Name: intranasal delivery of therapeutic stem cells to glioblastoma in a mouse model
Uploaded: 2017-06-04T15:00:00.000+00:00
Duration: 9 min 57 s
Description: Watch this Scientific Journal Video about Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model at JoVE.com

这项工作得到NIH R01NS087990（MSL，IVB）的支持。

所有动物手术必须经机构动物护理和使用委员会（IACUC）批准或等同。如果本文所述的具体步骤有任何不确定性，请勿继续。向机构的IACUC和指定的兽医工作人员澄清。 1.确保培养细胞的无菌性，并遵循无菌技术原理<ol><li>在给予小鼠9,10之前，遵循所有细胞培养程序和IACUC细胞测试和无病原体状态确认的标准良好实验室实践。 </li><li>遵循NIH要求进行细胞鉴定，以确保可重复的结果11 。 </li><li>遵循本手稿中概述的已建立的小动物手术无菌技术4 。 </li></ol> 2.制备胶质瘤细胞体内实验2，4 <ol><li> 含有血清的培养物（GBM43，GBM6和U87MG）（Dulbecco&#39;s Modified Eagle&#39;s培养基[DMEM]中的10％胎牛血清）或无血清培养基（含有B27，N2，肝素，表皮生长因子的神经巴氏培养基[ EGF]，碱性成纤维细胞生长因子[bFGF]，抗生素和L-谷氨酰胺12 ），在加湿的CO 2细胞培养箱中。 <ol><li>通过从商业服务提供商运行的鼠标清除面板测试所有细胞系。 </li></ol></li><li> 通过去除培养基收集贴壁细胞。在室温下与0.05％胰蛋白酶孵育5分钟（T25烧瓶1 mL胰蛋白酶，T75烧瓶2 mL，比表面积较大的培养瓶分级），然后用Ca 2+和Mg 2+游离磷酸盐洗涤细胞缓冲盐水（PBS）。 <ol><li>点击烧瓶以移出细胞，并立即用过量的含血清培养基（8-9mL）中和。然后使用血清移液管将细胞悬液吸入离心管中。 </li><li>以400×g离心细胞悬液5分钟。通过反复离心用10mL PBS洗涤细胞至少两次。 </li><li>通过离心（相同的速度和持续时间）离心收集在无血清培养基中作为肿瘤球生长的胶质瘤细胞;通过在1-2mL细胞分离溶液中在37℃温育5分钟来分离细胞沉淀，然后通过离心（400xgx5分钟）用10mL PBS洗涤两次。 </li><li>重新悬浮胶体瘤无菌生理盐水浓度为每个2.5μL50,000 - 200,000个细胞。用于注射到小鼠脑中的细胞数量取决于每个胶质瘤细胞系建立的生长速率。在这里，GBM43和GBM6患者来源的肿瘤细胞分别使用25,000和100,000。 </li><li>准备两倍的nece用于植入的ssary细胞编号以解释手术期间损失的分数。将注射细胞悬浮液转移到无菌微量离心管中，并将管置于冰上。在手术过程中将细胞留在冰上2小时，如果计划进行扩展手术，准备新的批次。 </li></ol></li></ol> 3.创建异种移植小鼠模型的颅内植入（ 图1A ）2，4，13 <ol><li>在手术之前，对高压灭菌器中的所有手术工具进行消毒，并按照IACUC批准的方案准备所有手术前和手术后的动物护理材料。灭菌立体定位框架和外围设备，以确保术中无菌条件，从而最大限度地减少并发症。 </li><li>组织操作区域，尽量减少杂乱和污染的可能性。 </li><li>穿着适当的个人防护装备（PPE）每IACUC要求。 </li><li>设置适当的术后恢复室。确保使用未过期的试剂新鲜制备麻醉剂和镇痛药。根据IACUC批准的协议设置适当的体温保养仪器。 </li><li>为了建立人类患者衍生的胶质瘤细胞异种移植物来测试人类神经干细胞的鼻内递送，在约6周龄时使用任何性别的免疫受损小鼠种类，例如nu / nu无胸腺小鼠。 </li><li> 使用批准的试剂， 即通过腹膜内（ip）注射氯胺酮（50-100mg / kg;咨询制剂IACUC用于经批准的剂量范围）和赛拉嗪（5-10mg / kg）混合使用体重麻醉动物， kg;咨询制剂IACUC批准的剂量范围），200-250μL无菌盐水，或使用吸入装置通过4-5％异氟烷麻醉。 <ol><li>确保动物已达到手术麻醉平面b在皮肤切口之前趾部捏。