，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。这个工具允许更快，更准确的生产microtumors的。此外，小的修改对维持microtumors的帮助加快变换媒体和给药的细胞的过程。几个这些修改包括，但不限于，用多电子移液器除去，并从96孔板并更换介质预混合新鲜药物给药方案，并在一个相应2毫升96孔板布置2个解决方案。 在3D建模的培养条件主要优化包括确定药物化合物合适的剂量，因为有2D建模和3D建模之间的差的关系。因此，连续稀释建立了剂量滴定曲线通常是必要为移动二维模型系统到3D microtumor模型。 考虑与从鼠标收获xenoline肿瘤细胞microtumor产生的潜在问题包括细菌或真菌污染，主要xenolines不一致的可用性，在小鼠的独特生长特性可以生产从不同的解离细胞产量在收获时间和方差的差异。用每个细胞系，获细胞的数量，同样，多少microtumors可以产生无线会有所不同。因此，有必要优先需要哪些测定法和哪些是"可选的"应microtumor收率不充分。与microtumors的kinomic分析潜在限制包括在校正惰性蛋白质材料装载的困难，如样品的方式装载以校正BCA测定蛋白水平，这可能不是基于biogel蛋白（ECM蛋白）和那些的区分被测定，旨在细胞激酶成分。通过钙黄绿素-AM毛microtumor质量测量，或使用一个内务蛋白质作为校正因子可以减轻此，虽然被改变可能混淆本肿瘤与持家蛋白水平的问题。对于短时程信号的实验，假设同样大小的（含活细胞数量激酶）和配对处理和未经处理的样品，这是不成问题的。 与此研究的目标是提供一种更具成本娥相较于原位患者来源的移植进行精确的药物治疗试验ffective和疾病临床前的代表机型，目前的黄金标准模型GBM测试。近年来，几组试图用于药物测试目的的体内 GBM环境建模。一些团体试图简单地将增长GBM肿瘤细胞的分化的非条件保留GBM干细胞或至少脑肿瘤启动细胞（BTIC）24,25。这些tumorsphere或神经球培养物可用于药物测试，并有可能优于传统的模型。然而，由于它们仍然缺乏的是在我们的模型microtumor保存肿瘤基质相互作用中，tumorsphere方法仍然是有限的。其他团体都试图运用工程学的原理，以提高GBM模型26-28。这些方法包括流动腔，变ECM蛋白与肿瘤细胞/正常细胞的混合物。同时承诺，几乎所有的这些回购RTS已经使用与前面提到的告诫永生化细胞系。因此，我们认为，在这里描述的基于PDX-microtumor模型更加临床相关的。 在将来，microtumor模型可以被用作一个真正的肿瘤化身或"先证者的系统。与先证系统，对PDX从特定患者开发不直接告知关于该特定病人的治疗的临床医生，与肿瘤化身系统。相反，一个病人的肿瘤是"匹配"，以一个预先存在的，充分表征（包括分子和表型）PDX 29。事实上，该模型可以作为一个"去到"化身或驻留在一个先证者库作为比较轮廓。与传统的替身，在小鼠中在平行于患者的治疗日益患者的肿瘤细胞中不仅是耗时的，因为它可能需要数个月的主通路，而且成本高昂，因为测试多个D在小鼠地毯疗法产生了压倒性的价格标签。为了解决这个问题，主要的患者肿瘤可以直接植入到biogel矩阵。与microtumors，可以迅速并以较低的成本产生的结果。 作为先证者模式，microtumor激酶组数据分布和药物反应可以从体外模型先前3D，现有PDXs，动物研究，其他患者等个人资料和数据一起加入到先证者&#39;库&#39;。在理论上，病人的肿瘤细胞可以接着是"omically&#39;（基因组，kinomically，transcriptomically 等 ）相 ​​匹配的一个类似的现有microtumor信息，以确定最佳的可能的结果准确的治疗。 总结:在这里，我们确定这些microtumor模型可以用于同时测量表型的生长，并且可以在这两个基础激酶活性水平，并且响应于药物治疗的可能有用的查询作为进一步的临床前研究的平移工具。人类肿瘤发展中准确，有效的分子模型测试的当务之急是有效的药物开发。

