Name: ultrasonic-augmented primary adult fibroblast isolation
Uploaded: 2019-07-29T14:00:00
Description: Watch this Scientific Journal Video about Ultrasonic-augmented Primary Adult Fibroblast Isolation at JoVE.com

我们感谢罗米·肯佩女士和安妮特·奥皮茨夫人提供专家技术支持。我们还感谢比约恩·宾诺格先生的 IT 支持。这项工作得到了Fürerkreis Dresdner Erz-Kreislauf-Tage e.V.的赠款支持,b) "哈比利特费德·弗劳恩",卡尔·古斯塔夫·卡鲁斯·德累斯顿医学院,以及科尔塞·克鲁纳-福森斯科莱格(EKFK)医学院卡尔·古斯塔夫卡鲁斯德累斯顿我们感谢资金和支持。

以下协议遵循德国德累斯顿理工大学的机构动物护理指南(文件号:T 2014/4)以及国际公认的动物护理指南(FELASA)17。图 1可视化细胞分离过程。
1. 准备设置、材料和介质
<ol><li>制备细胞培养基、PBS溶液、胶原酶混合库存溶液(在12 mL无菌超纯水中重组50mg冻胶酶混合物)和0.25%胰蛋白酶溶液。</li>
	<li>将介质、PBS 和胰蛋白酶溶液预热至 37°C。</l...

<ol>
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A., Bertrand-De Haas, M., Schuurman, A. H., Lamme, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+enzymatic+isolation+of+fibroblasts+for+the+creation+of+autologous+skin+substitutes.">Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes.</a> In Vitro Cellular &amp; Developmental Biology. Animal. 40 (8-9), 268-277 (2004).</li><li>El-Armouche, 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=Phosphatase+inhibitor-1-deficient+mice+are+protected+from+catecholamine-induced+arrhythmias+and+myocardial+hypertrophy.">Phosphatase inhibitor-1-deficient mice are protected from catecholamine-induced arrhythmias and myocardial hypertrophy.</a> Cardiovascular Research. 80 (3), 396-406 (2008).</li><li>Guillen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FELASA+Guidelines+and+Recommendations.">FELASA Guidelines and Recommendations.</a> Journal of the American Association for Laboratory Animal Science. 51 (3), 311-321 (2012).</li><li>Seluanov, A., Vaidya, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+primary+adult+fibroblast+cultures+from+rodents.">Establishing primary adult fibroblast cultures from rodents.</a> Journal of Visualized Experiments. (44), 2033 (2010).</li><li>Masur, S. K., Dewal, H. S., Dinh, T. T., Erenburg, I., Petridou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblasts+differentiate+from+fibroblasts+when+plated+at+low+density.">Myofibroblasts differentiate from fibroblasts when plated at low density.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (9), 4219-4223 (1996).</li><li>Rohr, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblasts+in+cell+culture+systems:+myofibroblasts+all+along.">Cardiac fibroblasts in cell culture systems: myofibroblasts all along.</a> Journal of Cardiovascular Pharmacology. 57 (4), 389-399 (2011).</li><li>Cell lines: Valuable tools or useless artifacts. PubMed - NCBI Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22553484">https://www.ncbi.nlm.nih.gov/pubmed/22553484</a> (2019)</li><li>Copp&#233;, J. P., 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=Senescence-associated+secretory+phenotypes+reveal+cell-nonautonomous+functions+of+oncogenic+RAS+and+the+p53+tumor+suppressor.">Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.</a> PLoS biology. 6 (12), 2853-2868 (2008).</li><li>Childs, B. G., Durik, M., Baker, D. J., van Deursen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence+in+aging+and+age-related+disease:+from+mechanisms+to+therapy.">Cellular senescence in aging and age-related disease: from mechanisms to therapy.</a> Nature Medicine. 21 (12), 1424-1435 (2015).</li><li>Singh, M., Sharma, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outgrowth+of+fibroblast+cells+from+goat+skin+explants+in+three+different+culture+media+and+the+establishment+of+cell+lines.">Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines.</a> In Vitro Cellular &amp; Developmental Biology. Animal. 47 (2), 83-88 (2011).</li><li>Linge, C., Green, M. R., Brooks, R. 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与不朽的成纤维细胞系相比,原纤维细胞具有几个优点。它们可以以高质量和数量经济高效地隔离。此外,初级培养物提供了研究来自多个个体的细胞的可能性,从而提高了获得的结果的可靠性,并降低了仅仅研究细胞培养物的可能性。持续产生新的初级文化可以防止基因改变,这通常发生在反复传递21之后。此外,细胞衰老22,23或增加的肌纤维细胞分化在高<sup...

