，脑肿瘤SPORE奖（PD:GY吉莱斯皮，P20CA 151129-03）和SBIR合同（PI:R.辛格，N43CO-2013-00026）:由美国国立卫生研究院资助R21（C.威利，CA185712-01 PI）的支持。

注:所有异种移植治疗的评价是使用胶质母细胞瘤原位肿瘤模型上的机构动物护理和使用委员会批准的方案进行。 1.患者来源的GBM异种移植细胞的分离<ol><li> 试剂的制备 <ol><li>重新构成胶原酶我在无菌水中的5毫克/毫升和无菌过滤的浓度。在-20℃1ml等份存储（终浓度为100毫升的酶溶液50微克/毫升）中。 </li><li>在2ml溶解100微克表皮生长因子（EGF）的无菌磷酸盐缓冲盐水（PBS）中。商店在100μl（5微克/ 100微升）在-20℃等份为10纳克/毫升的最终浓度。 </li><li>溶解100微克在2毫升的FGF-β的无菌PBS中。商店在100μl（5微克/ 100微升）在-20℃等份为10纳克/毫升的最终浓度。 </li><li>添加下面的一一瓶500毫升的Neurobasal医学的IA（NBM），以制备完整的NBM:不含维生素10ml的B-27补充，将5ml N2添加剂，100μl的EGF，100微升成纤维细胞生长因子（FGF） - 基本，将5ml两性霉素B，0.5 ml庆大霉素，加入5ml L-谷氨酰胺。 </li><li>结合配制新鲜酶液98.5毫升的PBS，1 ml胶原酶-1加入0.5ml 10倍胰蛋白酶/ EDTA，并通过0.22微米孔径过滤器进行过滤消毒。 </li><li>制备或获得的小体积的无菌的Dulbecco改良的Eagle培养基（DMEM），用7％胎牛血清（FBS）和1×PBS中无钙或镁/ F12细胞培养基。 </li></ol></li><li> 肿瘤解体 注:无胸腺NU / NU小鼠先前与肿瘤细胞在右胁从一个可触知的肿瘤块注入并使其是必要的此过程。在这里所示的例子中，我们使用源自患者的异种移植物的GBM细胞，JX10和JX12 17。 <ol><li>通过喷施2％洗必泰消毒工作区和设置工作区机智ħ必要的仪器/用品包括一次性夹头，镊子，解剖刀，玻璃培养皿，酶溶液，PBS和洗必泰（参见图2A）。 </li><li>安乐死鼠标/鼠标窝藏一个侧面肿瘤。瞄准的 CO 2，用30％ 的 CO 2 20％/分钟的位移速率。鼠标显示呼吸骤停后，保持约1分钟（一般为3分钟计）CO 2的流动。小鼠被从室中取出，并颈脱位是作为二次安乐死确认执行。喷动物用3％氯己定消毒皮肤。 </li><li>同时按住鼠标稳定，使半圆形切开皮肤〜使用无菌一次性解剖刀以＃11刀片从肿瘤块（侧面植入肿瘤）1.5厘米开始颅到肿瘤块和朝向鼠标的腹部切开并绕到尾端。 </li><li>反映在皮肤用手指对切口的尾端温柔的牵引肿块，然后按在皮肤上表面提升肿瘤有而不触及肿瘤最佳的访问（ 见图2b）。 </li><li>使用钝性分离用钳子轻轻地从腹腔壁及皮肤无肿块（ 见图2C）。 </li><li>转移肿块用镊子无菌玻璃培养皿（不要用塑料盘，以防止破切碎期间），并妥善丢弃尸体和一旁把这些仪器。 </li><li>使用新的消毒器械（含第11刀，半弯钳手术刀）到清创坏死组织和膜主机结缔组织覆盖肿瘤的肿瘤组织。 </li><li>部署15 mL酶溶液与搅拌棒的无菌通风-胰蛋白酶烧瓶中，并开始在引擎盖慢慢搅拌。 </li><li>在无菌玻璃培养皿，洗收获肿瘤3 - 5次用无菌PBS，去除多余血液（可能会倒或使用注射器，用PBS冲洗一下）。轻轻摇晃培养皿吸管吸出或洗材料。