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 border="0" cellpadding="0" cellspacing="0" width="1142">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Collagenase-I&#160;</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">CO130</td>
			<td style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Trypsin EDTA (10X)</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:119px;">15400-054&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Neurobasal-A</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10888-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">N-2 Supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td>1x final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">B-27 Supplement w/o Vitamin A</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">12587-010</td>
			<td>1x final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Recombinant Human FGF-basic</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">PHG0266</td>
			<td>10 ng/mL final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Recombinant Human EGF</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">PGH0315</td>
			<td>10 ng/mL final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">L-Glutamine</td>
			<td style="width:184px;">Corning Cellgro Mediatech</td>
			<td style="width:119px;">25-005-CI</td>
			<td>2 mM final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Fungizone</td>
			<td style="width:184px;">Omega Scientific</td>
			<td style="width:119px;">FG-70</td>
			<td>2.5 ug/mL final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Penicillin Streptomycin</td>
			<td style="width:184px;">Omega Scientific</td>
			<td style="width:119px;">PS-20</td>
			<td>100 U/mL Penicillin G, 100 ug/mL Streptomycin final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Gentamicin</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">15750-060</td>
			<td>50 ng/mL final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">MTT</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">M6494</td>
			<td>prepared to 5 mg/mL in PBS and sterile filtered, 1 mg/mL in well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">SDS</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">BP166</td>
			<td>for MTT lysis buffer, prepared to 10% in 0.01M HCL, 5% in well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">HCl</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">A144SI-212</td>
			<td>for MTT lysis buffer, prepared to 0.01M with SDS, 5 mM in well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Calcein AM</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">C1430</td>
			<td>1 mM in DMSO stock, 2 uM in PBS staining solution, 1 uM in well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Halt&#8217;s Protein Phosphatase Inhibitor cocktail&#160;</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td style="width:119px;">78420</td>
			<td>1:100 ratio in MPER&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Halt&#39;s Protein Protease Inhibitor&#160;</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td style="width:119px;">87786</td>
			<td>1:100 ratio in MPER</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Mammalian Protein Extraction Reagent (MPER)</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td style="width:119px;">PI78501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Trypan Blue</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DMSO</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">BP231</td>
			<td>for dissolution of calcein AM &#38; compounds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Phosphate-Buffered Saline without Ca/Mg</td>
			<td style="width:184px;">Lonza</td>
			<td style="width:119px;">17-517Q</td>
			<td>diluted to 1X with MiliQ ultrapure water and sterile filtered (for cell culture)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Dulbecco&#39;s Phosphate-Buffered Saline with Ca/Mg</td>
			<td style="width:184px;">Corning Cellgro Mediatech</td>
			<td style="width:119px;">20-030-CV</td>
			<td>diluted to 1X with MiliQ ultrapure water (for pre-fixation wash)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">10% Neutral Buffered Formalin</td>
			<td style="width:184px;">Protocol</td>
			<td style="width:119px;">032-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Trypan Blue</td>
			<td style="width:184px;">Pierce ThermoScientific</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">High Density Hubiogel</td>
			<td style="width:184px;">Vivo Biosciences</td>
			<td style="width:119px;">HDHG-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Halt&#39;s Protein Phosphatase Inhibitor</td>
			<td style="width:184px;">Pierce</td>
			<td style="width:119px;">78420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Halt&#39;s Protein Protease Inhibitor</td>
			<td style="width:184px;">Pierce</td>
			<td style="width:119px;">87786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Mammalian Protein Extraction Reagent (MPER)</td>
			<td style="width:184px;">Thermo Scientific</td>
			<td style="width:119px;">78501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Protein Tyrosine Kinase (PTK) Array Profiling chip</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">86312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">PTK kinase buffer</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">36000</td>
			<td>300 &#181;l 10X PK buffer stock in 2.7 ml dH20, catalog number for PTK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">ATP</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">36000</td>
			<td>catalog number for PTK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PY20- FITC-conjugated antibody</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">36000</td>
			<td>catalog number for PTK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTK Additive</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTK-MM1 tube (10X BSA)</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">36000</td>
