，这将确定自动切割的范围的结束。 </li><li>按"SINGLE / CONT"按钮一次，第l飞行由CONT应该继续下去。切割厚度设定为400-450微米，振动频率以5.5-6，以及速度为4。这将是剪裁设置。 </li><li>按"启动/停止"键一次，自动切割应该开始。按"暂停"用勺孔，因为需要收集组织。它大约需要5-10分钟，以达到接近中线。在这一点上，按"暂停"，改变速度为3，厚度改为200微米。不要收集进行此更改后产生的第一片。 </li><li>为200μm厚度所需的切片将不得不通过小脑嗅球很好地限定。转移的每个期望的切片，因为它们切成6孔板填充在冰上解剖缓​​冲器。通过移动的缓冲区切片室组织周围漂浮片，触摸它尽可能少的，将其从缓冲区时，它是正视在漏勺做到这一点。通常约为12片具有所需的结构可以收集。切片可留在解剖上冰缓冲液中达20分钟。 </li><li>而切片是在冰上，取出6孔板带插入涂覆有从37℃培养箱层粘连蛋白。在夹层罩丢弃层粘连蛋白，注意不要损坏刀片。添加3.5 ml的切片培养介质的每个刀片的顶。媒体的顶部会形成一个圆顶形状而不溅出入井。 </li></ol> 9.镀切片到插入<ol><li>使用漏勺工具，将脑切片到媒体上的插入，轻轻推片被完全淹没。重复所有切片。 </li><li>使用P1000绘制出1 ml的切片培养介质从刀片的顶和分配入井的底部。卸下并丢弃多余的介质，直到周围的大脑琼脂糖边缘slicebecome可见。这样做对其余切片。 </li><li>拿起吨他用钳，倾斜的边缘插入和取出多余的介质。然后，插入迅速转移到35毫米2道菜含有1ml片培养基。使用2对尖锐钳去除周围组织琼脂糖，注意不要伸/损坏片或戳一个孔膜（最简单的，如果你犯了一个催泪在琼脂糖的边缘，然后将除了琼脂糖任何一方）。然后将插入物回6孔板并重复对其余切片。 </li><li>在组织培养罩与鼓风机关闭（以防止变干片）从每个膜除去琼脂​​糖片段。 </li><li>转移切片在步骤5.1中制备的6孔板和板存储于37℃。片孵育24-48小时。 </li></ol> 10.更改切片文化传媒<ol><li>用新鲜的6孔板重复步骤5.1。 </li><li>在组织培养罩与送风机关闭时，插入物转移到新的6孔板续癌宁新鲜切片媒体。 </li></ol>对11片镀肿瘤细胞<ol><li>超声处理的CM的DiI染料和旋转中速2分钟。 </li><li>被用于覆盖在201×g离心5分钟降速肿瘤细胞。然后悬浮在2ml片培养基。 </li><li>加7微升出Cm的DiI入2 ml的细胞和培养基的孵育在37℃下20分钟。 </li><li>在200μl切片媒体降速细胞在201 XG 5分钟，重悬。轻轻磨碎以解离细胞（10-20次，或直至沉淀消失），然后添加800微升切片媒体，使1000微升总体积。 </li><li>计数采用台盼蓝存活的肿瘤细胞。绿色荧光微球添加到肿瘤细胞，在相同的浓度的细胞。这些惰性微球将作为对照。在201×g离心降速细胞/微球体混合物5分钟，然后重悬在切片培养介质的所需量。板细胞以6000个细胞的密度/ SL冰在65微升的体积。细胞每片镀的数量可以根据喜好和可用性增加。 </li><li>在组织培养罩分配细胞在65微升媒体/切片置于该片中的中心。 </li></ol> 12.成像和修复注:尼康的Eclipse镍C2si直立共聚焦被用来取整矢状切片的大图像扫描4×红色和绿色荧光通道。如果扫描功能不可用，取多个图像顺序地穿过切片和后来拼接图像在Photoshop（使用微球的位置和片的边缘进行导航）。 <ol><li>第二天（1天成像），转移片分为六35毫米2菜1毫升片培养基。图像中的每个切片一次一个在荧光显微镜直立。采取全矢状切片在4X用红色和绿色荧光通道的大图像扫描。保持激光和增益设置相同的每个片段。在成像结束，全部转让切片回含有新鲜切片媒体6孔板。 </li><li>如果另外EDU，其它增殖标志物或药物情况的需要，建立分片介质的股票与将日常使用的介质改变了治疗病情的药物。治疗情况应该在步骤12.1（紧接第1天成像）被引入。任何EDU或增殖标记应添加到媒体也开始在12.1和每日刷新（使用EDU在10μM）。 </li><li>图片第7天再次使用相同的设置第1天的图像。 </li><li>经过7天的成像完成后，将每片到6孔板每孔含1.2毫升4％多聚甲醛（PFA）的。