Name: maturation of human stem cell-derived cardiomyocytes in biowires using electrical stimulation
Uploaded: 2017-05-06T13:00:00.000+00:00
Duration: 10 min 11 s
Description: Watch this Scientific Journal Video about Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation at JoVE.com

这项工作是由一个赠款的援助从心脏和中风支持加拿大（G-14-0006265）的基础，从健康研究（137352和143066）和普·比克尔基金会授予加拿大学院运营赠款（1013821 ）到SSN。

主设计与制作注意:使用软光刻技术进行器件制造。为二甲基硅氧烷（PDMS）成型制成两层SU-8主机。 <ol><li>使用设计和绘图软件设计设备（ 图1A ，左）。分别绘制主体的每一层。在20,000 dpi的两个光掩模上打印设备设计，对应于主板15的两层。将设备图案设置为透明，周围区域为黑色;掩模将用作通过光刻将器件图案光学转移到SU-8上的模板（如其他15所述）。 </li><li>将约4mL的SU-8 50分配到4英寸硅晶片的中心。使用旋涂机将SU-8 50均匀地涂在晶片上，以每分钟2000转（RPM）旋转30秒。在95°C的电热板上烘烤晶片10分钟。将晶片暴露于紫外线（UV）GHT以200mJ / cm 2以下。 </li><li>在2500 RPM 30秒倒约4毫升的SU-8 2050到晶片和旋转的中心。烤上95℃热板上15分钟。冷却至室温（RT）。 </li><li>重复步骤1.3两次。 </li><li>覆盖与所述第一层的透明度的光掩模的涂覆的晶片。暴露晶片于UV光，在270毫焦/厘米2，以使第一层。烤上95℃热板上15分钟。 </li><li>通过重复步骤1.3两次使SU-8 2050的第二层。 </li><li>对准使用掩模对准器在第一层上的第二层掩模的功能。然后，暴露于UV光，在240毫焦/平方厘米。烘烤在95℃下15分钟。 注:曝光时间由所需要的总UV剂量和在使用前的天测得的UV光输出强度来确定。 </li><li>通过在丙二醇单甲醚乙酸酯（PGMEA）淹没它，并以200RPM在定轨振荡器中振荡30分钟开发晶片。 </li><li>地点主到培养皿并小心倒入PDMS混合物（碱以10:1的比例与交联剂:按重量计1）在晶片的中心。覆盖所有的功能，避免气泡。 </li><li>热在在70℃下2小时的烘箱中。使用锋利的刀片切割围绕PDMS biowire模板的（ 图1A，右图）的边缘。剥离从主的PDMS。修剪与刀片装置。把PDMS biowire模板在灭菌袋中，蒸汽高压釜中于121℃进行20分钟。 </li><li>在生物安全柜（BSC），将biowire模板PDMS到培养皿中。用镊子保持，并通过在信道（ 图1B-I）的两端在槽安置它安装了一块在PDMS微孔的通道的中心无菌手术丝缝线（6-0）的。 </li></ol> 2.电刺激商会创作<ol><li>使一对矩形聚碳酸酯片（长×宽×厚度:2厘米×0.85厘米X0.35厘米），并利用它们作为电刺激室的框架（ 图2）。 </li><li>钻两个宽3毫米的孔相距2厘米沿着各聚碳酸酯框架件的中心线上。 </li><li>切为1.5厘米长炭棒两片。通过关于从端3毫米碳棒钻一个1mm宽的孔。线穿过碳棒孔铂丝和通过包装它的碳棒周围收紧导线。夹子连接到铂线，用于与电刺激器后连接的另一端。 </li><li>插入碳棒在聚碳酸酯框架件。