Name: mesenchymal stromal cell culture and delivery in autologous conditions: a smart approach for orthopedic applications
Uploaded: 2016-12-08T15:00:00.000+00:00
Duration: 10 min 30 s
Description: Watch this Scientific Journal Video about Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications at JoVE.com

This study was funded by the Tuscany Region (grant number 539999_2014_Petrini_CUCCS). The authors would like to thank Prof. S. Berrettini for authorizing the use of the Otolab laboratory and instruments, and the surgery staff from the Orthopedic Clinic II of the University of Pisa, for the fundamental cooperation in sample harvesting. The support provided by Dr. F. Scatena is gratefully acknowledged. Finally, many thanks are due to Mr. J.G. De La Ossa for his precious contribution to histologic processing.

本协议是根据世界医学协会的有关研究涉及人类的道德行为赫尔辛基宣言的发展。它被批准了Azienda Ospedaliero-Universitaria蜗牛的伦理委员会。 注意: （根据步骤1.1）15名来自正在进行例行全髋关节置换手术的患者获得骨髓;从自体外周血13得到的凝块制备等离子体;自体血清，作为培养基的补充，从自体的全血采收集。所有患者均收到的有关过程的详细信息，并签署书面同意书。 1.骨髓样本的收集<ol><li>收集股骨髓腔骨髓全髋关节置换术（用于研究研究）之后。简要地说，手术准备的髓管的至h中乌斯假肢茎，收集骨髓血液溢出（约10 - 15毫升，取决于假体大小）15。 要么</li><li>从局部麻醉下髂骨采集骨髓穿刺，以下约20标准血液学方法-提前30天（临床应用）16。 注:在肝素化的注射器中这两种情况下（以防止凝块形成）必须用于骨髓恢复。 </li></ol> 2.自体血浆的制备<ol><li>从在含K3 EDTA真空采血管的患者（5.4毫克的3mL管）和离心机2400×g的15分钟收集20毫升外周血。 </li><li>吸在一个15毫升管中的血浆成分，并在-20℃冷冻直到使用。 </li></ol> 3.自体血清的制备<ol><li>血浆的单位转移到使用峰值传输包。通过焊接和weig断开填充袋小时袋来计算血浆（ 图1A）的体积。 </li><li>通过经由端口连接器（ 图1B）注射葡萄糖酸钙100毫克/毫升10％w / v的凝固的自体血浆。混合袋后，将其放置在4°CO / N无晃动，以促进血栓形成。 </li><li>离心4900×克袋在室温下15分钟。就拿离心机斗出来的离心机，并小心地取出袋（遵循制造商的准则编制的血小板裂解液）（ 图1C）。 </li><li>隔离血块正好夹上方，以方便过滤。过滤试剂盒连接到使用放入过滤试剂盒的出口的尖峰转移袋。 </li><li>挂袋，打开蓝色夹子，让通过重力血清流。不施加压力的过滤器，以避免转印纤维蛋白的入袋。过滤后，密封管和取出袋（按照MANUFAC商的准备血小板裂解准则）。 </li><li>连接专用线到最终包和转移到血清50ml试管层流长椅下，避免微生物污染。 </li><li>取样品用注射器测试缺乏纤维蛋白原和需氧和厌氧细菌培养17进行无菌试验。正确标记管和冻结他们 - 20°C，直到使用。 </li></ol> 4.膨胀介质的制备<ol><li>准备500毫升完整的增殖培养基中。到的Dulbecco改良Eagle培养基 - 低葡萄糖（DMEM-LG）中，添加10％自体血清（如在第3节中所述获得），2mM谷氨酰胺，1％抗生素溶液和1％抗真菌溶液。无菌过滤完整的增殖培养基。 </li></ol> 5.隔离从骨髓的hMSCs <ol><li>从注射器 - （10毫升5）到50mL锥形涂传送骨髓样品是并用无菌盐水稀释（比率1:4）。 VORTEX 50毫升管30秒，以分解的细胞团。 </li><li>暖密度梯度（密度1.077克/升;见材料的表 ）至RT使用之前，轻轻地分层稀释的骨髓（以防止两层混合）加入20毫升样品，以15毫升的密度到密度梯度梯度为每个50毫升管（ 图2A）。 </li><li>离心机在没有在25℃下30分钟制动器400×克，然后收集在液 - 液界面的单核组分和使用无菌5毫升吸管将其转移到新的50毫升管中。 </li><li>完全增殖培养基洗收集的单核组分两次，并在25℃下离心，在400×g下管10分钟，得到细胞团。 </li><li>弃去上清液并轻轻悬浮在5mL完全扩散介质的每个细胞沉淀。稀释在1:100的比率为细胞计数。 </li><li>1稀释台盼蓝染色来评估细胞生存力:使用血细胞计数器1后计数细胞。 </li></ol> 6.的hMSCs的文化自体血清存在<ol><li>填充两个或更多个75cm 2的组织培养（TC）瓶中用15ml新鲜的完全增殖培养基，并将它们在37℃下转移到细胞培养孵化器和5％的CO 2，直到使用。 </li><li> 0.3种子&#160; - 0.5×10 6个细胞/ cm 2（37.5×10 6细胞/ 15毫升新鲜完全增殖培养基），在37℃下孵育，5％CO 2下48小时。 </li><li>同时，种子1×10 6个细胞在用5ml的菌落形成单位新鲜完全增殖培养基的25cm 2的TC烧瓶-成纤维细胞（CFU-F）的测定法。 （第12待续）培养2周。 </li><li>弃去培养基，轻轻地从步骤6.2完全扩散介质REM洗瓶奥雅纳非贴壁细胞和碎片。新鲜培养基加入到瓶中，并每周更换一半的两次，直到70％ - 80％汇合（原代培养物，0代（P0））（ 图2B）。 </li><li>通过使用特定的重组动物游离蛋白酶回收细胞（见材料的表 ;使用每个烧瓶中的溶液3毫升如制造商推荐的），在37℃下孵育，5％CO 2的10分钟。加6毫升每瓶完整的增殖培养基中，在一个50毫升管收集。重复步骤5.6。 注:重要提示:使用这种蛋白酶代替胰蛋白酶允许一些中胚层祖细胞（多用途储值卡）存在于文化，这是抗胰蛋白酶的恢复。 </li><li>为进一步扩大，replate细胞悬浮液以2,000 - 3,000细胞/ cm 2在新75cm 2的TC瓶（P1）（ 图2C）。 </li><li>使用细胞等分来评价干细胞的分化潜能对成骨细胞，脂肪细胞和软骨细胞谱系。执行按照制造商的建议（ 图2D）分化测定。 注意:不同的染色方法可以用于评估多向分化。 </li></ol> 7.制备制药补充的成骨细胞培养基中<ol><li>稀抗坏血酸溶液（溶液1A:抗坏血酸200毫克/毫升（见材料表 ）1:10用DMEM-LG，以得到20毫克/毫升溶液（溶液1B，工作液）的解决方案1A的库存等份图1B在-20℃直至使用。 </li><li>在10mL无菌水中重悬1克氢化可的松（溶液2A:氢化可的松100毫克/毫升（见材料表 ）稀溶液2A以1:1000用DMEM-LG，得到100微克/毫升溶液（溶液2B，工作溶液）中。溶液2A和2B在-20℃直到使用的库存等分试样。 </li><li>当使用prepare如下新鲜成骨培养基:有10％的自体血清，50微克/毫升抗坏血酸和0.4微克/毫升氢化可的松添加的DMEM-LG。无菌过滤成骨网上平台。 </li></ol> 8.成骨诱导前和的hMSCs恢复<ol><li>彻底清除增殖培养基和细胞收获前增加成骨细胞培养基96小时（即 80 - 90％汇合在7天通常是到达）。提供额外的成骨细胞培养基变化细胞收获前24小时。 </li><li>分离的细胞如步骤6.5中所述，轻轻重悬沉淀加入2毫升完全增殖培养基在50mL管中。 注:细胞生存力可以降低（约10％），为骨诱导的结果。细胞聚集可以观察到。 </li><li>计数后，洗完全增殖培养基和离心将细胞在300×g下5分钟。轻轻重悬沉淀在1 - 2×10 6个存活细胞/ 2自体血浆的溶液（见第2节）每50毫升管。 </li></ol> 9.准备的hMSC /纤维蛋白凝块的构造<ol><li>在100毫克/毫升添加150微升葡萄糖酸钙的从步骤8.3获得的各50毫升管中。重悬下温和摇动的细胞。 </li><li>孵育细胞在37℃和5％CO 2的15 - 30分钟，得到的hMSC /纤维蛋白凝块构建体（ 图3A）。制备纤维蛋白凝块构建体没有细胞被用作对照。 注:重要提示:这是值得推荐的细胞活力检测前至少2小时，准备这种控制血块，因为它需要交联30分钟以上。 </li></ol> 10.细胞活力检测<ol><li>从50 mL管中除去液体，并添加含10％v / v的细胞生存力试剂完全增殖培养基的1毫升（AB;参见材料的表 ），根据制造商的指导方针。 </li><li>第孵育在37℃È管和5％CO 2的3小时。测量在含有600nm的参考波长570纳米的吸光度（AB在时间0; T0）。 </li><li>弃去上清液，加入2毫升的新鲜完整的增殖培养基中。孵育在37℃，5％的CO 2 O / N。第二天（AB在时间24小时; T24），重复步骤10.1至10.3（ 图3B）。 </li></ol>细胞活力和钙沉积的纤维蛋白凝块内部构造11.组织学评估<ol><li>固定在4％的hMSC /纤维蛋白凝块构建w / v的中性缓冲的福尔马林O / N在4℃。洗涤4X在D-PBS中的70％乙醇15分钟，并存储。 </li><li>脱水的样品在40℃的一系列梯度乙醇:80％一次30分钟，95％两次45分钟和3×100％1小时。在二甲苯澄清两次在40℃下45分钟。 </li><li>冲洗液体石蜡的样品在60℃下2小时，将它们嵌入。用切片机切蜡块和挂载Ť他在载玻片切片（5微米）。 </li><li>染色使用标准苏木精和曙红（H＆E）方法（ 图3C）的脱蜡段。 </li><li>通过用1％硝酸银的部分15分钟，2分钟，0.5％邻苯三酚，以及2分钟的5％硫代硫酸钠进行冯Kossa染色。染液在含有5g硫酸铝5分钟的溶液稀释0.1％的核快红的部分和漂洗自来水中5分钟。安装部分有安装代理。 </li><li>评估矿物沉积如光学显微镜（放大400倍）（ 图3D）下黑色颗粒。 </li></ol> 12. CFU-F分析<ol><li> （来自步骤6.3）。用D-PBS洗为25cm 2烧瓶中。通过使用标准的可能 - 的Grunwald /吉姆萨方法染色。通过在光学显微镜下进单个菌落（ 图4）在视觉量化的hMSC号码。 注:如果骨髓被作为在步骤1.2。和2的章节 - 9在良好生产规范（GMP）提供必要的监管批准执行，所述的hMSC /纤维蛋白凝块构建体可被植入用于整形外科应用。 </li></ol>

