Name: isolation and expansion of mesenchymal stem/stromal cells derived from human placenta tissue
Uploaded: 2016-06-06T14:10:00.000+00:00
Duration: 12 min 40 s
Description: Watch this Scientific Journal Video about Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue at JoVE.com

RP是由国家健康与医学研究委员会（NHMRC）博士后奖学金的培训支持。 VS是由昆士兰国际硕士研究生大学的奖学金支持。 MRD是由NHMRC和内轮澳大利亚支持。 我们感谢临床及护理人员对病人的同意和样本采集协助。我们感谢Nickolas菲斯克教授克里·阿特金森教授和罗汉Lourie博士产科见地的讨论中，胎儿 - 胎盘发育和胎盘解剖。

昆士兰科技大学人类研究伦理委员会在母校卫生服务，皇家布里斯班妇女医院，和昆士兰大学认可的研究中使用的人胎盘样本的研究和收藏。所有协议符合国家研究的指导方针。患者提供了利用组织的以研究为目的知情同意书。 孕晚期胎盘是从下面，从在澳大利亚布里斯班上述医院足月例行剖腹产（CS）出生健康的母亲得到的。对于术语样品雄性不和谐怀孕在本研究中用于从母体细胞区分胎儿。胎儿性别已通过超声波产生和/或出生时由临床工作人员的新生儿的目视检查之前被确定。 原位杂交（FISH），X和Y染色体荧光用来进一步验证性别和胎儿或母体起源组织从保存原来胎盘。 1.收获前，准备以下注意:当进行了酶溶液，由制造商对每一个产品提供的具体要求和浓度应遵循。酶通常提供蛋白质的粗混合物，因此来自不同公司的类似的酶可能有不同的活动/浓度。由于这个原因，制造商的意见应该得到承认和解决方案的准备可能需要作相应修改。 <ol><li>制备或购买1.5到2升总无菌Hank氏平衡盐溶液（HBSS），用于该协议。 </li><li>通过称出I型胶原酶制备胶原酶我原液和溶解为1mg / ml的HBSS和涡流轻轻以确保完全溶解。确定的HBSS（含有钙和镁）的使胶原酶溶液到100单位（U）/微升（1000倍原液），然后用0.2筛选所需的体积＃181，M注射器过滤。直到需要分装1毫升卷成1.5毫升管并储存在-20°C。 </li><li>准备由无钙或镁的Dulbecco磷酸盐缓冲盐水（DPBS）以10毫克/毫升溶于非无菌分散酶分散酶原液。用DPBS进一步稀释没有钙或镁为2.4单位/毫升的最终浓度。过滤通过0.2μm的注射器过滤器进行消毒。直到需要1.5毫升管分装1毫升体积并储存在-20°C。 </li><li>通过在0.15M NaCl的10毫克/毫升溶解的DNase-I制备脱氧核糖核酸酶（DNA酶）-I原液。 thorugh 0.2微米的注射器滤膜过滤。直到需要1.5毫升管分装1毫升体积并储存在-20°C。 </li><li>通过组合100U / ml胶原酶类型制备工作的消化溶液I，I.5微克/毫升DNA酶I和2.4 U / ml的分散酶在无血清的DMEM中。新鲜解冻并稀释酶股在使用前立即。大约，1:消解液1的比例体积组织体积需要（ 如 10毫升组织的，在50ml管中测得的，需要10毫升消化溶液）。 </li><li>制备在PBS中的4％多聚甲醛（PFA）中的溶液，并调节pH至7.4 15。商店25毫升分装，在-20℃下长期或在4℃下1周。 </li><li>准备从补充有1×抗生素和抗真菌溶液和10％非热灭活的胎牛血清的DMEM低葡萄糖（DMEM-LG）的MSC培养基中。 MSC-级FBS可以从许多FBS的供应商购买。 </li><li>高压灭菌器清扫设备提前按照机构准则。这些措施包括剪刀，镊子，解剖刀和一个大的金属夹层托盘。收集其他无菌组织培养塑料器具和消耗品（ 例如手术刀片50毫升和15毫升试管，将25ml，将10毫升和5ml移液管，和75或175 平方厘米细胞培养烧瓶）。 </li><li>使用标准的原代细胞培养实验室设备:II b类适合初级细胞分离iosafety柜，细胞培养培养箱设定为5％的CO 2和37℃下，在37℃培养箱一个摇杆，并用50毫升管容量的离心机。 </li><li>使用适当的个人防护装备:实验室外衣，2个副手套（在任何时候），安全防护眼镜，并靠近趾鞋按照机构准则。 </li><li>事先准备的人的血液标本按机构的指导方针和液体生物废物容器适当净化解决方案。 </li></ol> 2.胎盘MSC的分离<ol><li>羊胎素的制备<ol><li>穿着所需的个人防护装备。设置了所需的材料，解决方案和废物容器生物安全柜开展过程可达酶消化（第2.5.6）。 </li><li>获得知情同意和伦理批准胎盘。通过收集剖宫产秒胎盘无菌手术条件下化，和包无菌袋或桶运送至实验室。 </li><li>在运输过程中和之前夹层，在室温下储存或4℃胎盘。 注:组织可以在不影响细胞培养性能被存储长达6小时。 <ol><li>限制胎盘的收集和组织处理在可能的情况之间的时间。这一建议是基于实际问题。在一些情况下，我们已经延迟了出生后长达6小时的细胞分离过程。在这些情况下胎盘已被保持在室温或4℃（在实验室或在收集中心）。没有观察到可检测的差异，上述的一般供体变型中，从胎盘中获得的MSC时，被储存在4℃或室温。 </li></ol></li><li>放置在容器与在生物安全柜胎盘。打开容器和胎盘转移到一个无菌的托盘。 </li><li>开出胎膜（羊膜或羊膜-绒毛膜绒毛的）8。成为与胎盘的部分定向。胎侧具有脐带插入，这是在一般情况下，在胎盘集中插入。产妇侧（与蜕膜内衬），这是相对的胎侧，具有明显的子叶（或裂片）。 </li><li>开始剥离，定向与在无菌托盘朝上脐带胎盘。 注意:此协议被设计为产生足够的细胞接种到从每三个组织类型之一T175烧瓶中。每个摘要始于约10克组织的。这瓶从20ml的骨髓穿刺发起MSC文化的一个类似的起始编号。如果有更多的细胞是期望的，则更多的组织可以收获和协议可线性缩放。将收获的组织的大小仅仅是指示性的东西与胎盘组织实用。很少有definin克特征在胎盘绒毛，它可以作为一个参考点。指示的尺寸作为一个估计和脐带被用作胎表面上的一个里程碑。 </li></ol></li><li>蜕膜（四）组织的解剖<ol><li>翻转胎盘过来，让母体面（底蜕膜）朝上。确保胎侧脐带插入点朝下。 </li><li>从胎盘（含底蜕膜组织）的母亲一边切割厚0.5cm的片。 </li><li>将在解剖组织块放入含有HBSS培养皿中，以保持水分的组织块。 </li><li>转移足够的组织填充管到10毫升标记在50ml管（这大约为10克组织的）。 </li></ol></li><li>绒毛膜板夹层（CP）组织<ol><li>机械移除胎盘（不羊膜囊​​）的胎儿面的羊膜，留下町叶状rionic（绒毛膜板）不变。 </li><li> 0.5厘米的绒毛膜板深裁片〜1厘米宽。收获来自该地区的绒毛膜板最近的脐带，从胎盘的边缘了。 </li><li>将组织块放入夹层中含有HBSS的培养皿，以保持他们水合。 </li><li>转移足够的组织填补50ml管到10 ml刻度线（〜10克组织的）。 </li></ol></li><li>绒毛的夹层（CV）组织<ol><li>解剖CV组织约1厘米2×0.5-1厘米从哪里CP已被移除胎盘组织深。再次，从最接近脐带的区域，从胎盘的边缘离收获的简历。尝试从胎盘母体的身边至少有1厘米的距离（含底蜕膜组织）;我们的目标是从胎盘绒毛组织的内部收获组织。 </li><li>将组织块放入狄氏过程中含HBSS培养皿ction让他们水合。 </li><li>转移足够的组织填补50ml管到10 ml刻度线（〜10克组织的）。 </li></ol></li><li>切碎和D，CP的酶消化，和CV胎盘组织<ol><li>由于有10〜克D-中，CP或CV组织在三个不同的50ml试管，填充每个管加入约40ml的HBSS，并多次反转管，以洗组织（重复〜10秒）。 </li><li>倒出上清液，并重复此洗涤步骤2-3次，直到溶液是从血液中基本上没有。用10毫升吸管或长镊子以确保倾析上清液，当组织片不会丢失出管（多个）。 </li><li>返回组织块到10cm的培养皿中以最小的液体传输。印章/剁碎组织成细片约1-5毫米的3剪刀或刀片。 </li><li>转移剁碎组织放回适当标记（D，CP或CV）50ml试管。 </li><li>添加新鲜prepar编消化介质中的至少一个1:1的比例与组织（ 例如 10毫升组织加10毫升消化介质的）。替换每个管盖并倒转试管几次以混合。 </li><li>孵育组织与在37℃下消化介质1-2小时的管中。湿润气氛和5％CO 2的不需要的这一步。 注意:快速振荡，需要以有效地分离来自组织的细胞和最大化MSC产量。通过在48小时和一个第一通过时间超过1-2周更长附着在培养瓶非常少数细胞有限产量将是显而易见的。温育时间和摇动法是依赖于37℃培养箱，可以在实验室和可能需要优化。 <ol><li>对于具有快速和可控制的摇动机构，地点组织培养箱并消化在培养箱50ml管或无菌锥形瓶中，地点中，设置振荡速度至​​250rpm。这将导致在一个完整的摘要所述组织在1小时。 </li><li>可替代地，对于具有温和的摇摆或无摆动机构的恒温箱，手动迅速并剧烈摇动该管通过手10秒为1.5至2小时，每30分钟。这种酶的潜伏期是一个合适的时间丢弃垃圾，清除不必要的项目的生物安全柜，改变实验室的外衣弄脏和程序的下一个部分做准备。 </li></ol></li></ol></li><li>单个核细胞采集和电镀成瓶 注意:从前面的步骤中，有三个50ml试管（D，CP或CV）与消化组织片。当组织消化溶液演变混浊外观和白色的血管组织中是显而易见的，消化完全。 <ol><li> 30ml含有胎牛血清的MSC介质添加到每个管灭活包含在消化溶液中的酶。 </li><li>到了单核细胞从大未消化的碎片分开，脉冲离心50毫升管。简单地说，允许离心机达到为340 x 克5秒，然后停止。这将迫使大碎片在试管的底部沉淀，同时使单核细胞中的液体悬浮液。 注意:单核细胞将保持在液相如果管是只简单地（脉冲）离心。如果离心延伸时，单核细胞也将沉淀在与组织片在管的底部，从而导致不​​希望的细胞的损失。 </li><li>收集上清液，将含有单核细胞，并用移液管转移到一个新的50ml管中此。 </li><li>加入30ml媒体或HBSS加入剩余的胎盘组织碎片，剧烈摇动。这将使剩余分离单个核细胞悬液成，使从组织公认的MSC的第二个收获。 </li><li>脉冲离心管中的第二次，并再次将上清液转移至新的50ml管中。重复此步骤的第三时间，最大限度地收集效率从每个组织来源的单核细胞。 </li><li>汇集来自每个个体的组织类型的上清液分为两50ml试管。这将产生6管总（2管每个D，CP和简历）。离心每个管在340×g下5分钟。此步骤将沉淀的单核细胞到管的底部。 </li><li>小心倒出或移液器关闭上清到浪费。这是不必要的尝试，以消除上清液每一滴。 注意:将细胞沉淀将脆弱由于大量红细胞。要小心，不要意外弃沉淀，如果你是直接从50毫升管调迁。 </li><li>除去大部分上清液后，轻轻几次用手指轻弹管撞出细胞沉淀。结合颗粒，使得存在用于每个D，CP或CV组织单管和在35毫升的MSC介质的悬浮每个。 </li><li>可选:通过100微米网状细胞滤网过滤的单核组分集U中的p 50ml管以除去细胞团块或纤维材料。 注意:作为过滤器可以很容易地堵塞和过度填充护理是必需的。此外，气泡可以从引流阻塞过滤器。如果发生这种情况，慢慢提起过滤出管的以允许空气在过滤器下通过与细胞培养基或交换流动，洗净过滤到一个新的过滤器。不包括滤波步骤似乎不改变在随后的MSC培养物的产率或质量。 <ol><li>任选地，红血细胞（RBC）裂解或密度梯度离心可在此阶段15进行。然而，我们的经验是，红细胞不与MSC附着或增殖，因此RBC裂解干扰是没有必要的。单核细胞可以在这一步，如果去除红细胞进行15计算。虽然，在这个阶段，绝大多数细胞不会MSC，但是胎儿起源滋养层，内皮细胞和造血细胞，以及马ternal起源造血细胞及蜕膜基质细胞。 </li></ol></li><li>在37℃下转移为每个D，CP或CV组织的细胞悬浮液到一个单一的T175烧瓶中，并培养在湿润的培养箱中和5％的CO 2。 </li></ol></li><li>交分离细胞膨胀<ol><li>在48小时之后的初始隔离，从每个烧瓶（D，CP和CV）的除去培养基并用35ml新鲜的MSC介质的替换。用过的培养基和分离的细胞可以被丢弃，因为推定的MSC被假定为连接于组织培养瓶16。返回烧瓶培养箱另外24小时。 </li><li>以下培养另外24小时后，用DPBS（25毫升）洗烧瓶两次，以除去碎屑和红细胞。漩涡围绕烧瓶撞出红细胞底部的液体。使用移液管除去的液体和碎屑。添加35毫升的MSC介质向瓶和烧瓶返回到培养箱中。 </li><li>更换介质每周两次，并在cubate培养直至单层细胞是80-90％汇合。此初始扩张需4-14天，取决于组织的质​​量，原料和效率，孵育时间与消化溶液的量。 </li></ol></li></ol> 3.私营军保公司的亚文化<ol><li>当文化是80-90％融合，用20毫升1X HBSS或DPBS洗两次，并丢弃洗。胰蛋白酶替代添加到每个烧瓶中（ 即使用5号毫升一T175瓶）。 </li><li>孵育5分钟的烧瓶在37℃以从组织培养表面解放的MSC。挖掘烧瓶的侧面每〜2分钟，以促进分离。 </li><li>当细胞从表面和在一个单一的细胞悬浮液分离，从MSC介质烧瓶洗细胞并收集细胞在试管中。海安媒体将淡化和取消胰蛋白酶替代品（使用每〜T175瓶15毫升MSC媒体）。 </li><li>转移的每个组织培养瓶的内容传送到一个​​50ml管。 </li><li>在340×g离心5分钟离心管以沉淀细胞。 </li><li>弃去上清液并在1毫升的MSC介质的重悬细胞沉淀。 </li><li>稀释10微升细胞悬浮于10μl台盼蓝。算使用血球细胞和计算单元15的总数。 </li><li>转移细胞到新的培养瓶中在1150细胞/ cm 2新鲜的MSC介质和在组织培养箱孵育。在此接种密度，种子200000 MSC到在35毫升的MSC介质的每一个新的T175烧瓶中。饲料细胞每周两次，直到80-90％汇合。在1150细胞/ cm 2或200000的MSC到每个T175烧瓶种子后续的传代。 </li><li>当通道1（P1）或P2细胞汇合，冷冻保存的大部分细胞以供将来使用，并且补种只有一个进一步繁殖和表征T175烧瓶中。所传播的细胞可以在P3或P4用于经由FL中胚层分化测定和细胞表征流式细胞仪。 注意:在早期传代，可以存在很少疏分离菌落，但每一集落内的局部细胞密度可以非常高。这是不理想的，因为细胞的局部致密填料会导致接触抑制，延缓生长和减少培养性能。我们建议当密菌落观察到的，细胞应收获再接种到新的烧瓶中。这个再分配方法提供细胞室增殖，并整体培养性能将得到改善。 </li><li>冻存的MSC，收集〜1×10 6个细胞在1ml 90％FCS和10％的二甲亚砜（DMSO）中，并使用标准方案冻结。 注意:在代表性的结果部分中，有使用流式细胞仪MSC表征，中胚层分化和XY FISH分析的描述，以确定三种不同的（D，CP和CV）扩增的细胞群的性别。在我们的研究中，胎盘分别来自男宝宝;因此，所有的胎儿细胞可以区分为男性（Y染色体）和所有母亲的细胞可以（只有X染色体）来区分的女性。 </li></ol>