百果˚Finely使用两个＃11手术刀片（ 见图2D）。 </li><li> 14毫升酶溶液中加入切碎的肿瘤和吸管轻轻向上和向下2 - 3倍。到排气-胰蛋白酶转印混合物烧瓶中并缓慢地搅拌20分钟。通过在以中和胰蛋白酶在步骤1.2.11加入1.5毫升FBS的每个准备5 50ml锥形管中。 </li><li> 20分钟后，从胰蛋白酶烧瓶收集14毫升的细胞溶液，并加入到一个管与FBS的。 ，增加14毫升的新鲜酶溶液到烧瓶中，并继续搅拌。 </li><li>离心收集的细胞在150×g离心在室温下8分钟并弃上清。加入45毫升完整NBM到颗粒，轻轻混匀，然后重复离心冲洗血清。重新暂停5毫升完整NBM颗粒并保持在冰上。 </li><li>总共5收成重复细胞收获步骤，所有收获的细胞在一个锥形管相结合的冰上。 </li><li>放置一个40微米的细胞滤网上的50ml锥形管的顶部。准备ŧ他由流过10A的10ml DMEM / F12 + 7％FBS的通过，然后用10ml PBS清洗细胞滤网。 </li><li>慢慢细胞悬液添加到过滤器，允许它滴落到（参见图2M）。轻轻抬起每个新除了破坏任何真空，使悬浮细胞自由通过的细胞过滤的选项卡（ 见图2N）。 </li><li>离心如上过滤细胞悬浮液。在10毫升重悬浮完整的NBM和计数，以确定使用0.04％台盼蓝和血细胞计数器存活细胞总数。保持细胞在冰上直到microtumor产生。 </li></ol></li></ol> 2.交替组织分解协议<ol><li>自动组织离解和分类系统。 <ol><li>请将分解管进入细胞的离解，选择程序"肿瘤_02_02"，并按下启动并运行32秒。传输管到旋转器，发生在37℃培养箱中40分钟。 </li><li>离心悬浮细胞，在150×g离心在室温下8分钟并弃上清。而离心细胞，将细胞悬浮液的处理设置的试剂。加入10 mL无血清NBM和轻轻磨碎停牌。 </li><li>离心后，得到含〜10细胞沉淀- 30×10 6个活细胞（〜0.3ml）中。再悬浮的沉淀在10ml无血清的NBM（ 见图20）。确定由排除测定的存活细胞（请参阅步骤3.2.2）。 </li></ol></li></ol> 3. Microtumor代<ol><li>试剂的制备<ol><li>准备完整的NBM为1.1.4及（3毫克/毫升HDHG）每内部协议准备中和高密度的人类biogel（HuBiogel）。类似biogel矩阵也可被使用。 </li></ol></li><li> Microtumor生产与文化<ol><li>获得新鲜分离PDX细胞作为完整的NBM单细胞悬液，在冰上。 </li><li>混合细胞素spension与等体积的台盼蓝溶液（在PBS中0.4％）和分析用血细胞计数器通过台盼蓝排斥18，以确定细胞数量和存活力。除去必要量，以产生50,000细胞/ microtumor，其中体积=（50,000个细胞/肿瘤*＃肿瘤）/（存活细胞计数/ 1毫升）中，并放置到一个新的锥形管中。 </li><li>在150 xg离心浓缩细胞通过离心，8分钟，在室温下。丢弃上清，重悬用冰冷HDHG溶液将细胞沉淀在FBS的1份细胞和4份HDHG的最终比例。 </li><li>使用电子多道移液器以分配每引脚细胞HDHG混合物10微升到96针钢板（带疏水涂层），以产生microtumors（各含50,000细胞2毫米珠）。 </li><li>放置组织培养箱（37℃，5％CO 2，加湿）的内部的3D肿瘤珠15分钟以胶凝珠粒。 </li><li>凝胶后，转移microtumors（10μ; l）可定制的悬浮培养室（50毫升）或大体积培养皿（10厘米）用电子多通道移液器和定制销装置包含完整的NBM。 </li><li>后1 - 在组织培养培养箱中2天后，转移microtumors到含有50微升的NBM /使用公广口分配移液管并执行各种测定和分析协议的96孔培养板中。 </li></ol></li><li>药物治疗和microtumors维护<ol><li>最终选择浓度的药物测试。 注意:如果药物已经在二维培养已知功效，选择2倍的IC 50为中剂量浓度，然后选择3倍以上及以下共5个剂量水平的连续稀释。例如，如果IC 50为9μm为在二维培养的药物，选择2微米，6微米，18微米，54微米，162微米，作为药物测试的最终浓度。 </li><li>从二sulfoxi完全NBM准备2个药物剂量的解决方案德（DMSO）的股票。稀释在1％DMSO的培养基给药溶液，以制备5个剂量，3倍连续稀释。 </li><li>加入50μl的给药溶液到孔含有在分析板50微升microtumor达到0.5％的最终DMSO浓度。重复在3.3.1测定每种药物的剂量，每个重复（ 如，4）。 </li><li>保持在37℃培养，5％的CO 2，为1加湿组织培养箱- 14天。饲料培养按上述刷新媒体和药物溶液，每周两次。 </li></ol></li></ol> 4.形态和Microtumors的表型分析<ol><li>使用标准的活细胞染色确定microtumors细胞形态。 <ol><li>制备钙荧光素-AM的1mM储备在DMSO中。分装和储存在-20°C。 </li><li>在所需的培养时间间隔（1,7，和14天），加入在PBS中制备的（不含Ca / Mg）的96孔板为1微米终浓度钙黄绿素-AM溶液。 </li><li>孵化20分钟使用在2X，4X荧光显微镜37℃，5％CO 2，潮湿的培养箱，和图像，和10X（激发:450 - 490 nm的带通，发射:515纳米长传，二色500纳米） 。 </li></ol></li><li> Microtumor生长/增殖测定<ol><li>溶于PBS MTT粉（无钙/镁）准备12毫米的股票。无菌过滤，分装，并储存在-20℃。 </li><li>在所需的培养时间间隔（1,7，和14天），加入20微升的MTT溶液，以每100微升培养液体积的96孔板。孵育在组织培养箱中2小时。 </li><li>准备10％SDS新鲜裂解液在0.01M HCl和等量培养体积增加板。孵育密封板37℃培养箱过夜。 </li><li>使用多板读数器在570nm处读取吸光度。 </li></ol></li><li> Microtumor准备Kinomic分析<ol><li> 100比每100倍临:副冷却预至4℃，再加入1制备的裂解缓冲液蛋白磷酸酶抑制剂（PPI）和100倍的蛋白质蛋白酶抑制剂（PI）。拌匀，并保持在冰上。 </li><li>传输2 microtumors，以3个1.5 ml离心管中。除去上清液，并在4℃下添加裂解缓冲液40微升含PPI和PI对每个管和溶胞30分钟。 </li><li>吸取样品大力打破肿瘤珠。离心在4℃和存储样本16000 XG 10分钟，在-80°C，直到kinomic分析。 </li></ol></li></ol> 5. Microtumors的Kinomic剖析<ol><li> 蛋白酪氨酸激酶（PTK）分析 <ol><li>解冻试剂（10倍蛋白激酶（PK）缓冲液和2％牛血清白蛋白（BSA）），以4℃负载的PK缓冲液（300μl的10倍的PK缓冲库存中2.7毫升卫生署2 O）到注射器在位置＃2上的分析平台。 </li><li>打开激酶检测软件程序并装入PTK协议文件，并按下"开始"，标注在软件样品是扫描后的芯片方案。广场上的芯片，每个阵列分析平台和负载25微升2％牛血清白蛋白（BSA），然后按LOAD开始软件控制的协议封闭步骤。 </li><li>稀释6微米100毫摩尔ATP的股票有54微升的dh 2 O，以10毫米三磷酸腺苷（ATP）。当软件开始的第三和最后的清洗步骤（显示在屏幕上）将15微克裂解带来高达28微升与卫生署2 O，混合，并添加到阵列。 </li><li>制备PTK主混合物（MM）（最​​多12个样品）。添加32.6微升的dh 2 O来重建二硫苏糖醇（DTT），加入6微升DTT和60微升10X PK的解决方案，以PTK-MM1管（已包含60微升10X BSA）。 </li><li>等到在激酶检测软件"加载"提示出现再加入126微升PTK-MM1的60微升PTK添加剂，和60微升10毫摩尔ATP至MM2（包含以前分到4.5微升PY20 FITC抗体），混匀。加入16微升这PTK预混每个样品裂解液管移液器混合5次。 </li><li>确保玻璃盖是干净的，然后加入35微升裂解液/每个阵列，靠近旋转木马盖子，按LOAD按钮预混。 </li></ol></li><li> 丝氨酸/苏氨酸激酶（STK）剖析: <ol><li>解冻试剂（10倍的PK缓冲液，10倍STK缓冲液和2％的BSA）至4℃。负载PK缓冲液（300μl，以2.7毫升卫生署2 0）到注射器位置＃1，1:10，STK缓冲（300μl，以2.7毫升卫生署2 0）到注射器位置＃2。 </li><li>打开激酶测定计算机软件程序并加载STK协议文件，按START，扫描芯片后在程序中注释的样本。广场上的芯片平台分析和负载25微升每个阵列2％BSA，然后按LOAD开始软件控制的协议封闭步骤。 </li><li>稀释6微米100毫米ATP与54微升卫生署2 O.在最后（第3）清洗步骤组合2微克裂解并带来高达32.6微升与卫生署2 O，混合，并添加到阵列。 </li><li>让STK预混:添加70微升10X PK到STK-MM1管（包含7微升100X BSA），再加入42微升的dh 2 O到STK-MM1管。 </li><li>等到在激酶检测软件"加载"的提示，则:加入18微升10毫摩尔ATP到STK-MM1，并加入9微升MM1到每个样品裂解液。拌匀。 </li><li>确保盖玻片干净，加入35微升裂解液/每个阵列，靠近旋转木马盖，然后按LOAD主结构。 </li><li>在决赛（3 次 ）清洗步骤 添加1.05微升STK-FITC二次抗体DMAB管（包含3.03微升STK主要抗体混合物）中，添加39.6微升AB缓冲区，以DMAB，增加356.0微升的dh 2 O，以DMAB，加入30微升至DMAB到每个磁盘阵列，按下LOAD 。 </li></ol></li><li> Kinomic数据19,20分析 <ol><li>打开分析软件和加载图像分析应用。硒LECT先前生成包含的PTK或STK数据，选择的文章相匹配号码阵列布局文件（86312对PTK和87102为STK）条形码和时间戳整个阵列图像的图像文件夹中，并负载阵列注释文件。 </li><li>验证所有图像正确网格化和运行曝光时间缩放应用。统计学上比较曝光/坡LOG2转换值，并与预洗动力学曲线19-21验证。 </li><li>在上游激酶预测软件来查询条件之间改变kinomic型材上游激酶22,23。 </li></ol></li></ol>