纤维细胞是扁平的,主轴状细胞,具有多个硬质处理和广泛的粗糙内质视网膜1,2。平均成纤维细胞测量30-100μm,寿命为57~3天1,3。人类成纤维细胞的平均细胞周期持续时间范围为16-48小时,取决于培养条件4。有证据表明,培养的原纤维细胞的复制能力和功能质量与捐赠年龄呈负相关,这表明,如果可能的话,应优先考虑较年轻的捐赠者(动物或患者).
纤维细胞是大多数哺乳动物身体组织的主要细胞类型。尽管成纤维细胞无处不在,但成纤维细胞的分子识别仍然是一个挑战。在胚胎发育期间,纤维细胞从不同来源迁移到发育中的组织和器官8。因此,在成纤维细胞中可以找到过多的标记蛋白,而独特的标记蛋白,存在于每个成纤维细胞种群中,并且独有成纤维细胞,仍然缺失。因此,几个识别标记的表达模式通常用于识别成纤维细胞。其中最知名的标记物包括维他命素、人纤维细胞表面蛋白(hFSP)、迪斯科丁域受体2(DDR2)和α平滑肌活动素(αSMA)。
纤维细胞是主要的细胞外基质(ECM)产生细胞类型。因此,成纤维细胞维持有序的组织结构,并为邻近细胞1提供机械支持。ECM 合成和降解之间的平衡是一个调节良好的过程。转向合成标志着过度ECM沉积的开始,如果不终止,会导致纤维化。纤维化由肌纤维细胞介导,肌纤维细胞起源于经过分子和表型变化的活化成纤维细胞。肌纤维细胞的一个标志是增强ECM和细胞因子的分泌和有序排列的表达[SMA微丝9。
原纤维细胞一直是最近研究的焦点,重点是纤维化,组织炎症和成纤维细胞-癌症细胞相互作用10,11。然而,为了有效地研究成纤维细胞在健康和疾病的特性,有必要定期分离可行的原发成人成纤维细胞。有几种方法可以分离成纤维细胞12,13,14。成纤维细胞分离的三种主要方法是从组织块12、酶组织消化15和中空器官的酶灌注9、13、16中生长。生长的优点是温和的隔离过程,没有酶细胞降解。另一方面,生长外培养物通常需要较长的培养期,直到细胞可用于实验。常见的酶消化是快速的,但承担与其他细胞类型(例如,内皮细胞)或细菌在搅拌过程中的污染风险,这是机械溶解组织所必需的。此外,这些方法往往是复杂的,需要时间和技能来学习。
关于原成纤维细胞在研究中的重要性,仍然需要在快速性、简单性和可靠性方面优化现有的细胞隔离方法。这里提供了一种提供高质量细胞的新型超声波酶成纤维细胞分离方法。

