作者感谢教授亨德里克Tevaearai心血管外科为研究的初始部分资金支持的部门。

注：刘易斯雄性和雌性大鼠，200〜220克的安置标准实验室条件下（12小时光照和黑暗周期， 随意的水和食物，IVC笼）下。所有动物均符合FELASA和瑞士法律对动物保护的建议处理。 1，细胞的制备：从骨髓间充质干细胞分离<ol><li>麻醉大鼠用异氟烷5％和O 2的5升/分钟的感应腔室5分钟。将动物的口鼻部到鼻锥相连的麻醉系统。执行脚趾或尾巴捏确认镇静。 </li><li>通过切割皮肤从脚踝到臀部除去从腿部的皮肤用剪刀。取出肌肉，减少经股动脉动物放血。显露髋关节脱位和股骨头。不损坏股骨头是很重要的。把骨头无菌PBS在50毫升的塑料管。 </li><li>移除所有亩scles和韧带从骨子里不破坏它们。注意：它是重要的骨保持完整，以避免冲洗溶液或污染，用乙醇联动。漂洗在70％乙醇中剥离骨头5分钟。 </li><li>在无菌层流罩，切骨头用剪刀的两个技巧。冲洗骨髓从骨端注入无菌PBS中。骨髓采集到15毫升管。离心7分钟300×克。 </li><li>除去上清液。暂停在3ml红细胞裂解缓冲液沉淀。再次纺丝在300×g下7分钟前孵育悬浮液在室温下放置1分钟。在150 ml培养瓶中，加入无菌细胞培养液（395毫升IMDM培养基中，加入5ml青霉素/链霉素100毫升FBS）和种子细胞。 </li><li>改变介质的第二天，除去非粘附细胞。 <ol><li>直到获得所需的细胞量为2-3周进行中的变化，每隔一天。 </li><li>另外，准备biograft种子根据需要和其它地方描述的3荷兰国际集团的细胞在固体基质中。 </li></ol></li><li>心脏移植的当天，制备细胞或刚心外膜应用之前收获biograft。 <ol><li>收集使用的Accutase细胞脱离液细胞。计数细胞。填1.5ml的离心管中以计算得到所需的细胞数量的细胞悬浮液的体积。离心机在300 XG 7分钟，除去上清液。 </li><li>或者，收获来自细胞培养基的biograft，冲洗用无菌PBS中，并保持在新鲜培​​养基中无血清。 </li></ol></li></ol> 2，第一次开胸结扎LAD <ol><li>称取鼠。打开加热垫在37℃。麻醉，用5％异氟醚和5升/分钟100％O 2的大鼠中的感应腔室为5-7分钟。执行脚趾或尾巴捏确认镇静。放置在加热垫的老鼠。 </li><li>插管的动物有14ğ静脉导管。插管导管连接到编程为2.5升/分钟的氧气，2.5％异氟醚，2毫升潮气量，以及90次/分钟的呼吸频率啮齿动物呼吸机。 </li><li>准备在0.1毫克/公斤丁丙诺啡的解决方案。注入皮下的溶液的三分之一。剃胸廓的左侧部分。消毒，用1％的聚维酮碘溶液。 </li><li>切开皮肤垂直于胸骨在第四肋间。分出3层胸肌肉（胸大肌，胸升和腹外斜肌胸大肌）。打开第四肋间（肋4和5之间）。 </li><li>用小拉钩传播肋骨和暴露的心脏。小心地打开心包。找到并结扎冠状动脉左前降支（LAD）与7.0缝线4毫米中庭下方。 </li><li>使用3.0缝合两针封闭肋间。从胸骨近端和远端放置两个缝合。首先，Tighten远端缝合。用镊子将呼吸机的排气管2秒，以抬高胸部，避免气胸。收紧第二缝合。肌肉层放置回原位（不需要任何缝线）。关闭皮肤用5.0缝合。 </li><li>消毒用1％的聚维酮碘溶液的缝合。注射丁丙诺啡溶液的剩余部分。关闭麻醉系统。移除插管导管。 </li><li>请在笼子里的老鼠在一个温暖的灯为1-2小时。保持温度计在笼和控制，以避免过度加热的暖灯和轿厢之间的距离。返回在大鼠下腔静脉单元。注入皮下丁丙诺啡0.05-0.1毫克/公斤后-operatively每6-12小时48小时。 </li></ol>在通过治疗3。心外膜政府当局二次开胸<ol><li>重复步骤2.1-2.3。 </li><li>切开皮肤垂直于胸骨在第五肋间。分离出3层胸肌肉S（胸大肌，胸升和腹外斜肌胸大肌）。打开第五肋间（肋5与6之间）。 </li><li>用小拉钩传播肋骨和暴露的心脏。如果有一些坚持，小心地用细镊子取出。 </li><li>找到出现结扎下方的浅区的梗死面积。注：必要时，可使用10厘米长的缝合（7.0）的先端，以更好地可视化的左心室。轻轻拉至通过夹具松散地保持着，露出心脏的缝合。 </li><li>应用下列治疗之一： <ol><li>定位biograft在心脏表面。使用该预填充纤维蛋白封闭剂注射器预热，在室温下放置10分钟。通过将25g鲁尔针应用纤维蛋白胶的biograft下降（50-100微升）。验证biograft是完全密封的。 </li><li>另外，申请用枪头收集细胞沉淀在表面ØF表示心脏上的绷带下面的苍白区。应用纤维蛋白胶的下降（50〜100μL）到细胞沉淀。 </li></ol></li><li>从顶点中取出一块缝合。 </li><li>重复步骤2.6-2.8。 </li></ol>

