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使用23，或用简单的柠檬酸盐涂层和适当的浓度18中使用。另外，当氧化铁纳米颗粒在类似条件下，如本文描述的那些（与MSC和软骨）以外，我们最近证明了nanoparti的快速和几乎完全降解克莱斯在在蜂窝掺入和MSC球体形成约10天的核内体中发生。有趣的是，这种大规模的降解与铁蛋白中的游离铁的高效存储相关，并导致对细胞铁代谢非常有限的影响，为今后的临床应用25好兆头。 另一个关键点是细胞压实启动软骨的要求。通常情况下，细胞的缩合是通过离心实现;然而，这种方法是由较低的细胞数量（不大于2.5×10个5细胞）的限制。过去，这数，营养物质和气体不能扩散到聚集体的中心，引发细胞坏死。这里，标记的干细胞的磁性缩合成聚集体表现为显著方法，以形成用于软骨组织再生的3D构建体。这种磁过程已被用于其他作者与放置在板的顶部的细胞23的或与铁销的解离来本地化的磁场26后的磁体由磁悬浮:建立三维球状体。但是，磁悬浮不会出现以适合于骨料融合进一步阶段。与此相对，这里提出的磁性的方法，我们可以控制所有的融合步骤以获得一个步骤一步软骨组织构建。简言之，这个多步过程与干细胞的限制进入骨料积木开始，之后是融合这些块成较大的结构。这个自由脚手架干细胞聚集过程的关键步骤如下:首先，必须形成具有细胞作为可能的，并且第二的尽可能小的体积中的每个集合，一个必须控制的融合步骤，以避免的一个单一的，大的形成骨料。这里获得的组织是富含胶原蛋白II和蛋白聚糖。它还提出了advantag具有柔性的几何形状和尺寸的和存在的支架 - 自由ES。 磁的方法也被用来引导又厚又大的支架内的干细胞;另一替代为各种形状和尺寸的设计。这里的关键步骤是与细胞适当体积的种子支架:既不太少有细胞的整体均匀的分布，也不会太多，以避免任何细胞损失。磁力以前用来吸引和支架中保持细胞，并增强细胞接种27,28。在这里，多糖支架的孔隙内的足够的细胞凝结导致成功软骨。当磁性细胞化的支架进行生物反应器由于其双向轴向旋转由设置在转导/剪切应力刺激细胞外基质的产量显着提高。它已经在MUL其他研究显示TI-轴向负载条件下提高从软骨细胞29形成组织的质量。相对于现有技术，其中只有压缩力被施加30，31，32时，这种新颖的生物反应器的概念提出了一个真正的增益。 总之，对于软骨细胞分化，使用标记的干细胞的磁约束的形成和操纵聚集体以及种子允许创建毫米大小的软骨细胞构建体的支架。此外，磁性细胞种植与动态相结合的分化提供了再生医学应用价值的新工具。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Iron&#160; oxide (maghemite) nanoparticules ( &#947;-Fe2O3)</td>
			<td>PHENIX - University Paris 6</td>
			<td>Made and given by C. M&#233;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 (10M NaOH). Porosity: 185-205&#181;m; Thickness: 7mm; Surface area: 1.8cm2.</td>
		</tr>
		<tr>
			<td>TisXell Regeneration System</td>
			<td>QuinXell Technologies</td>
			<td>QX900-002</td>
			<td>Biaxial bioreactor with 500 mL culture chamber&#160;</td>
		</tr>
		<tr>
			<td>Cage for scaffolds: Histosette II M492&#160;</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 CaCl2 w/o MgCl2</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 (10.000U/mL)/Streptomicin (10.000&#181;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 100mM</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&#176;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&#176;C</td>
		</tr>
		<tr>
			<td>TGF-beta 3 protein 10&#181;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 1M stock solution diluted in distilled water and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Pullulanase from Bacillus acidopullulyticus</td>
			<td>Sigma</td>
			<td>P2986</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextranase from Chaetomium erraticum</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&#160;</td>
			<td>Promega</td>
			<td>C1181</td>
			<td>500 &#181;g/mL: Use diluted 1/2 and put 1 &#181;L per sample</td>
		</tr>
		<tr>
			<td>Recombinant RNAsin ribonuclease inhibitor</td>
			<td>Promega</td>
			<td>N2511</td>
			<td>40 U/&#181;L: put 1 &#181;L per sample</td>
		</tr>
		<tr>
			<td>PCR nucleotide dNTP mix (10mM 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&#39;-TGCATCAGTAC 
			CCCATTCTATCAT-3&#39; ; 
			5&#39;-AAGGTGTAATC 
			CGTCTCCACAGA-3&#39;</td>
		</tr>
		<tr>
			<td>Aggrecan Primer for qPCR</td>
			<td>Eurogentec</td>
			<td></td>
			<td>5&#39;-TCTACCGCTGCGAGGTGAT-3&#39; ; 3&#39;-TGTAATGGAACACGATGCCTTT-5&#39;</td>
		</tr>