放置与炭棒组装帧分成6孔板的孔中。 </li><li>倾2mL的PDMS的入井。浸没聚碳酸酯帧的底部PDMS同时保持PDMS表面水平接近但不到达炭棒的底部。热在在70℃下2小时的烘箱中。 </li><li>就拿电刺激室出来的六-Well板，并把它在灭菌袋中用于蒸汽消毒在121℃下20分钟。 </li></ol> 3. HPSC-CM的酶解<ol><li>诱导HPSC分化成使用建立的协议4心肌细胞。 注:简单地说，通过产生建立胚状体的心肌细胞（EB）协议在通过顺序补充骨形态发生蛋白4，碱性成纤维细胞生长因子，活化素A，血管内皮生长因子，和同系物可夫1 4干亲介质。从分化的20-34天使用的EB做出biowires。 </li><li>收集从低安装板将EB与P1,000移液管尖端和转移到锥形管中。通过离心在125×g下和RT 5分钟沉淀。与弃上清，重悬沉淀预温，37含1％脱氧核糖核酸酶I℃的I型胶原酶（DNA酶I）。孵育在37℃下2小时消化。 </li><li>添加预热的，37℃的5毫升洗涤介质和离心机在125×g下和室温下5分钟。弃去上清液。重悬用2mL的胰蛋白酶/乙二胺四乙酸（EDTA）溶液中的沉淀，并在37℃下孵育5分钟。 </li><li>加入1毫升含3％光阑培养基（V / V）DNA酶I </li><li>附加一个20 G（0.9毫米）针5毫升注射器，并通过它传递EB悬浮液3-5次。 </li><li>在280×g下加入7毫升洗涤介质和离心机5分钟。弃去上清液。重悬的Iscove氏改良的Dulbecco氏培养基（IMDM）的丸粒并储存在冰上。确定与血球细胞数。取0.5×10 6细胞和沉淀通过离心，在280×g离心5分钟。除去上清液。 </li><li>在冰上在无菌管中混合它们之前预冷所有凝胶组分。混合以下的胶原凝胶的组分（最终浓度）:2.1 mg / mL的鼠尾I型胶原蛋白，24.9 mM葡萄糖，23.8毫将NaHCO 3，14.3毫摩尔的NaOH，10mM的HEPES，和在1×M199培养基10％的细胞外基质。添加胶原蛋白和最后的细胞外基质。吹打向上和向下混合。 </li><li>重悬0.5×10 6细胞与胶原凝胶的3.5μL拌匀。 </li><li>使用P10移液管尖，以4微升的细胞悬浮液加入到0.5厘米长的信道（ 图1B-II）。调整用无菌镊子如果需要缝合的位置。 </li><li>覆盖培养皿用无菌磷酸盐缓冲盐水（PBS）的底部，确保不要触碰凝胶。孵育在37℃下30分钟。 </li><li>用培养基的12毫升（ 例如干Pro或介质推荐的电池制造商），足以覆盖细胞（ 图1B-III）更换PBS。培养在37℃下1周的孵化器。改变介质隔日。 </li></ol> 4.电刺激Biowire栽培<ol><li>在所述BSC，加入2毫升的PBS到6孔板，将容纳电刺激室的每个孔中。使用无菌镊子蒸压电刺激装置轻轻地放入井中。从井中丢弃PBS。 </li><li>覆盖用5ml预温热培养基的炭棒。使用无菌镊子装入biowires在电刺激室和它们定向垂直于碳棒（ 图2）。 </li><li>放置在6孔板的顶部上的盖子，但留下足够的铂丝在到达板的外侧与电刺激器相连接。置于细胞培养箱六孔板中于37℃和铂丝连接到电刺激器。 </li><li>双相脉冲的重复，1毫秒脉冲持续时间，3 V / cm，并且每秒1个脉冲（PPS）:使用具有以下设置的电刺激器刺激biowires。提高PPS每24个小时为下列值:1.83，2.66，3.49，40.82，5.15，和6更改隔日媒体。 </li><li>在10倍放大倍率观察在显微镜下响应于电刺激的心肌收缩性。 注:刺激后一周，该biowires变得成熟（参见结果部分）。 </li></ol>

