支持博士后奖学金资助从匹兹堡组织工程​​倡议的SN。 DOZ的日内瓦基金会颁发的赠款支持。

1。分离脂肪干细胞（ASCs）1，5 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>分离大鼠肾周和附睾脂肪用无菌汉克的缓冲盐溶液，含1％胎牛血清（FBS），（5先前所描述的HBSS中）。这项研究已进行符合动物福利法“，实施动物福利条例，并按照实验动物护理和使用指南”的原则。 </li><li>百果组织和转移到1-2克25毫升含有500克50毫升管和离心机在室温为8分钟到1％胎牛血清的HBSS。 </li><li>收集的自由浮动的脂肪层转移到125毫升三角瓶和治疗胶原酶II型（200单位/毫升）25毫升37°轨道摇床（125转）在45分钟的HBSSÇ。 </li&gt; <li>仔细由pipeting删除的液体部分（油和脂肪层以下）和过滤器，它通过一个100顺序 - 微米和70 - 微米尼龙网过滤器。在500克，在常温下为10分钟，离心滤液，吸上清，用25毫升的HBSS沉淀两次。 </li><li>悬浮细胞沉淀在50毫升的培养基中补充MesenPRO RS生长补充，抗生素，抗霉菌（100 U / ml的青霉素G，100微克/毫升的硫酸链霉素，两性霉素0.25微克/毫升（MesenPRO RS基础培养基） B）和2毫米的L-谷氨酰胺和移液器细胞分为两个的T75瓶（25毫升/瓶）。 </li><li>文化在5％CO 2培养箱孵化ASC在37°C间（通过2-4 ASCs的所有实验中使用的）。 </li></ol> 2。制备壳聚糖微球（CSMS） 注：所有的程序，在室温下进行，除非另有说明。 <ol><l我&gt; CSMS准备由水油乳化过程中，随着离子的凝聚技术，用我们以前的协议5。乳化油相混合组成的大豆油，正辛醇（1:2 V / V）和5在100毫升的壳聚糖水溶液（6毫升3％W / V壳聚糖醋酸在0.5中号） ％山梨醇单油酸酯（跨度80）乳化剂，同时使用方向相反的开销（1700 RPM）和磁力搅拌（1000转）。这种混合确保年初之前，在交联形成的胶束可以留在溶液中，并没有解决的底部双方法。此外，磁力搅拌棒的艾滋病在聚合过程中胶束的形成和刚化壳聚糖。 </li><li>连续搅拌混合搅拌约1小时，直到获得一个稳定的中水乳化油。启动离子交联除了为4小时（共24毫升）1.5毫升1％W / V正辛醇，每15分钟的氢氧化钾</li><l我&gt;交联反应，慢慢倒出油相的混合物含有的CSM完成后，立即加入100毫升的丙酮，该解决方案将变得混浊，由于渣油。 5分钟后，倒出，加入100毫升的丙酮和在室温下孵育过夜。次日，再与丙酮洗五分钟的珠子。在执行这些串行洗涤溶液应转清，如果不继续冲洗被删除，直到所有的石油和丙酮的解决方案是明确的。 </li><li>在真空干燥器的干燥回收领域，没有进一步的处理和分析。你可以决定CSM的平均颗粒大小，每毫克的表面积，使用粒度分析仪和单位立方体体积。 </li><li>对于随后的实验中，用无菌水冲洗CSM的三倍，以除去残留的盐和隔夜5毫升无水乙醇洗涤消毒。 </li></ol> 3。确定在CSMS游离氨基酸组数 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>确定后离子交叉连接使用硝基苯磺酸（三硝基苯磺酸）酸法的Bubnis和Ofner 6 CSMS目前的氨基酸组的数量。用1 mL 0.5％三硝基苯磺酸溶液50毫升的玻璃试管中4小时在40°C孵育5毫克的微球和水解与另外3毫升6N盐酸在60°2小时。 </li><li>样品冷却至室温，加入5毫升的去离子水和10毫升乙醚提取自由硝基苯磺酸。 </li><li>温暖的水相的5 mL等分40°C的水浴蒸发冷却至室温，任何残余的乙醚，15分钟和15毫升水稀释。 </li><li>与壳聚糖不使用三硝基苯磺酸溶液分光光度计测量吸光度作为空白和壳聚糖为CSM带够，在345 nm处确定氨基酸组总数的赔偿。估计CSM的相对壳聚糖的游离氨基酸组的数量。 </li></ol> 4。在CSM加载升序 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>平衡消毒CSMS在一夜之间无菌的HBSS 2.5节从5毫克，并添加到一个8 - 微米孔径的膜培养板插入（24孔板）。 </li><li> CSMS已落户到膜后，小心吸的HBSS，并添加300μL培养基中的生长介质的插入和700微升内插入外。 </li><li>重悬的ASCs在适当的浓度（1×10 4 4×10 4）200μL生长介质和种子在培养板内插入的CSM。 ，播种后，内插入的文化语言的最终体积为500μL。 </li><li> incub吃了24小时的ASC上CSMS种子在5％CO 2培养箱孵化器在37°C。 </li></ol> 5。 ASC的装载和细胞活力的百分比确定在CSMS 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>经过孵化，在无菌的1.5 ml离心管吸管ASC加载，而不会干扰细胞已迁移到插入膜的CSM。 </li><li>去除残留的培养基，并添加250μL新鲜生长培养基管。 </li><li>每管中，加入25μLMTT法[3 - （4,5 - dimethylthiozole -2 -基）-2,5 -二苯基溴化铵溶液（5毫克/毫升）和5％CO 2培养箱中孵育4小时孵化器在37°C。 </li><li>孵化后，取出介质，添加250μL二甲基亚砜，涡2-5分钟，以溶解甲臜的复杂混合物。 </li><li> CSM的离心2700Ğ5分钟，关闭上清吸管，并确定使用标准酶标仪在570 nm和630 nm的波长的吸光度，根据MTT法制造商的规格。 </li><li>确定细胞数量与培养制定一个标准曲线从定义升序号码所得的值相对CSMS。 </li></ol> 6。嵌入式PEG - 纤维蛋白凝胶的ASC-CSM的制备和表征 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>萨格斯等制备了聚乙二醇（PEG）纤维蛋白凝胶（PEG-纤维素）溶解的琥珀戊二酸改性聚乙二醇PEG（3400大）7。使用4毫升Tris缓冲生理盐水公司（TBS，pH值7.8）和过滤消毒与刚刚在实验开始前的0.22微米的过滤器。溶解PEG是只有在此应用程序的第3-4小时有效。 </li><li>混合500万亩;纤维蛋白原股票升（40毫克/毫升在TBS，pH值7.8）和250μlPEG的股票在6孔板，孵育20分钟，在5％CO 2培养箱孵化文化在37°C。这种混合物构成的摩尔浓度比是1:10，SG-PEG-SG的：纤维蛋白原。 </li><li>采取的ASC-CSM 250μL，浓度5毫克的CSM（≈2×10 4细胞）和聚乙二醇化纤维蛋白原的解决方案组合。 </li><li>立即加入1毫升的，凝血酶股票（25U/mL）和快速磨碎移液器一次或两次。与PEG-纤维蛋白原，凝血酶混合后，立即将在12孔板细胞凝胶的混合物，在37°C在5％CO 2培养箱培养箱孵育10分钟，以便为完整的凝胶。由于凝胶时间快，不尝试按住超过5秒内枪头凝胶溶液。聚乙二醇化纤维蛋白原，凝血酶切割，形成了聚乙二醇的纤维蛋白凝胶。由于这样，THé最终凝胶产品被称为PEG-纤维。 </li><li>洗两次PEG-纤维蛋白凝胶的HBSS和孵化最小的重要媒体阿尔法（α-MEM）的补充5％CO 2培养箱孵化器在37与10％FBS°C。 </li><li>观察从CSM的细胞迁移到凝胶超过11天，期间使用标准光学显微镜技术。 </li></ol> 7。胶原凝胶中的嵌入式的ASC-CSM的制备和表征 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>组合的ASC-CSM（含5毫克≈2×10 4细胞），与1型胶原根据8伯恩斯坦和fibrillate后调节pH至6.8使用2N氢氧化钠的方法，从鼠尾肌腱中提取（7.5毫克/毫升）。 </li><li>纤胶原蛋白ASC-CSM的混合物加入到12孔板，孵育30分钟，在5％的CO 2 humidi田间孵化器在37°C </li><li>完成后房颤，在5％CO 2培养箱孵化至11天孵化，在37°C胶原蛋白ASC-CSM的凝胶</li><li>观察从CSM的细胞迁移到凝胶超过11天，期间使用标准的显微镜技术。 </li></ol> 8。双层聚乙二醇，纤维蛋白（ASC-CSM）的胶原蛋白凝胶构造的发展 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>开发的双层构造，准备的胶原蛋白和聚乙二醇纤维蛋白凝胶如上所述，稍作修改。简单地说，研究一个单一来源的干细胞，使用两个bioscaffolds候鸟和共同诱导的性质，“三明治”的ASC-CSMS之间的胶原蛋白和聚乙二醇纤维支架使用的四步过程：1）负载升序到CSM的，2）投在山坳纤胶原凝胶层的ASC-CSM珠演员拉根凝胶，3）PEG-纤维素凝胶以上的ASC-CSM-Ⅰ型胶原凝胶和凝胶固化，4）增加中等和插入来研究细胞在体外或删除从插入体内应用（ 见图1 ）。 </li><li>准备1胶原蛋白的混合物，如上所述，无需添加的ASC-CSM的混合物，在7.1（7.5毫克/毫升）1毫升的类型。将在6孔组织培养插入（8微米孔径）的混合物，并在37°C 30分钟在5％CO 2培养箱孵化器的孵化</li><li>经过完整的颤动，多一层表面的胶原蛋白5毫克的ASC-CSM（10，000细胞/毫克）文化传媒（200μL）暂停。已落户在凝胶微球后，准备如6.0节所述的聚乙二醇纤维蛋白胶，无添加的ASC-CSM的混合物，并层以上的ASC-CSM胶原层的聚乙二醇化纤维蛋白原/凝血酶溶液。当准备PEG - 纤维蛋白胶，使用地方的T细胞培养液中加入250μl他250μL含有细胞的媒介。 </li><li>一旦完成，5％CO 2培养箱培养箱中培养30分钟的结构，实现喂奶前用培养基构造完整的凝胶。 </li><li>完成后的凝胶，地点1毫升的中型以上的下腔的构造和3毫升培养基上腔。 </li></ol> 9。使库存解决方案 注：所有的程序，在室温下进行，除非另有说明。 <ul><li> 氯化钙股票（40毫米）：使用氯化钙2 .2 H 2 O 100毫升去离子水溶解588.4毫克氯化钙2·2H 2 O。消毒用0.22微米的过滤器。 </li><li> 聚乙二醇 PEG是高度与氧气反应，并可以成为室内空气接触氧化。因此，PEG应存放在氮气（N2）气氛 ​​。命中率urately权衡，直到准备使用32毫克2毫升离心管（与N 2，使用前务必清除管）和存储于-80°C间。溶于4毫升TBS的解决方案之前，使用32毫克的聚乙二醇。 以下解决方案必须每次实验前新鲜。 </li><li> TBS的解决方案：（7.75-7.77 pH值在25°C，pH值在25°C是非常重要的）。要准备的TBS溶液15毫升，仔细溶解过滤灭菌去离子水的缓冲片，并调整到所需的pH值。不存储解决方案，准备新鲜每次股票。 </li><li> 纤维蛋白原的解决方案：（40毫克/毫升）。在TBS溶解纤维蛋白原粉使纤维蛋白原浓度40毫克/毫升。一夜之间解散它使用磁力搅拌器在4°C。这是比较容易溶解所需金额的TBS整个1-G数量。翌日，从4℃（通常是阴天）纤维蛋白原，并允许它地暖水浴，直到得到一个同质的解决方案。最后，过滤消毒使用0.45微米的过滤器的纤维蛋白原。 </li><li> 凝血酶溶液（25单位/毫升）：为了使凝血酶溶液，称出所需的金额和化解的准备40毫米氯化钙2 .2 H 2 O它总是一个很好的做法，使用凝血酶整个小瓶，使股票。例如：200毫米氯化钙2·2H 2 O，分装和储存在-20°C溶解一瓶5 KU凝血酶</li></ul> 10。代表结果这里介绍的是该技术的总体目标，证明将作为运载工具使用多个表型的CSM矩阵驱动的同时ASC的分化潜力。我们证明在体外战略提供CSMS干细胞成双层的聚乙二醇胶原纤维支架。