<ol>
	<li>Gjesdal, O., Bluemke, D. A., Lima, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+remodeling+at+the+population+level--risk+factors,+screening,+and+outcomes.">Cardiac remodeling at the population level--risk factors, screening, and outcomes.</a> Nat. Rev. Cardiol. 8, 673-685 (2011).</li><li>Alcon, A., Cagavi Bozkulak, E., Qyang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+functional+heart+tissue+for+myocardial+repair.">Regenerating functional heart tissue for myocardial repair.</a> Cell Mol. Life Sci. 69, 2635-2656 (2012).</li><li>Guex, A. G., Fortunato, G., Hegemann, D., Tevaearai, H. T., Giraud, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+Protocol+for+the+Culture+of+Cells+on+Plasma-Coated+Electrospun+Scaffolds.">General Protocol for the Culture of Cells on Plasma-Coated Electrospun Scaffolds.</a> Methods Mol. Biol. 1058, 119-131 (2013).</li><li>Selye, H., Bajusz, E., Grasso, S., Mendell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+techniques+for+the+surgical+occlusion+of+coronary+vessels+in+the+rat.">Simple techniques for the surgical occlusion of coronary vessels in the rat.</a> Angiology. 11, 398-407 (1960).</li><li>Gomes, A. C., Falcao-Pires, I., Pires, A. L., Bras-Silva, C., Leite-Moreira, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+heart+failure:+an+updated+review.">Rodent models of heart failure: an updated review.</a> Heart Fail. Rev. 18, 219-249 (2013).</li><li>Horstick, G., 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=Surgical+procedure+affects+physiological+parameters+in+rat+myocardial+ischemia:+need+for+mechanical+ventilation.">Surgical procedure affects physiological parameters in rat myocardial ischemia: need for mechanical ventilation.</a> Am. J. Physiol. 276, 472-479 (1999).</li><li>Duchatelle, J. P., Vivet, P., Cortes, M., Groussard, O., Pocidalo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+and+Hemodynamic-Effects+of+Lateral+Thoracotomy+or+Sternotomy+in+Mechanically+Ventilated+Rats.">Respiratory and Hemodynamic-Effects of Lateral Thoracotomy or Sternotomy in Mechanically Ventilated Rats.</a> Eur. Surg. Res. 17, 10-16 (1985).</li><li>Giraud, M. N., 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=Hydrogel-based+engineered+skeletal+muscle+grafts+normalize+heart+function+early+after+myocardial+infarction.">Hydrogel-based engineered skeletal muscle grafts normalize heart function early after myocardial infarction.</a> Artif. Organs. 32, 692-700 (2008).</li><li>Siepe, M., 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=Myoblast-seeded+biodegradable+scaffolds+to+prevent+post-myocardial+infarction+evolution+toward+heart+failure.">Myoblast-seeded biodegradable scaffolds to prevent post-myocardial infarction evolution toward heart failure.</a> J. Thorac. Cardiov. Sur. 132, 124-131 (2006).</li><li>Guex, A. G., 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=Plasma-functionalized+electrospun+matrix+for+biograft+development+and+cardiac+function+stabilization.">Plasma-functionalized electrospun matrix for biograft development and cardiac function stabilization.</a> Acta Biomater. 10, (2014).</li><li>Terrovitis, J., 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=Noninvasive+Quantification+and+Optimization+of+Acute+Cell+Retention+by+In+Vivo+Positron+Emission+Tomography+After+Intramyocardial+Cardiac-Derived+Stem+Cell+Delivery.">Noninvasive Quantification and Optimization of Acute Cell Retention by In Vivo Positron Emission Tomography After Intramyocardial Cardiac-Derived Stem Cell Delivery.</a> J. Am. Coll. Cardiol. 54, 1619-1626 (2009).</li><li>Guo, H. D., Wang, H. J., Tan, Y. Z., Wu, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+marrow-derived+cardiac+stem+cells+carried+in+fibrin+improves+cardiac+function+after+myocardial+infarction.">Transplantation of marrow-derived cardiac stem cells carried in fibrin improves cardiac function after myocardial infarction.</a> Tissue Eng. Part A. 17, 45-58 (2011).</li><li>Qiao, H., 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=Death+and+Proliferation+Time+Course+of+Stem+Cells+Transplanted+in+the+Myocardium.">Death and Proliferation Time Course of Stem Cells Transplanted in the Myocardium.</a> Mol. Imaging Biol. 11, 408-414 (2009).</li><li>Conradi, L., Pahrmann, C., Schmidt, S., Deuse, T., Hansen, A., Eder, A., 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=Bioluminescence+Imaging+for+Assessment+of+Immune+Responses+Following+Implantation+of+Engineered+Heart+Tissue+(EHT).">Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT).</a> J. Vis. Exp. 52 (e2605), (2011).</li></ol>

