作者要感谢QuinXell技术和CellD，特别是洛塔尔·格兰尼曼和多米尼克·霍兹伦他们用生物反应器的帮助。我们感谢凯瑟琳原我，谁用普鲁兰多糖/葡聚糖多糖支架为我们提供。这项工作是由欧盟（ERC-2014-COG项目马蒂斯648779）和由AgenceNationalede LA RECHERCHE（ANR），法国（MagStem项目ANR-11 SVSE5）的支持。

1.磁性设备的建设注意:用于细胞接种的装置取决于应用（ 图1）。形成聚集体，细胞的数量被限制在2.5×10 5 /聚集体，所以磁性尖端必须非常薄（750微米直径）。种子的1.8厘米七分之二毫米厚的支架，磁体必须更大（直径3mm），并确保通过支架的孔中的细胞迁移。 <ol><li>与微磁体为聚集体形成的装置的结构（ 图1A） <ol><li>使通过铝板0.8毫米钻微孔（直径3厘米和6毫米厚）。 </li><li>插入一个磁性尖端（750微米直径）插入所述板的每个孔中。 </li><li>将这个盘在永久钕磁铁，保证磁化饱和。 </li></ol></li><li>设备的施工脚手架播种（ <str翁&gt;图1B） <ol><li>切割硬聚苯乙烯成2.4厘米2米的正方形。 </li><li>在相等的距离超过1.6 cm 2的表面积插入9个小磁铁（直径3mm，长6毫米）。 </li><li>将这个装置在永久钕磁铁。 </li></ol></li></ol> 2.干细胞标记注:干细胞用0.1mM的磁性纳米粒子进行标记30分钟（2.6±0.2皮克铁/细胞）以形成聚集体，当他们用0.2mM的磁性纳米颗粒标记30分钟（5±0.4皮克铁/细胞）接种支架。这些纳米颗粒的浓度和温育时间先前已经使用并发表了MSC和其它细胞18， 图19，和它已经确定的是纳米颗粒既不影响细胞生存力，也不MSC分化能力。由干细胞掺入的铁物料经由单CE测定LL磁19，20。 <ol><li>培养人间充质干细胞（MSC）在完整的间充质干细胞生长培养基（MSCGM），在37℃和5％CO 2，直至接近至汇合（〜90％）。 </li><li>无谷氨酰胺在的Roswell Park Memorial研究所（RMPI）改性的无血清的McCoy&#39;s5A培养基和含有5:;由0.1或0.2mM的磁赤铁矿柠檬酸被覆铁氧化物（8nm的直径的芯γFe2 O 3）混合制备磁性标记溶液mM柠檬酸钠。 </li><li>弃去培养基，用清洗无血清的RPMI培养基中的细胞没有谷氨酰胺，并添加10毫升每150-cm 2的培养瓶中，以覆盖所有细胞所需的最小体积的氧化铁纳米颗粒溶液。 </li><li>孵育在37℃下30分钟和5％CO 2，然后丢弃该纳米颗粒溶液。冲洗5分钟用无血清的RPMI培养基中，而不谷氨酰胺内化楠oparticles仍然附着至质膜。 </li><li>丢弃该RPMI培养基并添加25毫升每烧瓶完整MSCGM培养基。在37℃下孵育过夜，5％的CO 2。 </li></ol> 3.磁性细胞种植<ol><li>新鲜加入50μML-抗坏血酸-2-磷酸，0.1μM地塞米松，1mM丙酮酸钠，0.35毫摩尔L-脯氨酸，1％万向培养制备使用Dulbecco改良的Eagle培养基（DMEM）高葡萄糖L-谷氨酰胺的软骨形成培养基补充含有胰岛素，人转铁蛋白和硒酸（ITS-预混料），和10ng / mL的转化生长因子-β3（TGF-β3）。 </li><li>分离使用每150-cm 2的培养瓶中0.05％胰蛋白酶-EDTA和离心机的8毫升的解离的细胞的磁性细胞在260×g下5分钟。吸出介质并计数的重新悬浮的细胞。 </li><li>放置在两磁性设备的顶部玻璃底细胞培养的培养皿（35毫米）。 </li><li>到magnetically形成聚集体，加入3毫升软骨形成培养基到培养皿中，并轻轻地沉积含2.5×10 5个标记的细胞每个聚集体（最多16个聚集体可以被沉积）的最小体积可能的（不超过8微升）。离开培养皿20-30分钟而不移动它，允许它以形成球状体，然后将完整的设备，包括含有16个聚集体的培养皿，放入培养箱中在37℃和5％CO 2。 </li><li>以下形式控制聚集体相同的协议和与没有TGF-β3软骨形成培养基替换完全培养基。 <ol><li>为了产生3D聚集体构建体，放置2点聚集在第8天接触，以形成8个双峰并启动所述融合。第11天，合并2个双峰形成4四胞胎。最后，融合在第15天的4四胞胎，得到最终的结构。 </li><li>与此同时，通过离心分离2.5×10 5形成聚集体标记的干细胞在26015; （分别为样品和对照）克在15-mL管5分钟用1.5mL有或没有TGF-β3软骨形成培养基的。 </li></ol></li><li>以磁性种子支架，将每个干燥支架到培养皿。使用由支链淀粉/葡聚糖21的多糖多孔支架。