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

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

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作者什么都没有透露。

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

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

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

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

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

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

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

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

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

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

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

Research

JoVE Journal

Bioengineering

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

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

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

形状记忆聚合物主动细胞培养

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

科研

生物工程

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

自体内皮祖细胞播种技术和生物相容性在大动物模型的心血管设备的测试

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统:作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

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 ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

皮肤纹身作为一种新的方法DNA疫苗交货

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

在细胞和组织力学生物学应用一种新型的拉伸平台

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.