作者感谢PGS的合成，彼得Crapo见地的讨论，为生物反应器的设置，博士博士博士高瑾。穆罕默德Ezzelarab explanting狒狒颈动脉和魏吴。这项研究是由来自美国国立卫生研究院（R01 HL089658）的赠款支持。

1。管状支架制造<ol><li>准备玻璃管（长度为70毫米，外径= 9.0毫米，壁厚为1.0毫米;小零件，美丽华，佛罗里达州）作为支架模具。 </li><li>配制成10毫升去离子水溶解100毫克医管局1.0 WT /卷％透明质酸（HA）（Sigma - Aldrich公司，圣路易斯，密苏里州）。混合使用一个肩过夜，直到有解决方案，无气泡。 </li><li>玻璃管的顶部倒入1.0％的HA解决方案。让它流沿模具的内壁慢慢放下。翻转油管达成的解决办法时用肩模具的底部，并重复此步骤，直到模具内壁均匀地涂在解决方案。 </li><li>涂层后，干成真空烘箱，在37 ° C，24小时全玻璃管材</li><li>研磨和筛盐颗粒（大小= 25-32微米）作为致孔剂。 </li><li>组装一个玻璃管（1.0％的HA解决方案内涂层），顶杆（PTFE管包裹不锈钢），热 ​​缩（HS）套筒，PTFE环所述先前 8 。 </li><li>添加所需的大小的盐颗粒玻璃模具，通过使用硅橡胶漏斗抹刀。轻轻点击模具，以确保盐颗粒的均匀分布。刮去多余的盐颗粒整个模具用锅铲一边的顶部。 </li><li>杂交孵化器为转移的盐包装模具的前30分钟打开。关闭杂交孵化器和盐融合杂交孵化器放入盐包装模具。 </li><li>从杂交培养箱中取出模具，干成真空烘箱，在37 ° C为24小时</li><li>从真空炉中取出盐包装模具，使他们能够在室温下冷却。取出不锈钢芯棒推持PTFE环。如果芯棒模具太紧，用针钳将其拉出。从模具底部取出PTFE环。 </li><li>置于120℃5分钟的模具收缩协套筒放入烘箱。检查HS套筒缩水。删除从盐包装模具的HS套，并允许他们在室温下冷却。模具，直到它们被用来保持到dessicator。 </li><li> PGS的解决方案，准备通过溶解到15毫升的四氢呋喃（THF）（Sigma - Aldrich公司，圣路易斯，密苏里州）3克至PGS的形成20 WT / vol％的解决方案。 </li><li> PGS的解决方案添加盐包装模具内腔，在休谟罩的一个spoid下降。精益玻璃模具约45 °，慢慢旋转模具放入内腔PGS的解决方案。检查PGS的解决方案，如果模具壁沿流下来。如果干点，增加更多的解决方案。 </li><li>加入PGS的解决方案后，将在休谟罩的模具，至少30分钟蒸发四氢呋喃。 </li><li>放入预先确定的固化时间的真空干燥箱（例如，24 / 36 / 48小时）在100毫托和150 ° C下固化PGS的模具。 </li><li>固化后，取出模具，在室温下放置他们冷静下来。与垂直位置，慢慢的去离子水浸入每个模具。不要浸入水快，因为气泡可能导致撕裂脚手架。 </li><li>模具转移到水浴，并放置在他们的角度为1 h，使用硅管容易溶解医管局。检查如果房委会是从模具溶解。如果没有房委会是从模具释放，推动脚手架的一端，慢慢地用锅铲从模具释放。在脚手架的另一端重复这一步，在水浴慢慢来回摇晃模具。检查棚架的模具内移动。 </li><li>脚手架转移到另一个搅拌器水浴仔细。不要抢牢固的脚手架，这可能破解脚手架。变幻出盐颗粒水浸出至少3天，在水浴中放置的脚手架。 </li><li>出盐颗粒浸出后，传输每个支架充满去离子水15 mL离心管中，并放置在干冰中1小时冻结。离心管放入冻干机（必须打开所有管驾驶室）和冻干2天。 </li><li>放入dessicator前，直到它们被用于所有棚架。 </li></ol> 2。细胞种植支架的制备<ol><li>从dessicator支架和切割长度为25 - 30mm的。 </li><li>准备两个硅橡胶瓶塞（直径为20毫米，壁厚= 6.35毫米，中间孔的直径3毫米），并连接两个聚四氟乙烯管材（外径= 3.97毫米，内径= 2.38毫米，壁厚= 0.79毫米，长度= 60毫米和40毫米），通过每个塞子中间的孔。 </li><li>剪下一个HS环的长度为1.5毫米，并把它的一端棚架上。推入一个尽可能小的重叠部分的脚手架的一个PTFE管（连接的硅橡胶塞）。 </li><li>置于120 ° C下10分钟的脚手架和PTFE管收缩协环放入烘箱。检查协环收缩和棚架牢牢地连接到PTFE管。 </li><li>取出支架和PTFE管，并将其放置在室温下冷却下来。放入聚碳酸酯管（外径= 15.5毫米，内径= 9毫米，长度= 50毫米）作为生物反应器室脚手架PTFE管大会。 </li><li>步骤2.3）和2.4），PTFE管的另一端连接的脚手架。 </li><li>调整两个硅橡胶瓶塞，将其内表面的聚碳酸酯管两端。将两个硅橡胶瓶塞外表面的两个铝合金板（顶部和底部宽度为20毫米，长度为70毫米，厚度为6.35毫米）。板的侧孔放入两个螺纹杆和修复拇指板螺母拧紧螺丝的聚碳酸酯管（包括支架内）到板。 </li><li>测量每个支架seedable的长度（长度两房协环），并计算出每个支架的内表面面积（πx的内部直径x seedable长度），细胞种植。 </li><li>用铝箔包裹之一的生物反应器室单位和在120℃30分钟高压灭菌消毒。在120℃高压灭菌30分钟消毒生物反应器的每个部分（中型水库，泵管，气体交换，阀组，针形阀）和细胞培养罩内组装。 </li><li>治疗前和一系列灌注（70，50，和25％的乙醇为1小时，磷酸盐缓冲液（PBS冲洗支架; Mediatech，赫恩登，弗吉尼亚州）H 2，以及24小时的使用SMC培养基在1.0毫升/分钟的流电路的蠕动泵。 </li></ol> 3。细胞种植和文化<ol><li>隔离的主要从少年的雄性狒狒（狒狒阿努比斯）颈动脉血管平滑肌细胞（校董会）和前传代和播种以前 9所描述的，在二维（2D）文化特征。 </li><li>展开"校董会使用MCDB 131培养基（Mediatech，赫恩登，弗吉尼亚州），10％小牛血清（龙沙，Walkersville，MD），1％的L -谷氨酰胺（Mediatech），50微克/毫升抗坏血酸（费舍尔科学，公平的草坪"新泽西州），和一种抗生素antimycotic解决方案（10,000 IU / ml青霉素，10000微克/ ml链霉素和25微克/毫升两性霉素B; Mediatech）。 </li><li>在生物反应器，分离流电路的每一个生物反应器室。种子的校董会（通过4-6）脚手架用5 ml注射器注射到管腔内支架后，切断都abluminal（进口）和管腔（出口）流动的悬浮液密度的2 × 10 6细胞/厘米2。 </li><li>杂交孵化器放入生物反应器商会在37 ° C，2 4小时的转速旋转，以使细胞均匀扩散到支架。 </li><li>从杂交孵化器重新连接它们的生物反应器商会在引擎盖流生物反应器电路。转移到细胞培养孵化器的所有生物反应器系统。 </li><li>连接泵管（内径1.6毫米），蠕动泵，软骨和负载在水库的新鲜培养液。开始穿墙灌注（管腔abluminal）1.0毫升/分钟的15分钟仅管腔流。 </li><li>流量和压力逐渐增加，从1.0毫升/分钟（管腔剪应力:1.1达因/厘米2）和1至14.2毫升/分（15.3达因/厘米2）和120毫米汞柱，21天分别为20毫米汞柱上一天。更改在4天的泵管（内径3.2毫米）和中等每周一次。 </li></ol> 4。组织采伐和用于分析的样品制备<ol><li>经过为期21天的文化，停止泵的中型水库和钳油管（入口和出口）。蠕动泵和生物反应器系统转移到引擎盖软骨分离的泵管。 </li><li>夹具每个生物反应器腔的进气口和插座油管和分离的流动循环。打开入口管，并准备在出口管培养皿盘，里面收集的聚碳酸酯管培养基。慢慢打开出口管和室中删除。 </li><li>脱离室abluminal针头和管腔硅胶管和分离释放拇指螺丝铝板。