<table border="0" cellpadding="0" cellspacing="0" style="width:1103px;" width="1103">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.25% Trypsin-EDTA</td>
			<td style="width:339px;">Sigma-Aldrich, St. Louis, USA</td>
			<td style="width:143px;">T4049-100ML</td>
			<td style="width:405px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotics</td>
			<td>Gibco-Life Technologies, Carlsbad, USA</td>
			<td>Gibco LS15140148&#160;</td>
			<td>Penicillin/ Streptomycin (10000 U/ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell culture hood</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td>51023608</td>
			<td>HeraSafe KSP15</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell culture incubator</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td>50049176</td>
			<td>BBD 6220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture plates</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td style="width:143px;">depends on vessel</td>
			<td>6-, 12-, 24-wells Nunclon surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell culture suction</td>
			<td style="width:339px;">VACUUBRAND GMBH + CO KG, Wertheim, Germany</td>
			<td>20727400</td>
			<td style="width:405px;">BVC professional suction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell strainer (mesh)</td>
			<td>Corning, Tewksbury, USA</td>
			<td style="width:143px;">431750</td>
			<td>40 &#181;m Nylon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:339px;">Thermo Fisher Scientific, Waltham, USA</td>
			<td style="width:143px;">75007213</td>
			<td>Megafuge 8R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cordless pipetting controller</td>
			<td>Hirschmann, Eberstadt, Germany</td>
			<td>9907200</td>
			<td>Pipetus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable pipette tips</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td style="width:143px;">depends on volume</td>
			<td>SafeSeal tips for pipettes (10 &#181;l, 20 &#181;l, 100 &#181;l, 200 &#181;l, 1000 &#181;l)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable plastic pipettes</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td style="width:143px;">depends on volume</td>
			<td>5 ml, 10 ml, 25 ml, 50 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable sterile scalpel</td>
			<td>Myco Medical, Cary, USA</td>
			<td style="width:143px;">n.a.</td>
			<td>Techno cut</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dulbeccos Modified Eagle Medium (DMEM)</td>
			<td style="width:339px;">Thermo Fisher Scientific, Waltham, USA</td>
			<td>41965-062</td>
			<td>High glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf tubes</td>
			<td>Eppendorf, Hamburg, Germany&#160;</td>
			<td style="width:143px;">depends on volume</td>
			<td>50 &#181;l, 500 &#181;l, 1.500&#181;l, 2.000 &#181;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal calf serum (FCS)</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>F2442-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase blend</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td style="width:143px;">5401020001</td>
			<td>Liberase TL Research Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish 6 cm</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>P5481-500EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>D8537-500ML</td>
			<td>500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Senescence detection kit</td>
			<td style="width:339px;">Abcam, Cambridge, UK</td>
			<td>ab65351</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Shaker/ Vortex</td>
			<td style="width:339px;">IKA, Staufen im Breisgau, Germany</td>
			<td style="width:143px;">n.a.</td>
			<td>MS2 Minishaker (subsequent model: Ident-Nr.: 0020016017)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile plastic tubes</td>
			<td>Thermo Fisher Scientific, Waltham, USA</td>
			<td>Falcon 352095</td>
			<td>BD Falcon tubes (15 ml, 50 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultrasonic water bath</td>
			<td>BANDELIN electronic GmbH &#38; Co. KG, Berlin, Germany</td>
			<td style="width:143px;">312</td>
			<td>Sonorex RK100H</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical scissors (atraumatic)</td>
			<td>Aesculap AG, Tuttlingen, Germany</td>
			<td style="width:143px;">NR 82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical scissors&#160;</td>
			<td>Aesculap AG, Tuttlingen, Germany</td>
			<td style="width:143px;">eq 1060.09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical forceps</td>
			<td>Aesculap AG, Tuttlingen, Germany</td>
			<td style="width:143px;">BD577</td>
			<td></td>
		</tr>
	</tbody>
</table>

ultrasonic-augmented primary adult fibroblast isolation

原发性成人成纤维细胞已成为研究所有身体组织纤维化、成纤维细胞相互作用和炎症的重要工具。由于原纤维细胞不能由于肌纤维细胞分化或衰老诱导而无限期分裂,因此必须定期建立新的培养物。然而,在制定可靠的隔离协议和初级成纤维细胞隔离过程中,有几个障碍需要克服:该方法的难度程度(尤其是初学者),细菌污染的风险,所需时间,直到原纤维细胞可用于实验,以及随后的细胞质量和生存能力。在这项研究中,提供了一种快速、可靠和易于学习的协议,从小鼠心脏、肺、肝脏和肾脏中分离和培养原发性成人成纤维细胞,结合酶消化和超声波搅拌。

证明了该方案从固体鼠组织中分离成人成纤维细胞的能力。获得了可行的成纤维细胞,可用于后续实验,如免疫荧光染色或增殖实验(图2D-F,图5A)。
成人成纤维细胞是扁平主轴状细胞,具有多个细胞过程,通常在单层12、18中生长。所得结果反映了这些形态特征,表明不?...

Watch this Scientific Journal Video about Ultrasonic-augmented Primary Adult Fibroblast Isolation at JoVE.com

Ultrasonic-augmented Primary Adult Fibroblast Isolation

我们提出了一种方案,以简单、快速和可靠的方式分离主要成人成纤维细胞,由初学者(例如学生)执行。该程序结合酶组织消化和机械搅拌与超声波获得原成纤维细胞。该协议可以很容易地适应特定的实验要求(例如,人体组织)。

