The authors gratefully acknowledge Ms. Cynthia DeKay for the preparation of the figures.

所有小鼠实验按照美国动物福利法进行，明尼苏达大学的机构动物护理和使用委员会批准。 1.小鼠心脏提取方法<ol><li>安乐死断头一个新生小鼠用单次使用的刀片。 </li><li>擦拭用70％乙醇的胸部。 </li><li>通过削减它远离胸壁与标准的剪刀，而与一对＃5镊子横向拉扯皮肤解剖从胸部皮肤。 </li><li>穿孔腹部只是不如与由通过腹壁切割剪刀胸骨。抓剑突与＃5镊子，从主体吻侧缩回胸骨切割时虽然肋上用剪刀胸部的两侧。肋骨体现优用钳揭示心脏。 </li><li>通过用＃5钳侧向拉动直截了当地解剖胸腺两个主瓣，露出在主动脉弓，以及静脉和肺静脉。 </li><li>从样主动脉弓的主要动脉和主动脉本身，与10厘米春风似剪刀。保留心脏的基部和臂动脉之间的主动脉。抓住切断血管的两端加上＃5镊子反映心脏前行，从气管和食管分离出来。 </li><li>横切肺血管等主脉，由肺和心脏之间切断对心脏两侧与10厘米春风似剪刀。叶脉保持开放提供排水。 </li><li>从纵隔与＃5镊子取出心脏，抓大血管被切断的末端。放置心脏中含有导尿无菌磷酸盐缓冲盐水（PBS）60毫米培养皿。 </li></ol> 2.通过的Langendorff灌注脱细胞方法<ol><li>预先准备的导管组件。绘制4cm的段PE在一个小酒精火焰50管创造一个更小，更薄的导管。修剪的端部用刀片以满足尺寸（约300微米与约500微米OD的凸缘外径OD） 如图1所示，这将提供从拉每侧的两个对称的导管。 </li><li>指向管子的切割端入火焰短暂，在凸缘熔化到在薄端的开口。 </li><li>填充12毫升注射器用PBS，并通过将一个3路活塞的注射器组装导管。添加22克×1针活塞，通过隔膜驱动针。绘制的PE管导管推到针（ 图1）。 </li><li>灌溉装配的零件用PBS，确保所有气泡已被删除。 </li><li>插入导管，如上所述，进入分离的心脏的主动脉，不延伸过主动脉瓣和与7-0缝合线的一个连接结扎制备。休息的t凸缘他近侧导管上日领带，使得密封并防止心脏脱落导管。 </li><li>观察导管倍率下，一边轻轻用灌注含PBS注射器的心脏。保证不存在任何泄漏在系统中，作为潜血液除去组织均匀Blanches的。 </li><li>放置隔膜进入入口适配器的颈部和通过在玻璃折叠边密封。 </li><li>附上经管线足够的长度，以产生生成的如图 20所示毫米汞柱压力的柱填充用60毫升1％十二烷基硫酸钠（SDS）在蒸馏水中（DH 2 O） 的贮存器。 1，计算从基于1毫米汞柱的关系的液柱高度的压力等于1.3595厘米H 2 O 注意:SDS是具有刺激性高度絮状粉末。接触，应该避免。使用适当的防护服处理的粉末。 </li><li>灌注用1％SDS的心脏14小时。心脏就会在外观上没有观察到剩余的组织是半透明的。 </li><li>冲洗系统到剩余SDS溶液的活塞，用10毫升的dh 2 O替换灌注用10ml 1％的Triton X-100的心脏（在蒸馏水中稀释的），接着加入10毫升的dh 2 O，随后60毫升的PBS含有1x青霉素链霉素（青霉素-链霉素）。 </li><li>在4℃保存在PBS用1X青霉素 - 链霉素的心脏。如果预期交脱细胞的应用需要灌注然后在主动脉中的导管应保持，否则切断缝合领带和取出导管。 </li></ol> 3. DNA测定<ol><li>在50mM氯化钾制备使用200微克/毫升蛋白酶K脱细胞ECM或控制心脏的摘要，2.5mM的MgCl 2的，在10mM的Tris（pH值8.3）0.45％吐温20。 </li><li>在搅拌孵育在55℃的组织，直到组织被溶解，一般4-5小时。 </li><li>定量的匀浆用DNA结合测定4,11 DNA含量。 </li></ol> 4.固定和组织切片<ol><li>固定在PBS中的4％多聚甲醛脱细胞，recellularized或控制P3心中，在室温下30分钟。 </li><li>洗PBS 3倍的组织。 </li><li>放置在7.5％的蔗糖的0.1M磷酸盐缓冲液中的溶液的组织在4℃下直到它下沉。 </li><li>改变在0.1M磷酸盐缓冲溶液至15％的蔗糖，4℃，再次，直到它下沉。 </li><li>暖组织至37℃，用15％的蔗糖和0.1M磷酸盐缓冲明胶溶液更换一半体积的和平衡过夜。明胶的浓度通过1％，2.5％，5％逐步增加，最后7.5％的两倍。 </li><li>放置在样品中使用新鲜的7.5％明胶cryomolds。用来恢复脱细胞样品的形状，液体的明胶可以灌注到腔室中为t他心中成型。通过浮动cryomolds液氮冷冻。存储在-80℃。 </li><li>切口（10微米厚）的部分与低温恒温器。带来接近部表面滑动，并观察部关闭从刀在表面上移动，由于在静电处理幻灯片的电荷。隔夜干的幻灯片，并在-80℃，供日后分析存储。 </li><li>从冰箱中取出幻灯片，平衡至室温，然后放置在含PBS中20分钟，在37℃以溶解明胶一个科普林缸。 </li><li>从步骤4.7与HE染色切段。广场幻灯片在科普林罐子。暴露在步骤4.8水合苏木45秒的幻灯片，自来水1.5分钟，缓冲液3分钟，自来水1分钟，上蓝剂，1.5分钟，80％乙醇1分钟，酒精曙红Y为8秒， 80％乙醇1分钟，100％乙醇1分钟2倍，清除剂（二甲苯替代品），持续1分钟3次，盖玻片用树脂根据安装中。显微镜检查的幻灯片。 </li><li>以通过将水合玻片在加湿室中确认心脏ECM，染色为ECM结构蛋白例如胶原IV的ECM蛋白的反应性的保持和在磷酸盐缓冲盐水，用10％正常驴血清（NDS）治疗用0.1％的Triton -X100（PBST）在室温下（RT）1小时。使用足够体积，以覆盖组织切片。 </li><li>在5％NDS / PBST中的经验确定的稀释替换所选择的初级抗体溶液中。例如，IV型胶原蛋白抗体稀释为1:150。在4℃孵育过夜。 </li><li>洗用PBST 3倍的幻灯片和应用用荧光染料缀合的选择的次级抗体。由经验确定量稀释抗体。在室温下孵育1小时。用PBS洗3倍。 </li><li>染色与DNA结合染料的幻灯片如DAPI通过使用含DAPI mounti验证没有细胞核的纳克媒体时滑倒盖的幻灯片。 </li><li>检查，在50至400倍的荧光显微镜的幻灯片。 </li></ol> 5. P1新生儿鼠心肌细胞的Recellularization <ol><li>喷用70％乙醇各小狗，并用单次使用的刀片斩首。 </li><li>持在拇指和食指之间的每个小狗而胸腔在含有小无菌剪刀中线分开以暴露胸腔。施加压力，以使心脏，同时它是免费的大血管，并只留下心室组织心房削减从胸部凸出自如。 </li><li>直接将每个心脏到含20毫升冰冷的钙，碳酸氢游离Hank氏用20mM 4-（2-羟乙基）-1-哌嗪直到收集所有的心酸（CBFHH）缓冲盐溶液的50ml管中。 </li><li>吸出CBFHH并加入10-15毫升新鲜CBFHH（冰冷）到管。纷飞在T组织洗净组织他管好几次。 </li><li>倒出含有的心，60毫米的塑料培养皿的解决方案。 </li><li>通过调整它与弹簧剪刀的心中远离从解剖的心中潜伏的任何心房组织倍率下冰。剁碎了心室成均匀大小的小块。取下菜＃5镊子分离的任何非脑室组织。 </li><li>吸管尽可能多的CBFHH溶液是需要的组织碎片转移到含有无菌微搅拌棒的小的无菌小瓶。 </li><li>允许所述组织以沉淀到小瓶的底部和取出CBFHH溶液。 </li><li>使上涨50毫升CBFHH的酶溶液1.75毫克/毫升胰蛋白酶，20微克/毫升DNA酶II的。吸管5毫升此酶溶液并将其添加到含有在磁力搅拌器剁碎心室组织和地点的管。搅拌在室温下以恒定的速率（340转）为8分钟。 注:搅拌动作要轻柔，但允许用于悬浮液中的淤浆。 </li><li>除去从搅拌盘的小瓶中，并允许浆料沉降3分钟。吸管并丢弃初始消化上清液，因为它主要包含血细胞和组织碎片。 </li><li>吸管酶溶液的第二个5毫升一份，并把它添加到消化心室。搅拌8分钟，并允许其沉降另外3分钟。之前开始消化，制备两个50毫升管（标记为1和2），每个含有12毫升冰冷的胎牛血清（FBS）。放置一个40微米的细胞过滤各管之上。吸取上清通过管1过滤器。 </li><li>重复该消化过程的附加的8倍，交替含有FBS的两个管之间的消化上清液的位置。平分管1和2之间的最后的上清液，以确保在每个管等体积。 注意:每个采集管现在将包含32.5毫升细胞悬液总体积。 </li><li>离心管含有ING上清液150 xg离心6分钟，4℃。 </li><li>同时收集消化的等分试样，制备个100毫米的组织培养板6（含10％FBS，1×青霉素 - 链霉素，1×L-谷氨酰胺的Dulbecco氏改良的Eagle氏培养基（DMEM））每板毫升介质。在37℃，5％CO 2孵育这些等分试样进行30分钟至平衡介质。 </li><li>吸出CBFHH酶混合物和重悬细胞到30毫升培养介质的上述制备中，细胞来自收集管结合。 </li><li>回线的6 ml的细胞悬液，以每100mm的板，并在37°C孵育45分钟。 </li><li>在温育结束时，非贴壁细胞转移到5个新菜肴和重新孵育45分钟（成纤维细胞馏分已经开始粘附到菜肴和如果需要的话，可以单独生长出）。 </li><li>在第二轮预镀的结束时，收集来自菜媒体和非贴壁细胞，并在150旋媒体xg离心6分钟。 </li><li>抽出多余的介质带来的细胞的近似所需的浓度，（每个构建体4.0×10 5个细胞在100μl灌注液）。删除计数和生存能力或测试14等份。 </li></ol> 6. P3心矩阵的生物反应器Recellularization <ol><li>加入细胞前一夜预处理与文化传媒（见步骤5.15），离体心脏矩阵。 </li><li>组装的Langendorff系统的元件， 如图。 2，高压灭菌玻璃部件和环氧乙烷消毒的塑料零件，以确保无菌。该系统的各个子单元可以在一个层流罩被安装在支撑支架之前进行组装。循环水浴中并加热水流路已为清楚起见被省略。 </li><li>填用120ml组织培养基的装配系统（见步骤5.15）。 </li><li>广场步骤2.14心脏导管插入基质60毫米的小路在放大下TURE培养皿并用22克×1针从步骤5.20到导管装有细胞悬液连接1ml注射器。确保本次大会没有气泡，以避免栓塞的心脏。 </li><li>轻轻地灌注到100微升细胞悬液通过进入冠状动脉的心脏矩阵。完成后，取下注射器/针的组合。 注:约的缓慢灌注。 20微升每分钟是必需的，以防止细胞从流出静脉，过境心脏。 </li><li>随着固定在生物反应器心脏瓶盖底部的鲁尔配件的22克钝存根，推到导管存根。 </li><li>启动蠕动泵，并通过穿过气泡阱泵放置在主动脉在步骤2.5的导管（在图2红路径）观察流程进入如图2中所概述。从贮存流前进。观察出静脉的心脏循环的流动S和滴水从顶点，循环媒体水库。 注:灌注流量取决于个人因素，如构建物的血管阻力，和心脏的大小，但在50〜100微升/分钟的速度是一个很好的起点。在中间时间点，功能评估可以被执行。新生儿recellularized心脏的大小规模限制了评估，可以进行，但我们已经确定，基于光学的系统可以用来量化打浆行为就像他们在成年大鼠心脏中使用4的构建体可以被灌注扩展的时间周期（长达23小时）。 </li></ol>