应用充足的无菌眼科凝胶，以确保动物角膜的潮湿状态。 </li></ol></li><li>使用每个Pritchett-Corning 等人使用betadine和异丙醇的重复和交替的圆形擦拭物来消灭头骨。 14 。 </li><li>在使用外科手术刀切开前，通过sc注射Buprenex（0.05 - 2 mg / kg）提供切口前镇痛。 </li><li>使用灭菌的棉花头施放器帮助止血，同时暴露头骨;使用3％过氧化氢来促进偶发性出血的清除。 </li><li>使用电池供电的手持式电钻圆珠笔（宽度小于1毫米），可以在左侧或右侧向左侧或右侧向外侧创建一个2毫米的毛刺孔，并在Bregma后面创建2mm的位置坐标对于特定研究的肿瘤模型）。 </li><li> 将动物安置在立体定位框架上e头部姿势，使得插入的汉密尔顿微型注射器垂直于颅骨周围的颅骨表面。注意确保动物的麻醉平面处于良好的姿势，由脚趾捏住。观察呼吸频率，以确保在以下步骤中动物未受到疼痛压力。 *我们不强调入耳式酒吧刊登位置，因为: <ol><li>我们预先钻出钻孔来定位针头，因此不需要精确的头骨坐标; </li><li>鼠标耳道是脆弱的，耳塞的精确入耳放置需要更多的与我们的目的无关的训练。鼠耳道损伤的风险超过精确放置的好处; </li><li>脸颊支持更容易执行，并能够在定位期间实现相同水平的鼠标头稳定性和平衡。这对多种时间敏感的手术来说是一个额外的好处及时完成，细胞活力良好。 </li></ol></li><li>使用立体定位框架旋钮将平头微型注射器定位到钻孔，然后缓慢降低到硬脑膜表面下方3 mm。缩回0.5毫米以将针头保持在硬脑膜下方2.5毫米处。 </li><li>在1分钟内注入2.5μL预加载的胶质瘤细胞。让针头缓慢地保持其位置1-2分钟，在1分钟的时间内将针头从大脑中取出并注意使细胞回流最小化。使用无菌骨蜡避免颅外肿瘤生长。 </li><li>用不锈钢伤口夹（7毫米）关闭伤口。将皮下（sc）注射1 mL无菌乳酸林格氏溶液（预热至37°C），并将动物置于加热垫中途的回收室中。 </li><li>一旦他们恢复流动性，将动物送回通风的机架，并按照常规的后期护理来确保平滑h恢复。 </li></ol> 4. 体内生物发光成像（BLI）的肿瘤生长（ 图1B ） 15 注意:患者衍生的胶质瘤细胞被修饰以表达萤火虫荧光素酶。这使我们能够在颅内植入后跟随肿瘤进展。 <ol><li>在成像前10分钟，给予动物腹膜内注射D-萤光素，钾盐（150 mg / kg）。 </li><li>立即将动物放置在由IACUC批准的含氧异氟烷诱导室中。 </li><li>将动物置于加热（37°C）成像室内以使用软件设置捕获BLI信号（有关详细信息，请参阅制造商说明）。 </li></ol> 5.全脑照射（ 图1C ）2，4，13 <ol><li>使用腹腔注射氯胺酮和赛拉嗪混合物（分别为50-100mg / kg和5-10mg / kg，咨询制剂IACUC用于批准的剂量范围）与亚手术麻醉平面以适度诱导小鼠麻醉。 </li><li>使用样品托盘将鼠标放置在室内的引线屏蔽块之间，从而允许仅头部暴露于辐射路径（ 图2 ）。 </li><li>给予具有阳性BLI确认肿瘤生长的小鼠（通常在神经胶质瘤细胞植入后第7-8天）每天2Gy的照射剂量连续5天。在最后一次照射剂量后进行BLI评估肿瘤。 </li></ol> 6.人类神经干细胞的遗传修饰（NSCs; Figure 3A） 4 <ol><li>在95％室温和5％CO 2 （ N NSCs）下，在潮湿的37℃培养箱中，在具有10％FBS和1％青霉素 - 链霉素的DMEM培养基中维持人类NSCs（克隆HB1.F3.CD） 6,7 。 </li><li> 通过用编码人CXCR4的慢病毒转导，在hNSC中进行趋化性受体（如CXC基序趋化因子受体4（CXCR4））的遗传过表达。 <ol><li>当NSCs接近75-80％融合时，将CXCR4慢病毒与聚凝胺（8μg/ mL）一起加入到培养基中，并孵育8小时或过夜。 </li><li>用含有4μg/ mL杀稻瘟素的新鲜培养基代替含病毒培养基用于阳性选择转导的细胞。 </li><li>通过使用抗CXCR4抗体和匹配的同种型抗体和vi的流式细胞术验证修饰的hNSC（ CXCR4 NSCs）中CXCR4表达的水平使用扩增CXCR4和甘油醛-3-磷酸脱氢酶（GAPDH）cDNA的一对引物进行定量RT-PCR。 </li><li>使用相同的策略产生载体控制NSCs（ VC NSCs），RFP替代CXCR4基因，并且由于存在RFP荧光， 通过显微镜或流式细胞术验证。 </li></ol></li></ol> 7.人类NSCs的缺氧预处理（ 图3A ） 4 <ol><li>在如上所述的T75培养瓶中培养的培养的NSCs直至达到90％汇合。 </li><li>将细胞培养瓶置于保持1％O 2水平的加湿CO 2室/培养箱中24小时， 通过由自动内置流量计控制的N 2气体供应。这些细胞被称为H NSCs。在步骤7.1中描述了控制N个 NSCs。 </li></ol>胡装载具有溶瘤病毒的人NSCs（ 图3B- 3H） 5 <ol><li> 8.1。具有50个条件复制腺病毒（CRAd）-S-pk7病毒颗粒/细胞的N NSCs， H NSCs， VC NSCs和CXCR4 NSCs 4,16。 </li><li> 8.2。室温孵育2小时后，定期敲打，用培养基洗涤细胞三次以除去未结合的病毒颗粒，并将细胞悬浮于无菌盐水中进行注射。 小心:在具有适当生物安全等级名称的设施中进行与病毒相关的体外细胞培养程序，如BSL2。处理溶瘤病毒的研究人员必须佩戴适当的个人防护装备，每个机构动物设施指南4,16。 </li></ol> 9.用微量加载干细胞通过磁共振成像进行体内追踪的n型顺磁性氧化铁（MPIO）（MRI; 图3B- 3H） 2 <ol><li>为了标记干细胞，在转染试剂的存在下，以约80％融合的方式，将MPIO添加到干细胞培养物中进行过夜孵育。通过流式细胞术测定干细胞标记（NSCs 4或MSC 2 ）与MPIO（闪红染色）的效率。 </li><li>为了可视化胶质瘤动物脑中的干细胞迁移和分布，采用松弛增强自旋回波图像和多切片T1加权快速低角度射击（FLASH）梯度回波序列获得多切片高分辨率T2加权快速采集通过小鼠脑的冠状面和轴向平面评估结构2 。 </li></ol> 10.鼻内递送治疗性NSCs（ 图3D ） 2，4 <ol><li>根据第3节中描述的方案培养和收集NSCs（ N NSCs， H NSCs， VC NSCs和CXCR4 NSCs）。 小心:涉及OV载荷的神经干细胞的所有动物手术必须在具有适当生物安全等级的设施中进行，例如BL2或BSL2设施。 </li><li>根据步骤3.6通过ip注射氯胺酮和赛拉嗪混合物麻醉小鼠。 </li><li>将小鼠放在一个干净的悬垂物上，面朝上放置一块加热垫，以保持体温。用胶带调整由卷起的纸巾制成的填充枕头，以确保放置在鼠标头下方时鼻孔的直立角度。 注意:透明质酸酶预处理（总共100 U，以5分钟间隔四次重复接种，每只鼻孔3μL）鼻e前述2，17描述了上皮。 </li><li>使用微量移液管，将2μLNSC悬浮液分配给小鼠的每个鼻孔。在移动到下一个鼠标之前，目视确认液滴的抽吸。传送的单元总数可由用户确定;使用62,500-125,000个细胞/μL，使得可以以4×2μL剂量递送0.5-1,000个细胞。 </li><li>使用定时器确保每个细胞悬液给药间隔5分钟，最多4次。 </li><li>允许动物在恢复室恢复流动，整个仰卧位。 </li></ol> 11.生存分析（ 图3E ） <ol><li>在统计软件中输入动物的生存期数据，并使用具有以下有效指标的对数秩检验进行统计学比较:* p &lt; 0.05，** p &lt;0.01，*** p &lt;0.001。 </li></ol> 12.组织收集和组织学/免疫细胞化学验证鼻内干细胞脑输入2，4 <ol><li>在研究的终点， 通过 IACUC批准的方案安乐死小鼠。标准做法是使用流量控制的CO 2室来牺牲动物作为主要的安乐死策略，然后通过灌注固定放血。 </li><li>处死后，立即用PBS对动物进行心内灌注，以确保从血管中清除血液残留物并保存组织用于随后的组织学分析。 </li><li>将CO 2 -安乐死的动物置于仰卧位置的吸收垫的顶部，进行剖腹术以暴露与肋骨一起解剖的隔膜肌肉以暴露心脏。 </li><li>之后使用一对解剖剪刀在右心房使用一个切口，通过顶点的左心室加入3-5mL PBS注射液的放血。使用注射器将冰冷的4％多聚甲醛（PFA）注入循环;外周肌肉痉挛提供视觉确认，循环中没有严重的阻塞，会阻止适当的组织固定。 </li><li>手术去除胴体的头部，并在4℃的PFA中在50mL离心管中保存4℃过夜。第二天将溶液更换为30％的蔗糖，然后再进行脑组织切除24小时。 </li><li>将脑组织嵌入OCT化合物并在干冰上快速冷冻在异戊烷上。对所得组织块进行冷冻切片以产生组织切片和免疫细胞化学应用的10-20μm组织切片。 </li></ol>