对于每一个支架，在稀软骨形成培养基的350微升2×10 6标记的干细胞而不TGF-β3和小心移液管将细胞在支架上。 <ol><li>孵育5分钟，在37℃，以允许在支架内充满细胞穿透，然后轻轻地用或不用TGF-β3（分别用于样品或对照）的培养皿中添加软骨形成培养基的3毫升。 </li><li>孵育其磁性装置上的细胞化的支架在37℃和5％CO 2 4天，以允许通过所述支架的孔和禁闭细胞迁移。 </li></ol></li><li>与此同时，种子支架与2×10 6 </s向上&gt;以下标记的干细胞相同的方法和未经孵育所述磁体，以获得均匀接种的支架作为阳性对照。 </li></ol> 4.分化为软骨细胞注:温育后的第4天，除去磁体并继续成熟软骨或者在皮氏培养皿（静态条件）或在生物反应器（动态条件）。阴性对照样品的成熟与无TGF-β3软骨中静态条件。 <ol><li>在静态条件下，保持在同一个培养皿中的细胞化的支架或聚集体。一个星期更改软骨中两次21天。 </li><li>在动态条件下，制备的生物反应器。 <ol><li>根据制造商的方案在适当的长度切断硅树脂管。 </li><li>高压灭菌的所有材料:500mL的培养室，管道，2路旋转器，和笼子。 </li><li>生物反应器部件放入无菌就微生物逻辑安全站。管道连接到2路旋转器和培养室按照制造商的说明。 </li><li>小心地将细胞化的支架转移到使用无菌刮刀消毒的笼子。将每笼2个支架。当笼子里准备好了，将它们插入到盖的针，让他们进一步旋转过程中移动。填充软骨形成培养基的培养室，用含有笼中的盖子关闭。 </li></ol></li><li>打开蠕动泵，以填补软骨中的管道和消除气泡。 </li><li>放置和填充室固定到所述生物反应器的马达和打开计算机，其控制所述臂的与所述腔室的旋转上。 </li><li>在臂和腔室都应用每分钟5转数（RPM）的转速。调整以10rpm的流速为细胞化支架的连续进给的蠕动泵。 </li></ol>注意:在此之前RNA提取，消化支架与酶溶液。 <ol><li>通过添加100μL的支链淀粉酶（40U / mL）和葡聚糖酶的50微升（60毫克/毫升），以850毫升的无血清DMEM培养基中制备1毫升的酶溶液。 </li><li>用无血清的DMEM培养基冲洗两次支架，弃去培养基，并加入每支架的酶溶液的800μL。孵育在37℃下轻柔搅拌15-30分钟。 </li><li>当支架被完全溶解，转移在300×g下将含有细胞的1.5毫升管中的溶液，离心10分钟，小心地吸出培养基，用无菌1×磷酸盐缓冲盐水（PBS），离心机在冲洗两次300×g的10分钟，并重新悬浮在RNA分离溶液中的细胞。 </li><li>取出从聚集体RNA，将球状体中的RNA分离溶液和执行RNA提取之前用均化器粉碎完全它们。 </li><li>隔离根据制造商的说明使用试剂盒提取总RNA的RNA。 </li><li>根据制造商的说明从使用逆转录酶400纳克的总RNA合成互补DNA，使用250纳克随机引物，1微升dNTP混合物（每种10mM），和40U / ml RNA酶抑制剂;反应的最终体积为20μL。在反应结束时，加入80μL的蒸馏水中，得到100微升的最终体积。 </li><li>对于定量聚合酶链式反应（PCR），使用含有荧光试剂的PCR混合物中定量的感兴趣的基因，如聚集蛋白聚糖（AGC）和胶原II（山口II）的相对表达，用10×稀释的cDNA。标准化基因的表达水平与参考基因核糖体蛋白，大的，P0（RPLP0）。 ΔΔCT式-与2执行计算其中ΔΔCT=ΔCT的分化的条件 - 平均ΔCT的控制条件，并且每个ΔCT代表感兴趣的基因的CT - 参照基因（RPLP0）的CT。 </li><li>确定为平均值±平均值（SEM）的标准误差的统计测量。执行用正≥2个独立实验的分析。使用学生t检验来分析离心颗粒和磁性融合体（* P &lt;0.05）之间的统计学差异。确定与Kruskal-Wallis检验（单向ANOVA非参数）的意义进行分析有区别的支架控制支架之间并具有统计学差异（* P &lt;0.05）。 </li></ol> 6.组织学分析<ol><li>冲洗细胞化的支架或聚集体，用无菌1×PBS，修复它们在10％福尔马林溶液，在室温下1个小时，并用1×PBS冲洗。 </li><li>除去PBS，在最佳cuttin嵌入样品克温度化合物（OCT），并且在异戊烷浴浸入液氮中冷冻。存储在-20℃下的样品。用低温恒温器以获得所述聚集体或12微米的细胞化的支架8微米冷冻切片切割样品。 </li><li>染色用0.5％甲苯胺蓝溶液的冰冻切片2分钟，漂洗在自来水中，用100％乙醇脱水，澄清使用甲苯，并与用于光学显微镜的安装介质装入滑动。 </li></ol>