<ol>
	<li>Sun, X., Nunes, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+hydrogel-based+strategies+for+application+in+cardiac+tissue+regeneration.">Overview of hydrogel-based strategies for application in cardiac tissue regeneration.</a> Biomed Mater. 10 (3), 034005 (2015).</li><li>Sun, X., Altalhi, W., Nunes, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+strategies+of+engineered+tissues+and+their+application+in+cardiac+regeneration.">Vascularization strategies of engineered tissues and their application in cardiac regeneration.</a> Adv Drug Deliv Rev. 96, 183-194 (2016).</li><li>Hastings, C. L., 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=Drug+and+cell+delivery+for+cardiac+regeneration.">Drug and cell delivery for cardiac regeneration.</a> Advanced Drug Delivery Reviews. 84, 85-106 (2015).</li><li>Yang, L., 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=Human+cardiovascular+progenitor+cells+develop+from+a+KDR++embryonic-stem-cell-derived+population.">Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.</a> Nature. 453 (7194), 524-528 (2008).</li><li>Zhang, 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=Extracellular+matrix+promotes+highly+efficient+cardiac+differentiation+of+human+pluripotent+stem+cells:+the+matrix+sandwich+method.">Extracellular matrix promotes highly efficient cardiac differentiation of human pluripotent stem cells: the matrix sandwich method.</a> Circ Res. 111 (9), 1125-1136 (2012).</li><li>Lian, X., 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=Robust+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+via+temporal+modulation+of+canonical+Wnt+signaling.">Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.</a> Proc Natl Acad Sci U S A. 109 (27), E1848-E1857 (2012).</li><li>Snir, 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=Assessment+of+the+ultrastructural+and+proliferative+properties+of+human+embryonic+stem+cell-derived+cardiomyocytes.">Assessment of the ultrastructural and proliferative properties of human embryonic stem cell-derived cardiomyocytes.</a> Am J Physiol Heart Circ Physiol. 285 (6), H2355-H2363 (2003).</li><li>Dolnikov, K., 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=Functional+properties+of+human+embryonic+stem+cell-derived+cardiomyocytes:+intracellular+Ca2++handling+and+the+role+of+sarcoplasmic+reticulum+in+the+contraction.">Functional properties of human embryonic stem cell-derived cardiomyocytes: intracellular Ca2+ handling and the role of sarcoplasmic reticulum in the contraction.</a> Stem Cells. 24 (2), 236-245 (2006).</li><li>Yang, X., Pabon, L., Murry, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+adolescence:+maturation+of+human+pluripotent+stem+cell-derived+cardiomyocytes.">Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes.</a> Circ Res. 114 (3), 511-523 (2014).</li><li>Zimmermann, W. 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=Tissue+engineering+of+a+differentiated+cardiac+muscle+construct.">Tissue engineering of a differentiated cardiac muscle construct.</a> Circ Res. 90 (2), 223-230 (2002).</li><li>Radisic, 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=Functional+assembly+of+engineered+myocardium+by+electrical+stimulation+of+cardiac+myocytes+cultured+on+scaffolds.">Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds.</a> Proc Natl Acad Sci U S A. 101 (52), 18129-18134 (2004).</li><li>Schaaf, S., 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=Human+engineered+heart+tissue+as+a+versatile+tool+in+basic+research+and+preclinical+toxicology.">Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology.</a> PLoS One. 6 (10), e26397 (2011).</li><li>Tulloch, N. L., 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=Growth+of+engineered+human+myocardium+with+mechanical+loading+and+vascular+coculture.">Growth of engineered human myocardium with mechanical loading and vascular coculture.</a> Circ Res. 109 (1), 47-59 (2011).</li><li>Nunes, S. S., 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=Biowire:+a+platform+for+maturation+of+human+pluripotent+stem+cell-derived+cardiomyocytes.">Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes.</a> Nat Methods. 10 (8), 781-787 (2013).</li><li>Lake, 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=Microfluidic+device+design,+fabrication,+and+testing+protocols.">Microfluidic device design, fabrication, and testing protocols.</a> Protocol Exchange. , (2015).</li><li>Shiba, Y., Hauch, K. D., Laflamme, M. 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+applications+for+human+pluripotent+stem+cells.">Cardiac applications for human pluripotent stem cells.</a> Curr Pharm Des. 15 (24), 2791-2806 (2009).</li><li>Yang, X., 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=Tri-iodo-l-thyronine+promotes+the+maturation+of+human+cardiomyocytes-derived+from+induced+pluripotent+stem+cells.">Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells.</a> J Mol Cell Cardiol. 72, 296-304 (2014).</li><li>Zhang, D., 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=Tissue-engineered+cardiac+patch+for+advanced+functional+maturation+of+human+ESC-derived+cardiomyocytes.">Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.</a> Biomaterials. 34 (23), 5813-5820 (2013).</li><li>Radisic, 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=Oxygen+gradients+correlate+with+cell+density+and+cell+viability+in+engineered+cardiac+tissue.">Oxygen gradients correlate with cell density and cell viability in engineered cardiac tissue.</a> Biotechnol Bioeng. 93 (2), 332-343 (2006).</li><li>Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., Bongso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+from+human+blastocysts:+somatic+differentiation+in+vitro.">Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.</a> Nat Biotechnol. 18 (4), 399-404 (2000).</li></ol>