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Implementing Rigor and Transparency) in NIH &amp; AHRQ Research Grant Applications. , (2015).</li><li>Wakimoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+primary+tumor+phenotype+and+genotype+in+glioblastoma+stem+cells.">Maintenance of primary tumor phenotype and genotype in glioblastoma stem cells.</a> Neuro Oncol. 14 (2), 132-144 (2012).</li><li>Cheng, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+In+Vivo+SPECT+Imaging+of+Neural+Stem+Cells+Functionalized+with+Radiolabeled+Nanoparticles+for+Tracking+of+Glioblastoma.">Dynamic In Vivo SPECT Imaging of Neural Stem Cells Functionalized with Radiolabeled Nanoparticles for Tracking of Glioblastoma.</a> J Nucl Med. 57 (2), 279-284 (2016).</li><li>Pritchett-Corning, K. R., Luo, Y., Mulder, G. B., White, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+rodent+surgery+for+the+new+surgeon.">Principles of rodent surgery for the new surgeon.</a> J Vis Exp. (47), (2011).</li><li>Clark, A. J., Fakurnejad, S., Ma, Q., Hashizume, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+Imaging+of+an+Immunocompetent+Animal+Model+for+Glioblastoma.">Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma.</a> J Vis Exp. (107), (2016).</li><li>Ulasov, I. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivin-driven+and+fiber-modified+oncolytic+adenovirus+exhibits+potent+antitumor+activity+in+established+intracranial+glioma.">Survivin-driven and fiber-modified oncolytic adenovirus exhibits potent antitumor activity in established intracranial glioma.</a> Hum Gene Ther. 18 (7), 589-602 (2007).</li><li>Danielyan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+cells+to+the+brain.">Intranasal delivery of cells to the brain.</a> Eur J Cell Biol. 88 (6), 315-324 (2009).</li><li>Dhuria, S. V., Hanson, L. R., Frey, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+to+the+central+nervous+system:+mechanisms+and+experimental+considerations.">Intranasal delivery to the central nervous system: mechanisms and experimental considerations.</a> J Pharm Sci. 99 (4), 1654-1673 (2010).</li><li>Gross, E. A., Swenberg, J. A., Fields, S., Popp, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+morphometry+of+the+nasal+cavity+in+rats+and+mice.">Comparative morphometry of the nasal cavity in rats and mice.</a> J Anat. 135 (Pt 1), 83-88 (1982).</li><li>Marieb, E. N., Hoehn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Anatomy &amp; Physiology. , (2007).</li></ol>