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此协议关注的关键步骤使用成人血清和蛋白酶，允许获得生物安全hMSC的治疗。特别是，人的成人血清使得隔离和维护，而蛋白酶确保的MPC的收成。这些存在于骨髓中，可以以新鲜的hMSCs产生，从而保证沿着培养时间可行的hMSCs的储未成熟细胞。不是所有的的MPC可以收集，因为增加曝光时间来蛋白酶活性是有害的hMSC生存能力。出于这个原因，一个10分钟的蛋白酶孵育被选为的MPC的回收和的hMSCs的生存能力之间的最佳折衷。另一个关键步骤是osteodifferentiation时间。事实上，广泛的体外 osteodifferentiation将大大降低细胞活力，从而影响体内最终的骨形成。最后关键的一步在于自体生物可吸收支架援用，which的通过在从血浆凝固纤维蛋白凝胶包埋细胞（hMSCs和的MPC）中获得。 一个关键的步骤，以提高的MPC的产率在较高的浓度播种单核细胞。使用单采程序得到血浆和葡萄糖酸钙处理它使得有可能在大量达到自体血清。已经观察到，人血清作为介质补充相当于胎牛血清（FBS）中的hMSC培养物。然而，在我们的经验，辅以FBS的完全培养基有利于比成人快血清衰老。一个显著的步骤是给予最小的骨诱导到的hMSCs。事实上，这种处理导致细胞容易地朝向成骨谱系分化，因此是为了避免在体内进一步细胞转化中有用。为了维持最低限度osteoinduced的hMSCs良好的生存能力，建议仔细按照步骤6.5中所述的建议。一个Ð8.2，包括处理，离心和介质的量被添加。如果分离性输血过程不可用，仍然有可能通过执行多个抽血到患者，或在替代方案中，通过购买汇集AB血清来进行此协议。显然，为了从替补到床边带来这个协议中，它是强制性的，以具有GMP细胞工厂，或等同物，购。 限制这种技术关注贫血，血液肿瘤内科和骨科病人受骨髓炎的应用程序，绘制了大量的来自贫血患者血液中，应尽量避免。在肿瘤学的患者中，细胞样品的质量是通过化疗的影响，而在骨髓炎患者感染可影响最终结果。在所有在其中自体血清不足或不适合的情况下，汇集男性AB血清代表一个很好的选择。 使用Cell /纤维蛋白CLO对于可能的临床应用吨构建体是释放完全自体细胞治疗，这可能是很容易在手术过程中，以处理和模具，导致极好的结果来治疗骨非工会13是至关重要的。最小的操作和丰富的操作9:临床目的，是的hMSCs通常通过两种主要的程序管理。为了克服有关的广泛的体外培养物中的问题，如在细胞形态和大小18异常，我们进行了很短的时间离体细胞扩增和骨的分化（仅4天）。 本文与短信元扩张和osteodifferentiation倍描述一起自由异种协议，证明是在为了获得快速骨生产体内诊所相关，没有组织过度生长和转化的证据，从而证实其有效性和长在骨修复-term安全（图5） </stroNG&gt; 13。 本报告中提出的方法的目的是证明，在用于在整形外科手术可能使用自体的条件体外扩增的hMSC的疗效和安全性。该协议采用从骨髓并培养在补充有自体血清和嵌入式自体血纤维蛋白凝块，从而确保了完全自体细胞治疗的培养基中分离的hMSCs。两折骨诱导，就在第二支队之前，提高hMSC的分化为成骨细胞的能力。其结果是，该技术特别适用于骨缺损的应用，因为它并不限于假关节。未来可能的应用可能涉及距骨囊肿和骨损管理。