作者宣称，他们没有竞争的经济利益。

该手稿描述了改进hPSC-CMs成熟的设计平台的生物线的设置和实现。该装置可以在标准的微细加工设备中制造，并且生物线可以用普通的细胞培养技术和电刺激器生产。 据我们所知，没有报道类似于生物线的方法。该策略表明，改善的成熟性质取决于电刺激方案，如通过较高刺激速率上升方案（6Hz对3Hz）的生物线中获得的较大成熟水平所证明的。生物线中的心肌细胞与培养20天?...

细胞治疗是最有前途和研究战略，以实现心肌修复/再生一个。已经通过心脏组织工程和生物材料1，2的共递送辅助。大多数现有的细胞来源进行了研究，在动物模型为他们的损伤，患病或年老心3潜在的有利影响。尤其显著已经努力用人类多能干细胞（HPSC）衍生的心肌细胞（HPSC-CM），用于心脏组织工程可能无限的自体细胞来源。 HPSC-CMS可以使用多种已建立的方案4，5，6来制造。然而，所获得的细胞相比，成年心室肌7显示胚胎样表型，具有一定范围的未成熟的特性， </sup&gt; 8。这可能是一个障碍HPSC-CMS应用在药物研发和成人心脏疾病模型9的发展成人心脏组织的模型。 为了克服表型不成熟的这种限制，新方法已被积极地研究促进心肌细胞成熟。早期的研究通过循环机械10或电刺激11显露在新生大鼠心肌细胞的有效促成熟性质。凝胶压实和环状机械刺激还显示出改善HPSC-CM熟化12， 图13，与电生理和钙的处理性能的增强最小的某些方面。因此，所谓的"生物丝"（biowire）的平台系统是由同时提供结构线索和电场stimulatio设计n增强hPSC-CMs的成熟14 。该系统使用微制造平台来创建适合于电场刺激的对准的心脏组织。这可以用于改善hPSC-CM的结构和电生理成熟度。在这里，我们描述制作这样的生物线的细节。

<table><tbody><tr><td>L-Ascorbic acid</td><td>Sigma</td><td>A-4544</td><td>hPSC-CM culture media componet</td></tr><tr><td>AutoCAD</td><td>Autodesk, Inc</td><td></td><td>Software to design device</td></tr><tr><td>Carbon rods, &amp;Oslash; 3 mm</td><td></td><td></td><td>Electrical stimulator chamber component</td></tr><tr><td>Collagen, type 1, rat tail</td><td>BD Biosciences</td><td>354249</td><td>Collagen gel: 2.1 mg/mL of rat tail collagen type I in 24.9 mM glucose, 23.8 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 14.3 mM NaOH, 10 mM HEPES, in 1x M199 media with 10 % of growth factor-reduced Matrigel.</td></tr><tr><td>Collagenase type I&amp;nbsp;</td><td>Sigma</td><td>C0130</td><td>0.2% collagenase type I (w/v) and 20% FBS (v/v) in PBS with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;. Sterilize with 0.22 &amp;mu;m filter and make 12 mL aliquots. Store at -20 &amp;deg;C.</td></tr><tr><td>Deoxyribonuclease I (DNase I)</td><td>EMD Millipore</td><td>260913-25MU</td><td>Make 1 mg/mL DNase I stock solution in water. Filter sterile and store 0.5 mL aliquots at &amp;minus;20 &amp;deg;C</td></tr><tr><td>Drill &amp;amp; drill bits (&amp;Oslash; 1 mm and 2 mm)</td><td>Dremel</td><td></td><td>Drill holes in polycarbonate frames</td></tr><tr><td>Electrical stimulator</td><td>Grass</td><td>s88x</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>WISENT Inc.</td><td>080-450</td><td></td></tr><tr><td>D-(+)-Glucose&amp;nbsp;</td><td>Sigma</td><td>G5767</td><td>Collagen gel component</td></tr><tr><td>L-Glutamine</td><td>Thermo Fisher Scientific</td><td>25030081</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O</td><td>MilliQ</td><td></td><td>18.2 M&amp;Omega;&amp;middot;cm at 25 &amp;deg;C, ultrapure, to make all solutions</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td>Collagen gel component</td></tr><tr><td>Hot plate</td><td>Torrey Pines</td><td>HS40</td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium(IMDM)</td><td>Thermo Fisher Scientific</td><td>12440053</td><td></td></tr><tr><td>Mask aligner</td><td>EVG&amp;nbsp;</td><td>EVG 620</td><td></td></tr><tr><td>Matrigel, growth factor reduced&amp;nbsp;</td><td>Corning</td><td>354230</td><td>Collagen gel component</td></tr><tr><td>Medium 199 (M199)</td><td>Thermo Fisher Scientific</td><td>11825015</td><td>Collagen gel component</td></tr><tr><td>Monothioglycerol (MTG)</td><td>Sigma</td><td>M-6145</td><td>hPSC-CM culture media componet</td></tr><tr><td>Orbital shaker</td><td>VWR</td><td>89032-088</td><td></td></tr><tr><td>Penicillin/Streptomycin (P/S)</td><td>Thermo Fisher Scientific</td><td>15070063</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS) with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>14040133</td><td></td></tr><tr><td>Plate (6-well)</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Plate (6-well), low attachment</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>Platinum wires, 0.2 mm</td><td></td><td></td><td>Electrical stimulator chamber component</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>Sylgard 184</td><td></td></tr><tr><td>Propylene glycol monomethyl ether acetate (PGMEA)</td><td>Doe &amp;amp; Ingalls Inc.</td><td></td><td>To develop the wafer</td></tr><tr><td>Pouch, peel-open</td><td>Convertors</td><td>92308</td><td>For steam sterilization</td></tr><tr><td>Silicon wafer, 4 inch</td><td>UniversityWafer Inc.</td><td></td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma</td><td>S5761</td><td>Collagen gel component</td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>S8045</td><td>Collagen gel component</td></tr><tr><td>Sprin coater</td><td>Specialty Coating Systems</td><td>G3P-8</td><td></td></tr><tr><td>StemPro-34 culture medium</td><td>Thermo Fisher Scientific</td><td>10639011</td><td>hPSC-CM culture medium. To make 50 mL, add 1.3 mL supplement, 500 &amp;mu;L of 100&amp;times; L-Glutamine, 250 &amp;mu;L of 30 mg/mL transferrin, 500 &amp;mu;l of 5 mg/mL ascorbic acid, 150 &amp;mu;L of 26 &amp;mu;l /2 mL monothioglycerol (MTG), and 500 &amp;mu;L (1 %) penicillin/streptomycin.</td></tr><tr><td>Stop media</td><td></td><td></td><td>Wash medium:FBS (1:1)</td></tr><tr><td>SU-8 50&amp;nbsp;</td><td>MicroChem Corp.</td><td></td><td>photoresist, master component</td></tr><tr><td>SU-8 2050&amp;nbsp;</td><td>MicroChem Corp.</td><td></td><td>photoresist, master component</td></tr><tr><td>Transferrin</td><td>Roche</td><td>10-652-202</td><td>hPSC-CM culture media componet</td></tr><tr><td>Trypsin/EDTA, 0.25%</td><td>Thermo Fisher Scientific</td><td>25200056</td><td>hPSC-CM culture media componet</td></tr><tr><td>Wash medium</td><td></td><td></td><td>IMDM containing 1% Penicillin/Streptomycin</td></tr></tbody></table>

