Name: neonatal cardiac scaffolds: novel matrices for regenerative studies
Uploaded: 2016-11-05T14:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies at JoVE.com

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>

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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repopulation+of+decellularized+mouse+heart+with+human+induced+pluripotent+stem+cell-derived+cardiovascular+progenitor+cells.">Repopulation of decellularized mouse heart with human induced pluripotent stem cell-derived cardiovascular progenitor cells.</a> Nat Commun. 4 (2307), 1-11 (2013).</li></ol>

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?...

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><tbody><tr><td colspan="4">&lt;strong&gt;1. Materials for mouse heart isolation&lt;/strong&gt;</td></tr><tr><td>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><td>60 mm Culture dish</td><td>BD Falcon</td><td>353004</td><td>or equivalent</td></tr><tr><td>Phosphate buffered saline pH 7.4 (sterile)</td><td>Hyclone</td><td>SH30256.01</td><td>or equivalent</td></tr><tr><td>Single Use Blade</td><td>Stanley</td><td>28-510</td><td>or equivalent</td></tr><tr><td>Standard Scissors</td><td>Moria Bonn (Fine Science Tools)</td><td>14381-43</td><td>or equivalent</td></tr><tr><td>Spring Scissors 10 cm</td><td>Fine Science Tools</td><td>15024-10</td><td>or equivalent</td></tr><tr><td>Vannas Spring Scissors - 3 mm Cutting Edge</td><td>Fine Science Tools</td><td>15000-00</td><td>or equivalent</td></tr><tr><td>#5 Forceps</td><td>Dumnot (Fine Science Tools)</td><td>11295-00</td><td>or equivalent</td></tr><tr><td colspan="4">&lt;strong&gt;2. Materials for decellularization&lt;/strong&gt;</td></tr><tr><td>Inlet adaptor</td><td>Chemglass</td><td>CG-1013</td><td>autoclavable</td></tr><tr><td>Septum</td><td>Chemglass</td><td>CG-3022-99</td><td>autoclavable</td></tr><tr><td>1/8 in. ID x 3/8 OD C-Flex tubing</td><td>Cole-Parmer</td><td>EW-06422-10</td><td>autoclavable</td></tr><tr><td>Male luer to 1/8&quot; hose barb adaptor</td><td>McMaster-Carr</td><td>51525K33</td><td>autoclavable</td></tr><tr><td>Female luer to 1/8&quot; hose barb adaptor</td><td>McMaster-Carr</td><td>51525K26</td><td>autoclavable</td></tr><tr><td>Prolene 7-0 surgical suture&amp;nbsp;</td><td>Ethicon</td><td>8648G</td><td>or equivalent</td></tr><tr><td>Ring stand</td><td>Fisher Scientific</td><td>S47807</td><td>or equivalent</td></tr><tr><td>Clamp</td><td>Fisher Scientific</td><td>05-769-6Q</td><td>or equivalent</td></tr><tr><td>Clamp regular holder</td><td>Fisher Scientific</td><td>05-754Q</td><td>or equivalent</td></tr><tr><td>60 cc syringe barrel&amp;nbsp;</td><td>Coviden</td><td>1186000777T</td><td>or equivalent</td></tr><tr><td>Beaker</td><td>Kimble</td><td>14000250</td><td>or equivalent</td></tr><tr><td>22 G x 1 Syringe Needle</td><td>BD</td><td>305155</td><td>or equivalent</td></tr><tr><td>12 cc syringe</td><td>Coviden</td><td>8881512878</td><td>or equivalent</td></tr><tr><td>3-way stop cock</td><td>Smith Medical</td><td>MX5311L</td><td>or equivalent</td></tr><tr><td>PE50 tubing</td><td>BD Clay Adams Intramedic</td><td>427411</td><td>Must be formable by heat. Polyethylene recommended.</td></tr><tr><td>1% SDS</td><td>Invitrogen</td><td>15525-017</td><td>Ultrapure grade recommended. Make up fresh solution and filter sterilize before use.</td></tr><tr><td>1% Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T8787</td><td>Make up fresh solution from a 10% stock and filter sterilize before use.&amp;nbsp;</td></tr><tr><td>Sterile dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Hyclone</td><td>SH30538.02</td><td>Or MilliQ system purified water.</td></tr><tr><td>1x Pen/Strep</td><td>Corning CellGro</td><td>30-001-Cl</td><td>or equivalent&lt;br /&gt;&amp;nbsp;</td></tr><tr><td colspan="4">&lt;strong&gt;3. Materials for DNA quantitation&lt;/strong&gt;</td></tr><tr><td>Proteinase K</td><td>Fisher</td><td>BP1700</td><td>&gt;30 U/mg activity</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td>or equivalent</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Mallinckrodt</td><td>5958-04</td><td>or equivalent</td></tr><tr><td>Tween 20&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P1379</td><td>or equivalent</td></tr><tr><td>Tris base/hydrochloride</td><td>Sigma-Aldrich</td><td>T1503/T5941</td><td>or equivalent</td></tr><tr><td>Pico-Green dsDNA assay kit</td><td>Life Technologies&amp;nbsp;</td><td>P7589</td><td>requires fluorimeter to read</td></tr><tr><td colspan="4">&lt;strong&gt;4. Method for fixation and sectioning of tissue&lt;/strong&gt;</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>or equivalent</td></tr><tr><td>Gelatin Type