我们感谢安德烈Parmeggiani（L2C和DIMNP，蒙彼利埃），伯努瓦夏洛（IES，蒙彼利埃），以及实验室的有益讨论的成员，克里斯托夫Duperray与流式细胞仪分析，蒙彼利埃RIO成像设备（MRI）提供帮助对于FACS和共聚焦显微镜充足的环境。国行是从LabEx Numev（ANR公约-10-的LabX-20）一个研究生奖学金的支持。 AB是由来自华沙和欧洲联盟的大学本科生奖学金支持（N°POKL.04.01.02-00-221 / 12）。 MLV是从国家科学研究中心（CNRS）工作的科学家。

1天 1.间充质干细胞的标记（MSC）线粒体（可选） <ol><li>线粒体制备前两天，种子人MSC在100毫米培养皿中，在10毫升αMEM/ FBS 10％，以便具有在第1天在培养4×10 5个干细胞。 </li><li>冲洗的MSC用PBS（4毫升）中，并添加4毫升αMEM/ FBS 1％（预热至37℃）。 </li><li>添加所需的线粒体活体染料的量，并培育细胞30分钟，在37℃培养箱。 </li><li>除去线粒体染料溶液，用预热4毫升（37℃）αMEM/ FBS 1％冲洗细胞两次，并加回4毫升αMEM/ FBS 10％。孵育在37℃的细胞。 </li><li> 30分钟后，改变培养基（10毫升αMEM/ FBS 10％）和，另一次，2小时后。 </li></ol> 1天 2.标签胶质母细胞瘤干细胞（GSC）（可选，见讨论部分） <table border="1" fo:keep-together.within页="1"FO:保持与 - next.within页="总是"&gt; <tbody><tr><td> 细胞培养基 </td><td> 组成 </td></tr><tr><td rowspan="5"> GSC基础培养基</td><td> DMEM / F-12补充有</td></tr><tr><td>胰岛素20毫克/毫升</td></tr><tr><td> N2补充1X </td></tr><tr><td>葡萄糖3g / L的</td></tr><tr><td> L-谷氨酰胺2mM的</td></tr><tr><td rowspan="9"> GSC增殖培养基</td><td>添加到基础培养基: </td></tr><tr><td> B27的补充1X </td></tr><tr><td> EGF 10毫微克/毫升</td></tr><tr><td> bFGF的10毫微克/毫升</td></tr><tr><td>为Fungine 10毫克/毫升</td></tr><tr><td>两性霉素B 0.25毫克/毫升</td></tr><tr><td>肝素2mg / ml的</td></tr><tr><td>环丙沙星2微克/毫升</td></tr><tr><td>庆大霉素2微克/毫升</td></tr><tr><td rowspan="4"> MSC增殖培养基</td><td> ＆＃945; MEM补充有</td></tr><tr><td> L-谷氨酰胺2mM的</td></tr><tr><td> 10％FBS的</td></tr><tr><td> bFGF的2毫微克/毫升</td></tr></tbody></table> 表1:文化传媒。 <ol><li>分离生长为在聚HEMA包被的细胞培养烧瓶神经球GSCS（GB4细胞线32（10×10 6个细胞）（参见步骤3.1至3.12）。 </li><li>在10 5个细胞在48孔板种子GSCS /孔在GSC增殖培养基（500微升）（ 见表1）。 </li><li>离心板在270×g离心20℃5分钟。 </li><li>添加所需的细胞活体染料的量，并在37℃下孵育30分钟。 </li><li>加入500μlGSC基础培养基（ 表1）每48孔板的孔。 </li><li>离心在270 xg离心该板在20℃下5分钟。吸出上清液。 </li><li>重复步骤2.5至2.6。 </li><li>加入500μlGSC基础培养基中，并在37℃孵育30分钟。 </li><li>离心在270 xg离心该板在20℃下5分钟。吸出上清液。 </li><li>加入500μlGSC增殖培养基的每孔的48孔板，并在37℃培养箱孵育。 注意:GSCS的量表示（10×10 6个细胞）允许执行不同的剂量-反应实验和FACS对照。一旦实验条件更精确地定义此量可被缩减。 </li></ol>第2天 3.胶质母细胞瘤干细胞接种<ol><li>在50ml管中收集的GSC神经球（10×10 6个细胞）通过离心在270×g离心5分钟，在20℃。 </li><li>在20℃下洗涤细胞用5毫升的HBSS，和离心机在270×g离心5分钟。 </li><li>吸出上清液。 </li><li>轻轻重悬在100μl胰蛋白酶-EDTA（0.25％）（在GSC粒料每10×10 <s达&gt; 6个细胞） </li><li>孵育在37℃下3分钟。 </li><li>添加10微升的CaCl 2（20毫米）和2μlDNA酶I（10毫克/毫升）。 </li><li>通过与P200吸管轻轻吹打向上和向下（30-50x）游离神经球。避免气泡。检查所有GSCS解离的显微镜下。 </li><li>添加10微升胰蛋白酶抑制剂（5％）和10毫升的HBSS。 </li><li>离心机GSCS在在20℃下270×g离心7分钟。 </li><li>弃上清，加入10 mL GSC基础培养基（ 表1）。 </li><li>算GSCS用托马计数室，然后离心细胞以270×g离心7分钟，在20℃。 </li><li>添加GSC增殖培养基（ 表1）的适当的体积达到10 6 GSCS / ml的细胞浓度。 </li><li>种子10 5 GSCS每一个96孔板的孔（100微升细胞悬浮液）。 </li><li>离心在270×g离心7分钟将板在20℃在机器人以获得GSCS井汤姆。 </li><li>在37℃下的96孔板直到第5节。 </li></ol>第2天 4. MSC线粒体隔离<ol><li>调整微管离心机温度至4℃。 </li><li>准备2个1.5毫升管的"A"和"C"含有线粒体萃取（200微升试剂A和400μl的试剂C，二者均含有的不含EDTA的蛋白酶抑制剂）的试剂。准备其他两管，标有"地中海"和"美图"。置于冰上所有的管子。还准备为管线粒体连续稀释。 </li><li>洗的MSC与预热10毫升（37℃）的PBS。 </li><li>洗的MSC用2ml胰蛋白酶（无EDTA）中10秒，并加入1 ml的胰蛋白酶（无EDTA）中。孵育细胞5-10分钟，在37℃。 </li><li>通过加入10mlαMEM/ FBS 10％，转移到50ml管中收回的MSC。 </li><li>离心细胞于在20℃下270×g离心5分钟。 </li><li>弃去上清液，加入10毫升αMEM/ FBS 10％至细胞沉淀。 </li><li>计数和计时Malassez室中的干细胞。 </li><li>离心机的MSCs（4-5×10 5）在20℃270×g离心5分钟。 </li><li>弃去上清液，加入1米冰冷αMEM/ FBS 10％到细胞沉淀，将细胞转移到"MSC"标记的管（在步骤4.2中制备）。保持管冰。 </li><li>离心机包含的MSC在900×g离心5分钟的管中，在4℃下。 </li><li>取下管所有剩余的网上平台。 </li><li>加入200μl线粒体提取试剂A（含不含EDTA的蛋白酶抑制剂）的。旋涡中速，持续5秒，离开冰管整整2分钟。 </li><li>以最大的速度添加2.5微升线粒体分离试剂B涡流的10秒，然后离开冰管中。重复每30秒5分钟。 </li><li>加入200μl线粒体提取试剂C（含不含EDTA的蛋白酶抑制剂）的。通过倾斜混合管（ROughly 30次，不要旋涡）。离心管中，在700×g离心在4℃下10分钟。 </li><li>转移上清液（含有MSC线粒体）至"三刀"管（在步骤4.2中制备）。 </li><li>离心机在3000 XG，在4℃下15分钟，得到线粒体沉淀。弃去上清液。沉淀包含分离线粒体。 </li><li>冲洗用200μl试剂C的。然后，离心分离的线粒体沉淀以12,000 xg离心，于4℃下5分钟，以获得线粒体沉淀。 </li></ol> 5.转让隔离MSC线粒体到GSCS（MitoCeption） <ol><li>添加200微升预冷的（0℃）的GSC增殖培养基由干细胞（4×10 5）中分离出的线粒体沉淀。 </li><li>稀释线粒体制剂（在GSC增殖培养基），以20微升的线粒体悬浮液的一贯添加到GSCS。 </li><li>添加分离线粒体的体积到96-W的孔中ELL板含有GSCS（从步骤3.15），在所需的浓度（0.1至10微克）。添加线粒体慢慢靠近井的底部，覆盖了整个表面至少一次。 </li><li>用于控制MSC线粒体活体染料渗漏，添加相同量的线粒体制剂（0.1至10微克）的含有GSC增殖培养基只（无GSCS）（参见用于活体染料泄漏检测部6.2）的96孔平板的孔中。 </li><li>离心机含有与MSC线粒体（步骤5.3）和控制板（步骤5.4）的线粒体收件人GSCS在1500 xg离心在4℃下15分钟的96孔板中。 </li><li>放置在37℃细胞培养箱培养板离心后立即。 注意:线粒体传输协议依赖于在足够的离心力的培养细胞的线粒体悬浮液的离心分离，具有一批离心，可以根据不同的SYST调整线粒体供体/受体细胞的他们。 </li></ol>第3天 6.流式细胞仪和共焦成像的线粒体传递的分析<ol><li> GSC样品的制备用于FACS分析 注意:如果MSC线粒体用活体染料预先（第1）标记的，效率可通过FACS线粒体转移后24小时监视。 <ol><li>离心机的96孔板用在270×g离心5分钟MitoCepted GSCS在20℃。 </li><li>弃去上清液。 </li><li>在每个孔中加入100μl胰蛋白酶（无EDTA）中。孵育在37℃下3分钟。吸管上下解离形成在24小时时间段的神经球。 </li><li>加入100微升GSC基础培养基。 </li><li>离心板在270×g离心5分钟，在20℃，并丢弃上清液。 </li><li>悬浮在GSCS 300微升GSC基础培养基，并转移到FACS适应管。 </li><li>执行的FACSalysis。 </li></ol></li><li> 从分离线粒体线粒体活体染料泄漏控制（FACS） 注意:该步骤的目的是确定本底的FACS信号，不会反映真正的MSC线粒体转移，而是从用于MitoCeption（步骤5.3）标记的MSC线粒体活体染料的光泄漏。 <ol><li> GSC种子细胞在步骤3.1所描述的3.15。 </li><li>孵育所述细胞在37℃下1小时。 </li><li>离心机仅在1500 xg离心含有线粒体（来自步骤5.4到5.6）中5分钟，在20℃的96孔板中。 </li><li>从GSC 96孔板（步骤6.2.2）吸出培养基，并通过与线粒体只（上清液从步骤6.2.3）孵育培养基更换。 </li><li>孵育含有GSCS的96孔板在37℃下2小时。 </li><li>请按步骤6.1.1 6.1.7 FACS分析。 </li></ol></li><li> GSC SAMP的制备LES的共焦成像 注意:如果MSC线粒体转移到GSCS是通过荧光成像分析，无论是MSC和GSCS需要预先分别与线粒体（步骤1）和小区（步骤2）活体染料标记。 <ol><li>准备GSCS如步骤6.1.1到6.1.5。 </li><li>悬浮GSCS在2毫升含2％FBS和种子35毫米培养皿（玻璃底）GSC增殖培养基。 </li><li>用转让荧光MSC线粒体24小时后的GSCS执行共焦成像。 </li></ol></li></ol>

