Name: slicing and culturing pig hearts under physiological conditions
Uploaded: 2020-03-20T14:00:00
Description: Watch this Scientific Journal Video about Slicing and Culturing Pig Hearts under Physiological Conditions at JoVE.com

TMAM 得到 NIH 授予 P30GM127607 和美国心脏协会授予 16SDG29950012 的支持。RB 支持 P01HL78825 和 UM1HL113530。

所有动物程序都符合路易斯维尔大学的体制准则，并经机构动物护理和使用委员会批准。
1. 切片准备（切片前一天）
<ol><li>振动微托姆的准备
	<ol>
<li>按照以下步骤将陶瓷刀片放入其支架中：仔细展开刀片后，先将锋利边缘放入刀片工具的插槽中。然后，松开支架臂上的两个螺钉，将刀片滑到每个套牢器下方，并将其牢固地向后推到后止动器上，将刀片安装到?...

<ol>
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R., 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=Revisiting+Cardiac+Cellular+Composition.">Revisiting Cardiac Cellular Composition.</a> Circulation Research. 118 (3), 400-409 (2016).</li><li>Kanisicak, O., 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=Genetic+lineage+tracing+defines+myofibroblast+origin+and+function+in+the+injured+heart.">Genetic lineage tracing defines myofibroblast origin and function in the injured heart.</a> Nature Communications. 7, 12260 (2016).</li><li>Fu, 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=Specialized+fibroblast+differentiated+states+underlie+scar+formation+in+the+infarcted+mouse+heart.">Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart.</a> Journal of Clinical Investigations. 128 (5), 2127-2143 (2018).</li><li>Kretzschmar, 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=Profiling+proliferative+cells+and+their+progeny+in+damaged+murine+hearts.">Profiling proliferative cells and their progeny in damaged murine hearts.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (52), E12245-E12254 (2018).</li><li>Perbellini, F., 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=Investigation+of+cardiac+fibroblasts+using+myocardial+slices.">Investigation of cardiac fibroblasts using myocardial slices.</a> Cardiovascular Research. 114 (1), 77-89 (2018).</li><li>Watson, S. 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=Preparation+of+viable+adult+ventricular+myocardial+slices+from+large+and+small+mammals.">Preparation of viable adult ventricular myocardial slices from large and small mammals.</a> Nature Protocols. 12 (12), 2623-2639 (2017).</li><li>Kang, C., 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+Organotypic+Cultured+Cardiac+Slices:+New+Platform+For+High+Throughput+Preclinical+Human+Trials.">Human Organotypic Cultured Cardiac Slices: New Platform For High Throughput Preclinical Human Trials.</a> Scientific Reports. 6, 28798 (2016).</li><li>Ou, Q., 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=Physiological+Biomimetic+Culture+System+for+Pig+and+Human+Heart+Slices.">Physiological Biomimetic Culture System for Pig and Human Heart Slices.</a> Circulation Research. 125 (6), 628-642 (2019).</li><li>Jones, S. 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=The+NHLBI-sponsored+Consortium+for+preclinicAl+assESsment+of+cARdioprotective+therapies+(CAESAR):+a+new+paradigm+for+rigorous,+accurate,+and+reproducible+evaluation+of+putative+infarct-sparing+interventions+in+mice,+rabbits,+and+pigs.">The NHLBI-sponsored Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR): a new paradigm for rigorous, accurate, and reproducible evaluation of putative infarct-sparing interventions in mice, rabbits, and pigs.</a> Circulation Research. 116 (4), 572-586 (2015).</li><li>Crick, S. J., Sheppard, M. N., Ho, S. Y., Gebstein, L., Anderson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+pig+heart:+comparisons+with+normal+human+cardiac+structure.">Anatomy of the pig heart: comparisons with normal human cardiac structure.</a> Journal of Anatomy. 193 (Pt 1), 105-119 (1998).</li><li>Fischer, C., 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=Long-term+functional+and+structural+preservation+of+precision-cut+human+myocardium+under+continuous+electromechanical+stimulation+in+vitro.">Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro.</a> Nature Communications. 10 (1), 117 (2019).</li><li>Franke, J., Abs, V., Zizzadoro, C., Abraham, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+the+effects+of+fetal+bovine+serum+versus+horse+serum+on+growth+and+differentiation+of+primary+equine+bronchial+fibroblasts.">Comparative study of the effects of fetal bovine serum versus horse serum on growth and differentiation of primary equine bronchial fibroblasts.</a> BMC Veterinary Research. 10, 119 (2014).</li><li>Vuorenpaa, 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=Novel+in+vitro+cardiovascular+constructs+composed+of+vascular-like+networks+and+cardiomyocytes.">Novel in vitro cardiovascular constructs composed of vascular-like networks and cardiomyocytes.</a> In Vitro Cellular &amp; Developmental Biology - Animal. 50 (4), 275-286 (2014).</li><li>Qiao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+slice+culture+platform+for+the+investigation+of+human+cardiac+tissue+physiology.">Multiparametric slice culture platform for the investigation of human cardiac tissue physiology.</a> Progress in Biophysics and Molecular Biology. 144, 139-150 (2018).</li><li>Watson, S. 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=Biomimetic+electromechanical+stimulation+to+maintain+adult+myocardial+slices+in+vitro.">Biomimetic electromechanical stimulation to maintain adult myocardial slices in vitro.</a> Nature Communications. 10 (1), 2168 (2019).</li></ol>