<ol>
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The authors have nothing to disclose.

这种技术对心脏的重复灌注的依赖使得栓塞的回避取得成果的一个重要组成部分。从心脏在步骤2.2-2.6初始导尿，到步骤2.8-2.14之间溶液的变化，有操作，可以允许引入气泡而妥协灌注液流入心肌的。由于新生儿心脏的面积小，在血管甚至微小气泡可引起心肌梗死的技术，从而使脱细胞不完整的。另外，在以后的洗涤步骤中，不完整的灌注可导致洗涤剂残留有负面影响的生物相容性。此外，如在步骤2.14建议的快速变化的温度，例如除去4℃下贮存基质时一样，应小心接近作为与变更溶解气体移出的解决方案时，这也可以是气泡形成源温度。 W¯¯母鸡继续到recellularization，额外护理应适用，但在细胞悬液制备注射器时，为确保输液是无气泡（步骤6.4）。 可能有必要操纵这个协议的具体细节，以适应其他组织类型。有许多的脱细胞的协议报4,9,11这可能提供指导。任何脱细胞协议的最终目标（多个）应包括ECM蛋白质结构的维修及相关生物化学，和原生细胞成分充分去除残留的DNA作为例证。在此设置中，过度的灌注压的应用导致ECM的超微结构，其可以组织学进行可视化的破坏。 相比于成人组织的P3小鼠心脏的大小穿上单元管理的一些限制。虽然心中成人出示足够厚的心室壁，使透注射液呐可能细胞递送方式，P3的心脏是足够小的针，其大小将不会裂解的细胞悬浮液，确实对心脏严重受损。灌注是一种可行的递送策略，但依赖于目标的大小和形状的细胞被有效地传递。新生儿鼠心肌细胞在这方面的工作。其他小圆形细胞也显示出用于此目的11。这些心的大小规模排除了使用常规的生理功能分析，如压力容积导管。基于视频捕捉其他方法可能必须加以考虑。 这些数据支持脱细胞和新生小鼠心脏的recellularization的可行性。以前的研究已经证明了脱细胞/ recellularization研究的目的而使用成人的心。从这些成年人心中产生的矩阵已经被重新填充新生儿心肌4和人诱导多能干细胞（人iPS细胞）17。在人iPS细胞的情况下，重新填充心显示在多能性标志物例如NANOG，Sox2和Oct4和形成肌样结构的减少;所有暗示成熟。 ECM的细胞外线索，但是，已被证明在细胞，组织，和器官这促使我们试图产生从已知怀有再生能力的组织基质的发展中发挥了关键作用。在我们的研究中，我们证明recellularized心中仍然表达心肌标志物，即使长时间培养后。这些数据表明，使用新的矩阵，心肌细胞可维持较长时间，而不会失去其身份为心肌细胞。我们的数据支持脱细胞新生小鼠心脏技术和可行性。新生儿矩阵保持电位为复育研究提供新的构建体，以及用于制造凝胶的具有SUPerior再生能力。使用这些新生儿的ECM，我们现在解决这些支架的优越性，以形成具有多种细胞类型，包括人iPS细胞的功能性的组织。

