作者非常感谢来自麦肯纳克莱尔基金会，神经疾病与中风研究所（NINDS K08NS070926和R01NS092597）研究所的支持，国防部（NF140075），加州再生医学研究所（CIRM RB4-06093和RN3-06510），Alex的柠檬水站在基金会，治愈开始现在，基金会和DIPG协作，莱拉Nsouli基金会，解开儿科癌症，儿童脑肿瘤基金会，马修·拉尔森基金会，V基金会，戈弗雷家庭基金菲奥娜佩内洛普的韦兰维拉尔DIPG基金会，迪伦朱厄特的记忆康纳·约翰逊，Zoey的Ganesh神，迪伦·弗里克，阿比盖尔延森和Jennifer克兰兹纪念基金，N8基金会，弗吉尼亚州和癌症研究DK路德维希基金会，儿童健康研究所在斯坦福安妮T.和罗伯特·巴斯明德学院在儿科奖学金癌症和血液病。

该协议一直与我们同所有的病人数据的适当去鉴定机构审查委员会，并按照为人类福利的所有机构，国家和国际准则进行审批。获取与该协议的工作之前，所有适当的机构审查委员会的批准和知情同意从病人家属。 1.获得组织样本注:本协议的一个重要组成部分，要求组织捐赠在尸检时立即启动妥善处理。总体样本生存能力上尽量减少验尸间隔（PMI），立即组织转移到添加抗生素/抗真菌药合适的冷媒，在整个运输保持样品在湿冰，并在整个协议保持无菌技术显著依赖。 <ol><li>一旦通知即将捐赠，准备样品采集套件。使用装运箱与能够直接用于组织样品的返回运输隔热容器，包括下列物质: <ol><li>包括无菌制剂材料，包括无菌手套，窗帘和手术刀。 </li><li>包括8 - 包含在冰或冰袋冷敷隔热容器30毫升航运媒体10无菌50毫升锥形管。 注:虽然任何标准的细胞培养基能正常工作，与辅以抗生素/抗霉菌休眠-A获得了最好的样品生存能力。 </li><li>包括其他分析（如固定剂），任何进一步的样品管。 </li><li>如果尸检不会在调查的现场进行，包括文件，明确规定如何收集肿瘤样品。这包括详细信息，如从脑和延髓如涂保持无菌，保持在湿冰上样品管，并且包括样本铁道部细胞常侵入这些地区。 </li></ol></li><li>在验尸过程中保持无菌。考虑脑颅骨内消毒，这样遵守无菌技术（包括更换手套）一旦头盖骨是开放的。避免污染对皮肤和头发是至关重要的。 </li><li>从解剖腹侧肿瘤（脑桥的"肚子"，见参考图1）。用无菌手术刀，切开从肿瘤小1厘米块，并立即转移到冷的航运媒体（参见1.1.2步注），并把湿冰。放置10毫升组织到每个管中，导致组织和介质中的40毫升每管的终体积。 注:在DIPG的情况下，肿瘤浸润弥漫桥脑，经常脑干的其余部分。因此，收集尽可能多的样品尽可能的，通常包括3 - 桥 - （40毫升组织30）和1 - 4管2管（10 - 20毫升组织）分别从脑和髓质。 </li><li>收集SAM普莱斯从脑和延髓并列脑桥，常含有许多肿瘤细胞。 </li><li>样品采集后，船在湿冰样品O / N在隔热容器。不出货干冰样本，因为冷冻会杀死细胞。 </li></ol> 2.准备来样加工<ol><li>之前开始样品制备，消毒组织培养罩与刀片，弯曲的止血，以及任何其他非无菌工具沿着在UV光下进行1小时。执行所有在无菌条件下执行以下步骤，以防止培养物的污染。 </li><li>准备和无菌过滤器解决方案。 <ol><li>以制备1.8M的蔗糖溶液中，溶解于蒸馏水300mL的308.07克蔗糖。加入50毫升10×Hank&#39;s缓冲盐溶液（HBSS）中不含钙或镁和使总体积用蒸馏水500毫升。保存在4℃。 </li><li>为了准备酶消化解决方案，取50毫升HBSS 钙和镁，每10毫升组织糜，并添加500微升5毫克/毫升脱氧核糖核酸酶I（终浓度50微克/毫升），500微升2.5毫克/毫升胶原酶I / II和分散酶溶液（终浓度25微克/毫升），和500微升的1M HEPES缓冲液。在37℃水浴Prewarm消化解决方案。 </li><li>为了制备肿瘤干介质（TSM）的基础上，混合250毫升的Neurobasal-A中，250毫升Dulbecco氏改良的Eagle培养基:营养混合物F12（DMEM / F12），5毫升100×抗生素 - 抗真菌剂，5毫升200mM的L-丙氨酰基-L-谷氨酰胺二肽（GLUTAMAX-A），5毫升HEPES缓冲液，5毫升的100mM丙酮酸钠，和5mL 100×MEM非必需氨基酸。 </li><li>与生长因子准备完整的TSM，添加以下补充TSM基地:50X B27负补充维生素A（1:50），人表皮生长因子（H-EGF，20毫微克/毫升），人成纤维细胞生长因子（H- FGF，20毫微克/毫升），人血小板衍生生长因子AA（H-PDGF-AA，10毫微克/毫升），人血小板衍生生长因子BB（H-PDGF-BB，10毫微克/毫升），和肝素溶液（2微克/毫升）。 </li></ol></li><li>预冷配备有吊桶式转子4℃的实验室离心机。 </li></ol> 3.机械分离<ol><li>转移组织成高壁100毫米×20mm的细胞培养皿。航运取出介质下脚用10代替 - 15毫升冷培养基。 </li><li>使用了弯曲止血把握刀片，细剁组织，同时消除明显的血管或脑膜。 注:最终组织片段应小于1mm（ 见图1B）。 </li><li>转移组织到一个干净的50毫升锥形管中。用另外5毫升冷培养基洗涤细胞培养皿并转移到锥形管中。需要时，重复此清洗步骤转移剩余组织。 </li><li>使用10毫升血清吸管，磨碎轻轻（4 - 5次）。允许更大的做卷烟UE片段沉降到管底部。如果需要的话，短暂离心样品1分钟（350 XG，4℃）。 </li><li>收集机械分离的部分。 <ol><li>除去上清液，并通过100微米过滤器的过滤标成一个新的50 mL锥形管"机械分离"。颠倒了原锥形管过滤器和培养基洗恢复组织碎片。 </li><li>离心"机械分离"分数为5分钟（350 XG，4℃）。 </li><li>从沉淀的"机械分离"部分去除上清，重悬在寒冷的文化传媒组织。如果有超过5毫升的"机械解离"锥形管组织，分裂组织成其他50毫升锥形管中，使得没有管具有超过5毫升的组织。 </li></ol></li><li>储存在冰上机械离解分数直至蔗糖梯度离心步骤（步骤第5页）。可替代地，机械解离的部分可继续在酶解潜伏期（步骤4.4）步骤5。 </li></ol> 4.酶解<ol><li>如果有超过5毫升的组织中含有剩余较大的组织碎片在管，分裂组织成其他新50mL的锥形管中，使得没有管具有超过5毫升的组织。 </li><li>离心含有剩余组织碎片5分钟（350 XG，4℃）的锥形管（多个）。 </li><li>除去上清液，并添加预热的酶消化溶液，使得存在5为每1毫升组织毫升消化溶液（ 例如，25 5毫升组织毫升消化溶液）。 </li><li>密封该锥形管盖与实验室膜孵育旋转器上，反应在37℃下30分钟。 </li><li>培养后，磨碎的样品轻轻地（参见图1C）。 <ol><li>用10毫升血清吸管，吸管样品上下6 - 8倍。避免产生过气泡。 </li><li>一个1000微升吸管尖添加到吸管的端部和磨碎额外的6 - 8倍。 </li><li>允许任何剩余块沉降到管底部。删除和过滤通过一个100微米的过滤器仍然悬浮在标有"酶解"一个新的50 mL锥形管的细胞上清并存储在冰上。 </li><li>如果显著组织碎片依然存在，增加一个额外的10毫升HBSS钙和镁的碎片和重复步骤3.5.1和3.5.2。过滤通过100微米的过滤器进入"酶解"管这个最终的解决方案。 </li></ol></li><li>离心"酶解"管5分钟（350 XG，4℃）并继续蔗糖梯度离心。 </li></ol> 5.蔗糖密度梯度离心<ol><li>如果样品仍然悬浮在溶液，centrifuGE 5分钟（350 XG，4℃）。 </li><li>除去在20毫升冷HBSS的上清，重悬组织没有钙和镁。把音量高达25毫升总用冷HBSS。 </li><li>慢慢加入25毫升1.8M的蔗糖溶液，并翻转试管混合。这导致了0.9M的蔗糖梯度。 </li><li> 没有制动器 10分钟（800 XG，4℃）离心。使用离心制动会扰乱梯度和降低产率（ 见图1D为一个样品的一例前和离心后）。 </li><li>小心吸髓鞘碎片，并尽可能多的蔗糖溶液越好。通过加入30毫升冷HBSS无钙和镁，并轻轻混合洗样品。离心5分钟（350 XG，4℃）。 </li></ol> 6. ACK红细胞溶解<ol><li>取出洗净上清。加入5毫升的ACK裂解缓冲液，轻轻重悬细胞沉淀，漩管在室温1分钟。 </li><li>淬火第l通过加入30毫升冷HBSS无钙和镁ysis。 </li><li>离心5分钟（350 XG，4℃）。 </li></ol> 7.首次保养文化<ol><li>重悬最终的细胞沉淀在10 - 15毫升温暖完整的TSM用生长因子和使用台盼蓝排除血球量化活细胞密度。 注:活细胞的变化范围很大，取决于组织捐赠的情况下，定量可能很难在早期阶段，由于剩余的细胞碎片。在理想的情况下，目标是具有每采样1万居住单元。当过多的碎片依然存在，在板材在T175烧瓶密度低的情况下。 </li><li>转移的最终细胞悬浮液到一个新T75培养瓶。 Spike在另外的生长因子，隔日保持整体生长因子水平，并监测肿瘤细胞的神经球的发展（参见图1E和图2）。 </li><li>可替代地，过程酶促解离的细胞用于进一步的分析，包括流式细胞仪和荧光激活细胞分选。 </li><li>神经球的发展（其平均需要3 - 4周，但是从几天范围为只要2个月）后，使用100微米的尼龙网过滤器，以减少碎片的量反向滤波器的样本。 注意:滤液应在培养物中维持的情况下存活的单细胞或小球都存在。 </li><li>通过进行DNA指纹而传代，在比较初始患者样品确保培养物的完整性和纯度。 </li></ol>