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虽然药物递送的鼻内途径已被广泛地用于小分子，纳米医学和蛋白质化合物18 ，但是用于鼻内脑肿瘤靶向的治疗性干细胞的应用在开发中的脑肿瘤治疗剂谱2 中是非常新的， 4 。鼻腔干细胞的行为有内在的复杂性，分子细节尚不清楚。干细胞的大小及其与颅神经的分布机制与非细胞生物制剂或小分子显着不同，目前我们正在进?...

人类干细胞的低毒性，低免疫原性和内在的脑肿瘤趋向性是用于递送治疗剂的有吸引力的特征1 。用于恶性脑肿瘤的新型基于干细胞的治疗剂是近年来发展的有希望的创新，并且该治疗策略的鼻内适应代表了临床翻译的飞跃，因为非侵入性和反复给药可能大大降低患者应用的障碍，可适用于门诊服务，无需全身麻醉或与侵入性外科手术1,2,3,4相关的长期住院服务。 我们和其他人开创了干细胞传播到脑肿瘤的鼻内途径，并为一些基本原则奠定了基础使用小鼠异种移植模型2,3,4的翻译研究，以及通过磁共振成像（MRI）试剂载体研究干细胞在体内的迁移。通过这些试点探索，我们积累了丰富的经验，并获得了如何使用建立良好的恶性胶质瘤患者来源的异种移植（PDX）小鼠模型建立强大的临床前评估策略的洞察力，同时保持调查决议，以检查精细的机械细节，传递到鼻腔的治疗性干细胞的鼻内入脑的复杂生物现象。在这里，我们描述标准化操作方案的原理，以证明使用完善的人神经干细胞系HB1.F3.CD 5的实验研究的现状<sup&gt;， 6,7,8 ，其可以容易地修改以适应使用人干细胞作为治疗载体的特定肿瘤模型或策略。

<table><tbody><tr><td>Stereotaxic frame</td><td>Kopf Instruments</td><td>Model 900</td><td></td></tr><tr><td>Hypoxic Cell Culture Incubator</td><td>ThermoFisher Scientific</td><td>VIOS 160i</td><td></td></tr><tr><td>Cell culture supplies (Plastics)</td><td>ThermoFisher Scientific</td><td>Varies</td><td>Replaceable with any source</td></tr><tr><td>Legend Micro 21R Refrigerated Microcentrifuge</td><td>ThermoFisher Scientific</td><td>75002490</td><td>Replaceable with any source</td></tr><tr><td>Bench centrifuge Sorvall ST16R&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>75004240</td><td>Replaceable with any source</td></tr><tr><td>Micro syringe 702N 25 &amp;micro;l (22S/2&quot;/2)</td><td>Hamilton Company</td><td>80400</td><td>Flat tip</td></tr><tr><td>Sample Tray for Irradiator</td><td>Best Theratronics</td><td>A13826</td><td>To set up mice protection with lead shield</td></tr><tr><td>Leica DMi8 Microscope</td><td>Leica Microsystem</td><td></td><td>Custom setup</td></tr><tr><td>Leica CM1860 UV cryostat</td><td>Leica Microsystem</td><td></td><td>Custom setup</td></tr><tr><td>Exel International Insulin Syringe</td><td>ThermoFisher Scientific</td><td>14-841-31</td><td></td></tr><tr><td>Corning Phosphate Buffer Saline</td><td>Corning Cellgro/ThermoFisher</td><td>21-031-CV</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium&amp;nbsp;</td><td>Corning Cellgro/ThermoFisher</td><td>11965-084</td><td></td></tr><tr><td>Trypsin 0.05%</td><td>Corning Cellgro/ThermoFisher</td><td>25300054</td><td></td></tr><tr><td>Hyaluronidase from bovine testes</td><td>MilliporeSigma</td><td>H3506</td><td></td></tr></tbody></table>

intranasal delivery of therapeutic stem cells to glioblastoma in a mouse model

对脑恶性肿瘤的内在向性使得干细胞成为治疗恶性肿瘤的有希望的载体。通过鼻内途径递送治疗性干细胞是最近发现的替代策略，由于与颅内植入或通过系统途径递送相比，其具有非侵入性，具有很强的临床翻译潜力。缺乏血脑屏障进一步加强了进行鼻内入侵的干细胞的治疗潜力。本文概述了我们研究中使用的基本技术，并概述了使用颅内神经胶质瘤异种移植小鼠模型进行干细胞传播的鼻内策略的基本原理。我们展示优化的程序，通过具体的预定实验参数产生一致和可重现的结果，并为精简的工作流程提供准则，确保有效执行和可靠的实验总结。该文章旨在作为基于假设，干细胞类型或肿瘤特异性的进一步实验定制的基准。

低氧预处理（ 图4A ） 4和CXCR4过表达（ 图4B和4C ） 4显着上调了CXCR4受体的细胞膜存在，如流式细胞术所证实的。 NSCs表现出的肿瘤趋向性（蓝色箭头）显示在肿瘤组织组织学（红色圆圈）中。通过普鲁士蓝染色确认肿瘤中MPIO标记（ 图4D ）干细胞的存在（ <strong class="x...