心脏衰竭是常见的，致命的。这是一种渐进性疾病，导致心脏，它损害的血液流向内脏和叶身未满足的代谢需求的收缩力下降。据估计，570万美国人患有心脏衰竭和它是住院在美国9的首要原因。在美国治疗的患者心脏衰竭的集体费用超过$ 300十亿美元，每年9-10。对于终末期心脏衰竭，唯一确定的疗法是心脏移植。每年，估计需要在美国1-2心脏移植程序超过10万个捐助者的心。由于捐助者的数量有限，只有约2,400个移植每年在美国2执行。显然，这个器官短缺需要其他的策略时需出示了transplanta额外的器官加以解决化，理想的是这些器官是自体的，以避免与排斥和寿命免疫抑制有关的并发症。 哺乳动物的成年心肌细胞表现出在损伤有限再生能力，但最近的证据表明哺乳动物新生儿心脏维持损伤后5-8显着的再生能力。具体地讲，下面的部分的手术切除，再生窗口已被生产后天之间发现7.该再生周期的特征是缺乏纤维化瘢痕，形成新血管形成的，从心外膜血管生成因子的释放，和心肌细胞增殖5-8 ，11。这个再生的时间窗口提供了使用新生儿心脏作为材料的生物人工心脏的发展了一种新的源的电位。 细胞外基质是已知的以提供重要的线索以促进cardiomyocytË增殖和生长。在新生儿和成人的矩阵12和促进再生能力的分子的可用性明显的差异已探索13。脱细胞成人基质已在几个研究中用于提供蜂窝复育的ECM支架和生物人工心脏的产生。虽然这些研究，并在干细胞技术的新的发现，正在迅速推进，几个障碍尚未得到满足。例如，在维护基质的天然结构，蜂窝融入矩阵壁的限制，并能够支持增殖和生长都限制了这种方法的成功。而优异的再生属性已被归因于新生儿心脏，使用这样的组织的实际方面限制了它的探索。 根据新生儿心脏的证明再生能力，我们通过开发一个新的发展矩阵脱细胞为P3小鼠心脏的技术。之所以选择这些研究的P3心脏，因为它是心肌再生的窗口内，先前确定的6，但心脏够大丰收，decellularize和recellularize。这项研究的目的是证明创建从新生小鼠心脏的矩阵的可行性。我们的研究为脱细胞一分钟，新生儿心脏同时维持ECM的结构和蛋白质的完整性的可行性提供了证据。我们也展示给recellularize与mCherry表达心肌这种心脏ECM的能力，我们已经研究这些心肌细胞为以下recellularization各种心脏标志物的表达。该技术将允许用于生物人工心脏的发展新生儿矩阵的优越性的测试。