<ol>
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INSERM（国家研究所德拉桑特等德拉RECHERCHE MEDICALE），与CJ和ML.V.挂靠，对MitoCeption技术（EP14306154.7）申请了专利。

越来越多的研究表明，细胞可以交换线粒体，这些线粒体对靶细胞的代谢和功能产生深远影响。因此，重要的是掌握工具来定量从供体细胞中这些靶细胞转移线粒体使它们的生物效应的精确研究。 这里所描述的协议最初是摸索出从人间充质干细胞中分离出的线粒体转移到粘附癌细胞系MDA-MB-231 33。如这里所示，它可适于该成长为体外神经球癌症干细胞?...

线粒体是在他们中的养分吸收，以及在能量和代谢物生产中发挥中心作用的真核细胞中发现的细胞器。这些细胞器包含圆形线粒体DNA（mtDNA的），16.6 kb的长，编码电子传递链复合物，tRNA和个rRNA 1的蛋白质。这些细胞器的功能是对细胞稳态和几个病状已与线粒体功能障碍1，2，3相关联的关键。线粒体状态已经例如被链接到炎症，感染性疾病和癌症，与用于转移和电阻后果治疗4，5，6，7后面这种情况。 线粒体显示的非凡能力，<html> "供体"和"目标"细胞间的转移得到。这导致在靶细胞的能量代谢的变化，以及在其它功能上的修改，例如组织修复和对化疗药物抗性，如最近由不同实验室8，9，10，11，12，13，14，15中所示16。人类间充质干细胞（MSCs）显示这个能力到线粒体转移到各种各样的靶细胞，包括心肌细胞，血管内皮细胞，肺泡上皮细胞，肾小管细胞和癌细胞，导致这些细胞的功能特性的修改8，&gt; 9，10，12，17，18。 线粒体交换现在显示为一种广泛使用的机制，允许多个不同的细胞类型中与彼此通信并修改他们的生物特性。这种线粒体交换可以通过隧道纳米管（TNT）的形成发生，涉及连接蛋白含有43缝隙连接8或M-SEC / TNFaip2和exocyst复杂的19。可替代地，也被示出的线粒体转移到通过抑制蛋白结构域的蛋白1介导的微泡（ARMMs）20介导的。有趣的是，线粒体转移的功效是有联系的所述的Rho GTP酶1的表达率MIRO1 21，用于说明在iPSC-之间线粒体转移功效的差别的关键因素干细胞与成人骨髓间充质干22。 尽管这种财富有关的细胞 - 细胞线粒体交换数据的，相对知之甚少此线粒体转移的代谢和生物结果。因此，它完全值得设置适当的工具充分评估这种转移的生物效应。多年来，若干技术方法从供体转移到线粒体受体细胞已被提出。这包括直接注射的线粒体成卵母细胞23，24，25，细胞融合，以产生transmitochondrial胞质杂种26,27 并且，最近，利用光热分离的线粒体的转移nanoblades 28。 我们和其他人以前证明孤立的和线粒体的能力河口到由活细胞内化，因为无论是在体外和体内观察到的 29，30，31，通过机制，提出了涉及巨吞32。我们进一步开发出一种方法，叫做MitoCeption，以定量地转移（从干细胞）分离线粒体靶细胞，与（贴壁）中列举的MDA-MB-231乳腺癌细胞系31。该协议在这里被改编为分离人MSC线粒体的转移胶质母细胞瘤干细胞（GSCS）。 胶质母细胞瘤是大脑中迅速成为处理有抗性，主要是由于胶质母细胞瘤干细胞（GSC）的肿瘤33内本侵略性恶性肿瘤。这些GSCS成长为体外神经球并产生异种移植模型的肿瘤。胶质母细胞瘤中的肿瘤细胞具有容量，使细胞与细胞间的连接，如最近所示为星形细胞脑肿瘤细胞通过扩展的微管，配线，通过该线粒体（以及钙和细胞核）可以迁移，导致耐放疗星形网络34。胶质母细胞瘤可以招募肿瘤微环境内的许多不同的细胞，包括干细胞35，36。我们表明，干细胞可以与在共培养GSCS细胞 - 细胞连接，并传送其线粒体（数据未显示），这是预期到修改GSC功能特性。本协议说明了MitoCeption技术如何可用于从人MSC与确定它们的功能的生物结果的目的传送线粒体，分离的预先，对人类GSCS。的多能和高致瘤性GB4 GSC线37在本研究中使用。