超声波增强原成人成纤维细胞隔离

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

我们的协议为原发性成纤维细胞分离提供了简单快捷的方法，明确侧重于避免细菌污染，这是一个大问题。特别是对于初学者。我们技术的主要优点是它的简单性。因为它不需要广泛的手工技能或经验，而且每个人都可以在短时间内轻松学习。展示这项技术的将是曼贾·纽，一个来自实验室的技术员。在开始解剖之前，戴上两副一次性手套，另一副放在另一副手套上。将鼠标固定到聚苯乙烯垫上。用70%乙醇对毛皮进行消毒，确保动物被浸泡。并使用乙醇消毒手术钳和创伤剪刀杯上尿道上方的皮肤。从最初的切口到颈部，沿着中线切三到四厘米，增加四肢的浮雕切口。将皮肤固定在垫上，以获得最佳地接触覆盖腹腔的肌肉。然后，用新鲜70%乙醇对腹部肌肉进行两次消毒。乙醇干燥后，取下第一副手套。并使用一套新的无菌钳子和剪刀来插入肌肉层，以打开腹腔和胸部。要切除感兴趣的器官，请用手术钳轻轻抓住每个器官，而不刺穿组织。用剪刀小心地切断器官入口点附近的供血血管。将每个器官放入无菌、冰冷的 PBS 管中，并紧闭管。将每个管子放在湿冰上，因为器官被收集。当所有器官都收获后，先用70%乙醇喷洒管子，然后再将它们放入无菌细胞培养罩中。戴上一副新鲜的手套，使用无菌钳子将每个器官转移到无菌六厘米培养皿的一半上。用新鲜的PBS短暂清洗器官，以去除多余的血液。将冲洗过的器官转移到每个培养皿的后半部分，并去除多余的PBS。使用两个无菌手术刀，将器官切成一到两毫米的碎片。并使用无菌铲子将组织片段转移到新的、单独的、无菌的 15 毫升管中。在每个管中加入两毫升0.25%的三辛肌溶液。将管子放入37摄氏度的细胞培养箱中5分钟。在孵育结束时，每分钟旋转约1400次，旋转管子10秒。并阻止三普辛反应与四毫升的适当培养，辅以胎儿小牛血清每管。接下来，将适当体积的胶原酶混合溶液添加到每个管中。将管子放在37摄氏度的超声波水浴中10分钟。在声波的末尾，轻轻旋转管子10秒钟。将管子放回超声波水浴 10 分钟之前。在第二次声声结束时，将管子旋转10秒。用70%乙醇对管子进行消毒。在细胞培养罩中，通过单个40微米滤株将细胞悬浮液过滤成新的无菌15毫升管。通过离心收集细胞。将颗粒重新悬浮在每管一毫升新鲜介质中。并在适当的电镀密度下查看合适的细胞培养容器中的细胞。然后，将细胞放在细胞培养箱中过夜。第二天早上，用PBS清洗每个培养物三次，然后加入新鲜培养剂，将细胞返回培养箱。使用本协议，可获取可行的成纤维细胞，用于后续实验。如氨基荧光染色或增殖实验。成人成纤维细胞是扁平的主轴状细胞，具有多种细胞过程，通常以单层生长。然而，可以观察到不同器官的成纤维细胞种群的形态差异。例如，肾成纤维细胞比心脏、肺或肝成纤维细胞更小，密度更高。孤立的成纤维细胞具有很高的增殖率，在大约六天后达到超过90%的光汇合。在这里，可以观察到具有独特阿尔法平滑肌肉行为素微丝的分化肌纤维细胞。这些细胞在心脏、肾脏、肝脏和肺细胞培养中大量发现。虽然只有约20%至30%的分离和培养细胞表达有序，有安排的，阿尔法平滑肌肉行为素微丝，几乎所有的细胞是积极的维门丁和迪斯科丁域受体2后，七天培养。要记住的最重要的事情是在整个过程中使用正确的技术，因为必须避免细菌污染。原发性成纤维细胞分离后，细胞可用于各种精神病学实验和表征。如免疫细胞化学增殖或伤口愈合论文，或分子生物学评估。

Research

JoVE Journal

Medicine

Primary Culture of Human Vestibular Schwannomas

人体前庭神经鞘瘤的原代培养

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

科研

医学

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

原发灶和MEF细胞分离，研究肺转移

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

分离培养犬从动脉和静脉主血管内皮细胞

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

成年大鼠心肌细胞的分离与培养

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

足月胎盘胎膜中原发性人蜕细胞的分离

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

非放射性 l-azidohomoalanine 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Isolation of Atrial Myocytes from Adult Mice

从成年小鼠分离心膜肌细胞

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

使用左前部降冠动脉的永久结扎的月心肌梗死模型

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

在健康和受伤的老鼠心脏中为三维纤维细胞组织进行精炼的清晰组织清除

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

朗根多夫方法的修改，用于从成年小鼠中同时分离心房和心室肌细胞

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...