Watch this Scientific Journal Video about Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model at JoVE.com

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

干细胞是有希望的治疗载体，以治疗脑肿瘤由于其内在的肿瘤向性。非侵入性鼻内干细胞传播绕过血脑屏障，并且具有很强的临床翻译潜力。本文概述了胶质瘤小鼠模型中鼻内干细胞传递的基本原理。

在小鼠模型中将胶质母细胞瘤治疗干细胞的鼻内递送

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

intranasal-delivery-therapeutic-stem-cells-to-glioblastoma-mouse

Research

JoVE Journal

Medicine

12.1K 次观看.  Northwestern University. 干细胞是有希望的治疗载体，以治疗脑肿瘤由于其内在的肿瘤向性。非侵入性鼻内干细胞传播绕过血脑屏障，并且具有很强的临床翻译潜力。本文概述了胶质瘤小鼠模型中鼻内干细胞传递的基本原理。 

视频: Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

在原位小鼠脑肿瘤模型结合放疗

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

科研

医学

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

异位黏膜移植程序直接给药到小鼠的大脑

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Translational Orthotopic Models of Glioblastoma Multiforme

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors&#160;displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

多形性胶质母细胞瘤的转化原位模型

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...

癌症研究

<meta charset="utf8"></head><body>小鼠模型中注射后的颈总动脉修复

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there is an urgent need to develop methods for intracarotid injections in mouse models, including methods to repair the carotid artery in mice after injection to allow for subsequent injections. We developed a method of intracarotid injection in a mouse model to deliver therapeutics into the internal carotid artery (ICA) with two alternative procedures. 
During injection, the needle is inserted into the common carotid artery (CCA) after tying a suture around the external carotid artery (ECA) and injected therapeutics are delivered into the ICA. Following injection, the common carotid artery (CCA) can be ligated, which limits the number of intracarotid injections to one. The alternative procedure described in this article includes a modification where intracarotid artery injection is followed by injection site repair of the CCA, which restores blood flow within the CCA and avoids the complication of cerebral ischemia seen in some mouse models. 
We also compared the delivery of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) to intracranial tumors when delivered through intracarotid injection with and without injection site repair following the injection. Delivery of BM-hMSCs does not differ significantly between the methods. Our results demonstrate that injection site repair of the CCA allows for repeat injections through the same artery and does not impair the delivery and distribution of injected material, thus providing a model with greater flexibility that more closely emulates intracarotid injection in humans.

<meta charset="utf8"></head><body>作者聚焦:通过溶瘤病毒和 MicroRNA 的动脉内递送策略推进胶质母细胞瘤治疗

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of Willis.

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there ...

<meta charset="utf8"></head><body>氧化铁纳米颗粒用于小鼠胶质母细胞瘤的图像引导治疗递送

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

<meta charset="utf8"></head><body>作者聚焦:使用氧化铁纳米颗粒治疗胶质母细胞瘤的创新癌症疗法

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and has no cure. Laser interstitial thermal therapy (LITT) is a Food and Drug Administration (FDA)-approved treatment for GB and is used in patients who may not be candidates for conventional surgical resection. While the clinical efficacy of LITT has been established, research beyond clinical case studies and case series is limited and hindered by the lack of an established animal model. This protocol uses C57BL/6 mice and syngeneic CT2A glioma cancer cell line to closely recapitulate human GB&#160;while also using a 1064 nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser, such as is used in one of the two FDA-approved LITT systems, providing excellent pre-clinical relevance. The successful establishment of this LITT murine model will provide a valuable platform for investigating the unique features of LITT ablation and its effects on the tumor microenvironment, potentially leading to improved therapeutic strategies.

Glioblastoma is a devastating form of primary brain cancer, and laser interstitial thermal therapy is emerging as a promising alternative to conventional surgical resection for inoperable glioblastoma. This protocol describes an optimized pre-clinical mouse model that can be used to study treatment effects or adjuvant and combinatorial treatments.

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and ...

在小鼠模型中将胶质母细胞瘤治疗干细胞的鼻内递送

摘要

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此视频中的章节

在原位小鼠脑肿瘤模型结合放疗

异位黏膜移植程序直接给药到小鼠的大脑

多形性胶质母细胞瘤的转化原位模型

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma