这项研究是由美国国立卫生研究院主任的新的创新奖励计划，美国国立卫生研究院医学研究路线图的一部分，通过授予数量DP2 - 1 - OD004309 - 01，部分支持。 SBS - N。想感谢美国国家科学基金会研究生研究奖学金。

1。前处理组织准备<ol class="lalpha"><li>使用此协议之前，必须已经脱细胞的组织选择。对于这个例子，新鲜的猪和人类心包样品使用去离子（DI）水和十二烷基硫酸钠（SDS），低渗和高渗漂洗脱细胞。 </li><li>具体来说，首先在DI水冲洗猪心包30分钟，然后搅拌在1％SDS不断在磷酸盐缓冲液（PBS）为24小时，5小时DI水冲洗。对于人类pericardia，首先在DI水冲洗30分钟，然后搅拌在1％SDS的PBS连续60-65小时，其次是一个通宵的直接投资冲洗。删除所有标本，从他们的最终解决方案，并再次运行 DI水1下冲洗。 </li><li>为了验证脱细胞，取出一小块，新鲜冻结华侨城冻结介质，并采取10μm的组织切片样本在整个审查通过组织学分析，每100微米，如前呈报 34-36 。 </li><li>使用苏木精和曙红（H＆E）的污渍研究原子核的情况下组织。人们还可以使用一个荧光赫司特33342染色（1.0μg/ mL的）进行DNA验证的H＆Ë结果，简单地说，修复的部分补充水分，并在水中冲洗，染色10分钟，和然后冲洗以及和存储在黑暗中。另外，可以使用一个工具包，如Qiagen公司DNeasy血液和组织试剂盒，其目的是量化样品的总DNA含量。 </li></ol> 2。注射流脑的制备<ol class="lalpha"><li>冻干<ol class="lroman"><li>经过适当的准备，冻结脱细胞样品用液氮或存放于-80 ° C。冻干样品，直至完全干燥。根据您的系统和你的样品的水分含量，这可能需要12至72小时的任何地方。 </li></ol></li><li>磨<ol class="lroman"><li>一旦完全干透，WILEY小型轧机是用来研磨成细粉干燥ECM。你的目的选择合适的筛孔尺寸，在这里，40计网。一旦大多数的样品通过过滤器，删除与精流脑粉收集罐。如果只有一个小样本，其余的样本，可以从工厂中提取多种方式;在这里，一个Q - TIP是用来消除落后轧机固定叶片卡住了ECM。 </li><li>务必确保轧机是干净的，无碎片。同样重要的是，以确保轧机和您的样品完全干燥;任何剩余的水分会导致ECM的聚合和聚集在一起，防止成功的铣床。 ECM可能拿起空气中的水分，所以应该直接去磨冻干机。 </li></ol></li><li>消化<ol class="lroman"><li>要形成的注射流脑，粉是消化的胃蛋白酶。从Freyetes以下协议进行了修改， 等。（2）。 </li><li>重要的是要保持无菌尽可能产品，因为它会在体内注入和污染可能会引起并发症。因此，使用无菌小瓶和权衡匙和过滤器在使用前的所有解决方案。冻干后铣削和前消化步骤也将有助于保持无菌。 </li><li>称取到一个合适的闪烁瓶中的ECM的粉所需的金额。总量小于1毫升，建议您使用2毫升的样品瓶和更大的卷，20毫升的小瓶。这将确保有足够的液体在小瓶有效挑起的底部。 </li><li>闪烁瓶称取所需数量的胃蛋白酶。添加0.1 M的盐酸，胃蛋白酶在1毫克/毫升的浓度是。确保充分溶解胃蛋白酶（无颗粒）在使用前可以通过震荡的胃蛋白酶溶液赶紧。 </li><li>添加到与ECM粉的闪烁瓶中的解决方案，使胃蛋白酶/盐酸ECM的浓度为10毫克/毫升。 </li><li>不断搅拌的解决方案，为60-​​65小时，小瓶双方定期刮了一掂勺或锅铲。 </li></ol></li><li> pH值调整<ol class="lroman"><li>执行这一步是把液体基质材料生理pH值和灭活的胃蛋白酶，进一步裂解ECM的。再次，一定要使用微孔水过滤解决方案。 </li><li> 1 M氢氧化钠被添加到原体积的1 / 10。测试在这一点上的pH值，添加少量的氢氧化钠或盐酸等（2-10微升），达到所需的pH值（7.4）。保持跟踪失效或添加少量的pH值测试中删除的所有。一旦解决方案已瓦解，加10倍PBS将1 / 10的最后量（1 / 9目前的音量）。确定注射流脑的浓度，然后添加1X PBS，以达到理想的最终合作ncentration，这里6毫克/毫升。 </li><li>注射流脑可通过SDS - PAGE Blyscan色度聚糖含量，和质谱表征。 </li><li>在这一点上，基体材料可以被注入体内 ，形成心肌组织工程支架材料。 </li></ol></li><li>生物素标记<ol class="lroman"><li> ECM可与生物素注射前注射流脑容易可视化标签。 </li><li>准备一个10mm的解决方案和生物素的添加比例的0.3mg/mg注射流脑。 ECM的，应在注射所需浓度。如果在注射6 mg / ml浓度，添加40μLECM的每1毫升的生物素。注射前，保持至少1个小时的上冰素ECM解决方案。 </li></ol></li><li>所有加工步骤，可在室温下进行。为了保持凝胶注射基质，pH值调整的步骤可能会在冰上进行。 </li></ol> 3。心肌注射<ol class="lalpha"><li>注射液<ol class="lroman"><li>对于这里使用的雌性大鼠（225-250克），75μLpH值7.4注射流脑注射准备在0.1毫升注射器30表针放倒。如果使用雄性大鼠（375-400克），90μL可以被注入。 </li><li>手术前应消毒所有的手术用品，这里我们用灭菌手术包，它包含了所有必要的工具。 </li></ol></li><li>在手术当天，哈伦SD大鼠用5％的异氟醚麻醉，气管插管，使用耳镜，然后在整个过程中异氟醚2.5％保持。 </li><li>添加人工泪液软膏动物的眼睛，以防止干燥和头发。在手术过程中水化管理乳酸林格溶液3毫升。这项工作应通过在较低的腹部皮下注射。 </li><li>仰卧位放置在手术台下来四肢轻轻磁带上的动物。使用快船，​​以消除腹部和真空的头发，以洗刷之前的头发。 </li><li>设为一系列小（每个约50微升）注射2％利多卡因沿对角线zyphoid过程的右下角的侧腹部。然后，用优碘擦洗三次，在中间，并开始向外移动。重复用70％乙醇。封面的动物使用与手术前的圆形窗口悬垂;安全用毛巾夹，如果有必要。 </li><li>使用一个10号手术刀xyphoid过程的右下角的侧腹部3-4厘米的切口。定位xyphoid的过程，并剖析通过肌肉垂直向下的权利，要小心，以避免在右边的大血管。一旦你通过肌肉解剖，用剪刀横向穿过肌肉，露出隔膜。要小心，以避免肝脏。 </li><li>通过zyphoid过程中使用一个36英寸长3-0 Vicrile缝合和止血一双，驱动一针，拉缝合中途。带的两端缝合在一起，解决这个问题一个点上方和后方的动物，基本上解除了zyphoid和揭露隔膜。与其他36英寸长3-0 Vicrile缝合，驱动器通过最接近的xyphoid过程肌肉针，再次转危为安并修复完另一点手术台的权利，充分暴露术腔。 </li><li>此时，你应该能够看到通过隔膜的心。使用鼠齿microforceps小对膜片的中心，抢，拉你出来，使一个非常小的切口，钝剪刀。这是非常重要的，使用很钝的剪刀以避免戳肺。一旦这个洞已经取得，肺部会收回，让你做出一个2-3厘米的切口，通过膈肌，垂直可视化的核心。 </li><li>插入一个3英寸的棉条腔左侧推的方式流出肺部。使用巾钳固定到位的棉条。另一个棉条使用移动的方式，以便找到心包囊右肺。使用microforceps一双，一双大锯齿形钳，通过心包囊撕裂，露出了心尖。如果动物以前发生梗死程序，心包已经不存在。 </li><li>抓斗用鼠齿microforceps的心尖，注入液体的基体材料，从心尖三分之一的方式。插入针心外膜表面平行，并与斜边留针注入。一定要避免心大静脉。取出针前，等待几秒钟。您应该看到在注射部​​位的组织美白。 </li><li>清理腔中的血液，是右肺删除巾钳和棉条。使用弯曲microhemostats和大鼠牙microforceps关闭膜片。作出初步结，然后用锯齿钳和microhemostats关闭膈肌连续缝合和一个锥形针。之前完全关闭，插入PE160吸痰管，并用10毫升注射器撤离胸腔，缝合收紧。 </li><li>当正确的疏散和封闭，隔膜应凹。凸膜片表明，仍然有空腔中的空气。 </li><li>在这一点上，异氟醚可以打开下调至1％。 </li><li>取出两个3-0 Vicrile缝合腔打开。使用无菌水滋润的肌肉，与棉条或无菌纱布抹走多余的。使用一个新的长度3-0缝合关闭间歇缝合肌肉层。 </li><li>在这一点上，异氟醚可以关闭。 </li><li>用无菌水，并用订书钉接近皮肤的清洁区。如果主食会干扰的研究，也可用于5-0脯氨酸缝合。在这种情况下，使用反向切割针（局）密切与间歇缝合皮肤。在这两种情况下，当场在闭合切口手术胶水。 </li><li>使用棉条膏与三联抗生素软膏切口部位。 </li><li>让动物恢复100％的氧气下。 </li><li>当动物开始周围的呼吸机呼吸，去除气管套管。 </li><li>一旦动物胸骨，管理的盐酸丁丙诺啡0.05毫克/公斤皮下注射，并返回他们自己的家乡笼上的无菌巾。 </li><li>动物应该接受手术后的观察和护理。 </li><li>感兴趣的时间点，动物安乐死，心中的分析中删除。短轴截面，可以用苏木精和曙红（H＆E）染色总值的组织分析。在很短的时间点，注入区域将显示为粉红色，纤维网络传播间隙。在稍后的时间点，观察细胞的大量涌入，并最终降解注射2至3周后的矩阵。如果ECM注射前用生物素标记，它可以更直接地通过HRP标记的链霉素染色可视化。免疫组织化学染色可以用来识别特定的细胞或在注射区域结构，幻灯片已与FITC标记的isolectin结合内皮细胞和荧光绿色和抗平滑肌肌动蛋白抗体与Alexafluor中学合作染色红色的校董会的抗体报告。 </li></ol> 4。代表性的成果这种方式准备的基体材料的前鉴定证明保留各种大分子。具体来说，多种纤维蛋白和糖蛋白，被确定通过质谱（见表1）。如果根据此协议的矩阵处理材料被发现不再包含一系列复杂的大分子，它可能是脱细胞协议是过于苛刻。一旦完全冻干，干心包ECM看起来很生硬，皱巴巴的纸。其他组织类型将类似于包装花生。如果碾好，流脑应属于通过筛，并收集在罐子底部。如果有水分留在样本，ECM将聚集在一起，并获得停留在研磨腔。消化后，ECM解决方案应该是乳白色和完全不可见微粒的。它也将盐酸仅略多于粘性。如果ECM并不好消化，仍会有较大颗粒在小瓶;另外，ECM的解决方案可能会崩溃。 pH值调整后，有没有明显的变化解决方案中的，和ECM的仍然是一个阴天，均匀的液体。如果它开始凝胶，粘度会增加，这将是很难或不可能吸引到一个注射器的材料。如果发生这种情况，执行上冰的pH值调整步骤，预冷的解决方案。已准备一旦注射，继续，直到用冰。如果注入成功，针尖周围地区的美白和一个小的丸是可见的。如果这是不遵守，注射可能已经进入会议厅，而不是到墙上。当缝合动物注射后，小心关闭的隔膜是最重要的一步。如果做得正确，膜片将紧对肺部会出现凹。如果有肺部内留下任何的空气，膜片将保持凹凸有致，或有一个地方气球。当注射区域的组织学检查在早期的时间点（注射后30分钟至4小时），我们应该看到一个粉红色的，纤维的面积，是缺乏的细胞，这是重组后的基体材料注射后（图2） 。网络经常传播间隙，可能会在整个目前许多路段。一个POSS只看到一个非常小的矩阵凝胶面积ible的解释是，部分注射错过壁间的目标，要么注入低压室或泄露注射位置。此外，根据对凝胶时间，间隙的传播可能会有所不同。 <img src="/files/ftp_upload/2109/2109fig1.jpg" alt="图1" /> 图1。聚丙烯酰胺凝胶电泳（PAGE）的结果。 （一）分子量标准。 （二）鼠尾胶原蛋白I型（2.5毫克/毫升）溶解人类（C）和猪（四）心包ECM（7毫克/毫升）。注意胶原蛋白的存在，以及几个其他蛋白质和肽在心包基质样品。 <img src="/files/ftp_upload/2109/2109table1.jpg" alt="表1" /> 表1。ECM成分质谱确定。 <img src="/files/ftp_upload/2109/2109fig2.jpg" alt="图2" /> 图2心肌注射： 在体内的凝胶。 H＆E染色的人（A）和猪（二）心包基质凝胶在体内注射45分钟后。箭头表示矩阵的位置，染色打火机比心肌粉色。比例尺为500微米。 <img src="/files/ftp_upload/2109/2109fig3.jpg" alt="图3" /> 图3。血管细胞浸润。荧光染料注入人类（AC）和猪（DF）矩阵凝胶在两个星期的船只。内皮细胞标记为绿色（A，D），而平滑肌细胞标记为红色（B，E）。合并后的图像显示在C和F.比例尺为100μm。白色虚线表示矩阵注射的区域，如H＆E附近的一个部分分析确定。 <img src="/files/ftp_upload/2109/2109fig4.jpg" alt="图4" /> 图4。矩阵注射区域内的干细胞。一个赫司特染色细胞核（蓝色）和c - kit的（绿色）标识在人类（A）和猪（二）基质注射地区的干细胞。比例尺为50μm。