<ol>
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The authors have nothing to disclose.

胎盘是一个物理上大母儿的器官，从胎儿或母亲MSC可以分离出22。在此，我们提供的胎盘解剖和教学对如何具体剖析蜕膜（产妇），绒毛（胎儿）和绒毛膜板（胎儿）的组织（步骤2.2-2.4）的详细介绍。随后，我们提出了一个强大的协议，允许从上述三个组织（步骤2.5-2.6）的MSC隔离。胎盘MSC扩增是有效的，并培养出现类似的骨髓来源的MSC培养物（ 图3和4）。成骨分化是可靠（ 图5B），而脂肪形成分化通常是效率较低（ 图5C）5。 很多新人到外地将推定胎儿绒毛或绒毛板组织衍生的培养将富集胎儿MSC。然而，在我们的手中FETAL细胞富集仅短暂时标准的MSC扩增培养基如DMEM-LG + 10％FBS利用5。在这里，我们提供使用从男宝宝衍生胎盘组织代表性的结果。通过使用胎盘组织从一个男宝宝，胎儿的细胞很容易可识别具有XY染色体，而母体细胞的识别具有XX染色体， 图6显示了XY FISH结果具有代表性的文化。而胎儿MSC（XY）的富集的（高达80％），在从胎绒毛膜绒毛或绒毛膜板组织来源的初始培养物，这些相同的培养物迅速取代（〜100％）由母体（ⅩⅩ）的MSC在第一两个通道。在标准培养基，组成的DMEM-LG + 10％FBS，即污染胎儿组织outcompete胎儿来源的细胞在培养的少数母体衍生的MSC。 在这个协议中提出了一个关键的步骤是从哪儿胎儿胎盘解剖和升值与母体组织可以最有效地收获。正如代表结果部分所述，胎儿组织的解剖未使胎儿衍生MSC瞬态富集。在扩大培养基配方的改进，通过特定的外源性生长因子的培养基补充应让胎儿来源的MSC居的选择性扩张，生产一种富含胎儿而不是母体细胞（我们的团队目前正在开发这样中等制剂）的细胞产物。胎儿MSC群体的制造中可以具有许多优点，因为胎儿MSC被声称具有更大的血管生成和比同等母体MSC群体37免疫抑制性质。 在每个所描述的分离方案中，我们使用大约10克组织的。整整一个胎盘通常是500-750克，而在以前的工作中，我们证明，通过自动组织消化和细胞扩张的生物反应器PROCS弯，它应该是能够制造从单个胎盘4 7000临床细胞的剂量。无论MSC原点（胎儿或母体）的这些数字突出同种异体MSC疗法的胎盘衍生的MSC，而且这种方法的意义的潜在适用性。从治疗的角度来看，这是最关键的，用户必须在细胞产物和可靠地制造这种电池产品的能力有充分的了解。我们希望我们的视频将帮助研究人员了解胎盘解剖，从胎盘中分离出MSC，并预见他们的文化可能胎儿或母亲的细胞组成。