在这里，我们描述了我们最近发布的简化中等通量方法的详细视频协议（过程多达48片/设备），使猪心切片的培养时间足够长，以测试急性心毒性13。建议的条件模仿心脏的环境，包括电刺激的频率、营养供应和间歇性氧合。我们将生物仿生刺激培养中心片的长期生存能力归因于我们专注于重建完整心脏所经历的生理状况。这一概念得到我们的数据的支持，表明仅靠电刺激而不?...

药物诱发的心肌毒性是市场退出的主要原因1.在20世纪的最后十年里，有8种非心血管药物因心室心律失常导致突然死亡而退出市场。此外，几种抗癌疗法（在许多情况下有效）可导致多种心毒性作用，包括心肌病和心律失常。例如，传统（如安曲霉素和辐射）和靶向（如曲图祖马布）乳腺癌疗法都可能导致患者的一部分3的心血管并发症。心脏病学家和肿瘤学家之间的密切合作（通过新兴的"心肿瘤"领域）帮助使这些并发症易于管理，确保患者得到有效治疗。较新的药物，包括Her2和PI3K抑制剂的心血管效应不太清楚，尤其是当疗法被结合使用时。因此，在人体临床试验之前，人们越来越需要可靠的心血管毒性临床前筛查策略， 与新兴的抗癌疗法有关。人体心脏组织缺乏在功能上和结构上可存活超过24小时的培养系统，是可靠的心毒性测试的一个限制因素。因此，迫切需要开发一个可靠的系统，在生理条件下培养人类心脏组织，以测试药物毒性。
最近，人类诱导多能干细胞衍生心肌细胞（hiPSC-CMs）在心毒性测试中的使用，为解决这一问题提供了部分解决办法;然而，与心脏组织的多细胞性质相比，hiPSC-CM的不成熟性质和组织完整性的丧失是该技术的主要局限性4。最近的一项研究通过从水凝胶上制造hiPSC-CM的心脏组织，并使它们随着时间的推移逐渐增加电刺激，从而部分克服了这种限制。然而，它们的机电性能并没有达到成人心肌的成熟度。此外，心脏组织在结构上更为复杂，由各种细胞类型组成，包括内皮细胞、神经元和各种类型的基质纤维细胞，与细胞外基质蛋白6的非常特殊的混合物连接在一起。这种非心肌细胞群的异质性在成年哺乳动物,8,心脏中为使用单个细胞类型对心脏组织进行建模的主要障碍。这些主要限制强调开发方法的重要性，使完整的心脏组织培养，以最佳研究涉及心脏的生理和病理条件5。
培养人类心脏切片是一种完整的人类心肌的有前途的模型。该技术提供了获得一个完整的3D多细胞系统，类似于人类心脏组织，可以可靠地反映人类心肌的生理或病理状况。然而，它的使用受到文化生存时间短的严格限制，在2018年10、11、12,11,12之前，使用报告的最可靠的协议不会超过24小时。这一限制是由于多种因素造成的，包括使用空气-液体接口来培养切片，以及使用一种不支持心脏组织高能量需求的简单培养介质。我们最近开发了一个水下培养系统，能够提供连续的电刺激和优化培养培养培养成分，以保持心脏组织切片的活性长达6天13。该培养系统有可能成为急性心毒性检测的强有力的预测性人类原位模型，以缩小临床前和临床检测结果之间的差距。在本文中，我们将详细介绍使用猪心切片来切片和培养的规程。同样的过程也适用于人类、狗、绵羊或猫的心脏。有了这个协议，我们希望将这项技术推广到科学界的其他实验室。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1000ml, 0.22&#181;m, Vacuum Filter/Storage Systems</td>
			<td>VWR</td>
			<td>28199-812</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Butanedione monoxime (BDM)</td>
			<td>Fisher</td>
			<td>AC150375000</td>
			<td></td>
		</tr>
		<tr>
			<td>500ml, 0.22&#181;m, Vacuum Filter/Storage Systems</td>
			<td>VWR</td>
			<td>28199-788</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well C-Dish Cover (electrical-stimulation-plate-cover)</td>
			<td>Ion Optix</td>
			<td>CLD6WFC</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Fisher</td>
			<td>08-772-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioline USA</td>
			<td>BIO-41025</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Thermo</td>