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	<li>Seif-Naraghi, S. B., Salvatore, M. A., Magoffin-Schup, P. J., Hu, D. P., Christman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+characterization+of+an+injectable+pericardial+matrix+gel:+A+potentially+autologous+scaffold+for+cardiac+tissue+engineering.">Design and characterization of an injectable pericardial matrix gel: A potentially autologous scaffold for cardiac tissue engineering.</a> Tissue Engineering. , (2009).</li><li>Freytes, D. O., Martin, J., Velankar, S. S., Lee, A. S., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+rheological+characterization+of+a+gel+form+of+the+porcine+urinary+bladder+matrix.">Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix.</a> Biomaterials. 29, 1630-1630 (2008).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+tissues+and+organs.">Decellularization of tissues and organs.</a> Biomaterials. 27, 3675-3675 (2006).</li><li>Liao, J., Joyce, E. M., Sacks, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+decellularization+on+the+mechanical+and+structural+properties+of+the+porcine+aortic+valve+leaflet.">Effects of decellularization on the mechanical and structural properties of the porcine aortic valve leaflet.</a> Biomaterials. 29, 1065-1065 (2008).</li><li>Singelyn, J. M., DeQuach, J. A., Seif-Naraghi, S. B., Littlefield, R. B., Schup-Magoffin, P. J., Christman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+derived+myocardial+matrix+as+an+injectable+scaffold+for+cardiac+tissue+engineering.">Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering.</a> Biomaterials. 30, 5409-5409 (2009).</li><li>Christman, K. L., Vardanian, A. J., Fang, Q., Sievers, R. E., Fok, H. H., Lee, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+fibrin+scaffold+improves+cell+transplant+survival,+reduces+infarct+expansion,+and+induces+neovasculature+formation+in+ischemic+myocardium.">Injectable fibrin scaffold improves cell transplant survival, reduces infarct expansion, and induces neovasculature formation in ischemic myocardium.</a> J Am Coll Cardiol. 44, 654-654 (2004).</li><li>Christman, K. L., Fok, H. H., Sievers, R. E., Fang, Q., Lee, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+glue+alone+and+skeletal+myoblasts+in+a+fibrin+scaffold+preserve+cardiac+function+after+myocardial+infarction.">Fibrin glue alone and skeletal myoblasts in a fibrin scaffold preserve cardiac function after myocardial infarction.</a> Tissue Eng. 10, 403-410 (2004).</li><li>Huang, N. F., Sievers, R. E., Park, J. S., Fang, Q., Li, S., Lee, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rodent+model+of+myocardial+infarction+for+testing+the+efficacy+of+cells+and+polymers+for+myocardial+reconstruction.">A rodent model of myocardial infarction for testing the efficacy of cells and polymers for myocardial reconstruction.</a> Nat Protoc. (1), 1596-1609 (2006).</li><li>Ott, H. C., Matthiesen, T. S., Goh, S. K., Black, L. D., Kren, S. M., Netoff, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat Med. 14, 213-221 (2008).</li><li>Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+as+a+biologic+scaffold+material.">The extracellular matrix as a biologic scaffold material.</a> Biomaterials. 28, 3587-3593 (2007).</li></ol>