间充质干细胞/基质细胞（MSC）正在成为在基于细胞的疗法1使用一个有希望的候选者。大多数应用程序似乎目标MSC介导的组织修复或免疫调节2。在许多这些应用中，异基因的MSC可以是自体的MSC 3一样有效。使用同种异体的MSC具有可与多个细胞剂量的大规模制造从单一来源组织4来治疗许多患者相容的经济优势。 从历史上看，临床前和临床研究已经利用从骨髓4的MSC来源。骨髓通常由志愿者供体的髂嵴收集。这个过程是侵入性的，并且只骨髓（约20毫升）的小体积，通过一个单一的穿刺收集。产生MSC的临床意义的数字，需要在体外扩增广泛。细胞效力与通道数量减少<suP&gt; 3，产生其中增加了更多的细胞群体扩大了临床功效所需的细胞的理论数一个矛盾。在对比骨髓抽吸，足月胎盘是物理大的起动组织（一般为500-750克4-），其可以在无菌条件下，没有风险的施主剖宫产期间收获。从胎盘来源的MSC具有长期增殖5和免疫调节容量6，优于骨髓来源的MSC。在先前的研究中，我们证明了一个足月胎盘含有足够的MSC高达7000临床剂量4的制造。这些特性使得胎盘的理想来源组织异体MSC的制造。 胎盘是由胎儿和母体组织7的母胎器官，从而MSC胎儿或母亲起源可以，理论上隔离。下列参考文献提供德在发展和病理尾信息，以及人体胎盘和附属器8,9的微观和宏观检查。胎盘适当的由主 ​​要的胎儿血管和分泌，并呼吁滋养层支持细胞，弥补由叶状绒毛膜（板）8所覆盖的绒毛。支胎盘绒毛沐浴在从子宫螺旋动脉交付母亲的血液，使营养物质，激素和胎儿和母亲之间的气体交换。胎盘挂靠在母体通过蜕膜基质细胞和胎儿extravillious滋养子宫内膜穿插在细胞外基质8。绒毛融合到胎儿的绒毛板在那里形成脐带8。 第一次国际研讨会上胎盘干细胞（2008年）的结果是需要规范的隔离和characterizatio升值从人类足月胎盘10个细胞的n个。由于胎盘的生理结构，不同的组织解剖，MSC的分离和预期成果的文化可以压倒新人到外地。在这个协议中，胎盘绒毛膜组织的收成，接着由MSC分离和扩增是彻底详尽。通过流式细胞术，并在体外分化的MSC表征被认为是常规5,11-13，并因此在此仅简要地详述。 正如在最近的系统的文献回顾14突出，获得MSC从胎盘绒毛膜一般都假定为胎儿。虽然，只有18％的研究审查获得的MSC的来源，并且这些中，只有研究的一半报告胎儿MSC和另一半报道母体或混合的MSC群。每个本文所述的三个组织成分（绒毛，绒毛板和蜕膜）是排版osed主要胎膜/绒毛和子宫衍生母体细胞，它保持附着在递送胎盘的一小部分的。我们提供的数据表明，从胎盘的母体侧，而非胎盘的胎儿侧分离MSC，如我们先前报告的5,11，是如果母体MSC期望的更合适的起始材料。该协议还描述了XY FISH来验证到细胞培养胎儿或母亲的贡献。虽然这是从制造商的标准方案，这种分析经常被忽略和它的重要性低估14。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HBSS</td><td>Gibco/Invitrogen</td><td>14185-052</td><td>Long name: Hanks Balanced Salt Solution</td></tr><tr><td>FBS</td><td>Gibco/Invitrogen</td><td></td><td>Long name: Fetal Bovine Serum</td></tr><tr><td>trypsin-substitute</td><td>Gibco/Invitrogen</td><td>12563-029</td><td>Long name: TrypLE Select</td></tr><tr><td>DMEM-LG</td><td>Gibco/Invitrogen</td><td>11885-092</td><td>Long name: Dulbecco&amp;rsquo;s Modified Eagles Medium-Low Glucose</td></tr><tr><td>DMEM-HG</td><td>Gibco/Invitrogen</td><td>11965118</td><td>Long name: Dulbecco&amp;rsquo;s Modified Eagles Medium-HIgh Glucose (4.5 g/L)</td></tr><tr><td>PBS (Mg+Ca+ free)</td><td>Gibco/Invitrogen</td><td>14190-250</td><td>Long name: Dulbecco&#039;s Phosphate buffered saline, magnesium and calcium free</td></tr><tr><td>Anti- anti-</td><td>Gibco/Invitrogen</td><td>15240-062</td><td>Long name: antibiotic-antimycotic solution x100</td></tr><tr><td>paraformaldehyde powder or 4% solution</td><td>any&amp;nbsp;</td><td></td><td></td></tr><tr><td>Collagenase I</td><td>Invitrogen</td><td>17100-017&amp;nbsp;</td><td>2,500 U/ml</td></tr><tr><td>Dnase I</td><td>Sigma</td><td>D5025</td><td>10 mg/ml in&amp;nbsp; 0.15 M NaCl</td></tr><tr><td>Dispase</td><td>Invitrogen</td><td>17105-041&amp;nbsp;</td><td>10 mg/ml in water (20,000 U/ml).</td></tr><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Disposables:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>Falcon or any brand</td><td></td><td></td></tr><tr><td>petri dishes, sterile plactic (25 cm and 10 cm diameter)</td><td>Nunc</td><td></td><td></td></tr><tr><td>Cell strainers (100 &amp;mu;m)</td><td>Becton Dickinson</td><td>352340</td><td></td></tr><tr><td>175 and 75 cm&lt;sup&gt;2&lt;/sup&gt; (T175 and T75) tissue culture flasks</td><td>Nunc</td><td></td><td></td></tr><tr><td>5 ml, 10 ml, 25 ml sterile serolgical pipettes</td><td>any brand</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Centrifuge&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;tissue culture incubator 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>Biological safety cabinet</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Sterile scissors and tweezers</td><td></td><td></td><td></td></tr><tr><td>Tube racks&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Pipette-boy or equivalent</td><td></td><td></td><td></td></tr><tr><td>Gilson type pipetters and sterile tips 1,000 &amp;micro;l, 200 &amp;micro;l, 20 &amp;micro;l.</td><td></td><td></td><td></td></tr><tr><td>rocking or shaking incubator (37 &amp;deg;C)</td><td></td><td></td><td></td></tr><tr><td>personal protective equipment</td><td></td><td></td><td></td></tr><tr><td>cleaning solutions (suitable for blood)</td><td></td><td></td><td></td></tr><tr><td>waste containers, correct disposal bins for tissue/blood</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents for MSC characterization&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Antibody (Clone ID)&lt;/strong&gt;</td><td>&lt;strong&gt;Manufacturer&lt;/strong&gt;</td><td>&lt;strong&gt;Catalogue No.