The authors have nothing to disclose.

法援署的永久结扎造成不可逆的心肌损伤。第一个动物模型于1960年4被描述。从那时起，它已被认为是慢性心肌梗塞标准和合适的模型。其稳定性和重复性使治疗的实验评价心肌梗死5。继最初的描述改进程序报告了一个手术死亡率35-13％6。 正如预期的那样，LAD结扎诱导的2个星期内观察到受损的心脏功能。在心脏功能的进一步下降，记录长达6周，并与...

自19 世纪结束时，心血管疾病一直留在工业化国家的头号连环杀手。其中，冠状动脉疾病代表了主要病因。急性期会导致心肌梗死（MI），随后是不适应的重塑是逐步走向慢性期和严重的心脏衰竭的发展。尽管最近显著技术和治疗的进步，发病率和死亡率因心脏衰竭的进展仍在增长1。在这种情况下，细胞疗法已经获得了越来越多的关注作为一种新的治疗方案，以阻止疾病朝向心脏衰竭的恶化和刺激心肌的最近确定的再生能力。实验和临床研究已经提供的各种细胞类型的心脏移植后获得的有益效果令人信服的证据。主要成果包括改进CARDI交流收缩功能降低，左心室重塑，减少梗塞面积，并在梗死区血管密度增加。然而，细胞注射后低细胞数量保持仍然是一个重要的缺点。细胞与生物基质，以改善电池交付2协会最近促成研究者和临床利益。 左冠状动脉前降支（LAD）结扎是心肌梗死的小动物模型，导致透梗死和成熟的疤痕的参考方法。在心肌梗死慢性期应用细胞治疗还需要第二个手术干预。正中胸骨切开术通常执行，使心肌内注射细胞或biografts的心外膜植入。这种微创外科手术增加的死亡率，手术后的恢复时间，疼痛和感染的风险。这里介绍的微创手术方法不仅可以防止这样的偏见，但也提供了Ô心脏的治疗应用ptimal访问。心肌梗死和凝胶型生物基质相关的细胞外膜植入是通过左肋间开胸手术在跳动的心脏进行。