人类间充质基质细胞（hMSC）的培养表示的用于组织工程应用的最佳小区的来源之一是促进成骨1,2。它们很容易地从骨髓和其他成人组织中分离，并表达典型的表面标记如CD90，CD105，CD73 1。此外，它们可以分化成多种细胞类型，如成骨细胞，软骨细胞和脂肪细胞3。其治疗效果归因于他们的再生和营养特性4。的hMSCs可以在整形外科手术中使用，以及在其它的再生临床应用。它们优选与支架相结合，以提高临床结果5。 与其它材料相比，纤维蛋白凝胶显示令人感兴趣的性质，如粘合性，吸收和营养物，这使得它适用于各种组织工程应用6,7-极为有用的高效率的运输。 在我们的方法中，纤维蛋白胶，从患者自身的血液来源，自体血清， 用于体外的hMSCs培养，被用作在矫形场8一个可能的治疗方案。 用于临床目的，的hMSCs通常通过两种主要的方法施用:（i）该"一步法"的过程（ 即 ，最小的操作），它允许骨髓的自动移植，无论是整体或浓缩（ 即 ，单核细胞），在手术过程中;及（ii）中的"两步"的过程（ 即，大量的操作），它是基于的hMSCs的体外扩增以增加植入前的产量，并且需要ģMP设施9。有趣的是，用人类成人血清代替小牛血清培养细胞允许回收，用的hMSCs一起，将细胞的一个子集（1 -单核培养物的10％）被称为中胚层祖细胞器（MPC），能够在体外分化成新鲜的hMSCs 10,11。因此，hMPCs可能当单独12,13的hMSCs相比起到再生过程一个显著的作用。最后，短期骨诱导推的hMSCs开始它们分化成成骨谱系而不丧失其增殖潜能和生存能力12。这些结果证实，已经报道增强体内骨形成通过的hMSCs先前的研究，随后在成骨培养基中14预培养物。而且，自体血浆凝块作为用于细胞递送的支架可容易地由外科医生操纵和成形，以适应骨腔13的形状。 钍erefore，这种方法可以为那些研究人员和临床谁瞄准从替补翻译其基于的hMSC疗法在整形外科应用床边极为有用。