&lt;/strong&gt;</td><td>&lt;strong&gt;Isotype&lt;/strong&gt;</td></tr><tr><td>CD73 (AD2)</td><td>Miltenyi Biotec</td><td>130-095-183</td><td>Mouse IgG1</td></tr><tr><td>CD105 (43A4E1)</td><td>Miltenyi Biotec</td><td>130-094-941</td><td>Mouse IgG1</td></tr><tr><td>CD90/Thy-1 (AC122)</td><td>Miltenyi Biotec</td><td>130-095-403</td><td>Mouse IgG1</td></tr><tr><td>CD45 (5B1)</td><td>Miltenyi Biotec</td><td>130-080-202</td><td>Mouse IgG2a</td></tr><tr><td>CD34 (AC136)</td><td>Miltenyi Biotec</td><td>130-090-954</td><td>Mouse IgG2a</td></tr><tr><td>HLA-DR (AC122)</td><td>Miltenyi Biotec</td><td>130-095-298</td><td>Mouse IgG2a</td></tr><tr><td>CD31 (PECAM-1)</td><td>BD Pharmingen</td><td>555446</td><td>Mouse IgG1</td></tr><tr><td>CD146 (541-10B2)</td><td>Miltenyi Biotec</td><td>130-092-849</td><td>Mouse IgG1</td></tr><tr><td>CD44 (DB105)</td><td>Miltenyi Biotec</td><td>130-095-180</td><td>Mouse IgG1</td></tr><tr><td>&lt;strong&gt;Isotype Controls (Clone ID)&lt;/strong&gt;</td><td>&lt;strong&gt;Manufacturer&lt;/strong&gt;</td><td>&lt;strong&gt;Catalogue No.&lt;/strong&gt;</td><td></td></tr><tr><td>Mouse IgG1 Isotype (IS5-21F5)</td><td>Miltenyi Biotec</td><td>130-092-214</td><td></td></tr><tr><td>Mouse IgG1 Isotype (IS5-21F5)</td><td>Miltenyi Biotec</td><td>130-092-212</td><td></td></tr><tr><td>Mouse IgG1 Isotype (IS5-21F5)</td><td>Miltenyi Biotec</td><td>130-092-213</td><td></td></tr><tr><td>Mouse IgG2a (S43.10)</td><td>Miltenyi Biotec</td><td>130-091-837</td><td></td></tr><tr><td>Mouse IgG2a (S43.10)</td><td>Miltenyi Biotec</td><td>130-098-849</td><td></td></tr><tr><td>MACS Buffer</td><td>Miltenyi Biotec</td><td>130-091-221</td><td></td></tr><tr><td>&lt;strong&gt;Osteogenic differentation&lt;/strong&gt;</td><td>&lt;strong&gt;Manufacturer&lt;/strong&gt;</td><td>&lt;strong&gt;Catalogue No.&lt;/strong&gt;</td><td></td></tr><tr><td>DMEM-HG</td><td>Gibco/Invitrogen</td><td>11965118</td><td>Long name: Dulbecco&amp;rsquo;s Modified Eagles Medium-HIgh Glucose (4.5 g/L)</td></tr><tr><td>FBS</td><td>Gibco/Invitrogen</td><td></td><td>Long name: Fetal Bovine Serum</td></tr><tr><td>Anti- anti-</td><td>Gibco/Invitrogen</td><td>15240-062</td><td>Long name: antibiotic-antimycotic solution x100</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td></td></tr><tr><td>&amp;beta;-Glycerol Phophate</td><td>Sigma</td><td>50020</td><td></td></tr><tr><td>L-ascorbic acid 2-phosphate</td><td>Sigma</td><td>A8960-5G</td><td></td></tr><tr><td>Alizarin Red S</td><td>Sigma</td><td>A5533-25G</td><td>For calcium matrix staining</td></tr><tr><td>&lt;strong&gt;Adipogenic differentation&lt;/strong&gt;</td><td>&lt;strong&gt;Manufacturer&lt;/strong&gt;</td><td>&lt;strong&gt;Catalogue No.&lt;/strong&gt;</td><td></td></tr><tr><td>DMEM-HG</td><td>Gibco/Invitrogen</td><td>11965118</td><td>Long name: Dulbecco&amp;rsquo;s Modified Eagles Medium-HIgh Glucose (4.5 g/L)</td></tr><tr><td>FBS</td><td>Gibco/Invitrogen</td><td></td><td>Long name: Fetal Bovine Serum</td></tr><tr><td>Anti- anti-</td><td>Gibco/Invitrogen</td><td>15240-062</td><td>Long name: antibiotic-antimycotic solution x100</td></tr><tr><td>insulin</td><td>Sigma</td><td>I2643</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td></td></tr><tr><td>Indomethacin</td><td>Sigma</td><td>I7378</td><td></td></tr><tr><td>3-isobutyl-1-methyl xanthine</td><td>Sigma</td><td>I5879</td><td></td></tr><tr><td>Oil Red O solution</td><td>Sigma</td><td>O1391-250ML</td><td>For lipid vacuole staining</td></tr><tr><td>&lt;strong&gt;XY FISH kit to determine fetal or maternal origin of cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>XY chomosome FISH kit</td><td>Vysis (Abbott Molecular)</td><td>07J20-050</td><td>Long name: CEP X SpectrumOrange/Y SpectrumGreen Direct Labeled Fluorescent DNA Probe Kit</td></tr></tbody></table>