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作者什么都没有透露。

这个协议描述了验尸肿瘤组织的捐赠的快速处理，开发患者来源的细胞培养物，其可以在多种体外 或体内实验中使用的方法。因为尸检样品工作的性质，保持样品活力构成了显著挑战。几个促成因素，包括尸体现象尸体解剖或组织的运输所需的时间，应尽量减少尽可能但往往是难以控制。根据我们的经验，不到6死亡时间 - 8小时（从死亡到该组织被放置在冰冷的海运和运输媒体上的时间的时间）已经产生了建立一个持久的细胞培养的最佳机会。这是最好的，然后得到在实验室中组织24 h内，并立即对其进行处理文化在收到。 由于这些罕见的情况下的位置变化很大，和组织检索步骤的质量强烈影响成功的机会，进行尸检的物流可以是具有挑战性。在许多情况下，为了实现6-8小时的时间框架，它是不可行的在学术中心来进行组织收获。相反，有随叫随到组织恢复服务，以IRB批准协议下的殡仪馆恢复肿瘤组织往往是组织恢复更有利。提前死亡获得知情同意建议并促进后勤规划。强烈建议准备与组织检索（手套，窗帘，手术刀）清晰透彻的说明和消毒用品自足尸检套件。此外，建立明确的沟通点之间的研究者，病理学家，和殡仪馆是至关重要的。例如，研究者应当事先尸检讨论与病理学家的协议和突出常见错误。这些错误包括微组织检索，通过故障隔夜/加快航运和失败船舶上有足够的湿冰样品中绝热容器装运过程中污染BIAL（通常是酵母）。最后，我们建议使用门对门快递服务的样品出货，以减少运输时间。 护理还应组织离解期间采取保持细胞活力。例如，设计了利用媒体保护神经组织卫生运输（休眠-A辅以抗生素/抗真菌剂）将增加产生一个成功的文化的机会。同样重要的是通过始终转印在冰上的样品，以保持样品在低温下在整个协议。收集机械和酶解分数最小化研磨可以提高协议的成功，因为这两个步骤是创伤性细胞一起。根据我们的经验，而最成功的文化生长出两种组分，我们有过，其中只有两种制剂之一，建立一个持久的文化，可能反映了这些样品的微妙性实例。在协议中的另一个创伤性的步骤是研磨;一种策略，可以减少必要的研磨的程度，以确保样品在制剂的一开始彻底剁碎。旨在提高样品生存能力的协议的各方面的其它实例包括在整个协议，它是酶解过程中尤其关键加入缓冲剂（ 例如，HEPES）。 此协议，以进一步增加细胞活力的一种可能的修改是取消所述ACK红血细胞裂解步骤的。然而，在这种情况下，常常是然后必需在培养过程中的初始周来执行ACK裂解步骤以除去红血细胞。该协议可以被修改的另一个方面是髓鞘和细胞碎片的使用蔗糖DENSIT消除ÿ梯度。具体地讲，已报道以提高小胶质细胞的细胞生存力的其他策略也是可能在此步骤中，如不连续密度梯度18或磁性髓鞘去除珠。 最后，初始组织解离和神经球的外观之间的持续时间的样品之间变化，并且可以从一个星期的任何地方采取一个月以上。由于初始培养物通常含有死细胞和细胞碎片的一个显著程度，它可以是具有挑战性的，以确定存活细胞是否保持;培养应当维持至少4 - 8周（ 图2）。从剩余的碎片这里介绍的隔离生长的细胞的一种策略（ 即扭转新鲜培养基筛选细胞团和再电镀）。关于是否继续保持文化的决定是最终根据周围届因素逐案判决Ë尸检（ 例如，延长死亡时间，组织运输过程中的问题），组织分解（ 例如，过多的血液或碎片），以及如何将样品出现在文化。一旦培养被建立，身份应通过DNA指纹使用短串联重复分析或类似方法验证，将培养比较原始肿瘤保留用于此目的的一个示例。我们建议您定期验证通过的文化DNA指纹，以确保其他文化没有污染的发生，例如每3 - 6个月。 这些患者来源DIPG文化的产生代表了有效的治疗成功发展的重要一步。可用病人衍生DIPG文化和异种移植模型的扩展将是确定最有效的治疗策略，并最终克服这种破坏性疾病的关键。