嵌入式这个脚手架reveale内ASC的表征ð同时ASC装CSMS“夹心阶层”之间的胶原蛋白和PEG-纤维层和差异来自外环境的提示下茁壮成长的新情况。首先，我们的特点模型系统的能力，以维持细胞活力和迁徙能力。胶原蛋白的ASCs的支持能力，以维持他们的“干”，被证明STRO-1的表达和成纤维样形态（ 图2D和2F）。相比之下，PEG-纤维蛋白诱导的ASCs分化对血管型，为的是证明他们的筒状结构形态，其内皮细胞特异性表达von Willebrand因子（ 图2E，2G），和周细胞特异性表达NG2的和血小板衍生生长因子受体β（PDGFRβ）（数据未显示）。此外，这些观察到的表型出现初发生在文化和维持超过11天，是DEM图3 onstrated在。 表格和图表双层福利建设： <ul><li>无细胞支架单独作为一种生物活性支架可以执行。 </li><li> PEG-纤维蛋白可诱导干细胞分化生长因子除了没有。 </li><li>胶原蛋白能协助维持干细胞表型的ASCs的。 </li><li>一个双层结构可以用来作为其他类型的细胞迁移和增殖（如内皮细胞，成纤维细胞，角质细胞，平滑肌细胞，周细胞）的活性基底。 </li><li>它可以用来与羟基磷灰石或脱钙骨等硬组织工程支架的硬软组织再生。 </li><li>它可以用来发展一个多层次，多样化的组织工程的结构（如皮肤血管，血管上皮细胞，真皮血管，皮下等）。 </li><li>它使用一种单细胞的来源，同时发展多细胞车厢。 </li><li>它有可能与宿主组织的整合，因为它是天然来源。 </li><li>内的凝胶结构CSM提供了一个平台，为细胞迁移。 </li><li>壳聚糖，用于准备CSM的，是一个众所周知的活跃的化学引诱剂。 </li><li>在这个“矩阵驱动的干细胞分化”的协议，适用于整体概念可以应用到其它的干细胞类型。然而，进一步的调查是必要的，确定的矩阵驱动分化的可行性。双层凝胶支架可以作为水库细胞在持续的和可控的方式提供。 </li><li>重建后，凝胶仍然可以被分离成单个组件。 </li></ul><img src="/files/ftp_upload/3953/3953fig1.jpg" alt="图1"/> 图1。示意图描绘的总体目标和过程的技术。 1）脂肪干细胞（ASCs）劳交锋到壳聚糖微球。 2）胶原蛋白，然后倒入6孔插入，调整以fibrillate胶原蛋白的pH值，并放置到6孔板室插入。在胶原蛋白ASC装的CSM球然后分层。 3）聚乙二醇化纤维蛋白原，然后倒在胶原蛋白（ASC - CSM）和凝血酶此外胶凝。 4）最后的双层结构可以从插入的文化，并在体外或体内分析。 <img src="/files/ftp_upload/3953/3953fig2.jpg" alt="图2"/> 图2。表征的ASC内的胶原蛋白和PEG-纤维蛋白的三维矩阵的培养。一个隔离升序）相衬显微照片传代并保持使用常规二维细胞培养技术。显微B，D和F描绘的ASC-CSM在三维胶原凝胶中培养的，而C，E和G秀的ASC-CSM在3维PEG纤维蛋白胶，既培养第1天2。在B和C），ASCs的迁移从CSM的领域中的两个支架类型。 ASCs的出现有一个扁平的，纺锤状的胶原蛋白的形态（乙），同时保持其表达干细胞标记STRO-1（D组;箭头）。当聚乙二醇 - 纤维素的ASCs培养表现出更多的筒状结构和诱导表达血管内皮细胞标记物，如血管性血友病因子（E）。透射电子显微镜描绘了典型的形态，通过在每个支架的ASCs证明。在胶原蛋白凝胶的ASCs出现有小的丝状伪足（FL）从体细胞（F）的延长，而通常的ASCs形成食管腔结构（L），（G组;箭头）。 <img src="/files/ftp_upload/3953/3953fig3.jpg" alt="图3"/> 图3。之间的胶原蛋白和PEG-纤维素凝胶的双层ASC-CSMS形态分析。 ASC-CSMS“夹心阶层”之间的胶原蛋白和聚乙二醇纤维蛋白凝胶和文化保持11天。左colu百万描绘ASCs的迁移和增殖，胶原基质内出现主轴样形态。右列描述的ASCs迁移远离了CSMS，形成整个PEG - 纤维蛋白胶的筒状结构。