isolation and expansion of mesenchymal stem/stromal cells derived from human placenta tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

胎盘MSC分离步骤总结于图1中从其中的MSC中分离的胎盘解剖学的三个区域在图2中突出显示这些母体蜕膜，以及绒毛膜板和绒毛的大部分胎儿组织。许多文字的书籍，文章和在线资源详细的发展和各种胎盘组织的职能作用（请参阅参考8）。 文化隔离和清除组织碎片后48小时的形态。 以下培养48小时，所述的MSC将已附着于组织培养塑料，而红细胞和大多数其他细胞碎片不会有附着。此时，介质必须用35ml新鲜培养基更换。在此之前，培养基交换，这是难以使由于精确观测大量的红细胞，这将阻碍视觉评估。培养物上清液的外观可能胎盘供体之间有很大差异。这种变化可以目测实施例1和2（ 图3A）中看到。这两个MSC隔离，这似乎是非常不同的，同时从两个不同的胎盘进行。然而，一旦洗涤，两种培养物出现了类似（见洗涤实施例3， 图3A）。 在显微镜下，在48小时的媒体交换后，只有少数细胞将附着在烧瓶（ 图3B），并且将细胞会出现从更广泛的扩展的MSC显著不同。一些碎片和红细胞将可见为浮动或附团块，这些将不会与MSC的生长造成干扰。附着在烧瓶底部的较长的成纤维细胞有可能是细胞的混合，包括MSC，造血，滋养层或内皮细胞。再次，非MSC细胞不泄露的MSC培养物，因为这些细胞通常不会在MSC培养条件下生存超过1-2代。尽管在培养物的初始外观一些变型中，随后的膨胀结果大致一致。 MSC文化形态随着时间的推移。 在此代表性的例子，七天以下隔离，小的成纤维细胞的MSC菌落是可见的，尽管非MSC细胞也可以被看作是圆形或松散地附着的细胞（ 图4A）。将附着的细胞被什么最初称为"集落部形成-fibroblast"（CFU-F）17，后来被称为MSC 18。十三天以下隔离，成纤维细胞的MSC菌落大（ 图4B）。通常，单层将在此点80-90％汇合，将细胞应帕萨<html> GED。从第2代开始，胎盘MSC单层将开发在汇合（ 图4C）的特征旋涡状的形态。除非将细胞以低密度传代时，CFU-F的形成将不再被观察到。在低密度，胎盘衍生的MSC具有比成人骨髓衍生的MSC 1较小，外观平方。而胎盘衍生的MSC和骨髓衍生的MSC表现出相似的扩散速率，胎盘衍生的细胞是不容易快速衰老5。 体外胎盘MSC的表征。 每个扩增的细胞群必须被表征，以确保它符合标准的MSC条件16，包括:（1）塑料粘附（2）的间充质表面标志物的存在和缺乏造血表面标志物，及（3）经过中胚层的能力分化。 "FO:保together.within页="1"&gt;胎盘干细胞是贴胶。 如在图4中所示，在培养的干细胞是塑料贴壁，并具有成纤维细胞样形态;这证实这些细胞符合定义MSC 16的第一准则。 胎盘MSC间充质显示屏表面标志。 第二限定MSC特征是间质表面标记的存在和不存在的造血表面标记16。因为能够明确地识别的MSC没有单一的标记，标记的面板通常使用在与流式细胞分析结合，以确定是间充质细胞，但不是造血。在这里提供的具有代表性的数据集（ 图5A）中，我们评估间质标记物CD73，CD105，CD90，CD146，和CD44的造血标志物CD45和CD34，和HLA-DR，如逢的细胞中的表达L为血管内皮标记CD31。所有在此胎盘MSC表征方法中使用的抗体的列在材料/设备的表中。进行染色按照制造商的说明，这里描述19分析方法。 细胞呈阳性的间质标记物CD73，CD105，CD44，和负的细胞表面标记物CD45，CD34，HLA-DR和CD31，如预期5,20。约37％和在我们的代表性的数据组单元的57％分别为阳性CD90和CD146 21。这两个CD90和CD146是常用的标志物MSC 21。取决于MSC组织来源，培养基组成，或通道编号22的MSC细胞表面标记轮廓可以是不同的。在我们多年的经验，我们没有观察胎盘来源的MSC的长期污染与非间充质细胞以下1-2 passages 5,11。 胎盘MSC间充质显示分化潜能 根据定义，MSC必须在体外具有中胚层分化的能力5,13。中胚层分化潜力通过任一三 - 或双谱系分化测定法通常评估。碧谱系测定通常评估成骨和成脂分化能力，同时三系试验还评估软骨细胞分化的能力。在这里提出的代表性的结果，我们表明，展开的MSC群体形成两个钙沉积，表示成骨分化的，并且脂质空泡，表示脂肪形成（ 图5B）。 为了表征这里报告在MSC群体，我们在1ml诱导培养基中接种细胞到24培养孔在6×10 4个细胞。介质康波堂费都列在材料/设备的表中。而诱导培养基配方在文献中是常见的，有在公开的配方相当的可变性。为此，我们简要地列出我们诱导培养基配方和染色的方法在这里。成骨诱导培养基含有DMEM-HG，10％胎牛血清，1×抗生素 - 抗真菌溶液，10毫β甘油phophate，100nM的地塞米松，和50μM的L-抗坏血酸-2-磷酸。脂肪诱导培养基含有DMEM-HG，10％胎牛血清，1×抗生素 - 抗真菌溶液，10微克/毫升的胰岛素，100纳米地塞米松，200μM的吲哚美辛，和500μM的3-异丁基-1-甲基黄嘌呤。这里，将培养物维持在37℃，5％CO 2培养箱并培养14天。诱导培养基在培养期间，每周两次交换。以下归纳为14天，培养物表征要么骨样基质（骨）或脂质空泡（脂肪形成）按照我们上一个欠条出版5。成骨钙基质沉积分析通过第一吸出介质，定影用4％多聚甲醛的培养物20分钟，用DPBS洗涤单层，然后根据制造商的说明用茜素红S染色来实现。脂诱导通过根据生产商的说明吸出介质，定影用4％多聚甲醛的培养物20分钟，洗涤单层，和染色油红O溶液进行评估。染色钙沉积和石油空泡，然后用光学显微镜可视化和图像保存以供将来参考。 在以前的出版物，我们有特点更广泛的4,5胎盘来源的MSC的分化潜能。胎盘衍生的MSC的成骨类似于骨髓衍生的MSC，而脂肪生成是通常效率较低的胎盘衍生的MSC 5 &lt;/ SUP&gt;。我们不经常开展以下几个原因软骨分化，虽然这已经被我们以前胎盘-MSC 5日报道。首先，虽然胚层分化能力的MSC的一个限定特性，它很可能是次要的23-25，特别是在治疗益处是可能被从MSC旁分泌分泌物26而得。其次，虽然Dominici 等人提出最低标准为临床生产成人骨髓衍生的MSC 16的更近的研究表明MSC从不同龛具有不同的固有性质和分化能力5,13,27-32。事实上，Parolini 等人提出，胎盘衍生的MSC应该分化成"一个或多个胚层"系，而不是所有的三个谱系10。