取出聚碳酸酯管，PTFE管分离组织的一端建造。 </li><li>准备一个Petri菜充满了10毫升的PBS和分离的构造从PTFE管的另一端。把建设成的PBS冲洗三次。 </li><li>用刀片剪切长度为5毫米的构造。冻结在-80 ° C的冷冻生化分析或转移到15 mL离心管5毫升的新鲜培养基充满机械测试或组织TEK最佳切削温度化合物（樱花仁昌，托兰斯，CA公司的管理单元冻结）为组织学/ immunofluorescence染色。 </li></ol> 5。代表性的成果: PGS的管状支架采用可生物降解弹性体，盐融合方法（图1A）。每个生物反应器室提供管腔和abluminal流动棚架，并可以作为一个独立的单元从主流程循环（图1B）的分离。生物反应器系统的设计时间，控制和监视流量以及压力（ 图1C＆D）的培养四个支架。 生物反应器培养的示意图如图所示 。 2。播种校董会后，每个生物反应器腔旋转37 ° C为4小时，均匀分布在管腔内支架的细胞。然后，脉动流，直到第14天支架应用于流量逐渐增加（ 图2A）和压力（ 图2B）。 14天之后，流量和压力保持不变，直到文化的结束（21天）。 PGS的支架表面形态的扫描电子显微镜（SEM）进行了检查。扫描电子显微照片表明，脚手架有一致的壁厚（539 ± 18微米）（ 图 3A）和随机分布的宏观和微观孔隙壁面（ 图 3B ）。 microcomputed断层扫描（显微CT）和成像分析的支架的形态学参数测量。平均孔径为23.3 ± 3.9μm和孔隙互连为99.4 ± 0.62％，这意味着所有的毛孔都在脚手架互连。乙醇位移测量的孔隙率是75.6 ± 2.7％。 细胞形态PGS的构造进行了检查，扫描电镜（ 图4）。多层校董会完全覆盖壁面和他们流动方向垂直导向。这些结果表明，脉动流生物反应器的机械空调，支持在脚手架的SMC方向。 H＆E染色和弹性蛋白的自体荧光（图5），细胞外基质（ECM）和弹性纤维的存在进行了检查。 H＆E染色显示，细胞和ECM蛋白完全覆盖的PGS的管腔结构。弹性蛋白自体荧光也表现出圆周组织的弹力纤维，管腔表面的构造。 PGS的构造ECM蛋白的生产生化分析测量。不溶于水的弹性蛋白和胶原含量分别为20.2 ± 9.1微克/毫克组织和6.3 ± 1.9微克/毫克组织，。 <img src="/files/ftp_upload/2691/2691fig1.jpg" alt="图1"/> 图1。脚手架制造和生物反应器系统。 （一）管状支架制造示意图。 （二）生物反应器室。支架PTFE管连接，放入聚碳酸酯管，硅橡胶瓶塞和铝合金板固定。每个分庭有两个流动路径:管腔（硅胶管）和abluminal（针头）流动。 （三）生物反应器系统置于培养箱内。它包括中型水库，蠕动泵模块，气体热交换器，压力传感器，两个歧管（顶部和底部），和针形阀。 （四）生物反应器系统置于外的孵化器。它包括压力监测，流量控制单元，数据采集系统和计算机。 <img src="/files/ftp_upload/2691/2691fig2.jpg" alt="图2"/> 图2的生物反应器文化的原理图。 （一）文化协议。 （b）申请在每个时间点的压力分布（第1天，4，7和14）。 <img src="/files/ftp_upload/2691/2691fig3.jpg" alt="图3"/> 图3。PGS的支架的表面形貌。 （一）横截面。 （二）流明。 <img src="/files/ftp_upload/2691/2691fig4.jpg" alt="图4"/> 图4的PGS的细胞形态结构。 （一）流明。 （二）45 °切割断面。在这两个数字的箭头代表流动方向。 <img src="/files/ftp_upload/2691/2691fig5.jpg" alt="图5"/> 图5。病理和弹性蛋白的PGS的自体荧光的结构。 （一）H＆E染色。 （二）通讯的弹性蛋白自体荧光。 L:流明。放大倍数:40X。比例尺:50微米。