maturation of human stem cell-derived cardiomyocytes in biowires using electrical stimulation

人类多能干细胞衍生的心肌细胞（HPSC-CMS）是一个有前途的细胞来源，并从而鼓励他们在心脏研究潜力的应用，包括药物发现，疾病建模，组织工程和再生医学的研究。然而，由现有的协议产生的细胞显示与天然成年心室肌相比的范围不成熟。许多已作出努力以成熟HPSC-CMS，只有迄今达到中等成熟。因此，工程化的系统，称为biowire，已通过提供物理和电线索导致HPSC-CMS 在体外更加成熟的状态设计的。该系统使用微制造平台种子HPSC-CM的I型胶原沿着一个刚性模板缝合组装成对准的心脏组织（biowire），将其进行电场刺激具有逐渐增加的频率胶凝。相比未刺激控制，刺激biowired心肌细胞表现出的结构和电生理学成熟的增强程度。这样的变化是取决于刺激速率。这份手稿详细描述了biowires的设计和创作。

理性在biowires使用的缝合线的是，以作为在一个轴对准并模仿心肌纤维的形状的3D结构的形成的模板。我们发现，之后在biowire培养7天的细胞改造围绕缝合（ 图3A）的凝胶。沿着缝合线的轴线组装细胞形成对准心脏组织（ 图3）。后7天的预培养物，在biowires进行7天电场刺激的，并与心肌细胞的结构和功能的成熟兼容进一步表现出的性质。 <p class="...

Watch this Scientific Journal Video about Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation at JoVE.com

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

心脏biowire平台是使用三维细胞培养与电刺激结合成熟人胚胎和诱导的多能干细胞衍生的心肌细胞（HPSC-CM）的体外方法。这份手稿介绍了心脏biowire平台的详细设置。

使用电刺激人干细胞衍生的心肌细胞的成熟中Biowires

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

maturation-human-stem-cell-derived-cardiomyocytes-biowires-using

Research

JoVE Journal

Developmental Biology

9.1K 次观看.  University Health Network. 心脏biowire平台是使用三维细胞培养与电刺激结合成熟人胚胎和诱导的多能干细胞衍生的心肌细胞（HPSC-CM）的体外方法。这份手稿介绍了心脏biowire平台的详细设置。 

视频: Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

科研

发育生物学

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

使用多电极阵列（MEA）的人多能干细胞来源的心肌细胞（hPSC-CM）的电生理分析

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

在体外人骨髓间充质干细胞分化为心肌样细胞功能

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

同时进行机电刺激增强细胞心肌成功能的潜能

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

生物工程

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

用于循环作用电位记录的多井微电极阵列上的人类 iPSC 衍生心肌细胞网络

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

生物学

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

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 ...

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

使用成熟的人诱导多能干细胞来源心肌细胞单层进行高通量心脏毒性筛选

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

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.