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R.S. and R.S.S. are employees of Vivo Biosciences. J.C.A. is partially funded by the UAB Kinome Core.

在协议中的关键步骤主要涉及microtumor代，以及药物剂量和维护。因为microtumor珠粒是脆弱的，容易撕裂，需要在检测和维修都发育阶段特别小心。如果在这些过程之一中出现错误，实验解释可能会受到损害，从而导致延长或实验的不必要的重复，甚至排除的数据。 修改和排查，尤其是microtumor发展过程中，包括为使用使得microtumor珠的自定义工具疏水性的设计和生产。这个工具?...

最常见的原发性脑恶性脑肿瘤是III级星形细胞瘤和Ⅳ级胶质母细胞瘤 （成胶质细胞瘤或GBM）。这些肿瘤提供与12之间平均一年的生存预后差- 15个月，在美国的1-3电流疗法GBM。多模态治疗包括手术，放疗和化疗，包括替莫唑胺（TMZ）和激酶靶向剂。激酶信号经常失调在GBM，包括在表皮生长因子受体（EGFR），增加血小板源生长因子受体（PDGFR）信令，增加的磷脂肌醇-3-激酶（PI3K），并与扩增的肿瘤的子集或激活突变肿瘤通过血管内皮生长因子受体（VEGFR），以及其他激酶驱动路径4-6支承血管生成信令。当前在体外和体内模型中经常失去这些代表性的改变&lt;SUP&gt; 7。此外，遗传分析没有提供可能反映出遗传和表观遗传变化并不总是在蛋白活性的水平，其中大多数激酶靶向剂直接作用预测的变化，并且其中与其它作用机制的治疗可充当预期的效益间接的影响。 可以传代无限传统的永生化细胞系一直是药物测试标准，由于其易于维护和重现性。然而，该模型从选择用于快速增长，从原来的肿瘤有很大的不同的细胞的高营养（和人工的）的生长环境受到影响。因此，出现了在显影反映一个更复杂的肿瘤生物学系统如存在于病人更现实的模型系统相当大的兴趣。肿瘤异种移植物直接从小鼠生长的原发肿瘤（"xenoline，"以病人为衍生异种移植或PDX）provi开发德一多个反射模型系统，特别是在癌症治疗的设置，因为它们被认为更可靠地预测临床成功。8尽管多个反射生物学，这些模型是昂贵的，并且难以建立和保持。此外，它们不适合于高通量研究。有必要更好地发展生物模型，更准确地反映原发肿瘤的分子改变，以及个人资料，并使用激酶活性的直接措施，检验这些模型，而不是代孕遗传标记，是显而易见的。 它众所周知，不同于二维（2D）单层培养，三维或多细胞测定模型可以提供更多的生理学相关端点9-11。常见的三维培养方法涉及涂基质微载体和细胞球体的形成。肿瘤球状体可以通过蜂窝聚合使用旋转器烧瓶中，的pHEMA板与悬滴技术产生。限制在tHESE方法包括:不能对一些细胞形成稳定的球状体，变异成长和挑战，混合细胞型。可替代地，许多合成的（水凝胶，聚合物）和动物衍生Engelbreth-Holm的-群基质已用于3D培养开发从小鼠肉瘤（EHS）矩阵，牛胶原）研究12-14。鼠标EHS矩阵被广泛使用，但已知促进细胞生长和分化的体外 和体内 15。 为了复制3D肿瘤生物学，人类生物基质系统是由拉吉辛格博士等人的 16开发的。 自然，生长自由因子人biogel允许三维培养支架（珠片），它支持多种类型的细胞长期培养。一系列的三维人体biogel文化设计都建立了研究肿瘤的生长，黏附，血管生成和侵袭特性。优势和人力biogel的性能比普通鼠标EHS凝胶总结在表1和表2。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 资源: </td><td> 人类羊膜 （池组织） 无病原体，IRB豁免/批准</td></tr><tr><td> ECM性质: </td><td> 非变性Biogel（GLP-生产） </td></tr><tr><td> 键 组件: </td><td> COL-I（38％），层粘连蛋白（22％），COL-IV（20％），COL-III（7％），巢蛋白和HSPG（&lt;3％） </td></tr><tr><td> GF-免费: </td><td> 检测不到 EGF，FGF，TGF，VEGF，PDGF（非血管，无毒） </td></tr></tbody></table> 表1:相比于常见的EHS凝胶人力Biogel的性质。 <table border="1" fo:keep-together.within-page="1" fo:keep-wiTH-next.within页="总是"&gt; <tbody><tr><td> 人类Biogel </td><td> EHS凝胶 </td></tr><tr><td>人类自然矩阵</td><td>重组小鼠基质</td></tr><tr><td>控制细胞生长及分化</td><td>能促进细胞的生长和分化</td></tr><tr><td>生理基因表达</td><td>可变基因表达</td></tr><tr><td>三维组织样培养模型</td><td>板为基础的养殖模式</td></tr></tbody></table> 表2:相比于常见的EHS凝胶人力Biogel的优势。