<table><tbody><tr><td>Triple Select (1x)</td><td>GIBCO, LIFE TECHNOLOGIES</td><td>12563-029</td><td>cell culture</td></tr><tr><td>Trypan blue stain&amp;nbsp;</td><td>GIBCO, LIFE TECHNOLOGIES</td><td>15250061</td><td>cell culture</td></tr><tr><td>DMEM-LG, no glutamine, no phenol red</td><td>GIBCO, LIFE TECHNOLOGIES</td><td>11880-028&amp;nbsp;</td><td>cell culture</td></tr><tr><td>Glutamax (100x)</td><td>Gibco, Life Technologies</td><td>35050038</td><td>cell culture</td></tr><tr><td>Penicillin/Streptomycin sol.</td><td>Gibco, Life Technologies</td><td>15140122</td><td>cell culture</td></tr><tr><td>Alamar Blue (AB)</td><td>Gibco, Life Technologies</td><td>DAL1025</td><td>proliferation/viability assay</td></tr><tr><td>Ficoll-Paque Plus</td><td>GE Healthcare</td><td>17-14440-02</td><td>cell culture</td></tr><tr><td>D-PBS</td><td>Gibco, Life Technologies</td><td>14190-094</td><td>cell culture</td></tr><tr><td>StemMACS AdipoDiff Media</td><td>Miltenyi Biotech</td><td>130-091-677</td><td>cell culture</td></tr><tr><td>StemMACS ChondroDiff Media</td><td>Miltenyi Biotech</td><td>130-091-679</td><td>cell culture</td></tr><tr><td>hMSC Osteogenicdiff BulletKit</td><td>Euroclone&amp;nbsp;</td><td>LOPT3002</td><td>cell culture</td></tr><tr><td>Vitamin C (ascorbic acid)</td><td>Bayer</td><td>ATC Code: A11GA01</td><td>Pharmaceutical grade drug</td></tr><tr><td>Flebocortid Richter (hydrocortisone)</td><td>Aventis Pharma</td><td>ATC Code: H02AB09</td><td>Pharmaceutical grade drug</td></tr><tr><td>Victor X3, reader</td><td>PerkinElmer</td><td>2030 multilabel reader</td><td>proliferation/viability assay</td></tr><tr><td>Silver nitrate</td><td>Sigma Aldrich</td><td>209139</td><td>histological assay</td></tr><tr><td>Pyrogallol</td><td>Sigma Aldrich</td><td>16040</td><td>histological assay</td></tr><tr><td>Sodium Thiosulphate</td><td>Sigma Aldrich</td><td>S8503</td><td>histological assay</td></tr><tr><td>Histoplast LP</td><td>Thermo Scientific</td><td>8332</td><td>histological assay</td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>32213</td><td>histological assay</td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>2860</td><td>histological assay</td></tr><tr><td>Nuclear fast red</td><td>Sigma Aldrich</td><td>60700</td><td>histological assay</td></tr><tr><td>Formalin</td><td>Bio-optica</td><td>05-K01009-X40</td><td>histological assay</td></tr><tr><td>Eosin B</td><td>Sigma Aldrich</td><td>45260</td><td>histological assay</td></tr><tr><td>DPX</td><td>Sigma Aldrich</td><td>6522</td><td>histological assay</td></tr><tr><td>Haematoxylin</td><td>Sigma Aldrich</td><td>H3136</td><td>histological assay</td></tr><tr><td>Aluminium Sulphate</td><td>Sigma Aldrich</td><td>202614</td><td>histological assay</td></tr><tr><td>Xylol</td><td>Sigma Aldrich</td><td>534056</td><td>histological assay</td></tr><tr><td>Microtome</td><td>Leika</td><td></td><td>histological assay</td></tr><tr><td>May Grunwald</td><td>Sigma Aldrich</td><td>MG1L-1L</td><td>histological assay</td></tr><tr><td>Giemsa</td><td>Sigma Aldrich</td><td>32884-250ML</td><td>histological assay</td></tr><tr><td>Transfer bag</td><td>Biom&amp;eacute;rieux</td><td>BC0300031</td><td>cell culture</td></tr><tr><td>Filtration kit</td><td>Macopharma</td><td>BC0800010</td><td>cell culture</td></tr><tr><td>Calcium Gluconate</td><td>Galenica Senese</td><td></td><td>Pharmaceutical grade drug</td></tr><tr><td>Bact Alert</td><td>Biom&amp;eacute;rieux</td><td>259790 anaerobic&amp;nbsp;</td><td>microbiological assay</td></tr><tr><td>Bact Alert</td><td>Biom&amp;eacute;rieux</td><td>259789 aerobic&amp;nbsp;</td><td>microbiological assay</td></tr><tr><td>Steryle Luer-Lok Syringe 50 mL</td><td>BD Plastipak</td><td>300865</td><td>cell processing</td></tr><tr><td>Heraeus Cryofuge 6000i</td><td>Thermo Scientific</td><td></td><td>blood bag centrifuging</td></tr><tr><td>SL16R Centrifuge</td><td>Thermo Scientific</td><td></td><td>blood tube centrifuging</td></tr></tbody></table>