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H., Hur, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold-free+formation+of+a+millimeter-scale+multicellular+spheroid+with+an+internal+cavity+from+magnetically+levitated+3T3+cells+that+ingested+iron+oxide-containing+microspheres.">Scaffold-free formation of a millimeter-scale multicellular spheroid with an internal cavity from magnetically levitated 3T3 cells that ingested iron oxide-containing microspheres.</a> Biotechnol Bioeng. 111 (5), 1038-1047 (2014).</li><li>Mattix, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+magnetic+cellular+spheroids+as+building+blocks+for+tissue+engineering.">Biological magnetic cellular spheroids as building blocks for tissue engineering.</a> Acta Biomaterialia. 10 (2), 623-629 (2014).</li><li>Mazuel, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massive+Intracellular+Biodegradation+of+Iron+Oxide+Nanoparticles+Evidenced+Magnetically+at+Single-Endosome+and+Tissue+Levels.">Massive Intracellular Biodegradation of Iron Oxide Nanoparticles Evidenced Magnetically at Single-Endosome and Tissue Levels.</a> ACS Nano. 10 (8), 7627-7638 (2016).</li><li>Kim, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+generation+of+spheroids+using+magnetic+nanoparticles+for+three-dimensional+cell+culture.">High-throughput generation of spheroids using magnetic nanoparticles for three-dimensional cell culture.</a> Biomaterials. 34 (34), 8555-8563 (2013).</li><li>Shimizu, K., Ito, A., Honda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+cell-seeding+into+3D+porous+scaffolds+by+use+of+magnetite+nanoparticles.">Enhanced cell-seeding into 3D porous scaffolds by use of magnetite nanoparticles.</a> J Biomed Mater Res B Appl Biomater. 77 (2), 265-272 (2006).</li><li>Sensenig, R., Sapir, Y., MacDonald, C., Cohen, S., Polyak, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticle-based+approaches+to+locally+target+therapy+and+enhance+tissue+regeneration+in+vivo.">Magnetic nanoparticle-based approaches to locally target therapy and enhance tissue regeneration in vivo.</a> Nanomedicine (Lond). 7 (9), 1425-1442 (2012).</li><li>Waldman, S. D., Couto, D. C., Grynpas, M. D., Pilliar, R. M., Kandel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-axial+mechanical+stimulation+of+tissue+engineered+cartilage:+review.">Multi-axial mechanical stimulation of tissue engineered cartilage: review.</a> Eur Cell Mater. 13, 66-74 (2007).</li><li>Takahashi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compressive+force+promotes+sox9,+type+II+collagen+and+aggrecan+and+inhibits+IL-1beta+expression+resulting+in+chondrogenesis+in+mouse+embryonic+limb+bud+mesenchymal+cells.">Compressive force promotes sox9, type II collagen and aggrecan and inhibits IL-1beta expression resulting in chondrogenesis in mouse embryonic limb bud mesenchymal cells.</a> J Cell Sci. 111 (14), 2067-2076 (1998).</li><li>Campbell, J. J., Lee, D. A., Bader, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compressive+strain+influences+chondrogenic+gene+expression+in+human+mesenchymal+stem+cells.">Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells.</a> Biorheology. 43, 455-470 (2006).</li><li>Vunjak-Novakovic, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreactor+cultivation+conditions+modulate+the+composition+and+mechanical+properties+of+tissue-engineered+cartilage.">Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage.</a> J Orthop Res. 17 (1), 130-138 (1999).</li></ol>