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+cell+microintegrated+blood+vessel+constructs+through+electrohydrodynamic+atomization.">Fabrication of cell microintegrated blood vessel constructs through electrohydrodynamic atomization.</a> Biomaterials. 28, 2738-2746 (2007).</li><li>Nieponice, A., Soletti, L., Guan, J., Deasy, B. M., Huard, J., Wagner, W. R., Vorp, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+tissue-engineered+vascular+graft+combining+a+biodegradable+scaffold,+muscle-derived+stem+cells+and+a+rotational+vacuum+seeding+technique.">Development of a tissue-engineered vascular graft combining a biodegradable scaffold, muscle-derived stem cells and a rotational vacuum seeding technique.</a> Biomaterials. 29, 825-833 (2008).</li></ol>

使用这里描述的一种可生物降解弹性体的制造技术有几个特点。 （1）我们作为一个脱模透明质酸（HA）。由于房委会是易溶于水，支架容易释放入水浸泡后的玻璃模具。在这份报告中，我们使用的HA解决方案的1.0％重量/体积，因为低浓度（&lt;0.5 / VOL％）的解决方案是不粘性和流下来的这么快，当我们浇筑玻璃管上。均匀的涂层HA解决方案，我们翻转过来当溶液的玻璃管飞下来的油管底部，并重复此步骤。这HA涂层是为最终支架释放的关键，我们的制造过程。 （2）我们保留在玻璃管的盐用于热缩（HS）套筒。由于盐密集在玻璃管和房协套的内壁之间的空间包装，恒生套保留芯棒和PTFE环取出后，在管底部的盐。我们可以轻松删除协袖模具放入一个烤箱在120℃5分钟，然后得到管状盐模板。 （3）我们所用的盐融合方法。这是众所周知的，盐融合方法，可以提高通过不同的融合时间 10孔互连和机械性能。此外，由于我们使用的PGS，宏观毛孔生产在浸出过程中的盐颗粒，而微孔隙可能甘油蒸气生成在固化正如我们前面描述的 11 PGS的形成。因此，这种方法有一个潜在的编造不同的宏观和微观结构的多孔管状支架，由不同的盐颗粒以及PGS的固化条件。 从生物反应器的机械空调提供的搏动血流灌注（最大平均流量= 14毫升/分钟，最大剪应力= 15.3达因/ 厘米 2，频率= 0.5 - 1.7 Hz）和生理有关的压力，从而导致与PGS的脚手架SMC增长和方向（ 图4）。这些结果与以往的研究报告，在此频率循环拉伸和剪切应力增加12，SMC增殖和ECM蛋白生产 13,14一致。此外SMC增长和方向，PGS的构建支持ECM蛋白的生产，尤其是周组织生物反应器中的弹性纤维，在为期3周的文化（ 图 5 ）。使用一个小直径的动脉构建一个弹性支架的一些研究已经证明与本土动脉15，并使用符合飞旋烧瓶16,17棚架的SMC的快速集成的机械强度和爆破压力，而没有弹性纤维，在这些构造中发现的。我们的研究结果表明，循环生物反应器的径向腹胀更有效地提高机械信号转导在PGS的脚手架，这可能导致弹性蛋白的合成和组织校董会。 由于血管平滑肌细胞产生ECM蛋白，在我们的方法，静态内皮细胞，并有必要制定临床上成功的小直径的动脉结构，提高机械强度。我们曾报道，内皮细胞共同培养，与校董会产生一个融合的单层和支持的表型蛋白表达下我们的文化条件和机械空调9。因此，基于我们的做法，在这里，共培养的实验条件下的修改将由此产生的构造，以改善功能和产生nonthrombogenic，强大的下一个步骤，并符合动脉结构，类似原生动脉的。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>透明质酸钠盐</td><td> Sigma - Aldrich公司</td><td> H7630 </td></tr><tr><td>四氢呋喃</td><td> Sigma - Aldrich公司</td><td> 401757 </td></tr><tr><td> MCDB 131 </td><td> Mediatech </td><td> 15 - 100 - CV </td></tr><tr><td>胎牛血清</td><td>龙沙</td><td> BW14 - 502F </td></tr><tr><td> L -谷氨酰胺</td><td> Mediatech </td><td> 25 - 005 - CV </td></tr><tr><td>抗坏血酸</td><td> Fisher Scientific则</td><td> A62 - 500 </td></tr><tr><td>抗生素antimycotic的解决方案</td><td> Mediatech </td><td> 30 - 004 - CI </td></tr><tr><td>磷酸盐缓冲液（PBS） </td><td> Mediatech </td><td> 21 - 031 - CV </td></tr><tr><td>组织- Tek的最佳切削温度的化合物，4583 </td><td>樱花仁昌</td><td> 25608-930 </td></tr></tbody></table>