mesenchymal stromal cell culture and delivery in autologous conditions: a smart approach for orthopedic applications

人类间充质基质细胞（hMSC）的培养是在与不同的介质体外培养。他们在临床环境中的使用限制，但主要依赖于异种养分他们的文化产生潜在的生物危害和炎症的风险。人的衍生物或重组材料是第一志愿考生，以减少这些反应。因此，培养补充剂和自体来源的材料代表了最好的营养物质和最安全的产品。 在这里，我们描述了用于分离和骨髓的hMSCs培养新的协议在自体的条件 - 即源自患者的血清作为培养基和纤维蛋白作为的hMSC给药的脚手架补充。事实上，修复hMSC /纤维蛋白凝块结构可能有多种临床应用极为有用。特别是，我们专注于自己的矫形外科使用，在允许的情况从捐赠者的自己的血源性纤维蛋白凝块有效的细胞输送养分/废物交换。为了确保最佳的安全条件下，它是极为重要的，以避免的hMSC转化和组织过度生长的风险。由于这些原因，在本文中所描述的方法也指示一个最小体外的hMSC扩张，以减少细胞衰老和形态学变化，和短期骨分化植入前，以诱导成骨谱系说明书中，从而减少随后的不受控制的风险增殖。

自体血清的制备血浆凝固，通过添加葡糖酸钙到转印袋进行，允许自体血清的大量的回收，如所需的hMSC的培养。事实上，使用这种技术，能够实现高达200毫升血清的（ 图1）。 HMSC分离，培养和分化用自体血清骨髓单核细胞使用密度梯度，这引起了一个中间环层（ 图2A）分离。通过电镀这些细胞，并用洗涤除去非粘附的人，它可以分离的hMSCs（ 图2B）。下的自体培养条件下，细胞的子集，被称为的MPC，是在P0和百分比高达Ť检测O 10％，这取决于患者变异性（ 图2B）。的MPC仍然如果用于细胞传代（ 图2C）的蛋白酶replating（即 P1）的后存在。的hMSCs分离和自体环境培养维持他们的能力，对三个著名的中胚层谱系（成骨细胞，脂肪细胞，软骨细胞和）区分。进行分化实验以比较使用标准（nonautologous）培养条件下获得的那些的hMSC人口身份。作为该协议，冯Kossa染色，四氧化锇染色，并爱茜蓝染色，在pH 1的功能测定法代替那些由分化培养基制造商建议的被选择（参见材料的表 ）（ 图2D）。 准备，可行性和MSC /纤维蛋白凝块结构的组织学特征图3A）所包围的形成。内这些血浆凝块，所述的hMSCs是可行的，通过从蓝色细胞活力染料T24的颜色变化（对照不含细胞），为表现出粉红色（细胞化凝块）（ 图3B）。血块内细胞活力是由H＆E染色证实，呈现出保存完好的细胞形态（ 图3C）。此外，一个最小的骨诱导只是第二小区收获前产生了初步钙沉积由刺激的hMSCs（ 图3D）。 菌落形成单位的hMSC菌落2周培养后，存在由CFU-F测定（ 图4）示出。 S ="jove_step"FO:保together.within页="1"&gt;构建骨不愈合MSC /纤维蛋白凝块中的应用在这种方法（步骤1.1）中描述获得HMSC /纤维蛋白凝块结构已成功应用在8体恤案件13来治疗严重上肢骨折不愈合。长期（最大7.6个月）评估确认为所有患者成功的临床和功能结果，没有组织增生或肿瘤形成的证据。 <img alt="图1" src="/files/ftp_upload/54845/54845fig1.jpg" /> 图1.自体血清的制备。 （一）等离子体装置在使用秒杀为中转行李传送; （B）中的等离子体是由注入通过端口连接器葡萄糖酸钙凝固; （C）血液袋离心机用于分离血清。/files/ftp_upload/54845/54845fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54845/54845fig2.jpg" /> 图2. hMSC的分离，培养和分化的自体血清。 经过骨髓密度梯度离心获得的（A）的单核细胞层（蓝线窗口）; （B）中 ，它出现在P1的细胞培养; （C）细胞培养，因为它出现在P0（小学文化）; （B - C）箭头显示在海安文化多用途储值卡的存在。比例尺为100微米; （D）多向分化实验的组织化学结果。成骨分化采用冯·科萨染色黑，脂肪细胞分化矿物基质沉积进行评估是由脂肪空泡存在所示，锇tetroxi黑色染色德，和软骨细胞分化是通过硫酸化糖胺聚糖，其在青色用爱茜蓝染色，在pH 1比例尺染色的存在显示是50微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54845/54845fig2large.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/54845/54845fig3.jpg" /> 图3.准备，MSC /纤维蛋白凝块构造的活力和组织学表征。 （一）hMSC的/纤维蛋白凝块构造，因为它出现的交联后; （B）的 AB测定的结果:所述的MSC是血纤维蛋白凝块内可行的，通过细胞活力染料（T24）的颜色变化所证明，从蓝色（对照不含有细胞的右侧）到粉红色（细胞化凝块，在剩下）。 （C - D）的hMSC /纤维蛋白凝块结构的组织化学结果。比例尺是50微米。 （C）的苏木精和示出嵌入在纤维蛋白基质的活细胞曙红染色; （D）矿物基质沉积冯Kossa染色，这证实了最初的成骨黑染色。 <a href="http://ecsource.jove.com/files/ftp_upload/54845/54845fig3large.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/54845/54845fig4.jpg" /> 图4. CFU-F试验。 与五月 - 格伦沃尔德/姬姆萨染色的方法培养的hMSCs烧瓶中的图片。在放大区域是表示一个的hMSC菌落的形态的光显微照片。比例尺为100微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54845/54845fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 在构建骨不连图5的hMSC /纤维蛋白凝块的应用。 （A）的受假关节的患者的上肢的X射线; （B）只植入前的hMSC /纤维蛋白凝块结构; （三）hMSC的/纤维蛋白凝块结构的手术应用; （D）的同一患者的上肢的5年后的X射线。这个数字是从Giannotti S. 等再版。以最小的修改13。 <a href="http://ecsource.jove.com/files/ftp_upload/54845/54845fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications at JoVE.com