<table border="0" cellpadding="0" cellspacing="0" width="1054">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:451px;">1. Materials For Mouse Heart Isolation</td>
			<td style="width:323px;"></td>
			<td style="width:123px;"></td>
			<td style="width:157px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P1 mouse pups (as shown; B6;D2-Tg(Myh6*-mCherry)2Mik/J)</td>
			<td>Jackson Laboratories</td>
			<td>21577</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm Culture dish</td>
			<td>BD Falcon</td>
			<td>353004</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline pH 7.4 (sterile)</td>
			<td>Hyclone</td>
			<td>SH30256.01</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Single Use Blade</td>
			<td>Stanley</td>
			<td>28-510</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Scissors</td>
			<td>Moria Bonn (Fine Science Tools)</td>
			<td>14381-43</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors 10 cm</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas Spring Scissors - 3mm Cutting Edge</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#5 Forceps</td>
			<td>Dumnot (Fine Science Tools)</td>
			<td>11295-00</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2. Materials For Decellularization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inlet adaptor</td>
			<td>Chemglass</td>
			<td>CG-1013</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Septum</td>
			<td>Chemglass</td>
			<td>CG-3022-99</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1/8 in. ID x 3/8 OD C-Flex tubing</td>
			<td>Cole-Parmer&#160;</td>
			<td>EW-06422-10</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Male luer to 1/8&#34; hose barb adaptor</td>
			<td>McMaster-Carr</td>
			<td>51525K33</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Female luer to 1/8&#34; hose barb adaptor</td>
			<td>McMaster-Carr</td>
			<td>51525K26</td>
			<td>autoclavable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolene 7-0 surgical suture&#160;</td>
			<td>Ethicon</td>
			<td>8648G</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring stand</td>
			<td>Fisher Scientific</td>
			<td>S47807</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clamp</td>
			<td>Fisher Scientific</td>
			<td>05-769-6Q</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clamp regular holder</td>
			<td>Fisher Scientific</td>
			<td>05-754Q</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 cc syringe barrel&#160;</td>
			<td>Coviden</td>
			<td>1186000777T</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beaker</td>
			<td>Kimble</td>
			<td>14000250</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">22g x 1 Syringe Needle&#160;</td>
			<td>BD</td>
			<td>305155</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12 cc syringe&#160;</td>
			<td>Coviden</td>
			<td>8881512878</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-way stop cock&#160;&#160;</td>
			<td>Smith Medical&#160;</td>
			<td>MX5311L</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22 x 1 g needle&#160;</td>
			<td>BD</td>
			<td>305155</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE50 tubing&#160;</td>
			<td>BD Clay Adams Intramedic</td>
			<td>427411</td>
			<td>Must be formable by heat. Polyethylene recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1% SDS&#160;</td>
			<td>Invitrogen</td>
			<td>15525-017</td>
			<td>Ultrapure grade recommended. Make up fresh solution and filter sterilize before use.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1% Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Make up fresh solution from a 10% stock and filter sterilize before use.&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Sterile dH2O</td>
			<td>Hyclone</td>
			<td>SH30538.02</td>
			<td>Or MilliQ system purified water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Pen/Strep&#160;</td>
			<td>Corning CellGro</td>
			<td>30-001-Cl</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3. Materials For DNA Quantitation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K&#160;</td>
			<td>Fisher</td>
			<td>BP1700</td>
			<td>&#62;30U/mg activity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl*6H2O</td>
			<td>Mallinckrodt</td>
			<td>5958-04</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris base/hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>T1503/T5941</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pico-Green dsDNA assay kit</td>
			<td>Life Technologies&#160;</td>
			<td>P7589</td>
			<td>requires fluorimeter to read</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4. Method for fixation and sectioning of tissue.&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin Type A from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G2500</td>
			<td>must be 300 bloom or greater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5. Method for tissue histology</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomolds 10 x 10 x 5mm</td>
			<td>Tissue-Tek</td>
			<td>4565</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Hacker/Bright</td>
			<td>Model OTF</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Slides&#160; 25 x 75 x 1 mm</td>
			<td>Fisher Scientific</td>
			<td>12-550-19</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin 560&#160;</td>
			<td>Surgipath/Leica Selectech&#160;</td>
			<td>3801570</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Define</td>
			<td>Surgipath/Leica Selectech&#160;</td>
			<td>3803590</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blue buffer&#160;</td>
			<td>Surgipath/Leica Selectech&#160;</td>
			<td>3802915</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcoholic Eosin Y 515&#160;</td>
			<td>Surgipath/Leica Selectech&#160;</td>
			<td>3801615</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formula 83 Xylene substitute&#160;</td>
			<td>CBG Biotech&#160;</td>
			<td>CH0104B</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permount Mounting Medium&#160;</td>
			<td>Fisher Chemical&#160;</td>
			<td>SP15-500</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen IV Antibody</td>
			<td>Rockland</td>
			<td>600-401-106.1</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-Actinin Antibody</td>
			<td>Abcam</td>
			<td>AB9465</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mCherry Antibody</td>
			<td>Abcam</td>
			<td>AB205402</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NKX2.5 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-8697</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-mouse AF488 Antibody</td>
			<td>Life Technology</td>
			<td>&#160;A21202&#160;</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-chicken AF594 Antibody</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>703-585-155&#160;</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-goat CY5 Antibody</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>705-175-147</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fab Fragment Goat Anti-Rabbit IgG (H+L) AF594</td>
			<td>Jackson Immunoresearch&#160;</td>
			<td>111-587-003&#160;</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Gold Antifade Mountant with DAPI</td>
			<td>ThermoFisher</td>
			<td>P36930</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">6. Isolation of neonatal ventricular cardiomyocytes using pre-plating.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS (Ca, Mg Free)</td>
			<td>Hyclone</td>
			<td>SH30031.02</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1M)</td>
			<td>Corning CellGro</td>
			<td>25-060-Cl</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primeria 100 mm plates</td>
			<td>Corning</td>
			<td>353803</td>
			<td>Primeria surface enhances fibroblast attachment promoting a higher myocyte purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin</td>
			<td>Difco</td>
			<td>215240</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase II</td>
			<td>Sigma-Aldrich</td>
			<td>D8764</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (Delbecco&#39;s Minimal Essential Media)</td>
			<td>Hyclone</td>
			<td>SH30022.01</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vitamin B12&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>V6629</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibronectin coated plates&#160;</td>
			<td>BD Bioscience</td>
			<td>354501</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum&#160;</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heart bioreactor glassware</td>
			<td>Radnoti Glass Technology</td>
			<td>120101BEZ</td>
			<td>Must be sterilizable by autoclaving or gas.</td>
		</tr>
	</tbody>
</table>