A from porcine skin</td><td>Sigma-Aldrich</td><td>G2500</td><td>must be 300 bloom or greater</td></tr><tr><td colspan="4">&lt;strong&gt;5. Method for tissue histology&lt;/strong&gt;</td></tr><tr><td>Cryomolds 10 x 10 x 5 mm</td><td>Tissue-Tek</td><td>4565</td><td>or equivalent</td></tr><tr><td>Cryostat</td><td>Hacker/Bright</td><td>Model OTF</td><td>or equivalent</td></tr><tr><td>Microscope Slides&amp;nbsp; 25 x 75 x 1 mm</td><td>Fisher Scientific</td><td>12-550-19</td><td>or equivalent</td></tr><tr><td>Hematoxylin 560&amp;nbsp;</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3801570</td><td>or equivalent</td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>2701</td><td>or equivalent</td></tr><tr><td>Define</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3803590</td><td>or equivalent</td></tr><tr><td>Blue buffer&amp;nbsp;</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3802915</td><td>or equivalent</td></tr><tr><td>Alcoholic Eosin Y 515&amp;nbsp;</td><td>Surgipath/Leica Selectech&amp;nbsp;</td><td>3801615</td><td>or equivalent</td></tr><tr><td>Formula 83 Xylene substitute&amp;nbsp;</td><td>CBG Biotech&amp;nbsp;</td><td>CH0104B</td><td>or equivalent</td></tr><tr><td>Permount Mounting Medium&amp;nbsp;</td><td>Fisher Chemical&amp;nbsp;</td><td>SP15-500</td><td>or equivalent</td></tr><tr><td>Collagen IV Antibody</td><td>Rockland</td><td>600-401-106.1</td><td>or equivalent</td></tr><tr><td>&amp;alpha;-Actinin Antibody</td><td>Abcam</td><td>AB9465</td><td>or equivalent</td></tr><tr><td>mCherry Antibody</td><td>Abcam</td><td>AB205402</td><td>or equivalent</td></tr><tr><td>NKX2.5 Antibody</td><td>Santa Cruz Biotechnology</td><td>SC-8697</td><td>or equivalent</td></tr><tr><td>Donkey anti-mouse AF488 Antibody</td><td>Life Technology</td><td>A21202</td><td>or equivalent</td></tr><tr><td>Donkey anti-chicken AF594 Antibody</td><td>Jackson Immunoresearch</td><td>703-585-155&amp;nbsp;</td><td>or equivalent</td></tr><tr><td>Donkey anti-goat CY5 Antibody</td><td>Jackson Immunoresearch&amp;nbsp;</td><td>705-175-147</td><td>or equivalent</td></tr><tr><td>Fab Fragment Goat Anti-Rabbit IgG (H+L) AF594</td><td>Jackson Immunoresearch</td><td>111-587-003</td><td>or equivalent</td></tr><tr><td>Prolong Gold Antifade Mountant with DAPI</td><td>ThermoFisher</td><td>P36930</td><td>or equivalent</td></tr><tr><td colspan="4">&lt;strong&gt;6. Isolation of neonatal ventricular cardiomyocytes using pre-plating&lt;/strong&gt;</td></tr><tr><td>HBSS (Ca, Mg Free)</td><td>Hyclone</td><td>SH30031.02</td><td>or equivalent</td></tr><tr><td>HEPES (1 M)</td><td>Corning CellGro</td><td>25-060-Cl</td><td>or equivalent</td></tr><tr><td>Cell Strainer</td><td>BD Falcon</td><td>352340</td><td>or equivalent</td></tr><tr><td>50 ml tube</td><td>BD Falcon</td><td>352070</td><td>or equivalent</td></tr><tr><td>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><td>Trypsin</td><td>Difco</td><td>215240</td><td>or equivalent</td></tr><tr><td>DNase II</td><td>Sigma-Aldrich</td><td>D8764</td><td>or equivalent</td></tr><tr><td>DMEM (Delbecco&#039;s Minimal Essential Media)</td><td>Hyclone</td><td>SH30022.01</td><td>or equivalent</td></tr><tr><td>Vitamin B12&amp;nbsp;</td><td>Sigma-Aldrich</td><td>V6629</td><td>or equivalent</td></tr><tr><td>Fibronectin coated plates&amp;nbsp;</td><td>BD Bioscience</td><td>354501</td><td>or equivalent</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Hyclone</td><td>SH30910.03</td><td>or equivalent</td></tr><tr><td>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?...

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

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

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 ...

科研

生物工程

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

组织工程：建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

制作生肌工程的组织结构

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

生物学

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

胎儿和成人心脏组织外植体作为三维体外研究平台的可比较去角质

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

基于网模的无支架三维心脏组织创建方法

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

脱碳软骨衍生基质支架的制备

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

从人体 iPS 细胞中提取的网状工程心脏组织用于活体心肌修复

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

人诱导多能干细胞来源的心脏球体的生成、高通量筛选和生物样本库

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.

使用聚己内酯支架和 hiPSC 衍生的心肌细胞的熔融静电纺丝写入生成 3D 人体心肌组织

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and ...

体内心脏移植研究内皮到间充质转化

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

作者聚焦:用于研究内皮-间充质转化的新生儿异位大鼠心脏移植模型  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

新生儿异位大鼠心脏移植模型在内皮间充质转化研究中的应用

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...