		<tr>
			<td>Collagen II Primer for qPCR</td>
			<td>Eurogentec</td>
			<td></td>
			<td>5&#39;-ACTGGATTGACCCCAACCAA-3&#39; ; 3&#39;-TCCATGTTGCAGAAAACCTTCA-5&#39;</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4×10 6个细胞（约4毫米大小）（ 图2B）的。第二，相同的磁吸引力的技术已被用来形成支架内的细胞聚集体。支架接种在磁性设备和显示出稠密冷凝细胞支架的孔隙内，在确切的微磁体的位置（ 图2C）。与此相反，当细胞没有磁吸引力（被动接种）的支架内接种，发现它们被均匀地分布。然后细胞化的支架插入到安装于生物反应器室动态熟化条件下（ 图3B）下进行的软骨形成过程笼（ 图3A）。这样的生物反应器提高了营养物和气体交换以及通过转导提供机械刺激。臂和两个腔室的旋转速度调节至5转，所推荐的构造为软三维组织再生。提供介质的连续供给蠕动泵的速度设定在10rpm。 对于细胞组织的全部条件（稠合的聚集体和支架内接种）和组织成熟（在或不在生物反应器），第25天，分析基因表达通过磁聚变形成的组织表现出了显在Ⅱ型胶原表达着性增加相比，通过离心（ 图4A）中获得的粒料，与聚集蛋白聚糖表达的增加的趋势一起。对于细胞化的支架，我们获得了在增加的聚集蛋白聚糖和胶原II-II时，使用表达式显著胶原磁性播种，相比于支架接种无磁力。此外，这两个基因的表达要高得多（对于山口II显著）当磁种用动态分化（ 图4B）相结合。 组织学分析使用一甲苯胺蓝染色以显示糖胺聚糖（GAG）也执行，例如。的16个聚集体的连续磁聚变表现出GAG的丰富沉积，通过蓝-紫色（ 图5A）作为明证。对于支架，只有那些磁接种甲苯胺蓝染色。 GAG conten吨较高时，支架在生物反应器（ 图5B），而不是静态地（ 图5C）分化。总之，这些结果表明支架内的磁性聚集和磁种的潜力，以提高软骨。这也表明，在生物反应器内的动态成熟的条件是有效的差异化更为有利。 <img alt="图1" src="/files/ftp_upload/55221/55221fig1.jpg" /> 图1结构的磁器件。 （A），用于形成聚集体的磁性装置的实施例:将铝板钻（0.8直径微米孔）和提示插入在每个孔中，然后放置在永久钕磁铁，保证磁化饱和。 （B）磁性设备种子支架:硬聚苯乙烯（24 平方毫米），用9个手动进行孔是放置在永久钕磁铁，保证磁化饱和。然后小磁铁（直径3mm）插入每孔以形成装置。 <a href="http://ecsource.jove.com/files/ftp_upload/55221/55221fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55221/55221fig2.jpg" /> 图2.干细胞和接种的磁性标记。 （A）干细胞与铁氧化物纳米颗粒进行标记30分钟，在37℃。 （B）球状体由16个微磁体网络吸引标记的细胞形成的。聚集体是由前3天形成的双峰的融合合并成四胞胎在第11天。然后，四胞胎被融合在第15天，以构成最终的工程化组织。 （C）的支架，放入玻璃底培养皿，用或接种出磁力。在第4天，在磁接种的支架中观察到压实的干细胞的斑点，而细胞出现均匀分布在无磁体接种的支架。 <a href="http://ecsource.jove.com/files/ftp_upload/55221/55221fig2large.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/55221/55221fig3.jpg" /> 图3.细胞化支架的动态成熟。 （A）磁或被动接种后，细胞化的支架投入笼中，以避免干扰。 （B）笼，使用帽的针固定，放入充满了软骨形成培养基的生物反应器的容器中。所述生物反应器应用于双轴旋转被独立控制的速度（1-12转和1-35的转速分别臂和腔室）。蠕动泵连续提供中介嗯。 <a href="http://ecsource.jove.com/files/ftp_upload/55221/55221fig3large.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/55221/55221fig4.jpg" /> 图4的第25天特定软骨基因的表达（A）MSC的球状体的磁感应融合显示在II型胶原一个显著相比，增加通过离心形成沉淀。 *表示使用学生t检验（p值&lt;0.05）统计学差异。 （B）II与磁性播种和动态成熟在生物反应器的组合区分的支架明显增加的聚集蛋白聚糖和胶原蛋白的表达。基因表达标准化为RPLP0 mRNA和以任意单位表示相对于对照（〜1±SEM）。结果表示为平均值两至四次独立实验±SEM。 *杰诺使用Kruskal-Wallis检验（单向ANOVA非参数检验）（p值&lt;0.05）TES统计学差异。 （ - ）:无接种磁铁; （+）:有磁力播种。 <a href="http://ecsource.jove.com/files/ftp_upload/55221/55221fig4large.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/55221/55221fig5.jpg" /> 图5. 25.糖胺聚糖（GAG）押金由蓝紫色着色证实一天糖胺聚糖的组织学染色 。 （A）在从16个聚集体的顺序融合获得的最终结构软骨的8微米的冷冻切片中观察到的正甲苯胺蓝染色。支架静态的（B）之后磁性接种或动态的（C）条件下的12-μm的冷冻切片清楚地表明这GAG含量在生物反应器中分化的支架较高。箭头表示分化细胞的聚集体。 <a href="http://ecsource.jove.com/files/ftp_upload/55221/55221fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

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

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 ...

科研

生物工程

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

非人灵长类龙脱细胞和Recellularization使用专门的大型器官生物反应器

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

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 ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

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 ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...