<table><tbody><tr><td>Collagenase-I&amp;nbsp;</td><td>Sigma-Aldrich</td><td>CO130</td><td></td></tr><tr><td>Trypsin EDTA (10x)</td><td>Invitrogen</td><td>15400-054&amp;nbsp;</td><td></td></tr><tr><td>Neurobasal-A</td><td>Life Technologies</td><td>10888-022</td><td></td></tr><tr><td>N-2 Supplement</td><td>Life Technologies</td><td>17502-048</td><td>1x final concentration</td></tr><tr><td>B-27 Supplement w/o Vitamin A</td><td>Life Technologies</td><td>12587-010</td><td>1x final concentration</td></tr><tr><td>Recombinant Human FGF-basic</td><td>Life Technologies</td><td>PHG0266</td><td>10 ng/ml final concentration</td></tr><tr><td>Recombinant Human EGF</td><td>Life Technologies</td><td>PGH0315</td><td>10 ng/ml final concentration</td></tr><tr><td>L-Glutamine</td><td>Corning Cellgro Mediatech</td><td>25-005-CI</td><td>2 mM final concentration</td></tr><tr><td>Fungizone</td><td>Omega Scientific</td><td>FG-70</td><td>2.5 &amp;micro;g/ml final concentration</td></tr><tr><td>Penicillin Streptomycin</td><td>Omega Scientific</td><td>PS-20</td><td>100 U/ml Penicillin G, 100 &amp;micro;g/ml Streptomycin final concentration</td></tr><tr><td>Gentamicin</td><td>Life Technologies</td><td>15750-060</td><td>50 ng/ml final concentration</td></tr><tr><td>MTT</td><td>Life Technologies</td><td>M6494</td><td>prepared to 5 mg/ml in PBS and sterile filtered, 1 mg/ml in well</td></tr><tr><td>SDS</td><td>Fisher</td><td>BP166</td><td>for MTT lysis buffer, prepared to 10% in 0.01&amp;nbsp;M HCl, 5% in well</td></tr><tr><td>HCl</td><td>Fisher</td><td>A144SI-212</td><td>for MTT lysis buffer, prepared to 0.01&amp;nbsp;M with SDS, 5 mM in well</td></tr><tr><td>Calcein AM</td><td>Life Technologies</td><td>C1430</td><td>1 mM in DMSO stock, 2&amp;nbsp;&amp;micro;M in PBS staining solution, 1&amp;nbsp;&amp;micro;M in well</td></tr><tr><td>Halt&amp;rsquo;s Protein Phosphatase Inhibitor cocktail&amp;nbsp;</td><td>Pierce ThermoScientific</td><td>78420</td><td>1:100 ratio in MPER&amp;nbsp;</td></tr><tr><td>Halt&#039;s Protein Protease Inhibitor&amp;nbsp;</td><td>Pierce ThermoScientific</td><td>87786</td><td>1:100 ratio in MPER</td></tr><tr><td>Mammalian Protein Extraction Reagent (MPER)</td><td>Pierce ThermoScientific</td><td>PI78501</td><td></td></tr><tr><td>Trypan Blue</td><td>Pierce ThermoScientific</td><td>15250-061</td><td></td></tr><tr><td>DMSO</td><td>Fisher</td><td>BP231</td><td>for dissolution of calcein AM &amp;amp; compounds</td></tr><tr><td>Phosphate-Buffered Saline without Ca/Mg</td><td>Lonza</td><td>17-517Q</td><td>diluted to 1x with MiliQ ultrapure water and sterile filtered (for cell culture)</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline with Ca/Mg</td><td>Corning Cellgro Mediatech</td><td>20-030-CV</td><td>diluted to 1x with MiliQ ultrapure water (for pre-fixation wash)</td></tr><tr><td>10% Neutral Buffered Formalin</td><td>Protocol</td><td>032-060</td><td></td></tr><tr><td>Trypan Blue</td><td>Pierce ThermoScientific</td><td>15250-061</td><td></td></tr><tr><td>High Density