			<td>catalog number for PTK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serine/Threonine Kinase (STK) Array Profiling chip</td>
			<td style="width:184px;">PamGene</td>
			<td>87102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STK kinase buffer</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32205</td>
			<td>catalog number for STK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STK Primary Antibody Mix (DMAB tube)</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32205</td>
			<td>catalog number for STK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">FITC-conjugated Secondary Antibody</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">STK-MM1 tube (100X BSA)</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32205</td>
			<td>catalog number for STK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">STK Antibody Buffer</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;">32205</td>
			<td>catalog number for STK reagent kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Equipment</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">#11 Blades, sterile</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">3120030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">#3 scalpel handles, sterile</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">08-913-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">100mm glass Petri dishes</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">08-748D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Semicurved forceps</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">12-460-318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Trypsinizing flask</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">10-042-12B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Magnetic stirrer</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">14-490-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">3/4&#34; stir bar</td>
			<td style="width:184px;">Fisher</td>
			<td>14-512-125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">B-D cell strainer&#160;</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:119px;">#352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">B-D 50ml Centrifuge tube</td>
			<td style="width:184px;">Fisher</td>
			<td>#352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">PamStation 12</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BioNavigator 6.0 kinomic analysis software&#160;</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Evolve Kinase Assay Software</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">UpKin App software (upstream kinase prediction)</td>
			<td style="width:184px;">PamGene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">gentleMACS Dissociator</td>
			<td style="width:184px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Rotary Cell Culture System (RCCS)</td>
			<td style="width:184px;">Synthecon</td>
			<td style="width:119px;">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 microtumors维持&gt;3周多的组织和高活力（&gt; 80％）。我们认为，这在体内样生物学是由于维护三维细胞矩阵架构（不可能的2D或球体文化）。它也被观察到，3D肿瘤难以生产具有脆弱的凝胶状蛋白质骨架，这可能是由于缺少Ⅰ型胶原蛋白14。的话筒的工作方案rotumor使用PDX细胞和三维biogel系统生产总结于图1和离解过程是在图2中描述。 经由钙黄绿素-AM活细胞成像，我们确定细胞生长和生存力以及来自PDX肿瘤JX10导出的GBM细胞的药物效果，通过在荧光的降低所指示的，信令细胞死亡。肿瘤的生长，在0天，第7和14，用于显示连续增长microtumors测定， 如图3A所示 。此microtumor，JX10，已知是向TMZ抗性。当暴露于1 - 10μMTMZ，最小生长抑制指出（ 图3B）。然而，测试PDX肿瘤JX12，这是众所周知的是TMZ敏感，表明通过MTT测定灵敏度14天，是由钙荧光素-AM成像（ 图 4）确认。 er.within页="1"&gt;为了探索耐药性的潜在机制，我们在TMZ耐microtumors（JX10）测量激酶信号。 144酪氨酸和丝氨酸144型材Kinomic /苏氨酸磷酸化的目标被抓获的DMSO和10μMTMZ治疗microtumors。对于所有的肽目标Kinomic磷酸强度，每个周期和每个多重曝光时间被捕获（数据未示出）。这显示了显著改变与10μMTMZ治疗强度肽代表热图（P &lt;0.05，非配对学生T检验）显示在图5A。在TMZ被改变该Kinomic型材处理使用鉴定SYK，LCK，并且CTK激酶如TMZ增加上游激酶预测软件处理过相对于DMSO中（ 图5B）样品JX10 microtumors进行分析。还进行（ 图5C）丝氨酸/苏氨酸激酶组上游激酶分析。 内容"FO:保together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/54026/54026fig1.jpg" /> 图1:工作方案Microtumor生产 GBM-PDX肿瘤从鼠宿主分离，并单细胞被包埋在biogel基质以形成microtumors。分析，然后在microtumors执行。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54026/54026fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54026/54026fig2.jpg" /> 图2:PDX肿瘤细胞的解离肿瘤从宿主小鼠取出之前用酶溶液（AG）的混合切碎。离解后40分钟（H）所示。肿瘤细胞离解器中示出（Ⅰ）。在媒体和过滤（JN）研制显示与最终地区环境部门一道含有29.5×10 6活细胞（P）的形式在球体在NBM 24小时（Q）0.3 ml颗粒（O）的LT。比例尺为20微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54026/54026fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54026/54026fig3.jpg" /> 图 3:通过MTT和代表性图像的光密度（OD）超过14天测定Microtumors JX10 microtumor 生长生长 ，无论基部（A）中 ，并且响应于1-10μMTMZ处理（B）。图像以放大4倍（比例尺= 500微米）所示。表示标准偏差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54026/54026fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p&gt; <img alt="图4" src="/files/ftp_upload/54026/54026fig4.jpg" /> 图 4:通过MTT和代表性图像的外径超过14天测定Microtumors JX12 microtumor 生长生长 ，无论基部（第1天），并且响应于0，1或10μM的TMZ治疗（比例尺= 500微米）。表示标准偏差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54026/54026fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54026/54026fig5.jpg" /> 图5:TMZ的Kinomic概况治疗Microtumors热图曝光积分值的（LOG2转化斜率（10，20，50，100，和200毫秒中值信号减去背景曝光值×100））被显示为显著TMZ改变的。改变的肽（p &lt;0.05）。红色表示增加，蓝色表示相对于DMSO％下降肽的意思。 TMZ改变型材分别使用上游激酶预测软件和改变的激酶进行比较。标准化激酶统计（NKS）评分&gt; 1.0和特异性得分&gt; 1.3（红色）被认为是高度发生变化。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54026/54026fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

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

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

慢分裂的细胞在人脑胶质瘤羧基荧光素琥珀酰亚胺酯（CFSE）的分离与鉴定

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

科研

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

前列腺癌患者产生派生异种移植模型从循环肿瘤细胞

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

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.

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 ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

利用人体肝干细胞人源化小鼠肝脏生成

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

药品再利用产生假设使用"RE:精药品"系统

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...