小心缓慢地滴上每一片的顶部额外1毫升煤灰。留在RT 1小时。 </li><li>从每个孔，并从每个切片的顶部除去煤灰。用PBS洗涤每口井的3倍。存储在PBS切片在4℃染色或安装在载玻片上。 注意:ImageJ的设置可能需要调整和优化，以考虑图象和显微镜质量以及肿瘤细胞大小。 </li></ol>图片13定量（使用ImageJ） 注意:ImageJ的设置可能需要调整和优化，以考虑图象和显微镜质量以及肿瘤细胞大小。 <ol><li>在ImageJ的，使用多边形工具来勾勒整个切片或要量化的区域。加入此选择的投资回报率经理，你可以将其重命名并保存它。 </li><li>右键单击选定区域，然后选择复制。名称与本地区，分片，一天，这个重复的图像。按CTL + Shift键+ E显示轮廓，然后选择过程 - 扣除背景 - 风水球半径= 4。 </li><li>经过背景减法，选择"图像"下拉菜单中选择"调整门槛"。检查黑暗的背景 -;看图像放大和设置的阈值。当你通过移动阈值条设置的阈值，确保所有细胞都包含/突出，但非常小圆点是碎片（或小于电池）不包括在内。使用相同的阈值的所有图像。移动阈值窗的一侧。 </li><li>选择"程序" - "查找千里马"，并设置5至10的噪声容限和检查以下内容:预览点选择，输出类型为分段颗粒，排除边缘最大值，高于下限阈值。然后按CTL + Shift键+ E重新选择感兴趣的区域。 </li><li>选择从分析下拉菜单"分析粒子"。设置像素大小到4-40和圆形到0.00-1.00。也可以点击"显示叠加轮廓"，"显示结果"和"汇总"，然后按确定。 </li><li>记录所有的原始数据，并在Microsoft Office文档的摘要。 "计数"为每个区域我Ş需​​要计算在周文化的倍数变化。 </li><li>重复此过程为所有图像和感兴趣的领域，确保门槛和其他设置保持一致。 </li><li>周期间计算细胞数量的倍数变化在该切片上培养由细胞的第1天同样的切片上数除肿瘤细胞的数量的片上在第7天，每个区域内的倍数变化在细胞数通过在第7天分裂细胞的数量在该区域由细胞的数目在该区域第1天计算的。 </li><li>执行的微步13.8为好。片上微球数量应该是在第7天的相同的第1天（倍数变化= 1）。类似地，每个区域内的微球数量应该是在第7天的相同的第1天（倍数变化= 1）。如果折变化不同于1，表示随时间的变化片形貌。 </li></ol> 14.染色<ol><li>磁带在​​th一块封口膜Ë实验台，并绘制六界与液体阻滞剂PAP笔（每个分片，只是比片的大小）。放置约400微升PBS中在每个圆的中间的点。号或标记的圆圈，以确定每个切片。 </li><li> 1毫升的PBS，用刀片切割片围绕一个正方形，留下膜切片周围的一些空间。使用镊子将切出片到第一圈，仔细躺在PBS的泡沫顶部的切片。重要的是要确保切片不翻转折迭或得到翻转在此过程颠倒。然后，从切片下方取出PBS，并把它放在片的顶部，如果需要的话，以确保它仍然淹没添加更多的PBS。重复每个片这一步。 </li><li>使10个ml的在PBS中的3％BSA的。取输出2毫升，并加入100微升毛地黄皂苷的吧。混合，并加入到切片以阻止和透化，在室温30分钟。 注意:使用的其他透剂如三吨X-100将导致的CM-的DiI标记的损失。 </li><li>除去透化/封闭溶液和冲洗3次，用PBS 10分钟。 </li><li>如果染色EDU，按照提供的套件组装混合物的方向。 45分钟涂敷混合物。如果与其它抗体染色，协议必须为初级和次级抗体的浓度（〜1小时RT下主和次）进行优化。冲洗3次，用PBS 10分钟。 </li><li>染色用DAPI 1:1,000在PBS 5分钟室温。漂洗2次，用PBS。 </li></ol> 15.安装了片<ol><li>切片转移至显微镜载玻片。确保与片膜的一面仍然朝上，并且不翻转折迭。绘制切片周围的边框有液体阻滞剂PAP笔。 </li><li>放置一个小玻璃盖玻片（5毫米）上的片的每一侧（保持被损坏的切片）。上的顶部落下的两个或三滴Fluoromount-G（对于免疫荧光）切片只是勉强覆盖。然后盖片长盖玻片（24×50毫米）。 </li><li>重复每个切片。密封用指甲油和存储幻灯片的边缘，在4℃。做染色共聚焦成像。 </li></ol>