DIPG是一个积极的中枢神经系统恶性肿瘤中出现的腹侧脑桥，通常在中期童年1,2。尽管几十年来的临床试验，目前的治疗是有限的，以放射疗法，这提供了临时提高或症状稳定和仅由平均3个月延伸位生存。甚至与放射疗法，中位总体生存仅9个月的儿童在2年内初步诊断死于该病的90％。由于肿瘤和脑干的关键功能的浸润性，手术切除是不可能的。此外，在美国，获得外科活组织检查为DIPG历来没有进行3中 ，作为组织病理学分析在指导临床治疗和诊断通常可以通过单独神经影像进行无电流的作用。因此，肿瘤组织f的可用性或学习受到限制，限制的努力进行候选药物分子和底层的肿瘤生物学研究。值得注意的是，在神经影像学和立体定向技术的改进，近年来4，其中，当在分子生物学的进步相结合，已经改变了我们的疾病的认识已经允许DIPG的安全活组织检查的发展。目前，使用前期活检及DIPG分子剖析了个体化的治疗计划多项临床试验正在进行（NCT01182350，NCT02274987）5。 DIPG和其他儿童高级别胶质瘤代表不同的疾病，从他们的成人胶质母细胞瘤的同行6， 第7和DIPG的忠实因此实验模型是必要了解其独特的病理生理机制，并探索有效的治疗策略。近年来，集中精力，努力获得DIPG肿瘤Ť问题在尸检时研究或较少见，活检已经彻底改变了我们的理解和学习能力DIPG。全基因组测序工作揭示了在H3组蛋白，家族3A（H3F3A）和组蛋白簇1，H3B（HIST1H3B），较引起的组蛋白的突变编码蛋白质8，9，10，11人类疾病的第一例的复发性突变。此外，DIPGs的一个子集表示在ACVR1基因突变，其以前没有报道癌症，但是相同于先天童年发育障碍进行性骨化性肌炎8，9，10，11中发现的突变。同时，第一个病人衍生DIPG细胞培养和异种移植原位莫现在德尔斯已经建立1，2，12，13，14，以及通过肿瘤细胞的直接异种移植建立到免疫缺陷小鼠的小鼠模型以生成串行异种移植物3，14，15。使用这些患者来源模型初始药物筛选努力已经确定有前途的新药剂用于临床翻译4,14和奠定了基础的至少一个临床试验（NCT02717455）。 这些走向进步的第一步仅仅是个开始，无数来自患者的组织样本和文化车型将有必要界定疾病亚型和发展最有效的治疗策略。基因引擎DIPG的ERED小鼠模型也将促进旨在促进我们对疾病的基本认识研究。制成以产生用于DIPG遗传模型的努力将通过上述讨论的基础基因组学研究来辅助，但目前的选项的数量有限。一个这样的模型，它产生组织学上相似高档脑干胶质瘤，采用病毒RCAS /电视系统来驱动PDGF-B表达和INK4A-ARF损失在巢蛋白阳性细胞在新生小鼠5,16的后颅窝。另一种方法涉及扼要在从人胚胎干细胞，这是足以使这些细胞的致瘤6，7，17衍生的神经前体细胞的几个主要DIPG相关的突变（组蛋白H3.3 K27M突变，p53的损失和PDGFRA活化）。遗传方法和患者DERIV组合ED车型将使临床前试验，进一步有效的治疗方法的发展。 为了解决上文详述DIPG研究的挑战，这种快速尸检协议被开发，以产生用于调查8，9，10，11，12，14患者来源的细胞培养物。该协议旨在保护的组织的生存能力和最大化，尽管具有扩展宰后间隔和运输时间相关的挑战生成耐久培养的可能性。当成功生成，这些DIPG培养物可在后续体外操作和体内异种移植物实验中使用，允许在源自患者的细胞的潜在治疗分子的评价。

