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>

<ol>
	<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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen&#160;</td>
			<td>17504044</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Invitrogen</td>
			<td>17502048</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Pennicillin Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14185-052</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td rowspan="2">Neurobasal-A- Medium minus phenol red</td>
			<td rowspan="2">Invitrogen&#160;</td>
			<td rowspan="2">12349015</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Low Melting Point Agarose</td>
			<td>Promega</td>
			<td>V2111</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Slice Culture Inserts&#160;</td>
			<td>Milipore</td>
			<td>PICM0RG50</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>Invitrogen</td>
			<td>23017015</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Cm-DiI&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>V22888</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>EDU (Labeling and Detection)&#160;</td>
			<td>Life Technologies</td>
			<td>c10337</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Microspheres&#160;</td>
			<td>Life Technologies</td>
			<td>F-21010</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Vibratome&#160;</td>
			<td>Leica</td>
			<td>N/A</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Confocal Microscope&#160;</td>
			<td>Nikon Eclipse Ni C2si</td>
			<td>N/A</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Image J software&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>5mm Cover Glasses&#160;</td>
			<td>Fisher Scientific</td>
			<td>64-0700 (CS-5R)</td>
			<td colspan="3"></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

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

科研

医学

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

脓毒症动物模型，用于评估新型草药疗法

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

允许有针对性的大鼠模型血 - 脑屏障破坏神经血管治疗

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

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 ...

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 ...

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 ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...