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	<li>Heddleston, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioma+stem+cell+maintenance:+the+role+of+the+microenvironment.">Glioma stem cell maintenance: the role of the microenvironment.</a> Curr Pharm Des. 17 (23), 2386-2401 (2013).</li><li>Sasai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shh+pathway+activity+is+down-regulated+in+cultured+medulloblastoma+cells:+implications+for+preclinical+studies.">Shh pathway activity is down-regulated in cultured medulloblastoma cells: implications for preclinical studies.</a> Cancer Res. 66 (8), 4215-4222 (2006).</li><li>Louis, D. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2007+WHO+classification+of+tumours+of+the+central+nervous+system.">The 2007 WHO classification of tumours of the central nervous system.</a> Acta Neuropathol. 114 (2), 97-109 (2007).</li><li>Duffau, H., Capelle, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+brain+locations+of+low-grade+gliomas.">Preferential brain locations of low-grade gliomas.</a> Cancer. 100 (12), 2622-2626 (2004).</li><li>Chourmouzi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manifestations+of+pilocytic+astrocytoma:+a+pictorial+review.">Manifestations of pilocytic astrocytoma: a pictorial review.</a> Insights Imaging. 5 (3), 387-402 (2014).</li><li>Buonamici, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfering+with+resistance+to+smoothened+antagonists+by+inhibition+of+the+PI3K+pathway+in+medulloblastoma.">Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma.</a> Sci Transl Med. 2 (51), 51ra70 (2010).</li><li>Choi, Y., Borghesani, P. R., Chan, J. A., Segal, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+from+a+mitogenic+niche+promotes+cell-cycle+exit.">Migration from a mitogenic niche promotes cell-cycle exit.</a> J Neurosci. 25 (45), 10437-10445 (2005).</li><li>Chan, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteoglycan+interactions+with+Sonic+Hedgehog+specify+mitogenic+responses.">Proteoglycan interactions with Sonic Hedgehog specify mitogenic responses.</a> Nat Neurosci. 12 (4), 409-417 (2009).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37 (2), 173-182 (1991).</li><li>Kenny, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+high+throughput+screening+using+a+primary+human+three-+dimensional+organotypic+culture+predicts+in+vivo+efficacy.">Quantitative high throughput screening using a primary human three- dimensional organotypic culture predicts in vivo efficacy.</a> Nat Commun. 6, 6220 (2015).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK+inhibitor+and+feeder+cells+induce+the+conditional+reprogramming+of+epithelial+cells.">ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.</a> Am J Pathol. 180 (2), 599-607 (2012).</li></ol>