<table><tbody><tr><td>Hibernate-A</td><td>Gibco</td><td>A12475-01</td><td></td></tr><tr><td>HBSS with Calcium/Magnesium</td><td>Gibco</td><td>24020-117</td><td></td></tr><tr><td>HBSS</td><td>Corning</td><td>21-022-CV</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Liberase DH (Collagenase/Dispase)</td><td>Roche</td><td>5401054001</td><td></td></tr><tr><td>DNAse I</td><td>Worthington Biochemical</td><td>LS002007</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>Antibiotic/Antimycotic</td><td>Gibco</td><td>15240-096</td><td></td></tr><tr><td>Neurobasal-A</td><td>Gibco</td><td>10888-022</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11330-032</td><td></td></tr><tr><td>GlutaMAX-I (100x)</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>Gibco</td><td>11360-070</td><td></td></tr><tr><td>B27 Supplement Minus Vitamin A</td><td>Gibco</td><td>12587-010</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids (100x)</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Human PDGF-AA</td><td>Shenandoah Biotechnology</td><td>100-16</td><td></td></tr><tr><td>Human PDGF-BB</td><td>Shenandoah Biotechnology</td><td>100-18</td><td></td></tr><tr><td>Human EGF</td><td>Shenandoah Biotechnology</td><td>100-26</td><td></td></tr><tr><td>Human FGF</td><td>Shenandoah Biotechnology</td><td>100-146</td><td></td></tr><tr><td>Sterile scalpel blades</td><td>Bard Paker</td><td>372610</td><td></td></tr><tr><td>Curved hemostats</td><td>Fine Science Tools</td><td>13013-14</td><td></td></tr><tr><td>Razor blades</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>100 x 20 mm cell culture dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Sterile forceps</td><td>TWD Tradewinds</td><td>DF8988-5</td><td></td></tr><tr><td>Sterile drapes</td><td>3M</td><td>2037</td><td></td></tr><tr><td>Sterile gloves</td><td>Kimberly Clark</td><td>1182307</td><td></td></tr><tr><td>ACK Lysis Buffer</td><td>Gibco</td><td>A1049201</td><td></td></tr><tr><td>100 micron mesh filter</td><td>Fisher</td><td>352360</td><td></td></tr><tr><td>50 mL conical tube</td><td>Fisher</td><td>1495949A</td><td></td></tr></tbody></table>

a protocol for rapid post-mortem cell culture of diffuse intrinsic pontine glioma (dipg)