<table><tbody><tr><td>Mitochondria Isolation Kit for Tissue&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>10579663</td><td></td></tr><tr><td>N-2 Supplement (100x)</td><td>Fisher Scientific</td><td>11520536</td><td></td></tr><tr><td>B-27 Supplement W/O VIT A (50x)</td><td>Fisher Scientific&amp;nbsp;</td><td>11500446</td><td></td></tr><tr><td>HBSS w/o Ca&lt;sup&gt;2+&lt;/sup&gt; w/o Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>&amp;nbsp;Sigma</td><td>H4385</td><td></td></tr><tr><td>poly Heme&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>P3932</td><td></td></tr><tr><td>aMEM w/o glutamine</td><td>Ozyme</td><td>BE12-169F</td><td></td></tr><tr><td>DMEM/F-12 without glutamine</td><td>Fisher Scientific&amp;nbsp;</td><td>11540566</td><td></td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>25030-024&amp;nbsp;</td><td></td></tr><tr><td>Glucose&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>G7021</td><td></td></tr><tr><td>Insuline&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>I 1882&amp;nbsp;</td><td></td></tr><tr><td>Human&amp;nbsp;bFGF&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>233-FB-025</td><td></td></tr><tr><td>Human EGF&amp;nbsp;</td><td>Peprotech&amp;nbsp;</td><td>AF-100-15&amp;nbsp;</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3149&amp;nbsp;</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>MERCK</td><td>2382</td><td></td></tr><tr><td>Trypsine Inhibitor</td><td>Sigma&amp;nbsp;</td><td>T9003</td><td></td></tr><tr><td>DNase I</td><td>SIGMA&amp;nbsp;</td><td>10104159001</td><td></td></tr><tr><td>Trypsine 0.25% / EDTA 1&amp;nbsp;mM</td><td>Invitrogen&amp;nbsp;</td><td>25200056</td><td></td></tr><tr><td>Trypsin</td><td>Gibco&amp;nbsp;</td><td>15090-046</td><td></td></tr><tr><td>Protease inhibitors EDTA free</td><td>Sigma</td><td>4693159001</td><td></td></tr><tr><td>Ciprofloxacine&amp;nbsp;</td><td>Sigma</td><td>17850-5G-F</td><td></td></tr><tr><td>Fungine&amp;nbsp;</td><td>Invivogen</td><td>ant-fn-1</td><td></td></tr><tr><td>Fungizone&amp;nbsp;</td><td>Thermofisher</td><td>15290018</td><td></td></tr><tr><td>Gentamycin</td><td>Euromedex</td><td>EU0410</td><td></td></tr><tr><td>MitoTracker Green FM</td><td>Molecular Probes</td><td>M7514</td><td></td></tr><tr><td>MitoTracker Red CMXRos</td><td>Molecular Probes</td><td>&amp;nbsp;M7512</td><td></td></tr><tr><td>MitoTracker Deep Red FM</td><td>Molecular Probes</td><td>&amp;nbsp;M22426&amp;nbsp;</td><td></td></tr><tr><td>CellTracker Green CMFDA</td><td>Molecular Probes</td><td>C7025</td><td></td></tr><tr><td>CellTracker Blue CMF2HC</td><td>Molecular Probes</td><td>C12881</td><td></td></tr><tr><td>RIPA</td><td>Santa Cruz</td><td>sc-24948</td><td></td></tr><tr><td>FluoroDish Sterile Culture Dish</td><td>World Precision Instruments</td><td>FD35-100</td><td></td></tr><tr><td>Hemacytometer</td><td>Fisher Scientific</td><td>267110</td><td></td></tr><tr><td>FACS tubes</td><td>Beckman Coulter</td><td>2,523,749</td><td></td></tr><tr><td>FACS apparatus</td><td>Gallios&amp;nbsp;</td><td>&amp;nbsp;3L 10C</td><td></td></tr><tr><td>LC FAST START DNA MASTER PLUS&amp;nbsp;</td><td>Roche</td><td>3515885001</td><td></td></tr></tbody></table>

mitoception: transferring isolated human msc mitochondria to glioblastoma stem cells