最后，因为它发生时通过一个类似的许多MSC研究排除软骨细胞分化细胞内信号通路作为骨（TGFβ家族途径）33-35。 胎盘的MSC是在使用培养的这种方法起源母体，尽管原料的解剖位置。 许多出版物认为在文化14从胎儿绒毛产量胎儿MSC分离细胞。然而，正如我们以前5日报道，所有的文化胎儿绒毛膜来源，使用此协议，迅速 ​​成为丰富产妇MSC作为将直观地预计产妇蜕膜MSC文化。在这些代表性的结果，我们使用了男婴的胎盘组织，这样，就可以很容易地划定在展开细胞群的胎儿和母体细胞的贡献。在这些研究中，我们使用的材料/设备的表中列出的XY FISH套件，并遵循制造商的说明。 图6） 。通过通道2，从母体组织的细胞群体为〜100％母体细胞（XX）和胎儿细胞（XY）检测不到。这表明，母体组织的物理剥离导致在随后的培养物母体细胞的富集。然而，关键是从胎绒毛和绒毛膜板衍生的培养物考虑培养的结果。在第0代，无论胎儿绒毛和绒毛膜板衍生的培养为〜85％XY，或胎儿起源，这表明有针对性的清扫富集胎儿细胞（ 图6）。在第0代，既培养物含有〜15％的产妇细胞（XX）的污染。出人意料的是，在通道2中，两个胎儿培养物填充〜100％母体细胞（XX）和胎儿细胞（XY）不再检测到。 XY FISH分析表明，母体细胞（XX染色体），并迅速接管一贯的文化从胎儿绒毛组织来源。这是一个关键的培养观察常常被忽视5。这种分析的细节被包括在这个协议，因为它表明，母体细胞迅速填充所有培养时的DMEM补充有10％FBS的无旨在支持胎儿衍生种群的附加因素是所使用的非常重要的观察。 <img alt="图1" src="/files/ftp_upload/54204/54204fig1.jpg" /> 图1:胎盘MSC分离过程中的概要 （ 步骤1）定向自己与胎盘解剖。 （ 步骤2）手动无论从蜕膜，绒毛或用剪刀绒毛膜板解剖10克组织。 （ 步骤3）百果Ť他解剖蜕膜，绒毛或绒毛膜板组织部分成件件精品，用剪刀或手术刀。 （ 第4步 ），通过在分散酶和胶原酶I.一个1-2小时消化解放从微细片的细胞 （ 步骤5）分离由脉冲离心和/或通过细胞过滤洗涤他们的纤维组织细胞。收集和悬浮在培养液中的细胞和置于培养瓶中。 <a href="https://www.jove.com/files/ftp_upload/54204/54204fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 间充质基质细胞（MSC）将根据他们的倾向塑料吸附和生存和在培养基中增殖的能力来选择。最后，在结果部分，所述扩大的MSC可被表征并存储在以后的实验中使用。 图2:在这个过程中分离出的人的足月胎盘和组织的解剖学将收获的第一组织是母体蜕膜。蜕膜是组织是从子宫壁（蜕膜由绿色标记标识）流下之后保持为胎盘的表面上的薄层。将从胎盘的内部被收获第二组织是胎儿绒毛（蓝色标记）。到收获的第三个组织是胎儿绒毛膜板（红色标记）（改编自参考36）。 <a href="https://www.jove.com/files/ftp_upload/54204/54204fig2large.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/54204/54204fig3.jpg" /> 图3:文化形态的分离和再经过48小时移动所述组织碎片（A）的培养上清的外观可以基本上胎盘供体之间洗掉碎片之前变化。实施例培养1和2说明这种变化。这两个隔离，同时实施，但是从两个不同的胎盘。一旦洗涤培养物将是清楚RBC和组织碎片的所示实施例3中的随后的膨胀结果大致一致。 （B）继48小时媒体交换，只有少数细胞将附着在烧瓶中。比例尺= 200微米。 <a href="https://www.jove.com/files/ftp_upload/54204/54204fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54204/54204fig4.jpg" /> 图4:MSC文化随着时间的推移形态 （A）隔离七天后，小Fibroblastic MSC菌落可见，尽管非MSC细胞也将到场为圆形或松散连接的细胞。 （B）隔离13天后，成纤维细胞克隆MSC大，往往MSC的单层汇合，并准备通过。 （C）从第2代开始，在MSC单层将开发在合流的特性旋涡状的形态。比例尺= 200微米。 <a href="https://www.jove.com/files/ftp_upload/54204/54204fig4large.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/54204/54204fig5.jpg" /> 图5:用流式细胞仪和中胚层分化的MSC特性 （A）胎盘绒毛来源的MSC显示，流式细胞仪分析的经典MSC标记谱，尽管这些细胞表面标记，EXPRES锡安是所有类型的人类MSC的相似。每个直方图显示与在y轴（最大值的％）规格化细胞计数的信号强度（x轴）。在该代表性数据集的细胞表达CD73，CD105和CD44，和阴性造血标志物CD45和CD34和HLA-DR。 （B）的胎盘的MSC通常经受健壮成骨分化，但是（C）的脂肪形成分化可以比与骨髓衍生的MSC的效率较低。标题中的B和C图像是放大40倍。 <a href="https://www.jove.com/files/ftp_upload/54204/54204fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54204/54204fig6.jpg" /> 图6:胎盘的MSC是在使用培养的这种方法起源母体，尽管原料的解剖位置。该图显示胎儿（男性，XY）和母亲每隔一个通道从蜕膜，绒毛和绒毛板组织中分离胎盘MSC文化（女，XX）细胞成分的定量。母体细胞（XX）快速，可重复接管从胎儿绒毛组织来源的文化。胎儿= =男性在单个细胞检测XY染色体，在单个细胞检测产妇=女= XX染色体。这里给出的数据是从男婴N = 3个独立的供体胎盘，用最少的100个细胞染色XY FISH和计算每个数据点。柱代表平均值，和误差条反映一个标准差。 <a href="https://www.jove.com/files/ftp_upload/54204/54204fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue at JoVE.com