漫内在脑桥脑胶质瘤（DIPG）是青梅竹马的脑干肿瘤携带普遍致命的预后。因为手术切除是不是一个可行的治疗策略，而不是常规进行活检，患者样品进行研究的可用性是有限的。因此，努力学习这种疾病已经被忠实的疾病模型的缺乏挑战。为了满足这一需要，我们在这里描述，以便产生可在体外测定法或体内原位异种移植实验中使用耐用患者来源的细胞培养模型验尸解剖组织样品的快速处理的协议。这些模型可用于筛选潜在药物靶标和DIPG内研究基本病理生物学过程。这个协议可以进一步扩展到分析和使用荧光激活细胞分选（FACS），使基因前的后续分析分离的肿瘤和微环境的细胞PRESSION，蛋白质表达，或者在散装细胞或单细胞水平的DNA的表观遗传修饰。最后，该协议也可以适合于产生用于其他中枢神经系统肿瘤患者来源的培养物。

描述的协议可以概括与图1在处理的不同阶段的组织的图像的五个步骤的工作流程。首先通过快速的无菌尸体解剖获得的样本。期间机械解离，而从样品中除去血管和脑膜组织被切碎，并通过100微米的尼龙网过滤器过​​滤。剩余的组织碎片在一个变暖的烤箱酶分解。接着，碎屑通过蔗糖梯度离心减少，分离从样品髓鞘的一个独立的层。此外，ACK裂解明显减少样品中存在的红细胞的量。最后，将细胞接种在补充有生长因子的无血清培养基。 补充图1是在尸检肿瘤的例子。肿瘤生长作为脑桥一个的弥漫性浸润D中的脑干的邻接区域。在样品制备，小约1cm×1cm的块应切断并立即放入冷水航运媒体冰上。 图2示出了如何可将样本从培养的早期阶段出现的持久培养各种实例。最初镀和早期培养物（A，B）通常不出现于包含许多幸存的细胞。此外，早期的细胞簇（C，D）往往不表现出的对比度通常与神经球相关的程度。值得注意的是，时间在培养细胞团的出现之前的持续时间可能要花几周到几个月。然而，这些细胞簇的初始通路，与细胞簇的反向滤波相结合，可以隔离能够形成球体（F）的健康的肿瘤细胞。滤液（E）通常包含剩余的杂物;然而，我们建议电镀和维持滤液和监测<html>细胞团的发展。这些患者来源的样品然后可以在培养多通道（G，H）被维持。 图3显示这些细胞是异种移植到小鼠中的能力。在每种这些情况下，DIPG细胞用GFP荧光素酶报告染以监测肿瘤的通过生物发光（A）中的发展。肿瘤也可以被组织学检查，例如，观察肿瘤植入（B）或特异性标记物（C）的表达。这些体内模型中 ， 在体外测定法组合可以用来评估各种药物或基因操作的影响（ 例如，8，9，10，11，14）。 LES / ftp_upload / 55360 / 55360fig1.jpg"/&gt; 图1.组织样本在不同加工阶段。 （A）获得通过无菌尸检程序，并在湿冰通宵航运的样本。 （B）从左至右依次为:（1行）含运费媒体肿瘤组织管;新鲜组织100毫米×20mm的细胞培养皿;组织初步切碎; （行2）部分切碎的组织;去除脑膜和血管;组织糜片最终大小。 （ 三 ）从左至右依次为:（1行）组织在37℃烘箱旋转工作台培养; （第2行），通过连接到一个10毫升移液管1000微升吸管尖组织研磨;通过100微米的尼龙网过滤器分离的组织的过滤。 （ 四 ）从左至右:离解组织悬浮在离心之前0.9M的蔗糖溶液;离解的组织离心后跨蔗糖梯度分离;可见层Ø在蔗糖梯度的顶部˚F髓鞘碎片; ACK裂解和洗涤后的最终的细胞沉淀。 （E）的最后的样品可以被放置到培养或在其他下游分析使用。 <a href="http://ecsource.jove.com/files/ftp_upload/55360/55360fig1large.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/55360/55360fig2.jpg" /> 图2.原代培养及早期传代细胞图像。 （A - B）文化解离后立即接种（SU-DIPG-XXX，A），或者说，还没有长大神经球，但（SU-DIPG-XXIX，B）。 （C - D）早在SU-DIPG-XXVIII（C）和SU-DIPG-XXIX（D）的原代培养神经球的出现。 （EF）从D-使用100微米的尼龙过滤器的主培养的第一通道，与网络连接ltrate（E）和反向滤液（F）电镀。 （G -高 ）从SU-DIPG -二十七（G）和SU-DIPG-XXV（H）的第三通道的第一通道熟女神经球文化发展。比例尺= 200微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55360/55360fig2large.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/55360/55360fig3.jpg" /> 图3.来自患者的细胞培养可以嫁接，并形成肿瘤的小鼠。与GFP的荧光素酶报告转染四种不同的患者来源的细胞培养物（A）生物发光图像。 （B）中从小鼠异种移植矢状截面，示出了在小鼠脑桥移入的肿瘤细胞。绿色:GFP，红色:髓鞘碱性蛋白。比例尺= 1毫米。 （C）为例immunofluores萤光图像表示小鼠脑移植的绿色荧光蛋白的肿瘤细胞。蓝色:DAPI，绿色:GFP，红色:IGF2R。比例尺= 40微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55360/55360fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 参考图1.脑桥肿瘤尸检样品 。弥漫性脑桥内在胶质瘤立即验尸的外观。肿瘤出现脑桥的腹侧表面上的大，富髓鞘质。 <a href="http://ecsource.jove.com/files/ftp_upload/55360/SupplementalFigure1.pdf" target="_blank">请点击这里下载此图像。</a>