neonatal cardiac scaffolds: novel matrices for regenerative studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

脱细胞 平均而言，时间使用这个协议的P3心脏的脱细胞大约为14小时。给出的平均心脏重量的23毫克为P3新生儿。 Acellularity 图3a说明了一个完全完好P3新生儿心脏（整装）。 图3b示出以下脱细胞相同的心脏; 图4a和4b分别显示了完整和脱细胞的心脏的苏木精和曙红染色。注意:如果没有苏木积极原子核和嗜酸性结构的脱细胞心脏的减少。此外，脱细胞心脏的DNA含量显著从68.08±2.25微克的完整心脏（N = 6）4.73±2.27微克脱细胞的心脏降低（N = 5）。 胶原蛋白免疫反应 以下脱细胞的胞外基质（ECM）的维持是至关重要的用外源性细胞和基体的功能，再增殖。为了评估在完整和脱细胞的ECM新生儿ECM的内容，进行免疫染色为IV型胶原。 图4c和d表明IV型胶原在两个完整的和脱细胞的心脏稳健表达，该蛋白的定位保持以下细胞的去除，而DAPI阳性核被有效地去除（ 图4e-h）中 。 DNA含量 DNA的存在被用作细胞构成的一个附加指示。在图5中 ，DNA被证明是由93％的新生儿心脏下列基于洗涤剂脱细胞减少。这种程度的DNA减少是一致使用基于洗涤剂脱细胞在其它组织15-16在文献中的报告。 Recellularization 我们已经进行免疫组织化学来确定recellularized心脏各种心肌标记物的表达。 图6示出了已经迁移到左心室的壁（ 图6A和6B）。 图6C展示了recellularized心脏的DAPI标记的细胞。 图图6D-G示出了阳性的分别NKX 2.5，mCherry，α辅肌动蛋白和DAPI，细胞。 NKX2.5已知标记心脏祖细胞，而α辅肌动蛋白是标记分化心肌一个肌节蛋白。我们已观察到大多数表达所有这些标记物的细胞（粉红色; 图6H），这表明这些细胞持续表达心肌标记物EV连接后灌注23天。 <img alt="图1" src="/files/ftp_upload/54459/54459fig1.jpg" /> 图1.原理脱细胞处理硬件。A的 。 60毫升注射器针筒水库洗涤液; B。导管组件用冲洗注射器尽可能详细。℃。绘制的PE管导管尖端的细节。ð。脱细胞腔和隔膜与流失。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54459/54459fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54459/54459fig2.jpg" /> 图2.心脏生物反应器的示意图。1。卡波金气体的加湿（绿线代表气体流）。 2.媒体灌注蠕动泵驱动器，氧合（紫色线代表通过氧合媒体流）。 3.薄壁氧合器。 4.表氧合器和媒体库。 5.预压腔和泡沫陷阱。 6.心腔（红色线代表媒体流和从心脏）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54459/54459fig2large.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/54459/54459fig3.jpg" /> 图3. P3新生小鼠心脏的整装前（A）和脱细胞（B）。如方法2.注意说明，心脏变得透亮这心脏使用的Langendorff灌注法脱细胞和轻度肿大以下灌注后 （B） 。规模= 2mm左右。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54459/54459fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <p类="jove_content"FO: - together.within页保留="1"&gt; <img alt="图4" src="/files/ftp_upload/54459/54459fig4.jpg" /> 本地和脱细胞P3新生小鼠心脏的图4.组织学研究。冰冻切片（10微米）与H＆E（A，B），IV型胶原蛋白（C，D，G，H）和DAPI（E，F，G，H染色）。相比于天然心脏（A）中时合并图像中G表示和H. H＆E染色示出了在去细胞的组织（B）一种无细胞核和细胞质的。而IV型胶原蛋白含量保持以下脱细胞（D）的DAPI染色（细胞核的标记）被废止。合并后的图像表明在天真心脏（G）和脱细胞心脏（H）缺席本次共定位的IV型胶原蛋白和DAPI的共存。这些数据表明，细胞没有<html>再填充心脏的胶原基质。规模= 500微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54459/54459fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54459/54459fig5.jpg" /> 图5.评价DNA含量。对照组（n = 6）和脱细胞（N = 5）测定由微微绿法DNA含量的心。定量表示为每心脏DNA±标准差微克。星号表示与对照相比P &lt;0.01。这些数据表明，脱细胞在P3新生儿心脏显著降低DNA含量。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54459/54459fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> OAD / 54459 / 54459fig6.jpg"/&gt; P3心脏矩阵的组织学图6.在与P1 mCherry表达心肌细胞。沾满H＆E（A，比例= 250微米，B，规模= 50微米），DAPI（C，比例= 250微米），NKX 2.5 recellularization 23天 ， mCherry，α辅肌动蛋白，DAPI和合并（D，E，F，G，H，规模= 50微米）。我们已证明用P1心肌（AC）的胶原基质的有效再增殖。此外，我们观察到，间 - 樱桃阳性心肌细胞表达的Nkx2.5和以下引入到胶原基质α辅肌动蛋白23天。这些数据表明，这些细胞保持长时间的心肌细胞的身份。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54459/54459fig6large.jpg" target="_blank">请点击此处查看该F的放大版本</a>igure。