None of the authors have competing interests or conflicting interests.

这个协议描述如何脑瘤细胞可荧光标记并接种在P6小鼠的矢状脑切片，然后在培养监视一周。这种脑肿瘤/器官切片共培养测定可用于确定区域微环境对肿瘤细胞数量的影响，并且也可以用作用于测量对人肿瘤生长的新的药物治疗的功效的系统。以前的研究已经使用了类似的策略，以评估脑微环境的神经前体细胞增殖7,8的作用。 该测定中，其中脑肿瘤细胞接种在一个器...

最近的癌症研究已发现遗传变异，分子变化和可能的治疗各种脑肿瘤取得显著的进步。尽管这一进展，脑肿瘤仍然是癌症相关死亡的成人和儿童的顶尖原因之一。限制因素脑瘤研究包括伯患者样本和细胞系的有限的可及在复制在可访问的实验系统的独特和异构脑微环境的难度。对于许多脑瘤维持肿瘤细胞随时间尚未得知所要求的条件。甚至对于可以在细胞悬浮液作为神经球来生长脑瘤，培养条件可影响肿瘤细胞1,2-。实际上，在加入碱性成纤维细胞生长因子或表皮生长因子，以鼓励增殖，抑制分化可以改变基因的表达1。其它方法对于肿瘤细胞的生长，例如如通过肿瘤细胞的原位或皮下异种移植物的小鼠的肿瘤传播是有价值的测定法，但由因子如肿瘤发展（尤其是对于低级别肿瘤），成本，和肿瘤细胞的数量的时间可以注射和研究是有限。因此，目前的方法种植人脑肿瘤细胞是不足以维持某些类型的肿瘤，而且还提供人工环境不这样做密切模仿体内肿瘤环境中。 不同类型的小儿脑肿瘤大脑内生长中高度专业化的位置[3,4]，这是可能反映肿瘤生长不同微环境的要求[5]。这个协议描述了一种新颖的系统，其中的细胞是难以在正常培养条件来传播可以在器官型脑微环境中生长该模拟在体内肿瘤生长的条件。在这种定量测定中，荧光标记的脑肿瘤细胞接种在幼年小鼠大脑器官切片，并随时间监测。该测定可用于研究微环境对肿瘤生长的影响，并在临床相关的脑微环境，以测试新的药物治疗方法。

<table><tbody><tr><td>HEPES</td><td>Invitrogen&amp;nbsp;</td><td>17504044</td><td></td></tr><tr><td>Glucose</td><td>Invitrogen</td><td>17502048</td><td></td></tr><tr><td>Pennicillin Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>HBSS</td><td>Life Technologies</td><td>14185-052</td><td></td></tr><tr><td>B-27</td><td>Life Technologies</td><td>17504-044</td><td></td></tr><tr><td>N2</td><td>Life Technologies</td><td>17502-048</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>Neurobasal-A- Medium minus phenol red</td><td>Invitrogen&amp;nbsp;</td><td>12349015</td><td></td></tr><tr><td>Low Melting Point Agarose</td><td>Promega</td><td>V2111</td><td></td></tr><tr><td>Slice Culture Inserts&amp;nbsp;</td><td>Milipore</td><td>PICM0RG50</td><td></td></tr><tr><td>laminin</td><td>Invitrogen</td><td>23017015</td><td></td></tr><tr><td>Cm-DiI&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>V22888</td><td></td></tr><tr><td>EDU (Labeling and Detection)&amp;nbsp;</td><td>Life Technologies</td><td>c10337</td><td></td></tr><tr><td>Microspheres&amp;nbsp;</td><td>Life Technologies</td><td>F-21010</td><td></td></tr><tr><td>Vibratome&amp;nbsp;</td><td>Leica</td><td></td><td></td></tr><tr><td>Confocal Microscope&amp;nbsp;</td><td>Nikon Eclipse Ni C2si</td><td></td><td></td></tr><tr><td>ImageJ software&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>5 mm Cover Glasses&amp;nbsp;</td><td>Fisher Scientific</td><td>64-0700 (CS-5R)</td><td></td></tr></tbody></table>

a brain tumor/organotypic slice co-culture system for studying tumor microenvironment and targeted drug therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

本节中举例说明了从利用所述脑瘤/器官切片共培养来研究区域微环境的偏好，以及测试新的疗法预期的结果的类型。我们表明，在测定被设计为复制的微环境脑肿瘤，作为组织的组织和切片的增殖状态被维持（图3）。我们还表明，增加了片随时间对肿瘤细胞的数目可以部分地是由于细胞到脑切片微环境的迁移（ 图4）。我们还表明，这个共培养可作为一个定量测定通过?...

Watch this Scientific Journal Video about A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies at JoVE.com

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

a-brain-tumororganotypic-slice-co-culture-system-for-studying-tumor

Research

JoVE Journal

Medicine

11.0K 次观看.  Dana-Farber Cancer Institute. Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice.  