Watch this Scientific Journal Video about A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG at JoVE.com

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

本协议描述为事后漫内在脑桥脑胶质瘤样品进行建立以病人来源的细胞培养模型或肿瘤微环境和细胞的直接表征的快速处理的方法。

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

a-protocol-for-rapid-post-mortem-cell-culture-diffuse-intrinsic

Universidad de Cartagena UDEC

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JoVE Journal

Cancer Research

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

16.8K 次观看.  Stanford University School of Medicine. 本协议描述为事后漫内在脑桥脑胶质瘤样品进行建立以病人来源的细胞培养模型或肿瘤微环境和细胞的直接表征的快速处理的方法。 

视频: A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

胶质瘤移植中体内成像的荧光分子层析成像研究

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

科研

癌症研究

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 ...

医学

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

多端根结核细胞迁移的实时监测

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

原正畸胶质血异草回顾渗透生长和同源脱氢酶 I 突变

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary ...

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 ...

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed electric fields (PEFs) are promising for the improvement of treatment efficiency. However, they rely on rigid electrodes that cause acute and chronic damage, especially in soft tissues such as the brain. In this work, flexible electronics were used to deliver PEFs to tumors and the biological response was evaluated with fluorescent microscopy. Interdigitated gold electrodes on a thin, transparent parylene-C substrate were coated with the conducting polymer PEDOT:PSS, resulting in a conformable and biocompatible device. The effects of PEFs on tumors and their microenvironment were examined using various biological models. First, monolayers of glioblastoma cells were cultured on top of the electrodes to investigate phenomena in vitro. As an intermediate step, an in ovo model was developed where engineered tumor spheroids were grafted in the embryonic membrane of a quail. Due to the absence of an immune system, this led to highly vascularized tumors. At this early stage of development, embryos have no immune system, and tumors are not recognized as foreign bodies. Thus, they can develop fast while developing their own vessels from the existing embryo vascular system, which represents a valuable 3D cancer model. Finally, flexible electrode delivery of PEFs was evaluated in a complete organism with a functional immune system, using a syngenic, orthograft (intracranial) mouse model. Tumor spheroids were grafted into the brain of transgenic multi-fluorescent mice prior to the implantation of flexible organic electrode devices. A sealed cranial window enabled multiphoton imaging of the tumor and its microenvironment during treatment with PEFs over a period of several weeks.

This work describes the development of flexible interdigitated electrodes for implementation in 3D brain tumor models, namely, in vitro culture, in ovo model, and in vivo murine model. The proposed method can be used to evaluate the effects of pulsed electric fields on tumors at different levels of complexity.

用于胶质母细胞瘤脉冲电场治疗的柔性有机电子器件

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

免疫与感染

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

动物的基因分型快速其次是建立大脑神经元原代培养

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

神经科学

在小鼠中使用 2P-IVM 实时跟踪脑肿瘤中的纳米颗粒

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

作者聚焦：优化胶质母细胞瘤治疗中药物递送和早期检测的多模式成像策略

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

小鼠模型中胶质母细胞瘤的双光子活体显微镜

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...