作者什么都没有透露。

首先，因为这里提出的技术依靠磁性纳米粒子的内化，一个重要的问题是纳米粒子的结果，一旦他们定位在细胞内。这是事实，铁纳米颗粒可以触发潜在毒性或取决于它们的大小，涂层，和曝光19，22的时间受损分化能力。然而，一些研究已经显示在细胞生理学没有影响当包封的铁纳米颗粒在magnetoferritin的形式，生物磁性纳米粒子24使?...

磁性纳米粒子在临床上用于磁共振成像（MRI）造影剂已经使用，其治疗应用不断扩大。例如，最近已经显示，标记的细胞可在体内使用的外部磁场在限定的注入1，2，3的部位和操作可被引导和/或保持。在再生医学中，它们可以被用于工程体外 4有组织的组织，包括血管组织5，6，7，8的骨和软骨9。 关节软骨浸渍在无血管的环境中，会发生损坏时使细胞外基质组分的维修非常有限。出于这个原因，researc目前h的重点，可以在缺损部位植入透明软骨替换的工程。为了生产自体替换，一些研究小组正在探索使用自体软骨细胞作为细胞源10，11的，而另一些强调间充质干细胞（MSC）分化成软骨细胞12，13的能力。在这里重现以前的研究中，我们选择MSC，因为他们的骨髓采样是相当简单，并不需要健康的软骨细胞，它可能失去其表型14的牺牲。 早期的步骤启动干细胞的软骨细胞分化的实质是他们的凝结。细胞聚集体使用的是离心或微团培养15共同形成;然而，这些冷凝方法neitheR有创建厚支架也不以控制聚集体的融合潜能内细胞团的潜力。在本文中，我们描述了一个创新的方法，利用冷凝MSC磁性标记和磁引力干细胞。该技术已被证明是形成通过聚集体的融合彼此自由支架-3D构建体，以获得毫米级软骨组织9。厚和大的脚手架磁种也让增加工程化组织的大小，用于植入更容易设计的形状是有用的，多样化用于软骨修复的临床应用的潜力的可能性。在这里，我们详细地对MSC的磁播种到天然多糖，支链淀粉，和葡聚糖组成的多孔支架的协议，先前使用的支架，以限制干细胞16,17。软骨分化finallÝ在生物反应器进行，以确保连续的营养物和气体扩散与细胞的高密度接种的支架的基质芯。除了提供营养物，软骨的生长因子，以及气体到细胞中，所述生物反应器提供机械刺激。总体而言，用于限制干细胞的磁性的技术，以在生物反应器的动态熟化组合，可以显着地改善软骨形成分化。