<ol>
	<li>Natesan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bilayer+construct+controls+adipose-derived+stem+cell+differentiation+into+endothelial+cells+and+pericytes+without+growth+factor+stimulation.">A bilayer construct controls adipose-derived stem cell differentiation into endothelial cells and pericytes without growth factor stimulation.</a> Tissue Eng. Part A. 17, 941-953 (2011).</li><li>Nuttelman, C. R., Tripodi, M. C., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+hydrogel+niches+that+promote+hMSC+viability.">Synthetic hydrogel niches that promote hMSC viability.</a> Matrix Biol. 24, 208-218 (2005).</li><li>Benoit, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-linked+kinase+production+prevents+anoikis+in+human+mesenchymal+stem+cells.">Integrin-linked kinase production prevents anoikis in human mesenchymal stem cells.</a> J Biomed Mater Res A. 81, 259-268 (2007).</li><li>Willerth, S. M., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+stem+cells+and+biomaterial+scaffolds+for+constructing+tissues+and+cell+delivery.">Combining stem cells and biomaterial scaffolds for constructing tissues and cell delivery.</a> , (2008).</li><li>Natesan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cell+delivery+into+collagen+gels+using+chitosan+microspheres.">Adipose-derived stem cell delivery into collagen gels using chitosan microspheres.</a> Tissue Eng. Part A. 16, 1369-1384 (2010).</li><li>Bubnis, W. A., Ofner, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+determination+of+epsilon-amino+groups+in+soluble+and+poorly+soluble+proteinaceous+materials+by+a+spectrophotometric+method+using+trinitrobenzenesulfonic+acid.">The determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid.</a> Anal. Biochem. 207, 129-133 (1992).</li><li>Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PEGylated+fibrin+patch+for+mesenchymal+stem+cell+delivery.">A PEGylated fibrin patch for mesenchymal stem cell delivery.</a> Tissue Eng. 12, 9-19 (2006).</li><li>Bornstein, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+rattail+collagen+used+as+substrate+for+tissue+cultures+on+coverslips+in+Maximow+slides+and+roller+tubes.">Reconstituted rattail collagen used as substrate for tissue cultures on coverslips in Maximow slides and roller tubes.</a> Lab Invest. 7, 134-137 (1958).</li><li>Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+differentiation+of+bone+marrow+stem+cells+is+directed+by+a+tunable+three-dimensional+matrix.">Vascular differentiation of bone marrow stem cells is directed by a tunable three-dimensional matrix.</a> Acta Biomater. 6, 3395-3403 (2010).</li><li>Rochon, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+epithelial+cells+regulate+the+size+and+morphology+of+tissue-engineered+capillaries.">Normal human epithelial cells regulate the size and morphology of tissue-engineered capillaries.</a> Tissue Eng. Part A. 16, 1457-1468 (2010).</li><li>Liu, H., Collins, S. F., Suggs, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+for+expansion+and+differentiation+of+mouse+embryonic+stem+cells.">Three-dimensional culture for expansion and differentiation of mouse embryonic stem cells.</a> Biomaterials. 27, 6004-6014 (2006).</li></ol>