视频: A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

测量组织肿瘤组织切片的实时药物反应

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

科研

癌症研究

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

一 种研究 并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

与器官型脑片和癌细胞的3D球体共培养系统

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

医学

<meta charset="utf8"></head><body>乐高：揭示脂质失调和药物靶点的人类 GBM 类器官模型

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and cellular characteristics of GBM have been a focal point of ongoing research, revealing that it is a group of heterogeneous diseases with variations in RNA expression, DNA methylation, or cellular composition. Despite the wealth of molecular data available, the lack of transferable pre-clinic models has limited the application of this information to disease classification rather than treatment stratification. Transferring the patients' genetic information into clinical benefits and bridging the gap between detailed descriptions of GBM, genotype-phenotype associations, and treatment advancements remain significant challenges. In this context, we present an advanced human GBM organoid model, the Laboratory Engineered Glioblastoma Organoid (LEGO), and illustrate its use in studying the genotype-phenotype dependencies and screening potential drugs for GBM. Utilizing this model, we have identified lipid metabolism dysregulation as a critical milestone in GBM progression and discovered that the microsomal triglyceride transfer protein inhibitor Lomitapide shows promise as a potential treatment for GBM.

<meta charset="utf8"></head><body>实验室工程胶质母细胞瘤类器官培养和药物筛选

Here, we outline a comprehensive protocol for generating and utilizing laboratory-engineered glioblastoma organoids (LEGO) to investigate genotype-phenotype dependencies and screen potential drugs for glioblastoma treatment.

实验室工程胶质母细胞瘤类器官培养和药物筛选

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and ...

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 ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

神经科学

Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the multicellular interactions of native tissue. Tissue&#160;slices immediately cut from freshly resected tumors preserve spatial aspects of intratumor heterogeneity (ITH), thus making them useful surrogates of in vivo biology. Careful optimization of tissue slice preparation and cultivation conditions is fundamental for the predictive diagnostic potential of tumor slice explants. In this regard, murine models are valuable, as these provide a consistent flow of tumor material to perform replicate and reproducible experiments. This protocol describes the culturing of murine lung tumor-derived tissue slices using a rotating incubation unit, a system that enables intermittent exposure of tissues to oxygen and nutrients. Our previous work showed that the use of rotating incubation units improves the viability of tissue compared to other culture methods, particularly floating slices and stagnant filter supports. Here, we further show that slice thickness influences the viability of cultured slices, suggesting that optimization of slice thickness should be done for different tissue types. Pronounced ITH in relevant oncogenic functions, such as signaling activities, stromal cell infiltration or expression of differentiation markers, necessitates evaluation of adjacent tissue slices for the expression of markers altered by drug treatment or cultivation itself. In summary, this protocol describes the generation of murine lung tumor slices and their culture on a rotating incubation unit and demonstrates how slices should be systematically analyzed for the expression of heterogeneous tissue markers, as a prerequisite prior to drug response studies.

We provide a method for the generation, cultivation and systematic analysis of organotypic slices derived from murine lung tumors. We also describe how to optimize for slice thickness, and how to select drug concentrations to treat tumor slices.

肿瘤切片提取物的建立与分析是诊断检测的前提

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the ...

免疫与感染

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 &#181;m thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

摘要

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测量组织肿瘤组织切片的实时药物反应

一种研究并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

与器官型脑片和癌细胞的3D球体共培养系统

实验室工程胶质母细胞瘤类器官培养和药物筛选

实验室工程胶质母细胞瘤类器官培养和药物筛选

实验室工程胶质母细胞瘤类器官培养和药物筛选

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

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

肿瘤切片提取物的建立与分析是诊断检测的前提

器官型切片培养作为原发性胰腺癌和转移瘤肿瘤微环境的临床前模型

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

摘要

探索更多视频

此视频中的章节

测量组织肿瘤组织切片的实时药物反应

一 种研究 并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型

与器官型脑片和癌细胞的3D球体共培养系统

实验室工程胶质母细胞瘤类器官培养和药物筛选

实验室工程胶质母细胞瘤类器官培养和药物筛选

实验室工程胶质母细胞瘤类器官培养和药物筛选

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

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

肿瘤切片提取物的建立与分析是诊断检测的前提

器官型切片培养作为原发性胰腺癌和转移瘤肿瘤微环境的临床前模型

一种研究并靶向乳腺癌脑转移性肿瘤生长的离体脑切片模型