Hubiogel</td><td>Vivo Biosciences</td><td>HDHG-5</td><td></td></tr><tr><td>Halt&#039;s Protein Phosphatase Inhibitor</td><td>Pierce</td><td>78420</td><td></td></tr><tr><td>Halt&#039;s Protein Protease Inhibitor</td><td>Pierce</td><td>87786</td><td></td></tr><tr><td>Mammalian Protein Extraction Reagent (MPER)</td><td>Thermo Scientific</td><td>78501</td><td></td></tr><tr><td>Protein Tyrosine Kinase (PTK) Array Profiling chip</td><td>PamGene</td><td>86312</td><td></td></tr><tr><td>PTK kinase buffer</td><td>PamGene</td><td>36000</td><td>300 &amp;micro;l 10x PK buffer stock in 2.7&amp;nbsp;ml dH&lt;sub&gt;2&lt;/sub&gt;O, catalog number for PTK reagent kit</td></tr><tr><td>ATP</td><td>PamGene</td><td>36000</td><td>catalog number for PTK reagent kit</td></tr><tr><td>PY20- FITC-conjugated antibody</td><td>PamGene</td><td>36000</td><td>catalog number for PTK reagent kit</td></tr><tr><td>PTK Additive</td><td>PamGene</td><td>32114</td><td></td></tr><tr><td>PTK-MM1 tube (10x BSA)</td><td>PamGene</td><td>36000</td><td>catalog number for PTK reagent kit</td></tr><tr><td>Serine/Threonine Kinase (STK) Array Profiling chip</td><td>PamGene</td><td>87102</td><td></td></tr><tr><td>STK kinase buffer</td><td>PamGene</td><td>32205</td><td>catalog number for STK reagent kit</td></tr><tr><td>STK Primary Antibody Mix (DMAB tube)</td><td>PamGene</td><td>32205</td><td>catalog number for STK reagent kit</td></tr><tr><td>FITC-conjugated Secondary Antibody</td><td>PamGene</td><td>32203</td><td></td></tr><tr><td>STK-MM1 tube (100x BSA)</td><td>PamGene</td><td>32205</td><td>catalog number for STK reagent kit</td></tr><tr><td>STK Antibody Buffer</td><td>PamGene</td><td>32205</td><td>catalog number for STK reagent kit</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>#11 Blades, sterile</td><td>Fisher</td><td>3120030</td><td></td></tr><tr><td>#3 scalpel handles, sterile</td><td>Fisher</td><td>08-913-5</td><td></td></tr><tr><td>100 mm glass Petri dishes</td><td>Fisher</td><td>08-748D</td><td></td></tr><tr><td>Semicurved forceps</td><td>Fisher</td><td>12-460-318</td><td></td></tr><tr><td>Trypsinizing flask</td><td>Fisher</td><td>10-042-12B</td><td></td></tr><tr><td>Magnetic stirrer</td><td>Fisher</td><td>14-490-200</td><td></td></tr><tr><td>3/4&quot; stir bar</td><td>Fisher</td><td>14-512-125</td><td></td></tr><tr><td>B-D cell strainer&amp;nbsp;</td><td>Fisher</td><td>#352340</td><td></td></tr><tr><td>B-D 50 ml Centrifuge tube</td><td>Fisher</td><td>#352098</td><td></td></tr><tr><td>PamStation 12</td><td>PamGene</td><td></td><td></td></tr><tr><td>BioNavigator 6.0 kinomic analysis software&amp;nbsp;</td><td>PamGene</td><td></td><td></td></tr><tr><td>Evolve Kinase Assay Software</td><td>PamGene</td><td></td><td></td></tr><tr><td>UpKin App software (upstream kinase prediction)</td><td>PamGene</td><td></td><td></td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>Rotary Cell Culture System (RCCS)</td><td>Synthecon</td><td>RCCS-D</td><td>with 10 ml disposable HARV</td></tr></tbody></table>