没有竞争的金融利益存在。 免责声明所载的意见或主张是作者的私人意见，是不被理解为官方或反映国防部或美国政府部门的意见。作者是美国政府的雇员，以及作为其执行公务的一部分准备工作。所有的工作是由美国陆军医学研究和装备司令部的支持。这项研究是由美国陆军医学研究和装备司令部机构审查委员会，并按照批准的协议的审查和批准了一项协议下进行的。

ASCs的是众所周知的，他们易于隔离和向多种细胞类型分化的能力。在这个手稿中描述的技术，我们能够利用这些细胞暴露多个biomatrices同时ASCs的可塑性。细胞迁移远离他们的CSM基地，并进入其周围的细胞外环境，细胞采取从断头台上的提示，并可以保持“干细胞”（胶原蛋白），或向血管和血管支持细胞类型（纤维蛋白）被诱导分化。由于我们的实验室是在皮肤及软组织伤口愈合的兴趣，我们?...

<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>汉克斯平衡盐溶液（HBSS中） </td><td> Gibco公司</td><td> 14175 </td><td>耗材</td></tr><tr><td>胎牛血清</td><td>胎牛血清</td><td> SH30071.03 </td><td>耗材</td></tr><tr><td>二型胶原酶</td><td> Sigma-Aldrich公司</td><td> C6685 </td><td>耗材</td></tr><tr><td> 70微米尼龙网过滤器</td><td> BD公司</td><td> 352350 </td><td>耗材</td></tr><tr><td> 100μm的尼龙网过滤器</td><td> BD公司</td><td> 352360 </td><td>耗材</td></tr><tr><td> MesenPRO生长介质系统</td><td> Invitrogen公司</td><td> 12746-012 </td><td>耗材</td></tr><tr><td> L-谷氨酰胺</TD&gt; <td> Gibco公司</td><td> 25030 </td><td>耗材</td></tr><tr><td>氯化钙2·2H 2 O </td><td>西格玛</td><td> C8106 </td><td>耗材</td></tr><tr><td>的T75组织培养瓶</td><td> BD公司</td><td> 137787 </td><td>耗材</td></tr><tr><td>壳聚糖</td><td> Sigma-Aldrich公司</td><td> 448869 </td><td>耗材</td></tr><tr><td>醋酸</td><td> Sigma-Aldrich公司</td><td> 320099 </td><td>耗材</td></tr><tr><td>正辛醇</td><td> Acros Organics公司</td><td> 150630025 </td><td>耗材</td></tr><tr><td>山梨醇单声道，油酸</td><td> Sigma-Aldrich公司</td><td> S6760 </td><td>耗材</td></tr><tr><td>氢氧化钾</td><td> Sigma-Aldrich公司</td><td> P1767 </td><td>耗材</td></tr><tr><td>丙酮</td><td> Fisher Scientific则</td><td> L - 4859 </td><td>耗材</td></tr><tr><td>乙醇</td><td> Sigma-Aldrich公司</td><td> 270741 </td><td>耗材</td></tr><tr><td>硝基苯酸</td><td> Sigma-Aldrich公司</td><td> P2297 </td><td>耗材</td></tr><tr><td>盐酸</td><td> Sigma-Aldrich公司</td><td> 320331 </td><td>耗材</td></tr><tr><td>乙醚</td><td> Sigma-Aldrich公司</td><td> 472-484 </td><td>耗材</td></tr><tr><td> 8微米的细胞培养板插入</td><td> BD公司</td><td> 353097 </td><td>耗材</td></tr><tr><td> 1.5毫升离心管</td><td>费舍尔</td><td> 05-408-129 </td><td>耗材</td></tr><tr><td> MTT法试剂</td><td> Invitrogen公司</td><td> M6494 </td><td>耗材</td></tr><tr><td>二甲基亚砜</td><td> Sigma-Aldrich公司</td><td> D8779 </td><td>耗材</td></tr><tr><td> qtracker细胞标记试剂盒（问跟踪655） </td><td>分子探针</td><td> Q2502PMP </td><td>耗材</td></tr><tr><td> 1型胶原</td><td> travigen </td><td> 3447-020-01 </td><td>耗材</td></tr><tr><td>氢氧化钠</td><td> Sigma-Aldrich公司</td><td> S8045 </td><td>耗材</td></tr><tr><td> 12孔细胞培养板</td><td> BD公司</td><td> 353043 </td><td>耗材</td></tr><tr><td>纤维蛋白原</td><td>西格玛</td><td> F3879 </td><td>耗材</td></tr><tr><td>凝血酶</td><td>西格玛</td><td> T6884 </td><td>耗材</td></tr><tr><td> benztriazole衍生的聚乙烯</td><td> sunbio </td><td> DE-034GS </td><td>耗材</td></tr><tr><td>平板Tris缓冲（pH值7.6） </td><td>西格玛</td><td> T5030 </td><td>耗材</td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td> 5417R </td><td>马巴贝西pment </td></tr><tr><td>轨道摇床</td><td>新不伦瑞克省Scienctific </td><td> C24的</td><td>设备</td></tr><tr><td>湿润的孵化器与空气，5％的CO 2 </td><td> Thermo Scientific的</td><td> 370型</td><td>设备</td></tr><tr><td>架空搅拌器</td><td>伊嘉</td><td> visc6000 </td><td>设备</td></tr><tr><td>磁力搅拌器</td><td>康宁</td><td> PC-210 </td><td>设备</td></tr><tr><td>真空干燥器</td><td> - </td><td> - </td><td>设备</td></tr><tr><td>粒度分析仪</td><td>马尔文</td><td> STP2000 Spraytec </td><td>设备</td></tr><tr><td>水浴</td><td> Fisher Scientific则</td><td> isotemp210 </td><td>设备</td></tr><tr><td>分光光度计</td><td>贝克曼</td><td>贝克曼DU 800UV/Visible分光光度计</td><td>设备</td></tr><tr><td>涡旋</td><td> diagger </td><td> 3030A </td><td>设备</td></tr><tr><td>酶标仪</td><td>分子器件</td><td> SpectraMax M2的</td><td>设备</td></tr><tr><td>光/荧光显微镜</td><td>奥林巴斯</td><td> IX71 </td><td>设备</td></tr><tr><td>共聚焦显微镜</td><td>奥林巴斯</td><td> FV-500激光共聚焦显微镜</td><td>设备</td></tr><tr><td>扫描电子显微镜</td><td>卡尔蔡司显微成像</td><td>狮子座435副总裁</td><td>设备</td></tr><tr><td>透射电子显微镜</td><td> JEOL </td><td> JEOL 1230 </td><td>设备</td></tr></tbody></table>