<table><tbody><tr><td>Iron oxide (maghemite) nanoparticules (&amp;gamma;-Fe&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;)</td><td>PHENIX - University Paris 6</td><td>Made and given by C. M&amp;eacute;nager</td><td>Mean diameter of 8.1 nm and negative surface charge</td></tr><tr><td>Polysaccharide Pullulan/Dextran scaffolds</td><td>LIOAD - University Nantes</td><td>Made and given by C. Le Visage</td><td>Prepared from a 75:25 mixture of pullulan/dextran in alkaline conditions (10&amp;nbsp;M NaOH). Porosity: 185-205&amp;nbsp;&amp;micro;m; Thickness: 7&amp;nbsp;mm; Surface area: 1.8&amp;nbsp;cm&lt;sup&gt;2&lt;/sup&gt;.</td></tr><tr><td>TisXell Regeneration System</td><td>QuinXell Technologies</td><td>QX900-002</td><td>Biaxial bioreactor with 500 mL culture chamber&amp;nbsp;</td></tr><tr><td>Cage for scaffolds: Histosette II M492&amp;nbsp;</td><td>VWR</td><td>720-0909</td><td></td></tr><tr><td>Mesenchymal Stem Cell (MSC)</td><td>Lonza</td><td>PT-2501</td><td>Three independant batches have been used</td></tr><tr><td>MSCGM BulletKit medium</td><td>Lonza</td><td>PT-3001</td><td>For the complete medium, add the provided BulletKit (containing serum, glutamine and antibiotics) to the MSCGM medium</td></tr><tr><td>DMEM with Glutamax I</td><td>Life Technologies</td><td>31966-021</td><td>No sodium pyruvate, no HEPES</td></tr><tr><td>RPMI medium 1640, no Glutamine</td><td>Life Technologies</td><td>31870-025</td><td>No sodium pyruvate, no HEPES</td></tr><tr><td>PBS w/o CaCl&lt;sub&gt;2&lt;/sub&gt; w/o MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Life Technologies</td><td>14190-094</td><td></td></tr><tr><td>0.05% Trypsin-EDTA (1x)</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Penicillin (10,000&amp;nbsp;U/mL) / Streptomycin (10,000&amp;nbsp;&amp;micro;g/mL)</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>ITS Premix Universal Culture Supplement (20x)</td><td>Corning</td><td>354352</td><td></td></tr><tr><td>Sodium pyruvate solution 100 mM</td><td>Sigma</td><td>S8636</td><td></td></tr><tr><td>L-Ascorbic Acid 2-phosphate</td><td>Sigma</td><td>A8960</td><td>Prepare the concentrated solution (25 mM) in distilled water extemporaneously</td></tr><tr><td>L-Proline</td><td>Sigma</td><td>P5607</td><td>Prepare the 175 mM stock solution diluted in distilled water and store at 4&amp;nbsp;&amp;deg;C</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td>Prepare the 1 mM stock solution diluted in Ethanol 100% and store at -20&amp;nbsp;&amp;deg;C</td></tr><tr><td>TGF-beta 3 protein 10&amp;nbsp;&amp;micro;g</td><td>Interchim</td><td>30R-AT028</td><td></td></tr><tr><td>Tri-sodium citrate</td><td>VWR</td><td>33615.268</td><td>Prepare the 1&amp;nbsp;M stock solution diluted in distilled water and store at 4&amp;nbsp;&amp;deg;C</td></tr><tr><td>Pullulanase from &lt;em&gt;Bacillus acidopullulyticus&lt;/em&gt;</td><td>Sigma</td><td>P2986</td><td></td></tr><tr><td>Dextranase from &lt;em&gt;Chaetomium erraticum&lt;/em&gt;</td><td>Sigma</td><td>D0443</td><td></td></tr><tr><td>NucleoSpin RNA Extraction Kit</td><td>Macherey-Nagel</td><td>740955.5</td><td></td></tr><tr><td>SuperScript II Reverse Transcriptase</td><td>Life Technologies</td><td>18064-014</td><td></td></tr><tr><td>Random Primer - Hexamer&amp;nbsp;</td><td>Promega</td><td>C1181</td><td>500 &amp;micro;g/mL: Use diluted 1/2 and put 1 &amp;micro;L per sample</td></tr><tr><td>Recombinant RNAs in ribonuclease inhibitor</td><td>Promega</td><td>N2511</td><td>40 U/&amp;micro;L: put 1 &amp;micro;L per sample</td></tr><tr><td>PCR nucleotide dNTP mix (10&amp;nbsp;mM each)</td><td>Roche</td><td>10842321</td><td></td></tr><tr><td>SyBr Green PCR Master Mix</td><td>Life Technologies</td><td>4368708</td><td></td></tr><tr><td>Step One Plus Real-Time PCR System</td><td>Life Technologies</td><td>4381792</td><td></td></tr><tr><td>Formalin solution 10% neutral buffered</td><td>Sigma</td><td>HT5012</td><td></td></tr><tr><td>OCT solution</td><td>VWR</td><td>361603E</td><td></td></tr><tr><td>Isopentane</td><td>Sigma</td><td>M32631</td><td></td></tr><tr><td>Toluidine blue O</td><td>VWR</td><td>1.15930.0025</td><td></td></tr><tr><td>Ethanol absolute</td><td>VWR</td><td>20821.310</td><td></td></tr><tr><td>Toluene</td><td>VWR</td><td>1.08323.1000</td><td></td></tr><tr><td>Mounting medium Pertex</td><td>Histolab</td><td>840</td><td></td></tr><tr><td>RPLP0 Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-TGCATCAGTAC&lt;br/&gt; CCCATTCTATCAT-3&#039;;&lt;br/&gt; 5&#039;-AAGGTGTAATC&lt;br/&gt; CGTCTCCACAGA-3&#039;</td></tr><tr><td>Aggrecan Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-TCTACCGCTGCGAGGTGAT-3&#039;; 3&#039;-TGTAATGGAACACGATGCCTTT-5&#039;</td></tr><tr><td>Collagen II Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-ACTGGATTGACCCCAACCAA-3&#039;; 3&#039;-TCCATGTTGCAGAAAACCTTCA-5&#039;</td></tr></tbody></table>