elastomeric pgs scaffolds in arterial tissue engineering

心血管疾病是在美国死亡的首要原因之一，特别是冠状动脉疾病的增加与人口老龄化和增加肥胖1。目前，搭桥手术使用的自体血管，移植，合成移植作为一种常用动脉的替代品使用2。然而，这些移植动脉的内径小于6毫米，由于低可用性，血栓性并发症，遵守不匹配，和内膜增生晚期3,4有限的应用。为了克服这些限制，组织工程已成功地应用于作为一个很有前途的替代发展小直径动脉结构nonthrombogenic，健壮的，符合。一些以前的研究已经开发了小直径三层状结构，优良的机械性能和爆破压力与 5,6本土动脉动脉结构。虽然高的拉伸强度和爆破压力，通过增加胶原蛋白，从刚性材料或细胞表脚手架的生产，这些结构仍然有低弹性蛋白的生产和遵守，这是一个重大的问题，植入后造成移植失败。考虑到这些问题，我们假设elastometric生物材料与机械空调相结合，将提供弹性和更有效地进行机械信号血管细胞，增加细胞外基质的生产和支持细胞的方向。 本报告的目的是引入多孔管状支架的制造技术和动态申请动脉组织工程机械空调。我们用一种可生物降解弹性体，聚（癸二酸丙三醇）从盐融合方法制备多孔管状支架（PGS）7 。成人小学狒狒平滑肌细胞（校董会）接种棚架，在我们设计的脉动流生物反应器培养3周的管腔。 PGS的支架有一致的厚度和随机分布的宏观和微观孔隙。从脉动流生物反应器的机械空调支持SMC方向和增强ECM在支架的生产。这些结果表明，弹性支架和生物反应器培养的机械空调可能是一个有前途的方法动脉组织工程。

Watch this Scientific Journal Video about Elastomeric PGS Scaffolds in Arterial Tissue Engineering at JoVE.com

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

弹性的PGS在脉动流生物反应器培养的血管平滑肌细胞的生物支架，可能会导致在一个相对较短的文化时期的本地流脑生产的直径有前途的小动脉结构。

弹性PGS的动脉组织工程支架

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

elastomeric-pgs-scaffolds-in-arterial-tissue-engineering

Research

JoVE Journal

Bioengineering

15.5K 次观看.  University of Pittsburgh. 弹性的PGS在脉动流生物反应器培养的血管平滑肌细胞的生物支架，可能会导致在一个相对较短的文化时期的本地流脑生产的直径有前途的小动脉结构。

视频: Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

小鼠模型肠组织工程

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

科研

生物工程

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

密度梯度多层聚合（dGMP的）:创建多室的，可定制的一种新技术，组织工程支架

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

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 ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

保利的静电纤维支架（甘油 - 十二双）工程神经组织从小鼠胚胎干细胞

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

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. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

三明治般的微环境来驾驭细胞/材料相互作用

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

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 ...

弹性PGS的动脉组织工程支架

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