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

这里我们描述了从人足月胎盘的胎儿和母体组织的解剖方法，随后分离并从这些组织的间充质干细胞/基质细胞（MSC）的扩展。

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

isolation-expansion-mesenchymal-stemstromal-cells-derived-from-human

Research

JoVE Journal

Developmental Biology

36.2K 次观看.  The University of Queensland. 这里我们描述了从人足月胎盘的胎儿和母体组织的解剖方法，随后分离并从这些组织的间充质干细胞/基质细胞（MSC）的扩展。 

视频: Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

科研

发育生物学

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

隔离和人力富集脂肪基质细胞的成骨增强

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

生物学

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

早产儿和足月婴儿脐带源性间充质干细胞的分离与鉴定

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

医学

从人脐带中解剖和分离 WJ-MSC

Isolation of Umbilical Cord-Derived Mesenchymal Stem Cells with High Yields and Low Damage

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) from the umbilical cord (UC) has gained increasing interest in the biomedical field as an outstanding source of MSCs. However, challenges such as limited supply and lack of standardization in existing methods have arisen. This article presents a novel method for enhancing MSC yield by dissecting intact WJ from the umbilical cord. The method employs blunt dissection to remove the epithelial layer, maintaining the integrity of the entire WJ and resulting in an increased quantity and viability of harvested MSCs. This approach significantly reduces WJ waste compared to conventional sharp dissection methods. To ensure the purity of WJ-MSCs and minimize external cellular influence, a procedure utilizing internal tension to peel off the endothelium after flipping the UC was conducted. Additionally, the Petri dish was inverted for a short time during explant culture to improve attachment and cell outgrowth. Comparative analysis demonstrated the superiority of the proposed method, showing a higher yield of WJ and WJ-MSCs with better viability than traditional methods. The similar morphology and expression pattern of cell surface markers in both methods confirm their characterization and purity for various applications. This method provides a high-yield and high-viability approach for WJ-MSC isolation, demonstrating great potential for the clinical application of MSCs.

作者聚焦:干细胞来源的血管化类器官的进展和沃顿胶的优化分离以增强 MSC 生产

This study presents a unique blunt dissection procedure to preserve the integrity of Wharton's jelly (WJ), resulting in less damaged WJ and a greater quantity and viability of the harvested mesenchymal stem cells (MSCs). The method demonstrates superior yield and proliferative ability compared to conventional sharp dissection methods.

高产量和低损伤分离脐带来源的间充质干细胞

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) ...

从人胎盘组织源分离和间充质干扩张/基质细胞

摘要

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此视频中的章节

人类骨髓间充质干细胞的分离及其培养的多孔骨基质

隔离和人力富集脂肪基质细胞的成骨增强

从胎盘中的造血干细胞的分离和分析

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

早产儿和足月婴儿脐带源性间充质干细胞的分离与鉴定

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

高产量和低损伤分离脐带来源的间充质干细胞

高产量和低损伤分离脐带来源的间充质干细胞

高产量和低损伤分离脐带来源的间充质干细胞

从人胎盘组织源分离和间充质干扩张/基质细胞

摘要

探索更多视频

此视频中的章节

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

隔离和人力富集脂肪基质细胞的成骨增强

从胎盘中的造血干细胞的分离和分析

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

早产儿和足月婴儿脐带源性间充质干细胞的分离与鉴定

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

高产量和低损伤分离脐带来源的间充质干细胞

高产量和低损伤分离脐带来源的间充质干细胞

高产量和低损伤分离脐带来源的间充质干细胞

人类骨髓间充质干细胞的分离及其培养的多孔骨基质