3d magnetic stem cell aggregation and bioreactor maturation for cartilage regeneration

软骨工程仍因创建类似于天然组织的体外功能植入物的困难挑战。最近探讨自体替代发展的方法涉及干细胞分化为软骨细胞。要启动这个软骨，一定程度的干细胞需要的压实;因此，我们证实磁冷凝细胞的可行性，既厚支架和自由脚手架内，使用小型化的磁场源如细胞吸引子。这种磁性方法也被用来引导聚集融合，打造脚手架免费的，有组织的，三维（3D）的大小组织中几毫米。除了具有增强的大小，通过磁性驱动的融合形成的组织示于胶原II的表达的增加显著，观察聚集蛋白聚糖表达有类似的趋势。作为天然软骨进行力吨帽子影响了它的三维结构，还进行动态成熟。提供机械刺激的生物反应器被用来培养磁接种的支架在21天的期间。生物反应器的成熟大大改善软骨进入细胞化的支架;这些条件下得到的细胞外基质是富含胶原蛋白II和聚集蛋白聚糖。这项工作概述在生物反应器用于改善软骨细胞分化，这两种支架和无内多糖支架标记的干细胞的磁性缩合和动态成熟的创新潜力。

首先，聚集体可以单独使用微磁体通过沉积2.5×10 5个标记的干细胞（ 图2A）形成的。这些单个聚集体（〜大小0.8毫米）然后可融合成由于连续的，磁感应融合较大的结构。例如，在成熟软骨的第8天，聚集体放置在对以形成双联体接触;四胞胎通过合并2个双重峰组装在第11天;最后，在第15天，将4四胞胎融合以形成包含最初形成的聚集体16的3D构建体，总4?...

Watch this Scientific Journal Video about 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration at JoVE.com

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

从干细胞软骨需要微调的培养条件。在这里，我们提出了冷凝细胞磁性的方法，一个必不可少的步骤启动软骨。此外，我们显示，在生物反应器中动态成熟施加机械刺激，以在蜂窝结构和增强软骨细胞外基质的产生。

3D磁干细胞聚集和生物反应器为成熟软骨再生

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

3d-magnetic-stem-cell-aggregation-bioreactor-maturation-for-cartilage

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.7K 次观看.  UMR 7057 CNRS and University Paris Diderot. 从干细胞软骨需要微调的培养条件。在这里，我们提出了冷凝细胞磁性的方法，一个必不可少的步骤启动软骨。此外，我们显示，在生物反应器中动态成熟施加机械刺激，以在蜂窝结构和增强软骨细胞外基质的产生。 

视频: 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

科研

生物工程

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

脐带血诱导的多能干细胞的软骨形成颗粒形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

发育生物学

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D水凝胶支架的关节软骨细胞培养及软骨生成

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

Ferumoxides植入人类间质干细胞在软骨缺损的标记

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

生物学

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources and are emerging as promising candidates for regenerative medicine, especially for regeneration of the tooth, bone, cartilage, and skin. The self-assembled approach of MSC aggregation, which notably constructs cell clusters mimicking the developing mesenchymal condensation, allows high-density stem cell delivery along with preserved cell-cell interactions and extracellular matrix (ECM) as the microenvironment niche. This method has been shown to enable efficient cell engraftment and survival, thus promoting the optimized application of exogenous MSCs in tissue engineering and safeguarding clinical organ regeneration. This paper provides a detailed protocol for the construction and characterization of self-assembled aggregates based on umbilical cord mesenchymal stem cells (UCMSCs), as well as an example of the cranial bone regenerative application. The implementation of this procedure will help guide the establishment of an efficient MSC transplantation strategy for tissue engineering and regenerative medicine.

This study describes a method to construct aggregates based on the self-assembly of human mesenchymal stem cells and identifies the morphological and histological characteristics for the regenerative treatment of cranial bone defects.

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources ...

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients&#8217; life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

应用三维单轴机械刺激生物反应器系统诱导肌生衍生干细胞的畸因分化

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited ...

医学

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

在重力和自制微型生物反应器下使用低粘附条件从各种软骨细胞生成球状体

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...