动物使用：在这项研究中所有实验均按照机构动物护理和使用委员会在加州大学圣迭戈分校和美国认可的实验室动物护理协会公布的指引执行。

这种方法允许代生物衍生，注射用心肌组织工程支架。虽然这些方法最初是为制造和开发在体内试验心肌基质凝胶，并提出与心包基质凝胶中，这种协议可以适应任何组织使用，提供了组织可适当的脱细胞。脱细胞应进行验证使用这些方法之前，基质凝胶中的DNA的存在，可能会造成有害的免疫反应。有多种方式decellularize材料，并有关于这一问题的3，4的书面的几个很好的评价。 ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Reagents:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pepsin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>p6887-1G</td>
<td>Lyophilized </td>
</tr>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>21217</td>
<td>&nbsp;</td>
<tr>
<td>Neutravidin-HRP</td>
<td>&nbsp;</td>
<td>Thomas Scientific</td>
<td>21130</td>
<td>&nbsp;</td>
<tr>
<td>Equipment: </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Wiley Mini Mill</td>
<td>&nbsp;</td>
<td>Thomas Scientific</td>
<td>3383L10</td>
<td>&nbsp;</td>
<tr>
<td>Labconco Lyophilizer</td>
<td>&nbsp;</td>
<td>Labconco, Inc</td>
<td>7670520</td>
<td>&nbsp;</td>
<tr>
<td>Surgical supplies:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Betadine</td>
<td>&nbsp;</td>
<td>Purdue Products, L.P.</td>
<td>67618-154-16</td>
<td>&nbsp;</td>
<tr>
<td>Lactated Ringers Solution</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>003966</td>
<td>&nbsp;</td>
<tr>
<td>KY Jelly</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>28658</td>
<td>&nbsp;</td>
<tr>
<td>Lidocaine, 2%</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>17767</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine hydrochloride</td>
<td>&nbsp;</td>
<td>Reckitt Benckiser Healthcare (UK) Ltd.</td>
<td>12496-0757-1</td>
<td>&nbsp;</td>
<tr>
<td>Artificial tear ointment</td>
<td>&nbsp;</td>
<td>Fisher </td>
<td>NC9860843</td>
<td>&nbsp;</td>
<tr>
<td>Triple antibiotic ointment</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>19082795</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>60307-120-25</td>
<td>&nbsp;</td>
<tr>
<td>Otoscope </td>
<td>&nbsp;</td>
<td>MWI</td>
<td>008699</td>
<td>&nbsp;</td>
<tr>
<td>Stop cock</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>006245</td>
<td>&nbsp;</td>
<tr>
<td>3-0 Vicrile suture</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>J327H</td>
<td>&nbsp;</td>
<tr>
<td>5-0 Proline suture</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>s-1173</td>
<td>&nbsp;</td>
<tr>
<td>Reverse cutting (RC) needle</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>8684G</td>
<td>&nbsp;</td>
<tr>
<td>Microhemostats </td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>13013-14</td>
<td>&nbsp;</td>
<tr>
<td>Rat tooth microforceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11084-07</td>
<td>&nbsp;</td>
<tr>
<td>No. 10 scalpel</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10110-01</td>
<td>&nbsp;</td>
<tr>
<td>Blunt scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14108-09</td>
<td>&nbsp;</td>
<tr>
<td>Sharp, curved scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14085-08</td>
<td>&nbsp;</td>
<tr>
<td>Large, serrated forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>1106-12</td>
<td>&nbsp;</td>
<tr>
<td>PE160 suction tubing</td>
<td>&nbsp;</td>
<td>BD</td>
<td>427430 </td>
<td>&nbsp;</td>
<tr>
<td>Clippers </td>
<td>&nbsp;</td>
<td>MWI</td>
<td>21608</td>
<td>&nbsp;</td>
<tr>
<td>Skin staples/stapler </td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>PRR35</td>
<td>&nbsp;</td>
<tr>
<td>General supplies:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Stir plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0.1 M HCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 M NaOH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10x PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1x PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>70% Ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0.1 mL syringes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 mL syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Q-tips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Surgical glue</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Surgical drape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Towel clamps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Small hand-held vacuum </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fabrication of biologically derived injectable materials for myocardial tissue engineering

该协议规定编制的注射细胞外基质（ECM）的心肌组织工程中的应用凝胶的方法。简单地说，是冻干脱细胞组织，精，酶消化，然后带来生理pH值。冻干删除所有从组织的含水量，导致干流脑可分为地面用小磨细粉。铣削后，ECM的粉是用胃蛋白酶消化，以形成一种注射矩阵。调整pH至7.4后，液体基质材料可注射到心肌。以往的定性分析的结果表明，脱细胞心包及心肌组织产生的基质凝胶保留原生的ECM成分，包括不同的蛋白质，肽和粘多糖。由于使用这种材料的组织工程，是体内特性特别有用;在这里，左心室壁内注射执行的方法（LV）的游离壁作为分析宿主反应基质凝胶的手段提出一个小动物模型。获得通过膈肌进入胸腔注射略高于在LV游离壁的心尖。生物衍生支架可以可视化和注射前生物素标记，然后与辣根过氧化物酶标记的neutravidin染色的组织切片，并通过二氨基联苯胺（DAB）染色可视化。注射区域的分析，也可以做组织学和免疫组织化学染色。这样，在先前研究的心包和心肌基质凝胶形成纤维，多孔网络和注射区域内促进血管的形成。

Watch this Scientific Journal Video about Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering at JoVE.com

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

准备脱细胞组织注射基质凝胶注入大鼠心肌的方法<em&gt;在体内</em&gt;描述。

心肌组织工程生物衍生的注射材料的制备

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

fabrication-biologically-derived-injectable-materials-for-myocardial

Research

JoVE Journal

Bioengineering

14.9K 次观看.  University of California, San Diego. 准备脱细胞组织注射基质凝胶注入大鼠心肌的方法在体内描述。

视频: Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

科研

生物工程

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

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. ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

设计和人工细胞外基质的构建（AECM）蛋白质<em&gt;大肠杆菌</em&gt;对皮肤组织工程

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

倒胶体晶体聚（乙二醇）脚手架制作：肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

可伸缩、双通道、微流控芯片制造

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...