engineering a bilayered hydrogel to control asc differentiation

多年来的天然聚合物获得了更多，因为它们的宿主的生物相容性和交互能力与细胞在体外和体内的重要性，认为在再生医学中的承诺的一个研究领域，是新型生物材料和干细胞的组合使用。在组织工程领域的一个基本战略是指导细胞的功能，使用三维支架（如脱细胞外基质，水凝胶，微/纳米颗粒）。这项技术已经从发现，细胞需要一个基板后，他们能够坚持，增殖，并表达他们分化 ​​的细胞表型和功能2-3。最近，它也已被确定，细胞不仅使用这些基板的坚持，但也互动，并采取从矩阵基板（例如，细胞外基质，细胞外基质）的线索。因此，细胞和支架有一个相互连接，为控制组织发展，组织，和最终功能。脂肪干细胞（ASCs）是间质，非造血干细胞存在于脂肪组织，可以表现出多谱系分化的细胞现成的源（即前血管内皮细胞和周细胞）作为。我们的假设是，脂肪干细胞可以向简单的合作，培养他们的不同表型，同时在双层矩阵1。我们的实验室专注于皮肤伤口愈合。为此，我们建立了从天然生物材料的复合材料基体，纤维蛋白，胶原蛋白，壳聚糖，可以模仿特定皮肤伤口愈合的ECM环境的特点和功能。

Watch this Scientific Journal Video about Engineering a Bilayered Hydrogel to Control ASC Differentiation at JoVE.com

Engineering a Bilayered Hydrogel to Control ASC Differentiation

该协议着重于利用干细胞的固有能力，其周围的细胞外基质的线索，被诱导分化成多个表型。这种方法手稿延伸我们描述一个利用双层水凝胶的模型，PEG-纤维蛋白和胶原蛋白的组成和特性，同时脂肪干细胞分化<sup&gt; 1</sup&gt;。

工程双层凝胶控制升序分化

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

engineering-a-bilayered-hydrogel-to-control-asc-differentiation

Research

JoVE Journal

Bioengineering

13.8K 次观看.  United States Army Institute of Surgical Research. 该协议着重于利用干细胞的固有能力，其周围的细胞外基质的线索，被诱导分化成多个表型。这种方法手稿延伸我们描述一个利用双层水凝胶的模型，PEG-纤维蛋白和胶原蛋白的组成和特性，同时脂肪干细胞分化 1。

视频: Engineering a Bilayered Hydrogel to Control ASC Differentiation

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

龙工程的程序

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

科研

生物工程

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

心肌在心脏组织工程中的纤维蛋白凝胶的封装

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

一个工具包，使水环境中的烃类转化

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

内含肽介导的人工蛋白水凝胶的合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

肿瘤微环境基质与应用癌细胞侵袭组织工程

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

可注射药物加载超分子凝胶局部导管注入猪心

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

基于状的不均匀 Nanopatterns 对局部控制表面粘附的研究--一种直接软骨分化的方法

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

凝胶序列: 一种同时测序库的方法用水凝胶基质制备 DNA 和 RNA

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...