线粒体发挥细胞代谢，能源生产和细胞凋亡的控制中心作用。不足的线粒体功能已经发现负责非常多样的疾病，从病症神经系统癌症。有趣的是，线粒体最近已显示，以显示有能力的细胞类型之间从人间充质干细胞（MSC）的共培养条件转移，特别是癌症细胞，代谢和功能的后果对线粒体受体细胞，进一步增强了当前兴趣这些细胞器的生物学性质。 评估在靶细胞的转移的MSC线粒体的效果是最重要的，以了解这样的细胞 - 细胞相互作用的生物学结果。这里所描述的MitoCeption协议允许从供体细胞中预先分离到靶细胞的线粒体的转移，使用MSC线粒体和胶质母细胞瘤干细胞（GSC）作为模型系统。该协议之前已经用于从干细胞转移线粒体，分离的，以贴壁的MDA-MB-231癌细胞。此线粒体传输协议此处适于呈递如体外神经球生长的具体特殊性GSCS。分离的线粒体的转移可以使用线粒体活体染料后跟荧光激活细胞分选（FACS）和共聚焦成像。使用线粒体供体和靶细胞具有不同的单倍型（单核苷酸多态性）的也允许基于其圆线粒体DNA（mtDNA的）的靶细胞的浓度被转印线粒体的检测。一旦该协议已经被验证这些标准，窝藏转移的线粒体的细胞可以被进一步分析，以确定对生物性能如细胞代谢，可塑性，增殖和对治疗的反应的外源线粒体的作用。

概述从间质干细胞（MSC）和由MitoCeption其转移到靶向胶质母细胞瘤干细胞（GSC）线粒体的分离的过程步骤示于图1。 GSCS生长神经球，以维护自己的干细胞特性的肿瘤干细胞。用于协议，GSCS接种为单细胞几小时线粒体（步骤3），以允许更高的线粒体转印效率的转印之前（见的FACS数据图3B）。以下第5（第3天），这些细胞被再次观察到作为形成神经球...

Watch this Scientific Journal Video about MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells at JoVE.com

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

这里，协议（MitoCeption）呈现给传送线粒体，从人类间质干细胞（MSC）隔离，胶质母细胞瘤干细胞（GSC），具有研究它们对GSC代谢和功能的生物效应的目的。类似的协议可以适于其它类型的细胞之间转移线粒体。

MitoCeption:转让人离MSC线粒体脑胶质瘤干细胞

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found ...

mitoception-transferring-isolated-human-msc-mitochondria-to

Research

JoVE Journal

Biology

12.7K 次观看.  Montpellier University. 这里，协议（MitoCeption）呈现给传送线粒体，从人类间质干细胞（MSC）隔离，胶质母细胞瘤干细胞（GSC），具有研究它们对GSC代谢和功能的生物效应的目的。类似的协议可以适于其它类型的细胞之间转移线粒体。 

视频: MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

人类胶质母细胞瘤肿瘤细胞的分离和扩大使用的神经球含量

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

科研

神经科学

来自悬浮生长癌细胞的传输软骨 Cybrid 生成

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (&#961;0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

作者聚焦:使用癌细胞系进行传递软骨 Cybrid 生成  

This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process.

使用癌细胞系的传递软骨Cybrid生成

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...