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

培养人骨髓间充质基质细胞（hMSCs）与自体血清，降低排斥异种材料等负面影响的风险。它也允许对中胚层祖细胞的子集，这可以提供新鲜的hMSCs的恢复。在自体纤维蛋白凝块嵌入的hMSCs可轻松处理和有效的手术植入。

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

mesenchymal-stromal-cell-culture-delivery-autologous-conditions-smart

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Bioengineering

9.9K 次观看.  University of Pisa. 培养人骨髓间充质基质细胞（hMSCs）与自体血清，降低排斥异种材料等负面影响的风险。它也允许对中胚层祖细胞的子集，这可以提供新鲜的hMSCs的恢复。在自体纤维蛋白凝块嵌入的hMSCs可轻松处理和有效的手术植入。 

视频: Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

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 ...

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

生物工程

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

生产和治疗间充质干行政/基质细胞（MSC）球体底漆在三维培养下无异物条件

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

修复的临界大小的颅骨缺损模型脂肪间充质干细胞的收获Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

医学

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Autologous Microfractured and Purified Adipose Tissue for Arthroscopic Management of Osteochondral Lesions of the Talus

In recent years, regenerative techniques have been increasingly studied and used to treat osteochondral lesions of the talus. In particular, several studies have focused their attention on mesenchymal stem cells derived from adipose tissue. Adipose-derived stem cells (ADSCs) exhibit morphological characteristics and properties similar to other mesenchymal cells, and are able to differentiate into several cellular lines. Moreover, these cells are also widely available in the subcutaneous tissue, representing 10 - 30% of the normal body weight, with a concentration of 5,000 cells per gram of tissue.
In the presented technique, the first step involves harvesting ADSCs from the abdomen and a process of microfracture and purification; next, the surgical procedure is performed entirely arthroscopically, with less soft tissue dissection, better joint visualization, and a faster recovery compared with standard open procedures. Arthroscopy is characterized by a first phase in which the lesion is identified, isolated, and prepared with microperforations; the second step, performed dry, involves injection of adipose tissue at the level of the lesion.
Between January 2016 and September 2016, four patients underwent arthroscopic treatment of osteochondral lesion of the talus with microfractured and purified adipose tissue. All patients reported clinical improvement six months after surgery with no reported complications. Functional scores at the latest follow-up are encouraging and confirm that the technique provides reliable pain relief and improvements in patients with osteochondral lesion of the talus.

The aim of this study is to report a protocol for the arthroscopic treatment of osteochondral lesions of the talus using microfractured and purified adipose-derived stem cells.

自体 Microfractured 与纯化脂肪组织在关节镜下治疗距骨软骨病变

In recent years, regenerative techniques have been increasingly studied and used to treat osteochondral lesions of the talus. In particular, several ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

大鼠双侧髌腱损伤模型中干细胞治疗的评价

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

制备脂肪组织基质血管部分用于潜在软骨再生的临床协议

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

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

人类血管周围干细胞用于骨再生

生产和治疗间充质干行政/基质细胞（MSC）球体底漆在三维培养下无异物条件

修复的临界大小的颅骨缺损模型脂肪间充质干细胞的收获Lipoaspirate

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

自体 Microfractured 与纯化脂肪组织在关节镜下治疗距骨软骨病变

大鼠双侧髌腱损伤模型中干细胞治疗的评价

制备脂肪组织基质血管部分用于潜在软骨再生的临床协议

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

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

人类血管周围干细胞用于骨再生

生产和治疗间充质干行政/基质细胞（MSC）球体底漆在三维培养下无异物条件

修复的临界大小的颅骨缺损模型脂肪间充质干细胞的收获Lipoaspirate

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

自体 Microfractured 与纯化脂肪组织在关节镜下治疗距骨软骨病变

大鼠双侧髌腱损伤模型中干细胞治疗的评价

制备脂肪组织基质血管部分用于潜在软骨再生的临床协议

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

人类骨髓间充质干细胞的分离及其培养的多孔骨基质