generation of microtumors using 3d human biogel culture system and patient-derived glioblastoma cells for kinomic profiling and drug response testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

我们已经表明，3D biogel培养系统支持多种细胞类型的长期生长和功能。在这个合作项目，来自患者的GBM xenolines（PDX）用于生产数百microtumors的。解离的细胞（3×10 5个 ），或神经球（40 - 50）包埋在biogel珠（2mm）和之后快速凝胶化它们在一个NB-介质填充定制的生物反应器中培养。细胞活力（钙黄绿素-AM），生长曲线（MTT），和kinomic基于阵列活性分析进行了测定。 PDX...

Watch this Scientific Journal Video about Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing at JoVE.

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

generation-microtumors-using-3d-human-biogel-culture-system-patient

Research

JoVE Journal

Medicine

9.0K 次观看.  University of Alabama at Birmingham. Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis. 

视频: Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

科研

癌症研究

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

在全融合微流体平台中增强患者衍生异种移植物的外体 3D 水凝胶培养物的生存能力

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

胶质母细胞瘤的3D球形模型

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

优化高等级胶质瘤细胞培养从外科标本用于临床相关的动物模型和3D免疫化学

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

医学

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular diversity, as well as complex interactions between tumor microenvironments, can make finding effective treatments a challenge. Traditional adherent or sphere culture methods can mask such complexities, whereas three-dimensional organoid culture can recapitulate regional microenvironmental gradients. Organoids are a method of three-dimensional GBM culture that better mimics patient tumor architecture, contains phenotypically diverse cell populations, and can be used for medium-throughput experiments. Although three-dimensional organoid culture is more laborious and time-consuming compared to traditional culture, it offers unique benefits and can serve to bridge the gap between current in vitro and in vivo systems. Organoids have established themselves as invaluable tools in the arsenal of cancer biologists to better understand tumor behavior and mechanisms of resistance, and their applications only continue to grow. Here, details are provided about methods for generating and maintaining GBM organoids. Instructions of how to perform organoid sample embedding and sectioning using both frozen and paraffin-embedding techniques, as well as recommendations for immunohistochemistry and immunofluorescence protocols on organoid sections, and measurement of total organoid cell viability, are all also described.

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Here, we describe a method of generating glioblastoma (GBM) organoids from primary patient specimens or patient-derived cell cultures and maintaining them to maturity. These GBM organoids contain phenotypically diverse cell populations and recreate tumor microenvironments ex vivo.

使用三维类器官培养物离 体 维持人胶质母细胞瘤细胞多样性

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

生成用于药物评估研究的 3D 肿瘤微球

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are&#160;modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,&#160;a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

<meta charset="utf8"></head><body>胶质瘤干细胞系中的球状体药物敏感性筛选

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

胶质瘤干细胞系中的球状体药物敏感性筛选

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

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 ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

水凝胶阵列可提高 3D 肿瘤模型中基质成分和治疗药物筛选效果的通量

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

摘要

探索更多视频

高通量三维肿瘤球体药物筛选的生成

在全融合微流体平台中增强患者衍生异种移植物的外体 3D 水凝胶培养物的生存能力

胶质母细胞瘤的3D球形模型

优化高等级胶质瘤细胞培养从外科标本用于临床相关的动物模型和3D免疫化学

使用三维类器官培养物离体维持人胶质母细胞瘤细胞多样性

生成用于药物评估研究的 3D 肿瘤微球

胶质瘤干细胞系中的球状体药物敏感性筛选

胶质瘤干细胞系中的球状体药物敏感性筛选

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

水凝胶阵列可提高 3D 肿瘤模型中基质成分和治疗药物筛选效果的通量

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

摘要

探索更多视频

高通量三维肿瘤球体药物筛选的生成

在全融合微流体平台中增强患者衍生异种移植物的外体 3D 水凝胶培养物的生存能力

胶质母细胞瘤的3D球形模型

优化高等级胶质瘤细胞培养从外科标本用于临床相关的动物模型和3D免疫化学

使用三维类器官培养物离 体 维持人胶质母细胞瘤细胞多样性

生成用于药物评估研究的 3D 肿瘤微球

胶质瘤干细胞系中的球状体药物敏感性筛选

胶质瘤干细胞系中的球状体药物敏感性筛选

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

水凝胶阵列可提高 3D 肿瘤模型中基质成分和治疗药物筛选效果的通量

使用三维类器官培养物离体维持人胶质母细胞瘤细胞多样性