癌症研究

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular mechanisms that maintain oocyte quality remain unclear, thus it is difficult to increase the birth rate of women over 35 years old at present. Oocytes contain more mitochondria than any type of cell in the body, and any mitochondrial dysfunction can lead to reduced oocyte quality. In the 1990s, oocyte cytoplasmic transfer resulted in great success in human reproduction but was accompanied by ethical controversies. Autologous mitochondrial transplantation is expected to be a useful technique to increase the quality of oocytes that have decreased due to age. In the present study, we used adipose-derived stem cells from aged mice as a mitochondria donor to increase the quality of oocytes of aged mice. Further development of autologous mitochondrial transfer technology will provide a new and effective treatment for infertility in aged women.

Here, we describe the isolation of mitochondria from mouse adipose-derived mesenchymal stem cells, and then transfer the mitochondria into aged mouse oocytes to improve the quality of the oocytes.

Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular ...

发育生物学

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

免疫与感染

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

超声引导下人骨髓间充质干细胞在实验性肠炎小鼠模型中的应用

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

医学

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

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

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 ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

影像引导切除治疗干细胞种子支架的胶质母细胞瘤和颅内植入术

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

<meta charset="utf8"></head><body>研究 MSC 和 ARPE19 共培养物中的线粒体转移

Mitochondrial Transfer Via Tunneling Nanotubes Between Mesenchymal Stem Cells and Retinal Pigment Epithelium In Vitro

Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important pathway for mitochondrial transport is through tunneling nanotubes (TNTs). There have been many studies reporting that mesenchymal stem cells (MSCs) can transfer mitochondria to other cells by TNTs. However, few studies have demonstrated the phenomenon of bidirectional mitochondrial transfer. Here, our protocol describes an experimental approach to study the phenomenon of mitochondrial transfer between MSCs and retinal pigment epithelial cells in vitro by two mitochondrial tracing methods.
We co-cultured mito-GFP-transfected MSCs with mito-RFP-transfected ARPE19 cells (a retinal pigment epithelial cell line) for 24 h. Then, all cells were stained with phalloidin and imaged by confocal microscopy. We observed mitochondria with green fluorescence in ARPE19 cells and mitochondria with red fluorescence in MSCs, indicating that bidirectional mitochondrial transfer occurs between MSCs and ARPE19 cells. This phenomenon suggests that mitochondrial transport is a normal physiological phenomenon that also occurs between MSCs and ARPE19 cells, and mitochondrial transfer from MSCs to ARPE19 cells occurs much more frequently than vice versa. Our results indicate that MSCs can transfer mitochondria into retinal pigment epithelium, and similarly predict that MSCs can fulfill their therapeutic potential through mitochondrial transport in the retinal pigment epithelium in the future. Additionally, mitochondrial transfer from ARPE19 cells to MSCs remains to be further explored.

<meta charset="utf8"></head><body>作者聚焦:MSC 和视网膜色素上皮细胞之间的双向线粒体转移 — 通路和体内挑战

Here, we present a protocol including mitochondrial tracing, direct co-culture procedures of mesenchymal stem cells (MSCs) and retinal pigment epithelial cells (ARPE19), as well as the methods for observing and statistically analyzing tunneling nanotubes (TNT) formation and mitochondrial transfer to characterize mitochondrial exchange via TNTs between MSCs and ARPE19 cells.

在体外通过间充质干细胞和视网膜色素上皮之间的隧道纳米管进行线粒体转移

Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important ...

MitoCeption:转让人离MSC线粒体脑胶质瘤干细胞

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此视频中的章节

人类胶质母细胞瘤肿瘤细胞的分离和扩大使用的神经球含量

使用癌细胞系的传递软骨Cybrid生成

使用癌细胞系的传递软骨Cybrid生成

使用癌细胞系的传递软骨Cybrid生成

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

超声引导下人骨髓间充质干细胞在实验性肠炎小鼠模型中的应用

在小鼠模型中将胶质母细胞瘤治疗干细胞的鼻内递送

影像引导切除治疗干细胞种子支架的胶质母细胞瘤和颅内植入术

在体外通过间充质干细胞和视网膜色素上皮之间的隧道纳米管进行线粒体转移