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:104px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:239px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:216px;">POLYSORB 3-0 suture</td>
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			<td></td>
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			<td height="21" style="height:21px;width:216px;">POLYSORB 5-0 suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">UL 202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SURGIPRO II 7-0 Suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">VP904X</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">CATHETER INSYTE 14G</td>
			<td style="width:104px;">BD</td>
			<td style="width:119px;">381267</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">557058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DUMONT #7 Forceps</td>
			<td style="width:104px;">FST Germany</td>
			<td style="width:119px;">11274-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibrin sealant</td>
			<td style="width:104px;">BAXTER</td>
			<td style="width:119px;">1501441</td>
			<td>TISSEEL Glue, Frozen -20&#176;C</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CASTROVIEJO EYE SPECULA</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">72-8925</td>
			<td>use as retractor for the ribs</td>
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		<tr height="21">
			<td height="21" style="height:21px;">IMDM Glutamax</td>
			<td>Gibco</td>
			<td>31980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>PAA Clone</td>
			<td>&#160;&#160;&#160;&#160;&#160; A15-101</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">bone scissors</td>
			<td style="width:104px;">Fine Science Tols</td>
			<td style="width:119px;">16044-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red Blood Cell Lysis Solution</td>
			<td>Gentra Systems</td>
			<td>D-50k1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accutase cell detachment solution</td>
			<td style="width:104px;">Stem cell technology</td>
			<td style="width:119px;">7920</td>
			<td></td>
		</tr>
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cell-based therapy for heart failure in rat: double thoracotomy for myocardial infarction and epicardial implantation of cells and biomatrix

心脏细胞疗法已经获得了越来越多的关注与细胞相关的生物材料和植入已成为一个主要的问题，优化心肌细胞传递。心肌梗死的啮齿动物模型（MI）由左前降支（LAD）结扎已普遍被通过开胸进行的;在传统上一直进行用于治疗心外膜应用程序通过胸骨第二心脏直视手术。由于LAD结扎模型的描述，手术后的死亡率已经从35-13％下降，但第二次手术依然是至关重要的。为了改善手术后的恢复，减少疼痛和感染，微创手术过程给出。两个开胸被执行，初始一个用于LAD结扎，而第二个用于治疗心外膜给药。 Biografts包括与固体或凝胶型的矩阵相关联的单元的方式进行涂敷在梗塞芳EA。 LAD结扎导致心脏功能的丧失证实了2周，6周后进行超声心动图。 Goldner对心脏切片进行三色染色证实透瘢痕形成。第一和第二次手术产生了小于10％，术后死亡率。

开胸手术后1小时内的所有动物恢复。伤口愈合快。无感染或水肿观察。 双左侧开胸允许的心脏最优访问（ 图1）。疼痛和手术后的死亡率较低。动物从手术中恢复迅速和体重增加（ 图2）。卡普兰梅厄成活率为96％，为第一开胸手术。四只超过104 通过第二开胸手术24小时以下处理应用程序内死亡。卡普兰-梅尔存活百分比从当动物接受第二次开?...

Watch this Scientific Journal Video about Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix at JoVE.com

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

àbiograft植入治疗心肌梗死的啮齿动物模型由法援署结扎诱发了常规需要两个开放式心脏手术。为了降低死亡率，并提供用于治疗与细胞相关的固体和胶状biomatrices固定最佳条件，微创手术已得到了发展。

基于细胞治疗心脏衰竭大鼠：双开胸心肌梗死和细胞与生物基质植入心外膜

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

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JoVE Journal

Bioengineering

11.8K 次观看.  University of Fribourg.  àbiograft植入治疗心肌梗死的啮齿动物模型由法援署结扎诱发了常规需要两个开放式心脏手术。为了降低死亡率，并提供用于治疗与细胞相关的固体和胶状biomatrices固定最佳条件，微创手术已得到了发展。 

视频: Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

生物发光成像评估的免疫反应，植入心脏组织工程（东隧）

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

科研

生物工程

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

管式膜网络在心肌细胞的心房和心室分析

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

小鼠模型中生物合成组织工程气管移植的育苗与植入

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

二维和三维 i 型胶原蛋白基质角质形成细胞增殖的评价

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

磁性、声学和光学三响应性微泡，用于磁性高温和波托托热组合癌症治疗

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

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 ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

心肌梗塞穆林模型中的 3D 生物打印贴片移植

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

通过心血管磁共振成像定量小鼠心脏左心室功能、心肌劳损和血流动力学力

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

2D人干细胞来源心肌细胞中心脏收缩力调节疗法的评估

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...