3D磁干细胞聚集和生物反应器为成熟软骨再生
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 与微磁体为聚集体形成的装置的结构（ 图1A） 使通过铝?…
Representative Results
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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Iron oxide (maghemite) nanoparticules ( γ-Fe2O3) | PHENIX – University Paris 6 | Made and given by C. Ménager | Mean diameter of 8.1 nm and negative surface charge |
| Polysaccharide Pullulan/Dextran scaffolds | LIOAD – University Nantes | Made and given by C. Le Visage | Prepared from a 75:25 mixture of pullulan/dextran in alkaline conditions (10M NaOH). Porosity: 185-205µm; Thickness: 7mm; Surface area: 1.8cm2. |
| TisXell Regeneration System | QuinXell Technologies | QX900-002 | Biaxial bioreactor with 500 mL culture chamber |
| Cage for scaffolds: Histosette II M492 | VWR | 720-0909 | |
| Mesenchymal Stem Cell (MSC) | Lonza | PT-2501 | Three independant batches have been used |
| MSCGM BulletKit medium | Lonza | PT-3001 | For the complete medium, add the provided BulletKit (containing serum, glutamine and antibiotics) to the MSCGM medium |
| DMEM with Glutamax I | Life Technologies | 31966-021 | No sodium pyruvate, no HEPES |
| RPMI medium 1640, no Glutamine | Life Technologies | 31870-025 | No sodium pyruvate, no HEPES |
| PBS w/o CaCl2 w/o MgCl2 | Life Technologies | 14190-094 | |
| 0.05% Trypsin-EDTA (1x) | Life Technologies | 25300-054 | |
| Penicillin (10.000U/mL)/Streptomicin (10.000µg/mL) | Life Technologies | 15140-122 | |
| ITS Premix Universal Culture Supplement (20x) | Corning | 354352 | |
| Sodium pyruvate solution 100mM | Sigma | S8636 | |
| L-Ascorbic Acid 2-phosphate | Sigma | A8960 | Prepare the concentrated solution (25 mM) in distilled water extemporaneously |
| L-Proline | Sigma | P5607 | Prepare the 175 mM stock solution diluted in distilled water and store at 4°C |
| Dexamethasone | Sigma | D4902 | Prepare the 1 mM stock solution diluted in Ethanol 100% and store at -20°C |
| TGF-beta 3 protein 10µg | Interchim | 30R-AT028 | |
| Tri-sodium citrate | VWR | 33615.268 | Prepare the 1M stock solution diluted in distilled water and store at 4°C |
| Pullulanase from Bacillus acidopullulyticus | Sigma | P2986 | |
| Dextranase from Chaetomium erraticum | Sigma | D0443 | |
| NucleoSpin RNA Extraction Kit | Macherey-Nagel | 740955.5 | |
| SuperScript II Reverse Transcriptase | Life Technologies | 18064-014 | |
| Random Primer – Hexamer | Promega | C1181 | 500 µg/mL: Use diluted 1/2 and put 1 µL per sample |
| Recombinant RNAsin ribonuclease inhibitor | Promega | N2511 | 40 U/µL: put 1 µL per sample |
| PCR nucleotide dNTP mix (10mM each) | Roche | 10842321 | |
| SyBr Green PCR Master Mix | Life Technologies | 4368708 | |
| Step One Plus Real-Time PCR System | Life Technologies | 4381792 | |
| Formalin solution 10% neutral buffered | Sigma | HT5012 | |
| OCT solution | VWR | 361603E | |
| Isopentane | Sigma | M32631 | |
| Toluidine blue O | VWR | 1.15930.0025 | |
| Ethanol absolute | VWR | 20821.310 | |
| Toluene | VWR | 1.08323.1000 | |
| Mounting medium Pertex | Histolab | 840 | |
| RPLP0 Primer for qPCR | Eurogentec | 5'-TGCATCAGTAC CCCATTCTATCAT-3' ; 5'-AAGGTGTAATC CGTCTCCACAGA-3' |
|
| Aggrecan Primer for qPCR | Eurogentec | 5'-TCTACCGCTGCGAGGTGAT-3' ; 3'-TGTAATGGAACACGATGCCTTT-5' | |
| Collagen II Primer for qPCR | Eurogentec | 5'-ACTGGATTGACCCCAACCAA-3' ; 3'-TCCATGTTGCAGAAAACCTTCA-5' |
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Tags
3D Magnetic Stem Cell AggregationCartilage RegenerationMesenchymal Stromal CellsCell CondensationCellular BioreactorDynamic MaturationMuscle RepairCardiac RepairVascular RepairMicromagnet DeviceScaffold SeedingIron Oxide NanoparticlesMagnetic Cell LabelingCell AggregationCell CultureTrypsin-EDTACell PelletCell CountingGlass Bottom Petri Dish
Cite This Article
Van de Walle, A., Wilhelm, C., Luciani, N. 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration. J. Vis. Exp. (122), e55221, doi:10.3791/55221 (2017).