Watch this Scientific Journal Video about Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies at JoVE.com

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

在这些研究中，我们提供了新颖，新生儿，鼠心脏的再生研究使用支架的方法。

新生儿心脏支架:再生研究新矩阵

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

neonatal-cardiac-scaffolds-novel-matrices-for-regenerative-studies

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.7K 次观看.  University of Minnesota. 在这些研究中，我们提供了新颖，新生儿，鼠心脏的再生研究使用支架的方法。 

视频: Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

投诉矩阵量化细胞收缩的准备工作

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

科研

生物工程

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

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 ...

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 ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

纳米谐波再生研究

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

静电纳米纤维支架与渐变的纤维组织

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

Here we present a fluorophore based imaging technique to detect cell viability on a non-transparent titanium scaffold as well as to detect glimpses of the scaffold impurities. This protocol troubleshoots the drawback of imaging cell-cell or cell-metal interactions on non-transparent scaffolds.

非透明的支架成像细胞活性 - 使用新型针织钛种植体为例

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

Anatomically Realistic Neonatal Heart Model for Use in Neonatal Patient Simulators

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff practice clinical interventions such as chest compressions to ensure patient survival in the case of bradycardia or cardiac arrest. The simulators used currently are of low physical fidelity and therefore cannot provide qualitative insight into the procedure of chest compressions. The embedding of an anatomically realistic heart model in future simulators enables the detection of cardiac output generated during chest compressions; this can provide clinicians with an output parameter, which can deepen the understanding of the effect of the compressions in relation to the amount of blood flow generated. Before this monitoring can be achieved, an anatomically realistic heart model must be created containing: two atria, two ventricles, four heart valves, pulmonary veins and arteries, and systemic veins and arteries. This protocol describes the procedure for creating such a functional artificial neonatal heart model by utilizing a combination of magnetic resonance imaging (MRI), 3D printing, and casting in the form of cold injection molding. Using this method with flexible 3D printed inner molds in the injection molding process, an anatomically realistic heart model can be obtained.

This protocol describes a procedure for creating functional artificial neonatal heart models by utilizing a combination of magnetic resonance imaging, 3D printing, and injection molding. The purpose of these models is for integration into the next generation of neonatal patient simulators and as a tool for physiological and anatomical studies.

在新生儿患者模拟器中使用的解剖现实新生儿心脏模型

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

一种新的三维打印解光矩阵处理方法

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...