			<td>15-240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>C-Pace EM (cell-culture-electrical-stimulator)</td>
			<td>Ion Optix</td>
			<td>CEP100</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic Blades for Vibrating Microtome</td>
			<td>Campden Instruments</td>
			<td>7550-1-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Cooley Chest Retractor</td>
			<td>Millennium Surgical</td>
			<td>63-G5623</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Fisher</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel #20</td>
			<td>Biologyproducts.com</td>
			<td>DS20X</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell Strainers, Sterile, Corning</td>
			<td>VWR</td>
			<td>21008-952</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo</td>
			<td>A3160502</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fisher</td>
			<td>NC9475675</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-50KU</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoacryl BLUE Tissue glue</td>
			<td>Amazon</td>
			<td></td>
			<td><a href="https://www.amazon.com/HISTOACRYL-FLEXIBLE-1051260P-Aesculap-Adhesive/dp/B074WB5185/" target="_blank">https://www.amazon.com/HISTOACRYL-FLEXIBLE-1051260P-Aesculap-Adhesive/dp/B074WB5185/</a></td>
		</tr>
		<tr>
			<td>Iris Spring scissors</td>
			<td>Fisher</td>
			<td>NC9019530</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Straight Scissors</td>
			<td>Fisher</td>
			<td>731210</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane, USP</td>
			<td>Piramal</td>
			<td>NDC 66794-017-25</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS Liquid Media Supplement</td>
			<td>Sigma-Aldrich</td>
			<td>I3146-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine HCl (500 mg/10 mL)</td>
			<td>West-Ward</td>
			<td>NDC 0143-9508</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride (MgCl2)</td>
			<td>Fisher</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo SuperCut Surgical Scissors</td>
			<td>AROSurgical Instruments Corporation</td>
			<td>AROSuperCut&#8482; 07.164.17</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199, Earle&#39;s Salts</td>
			<td>Thermo</td>
			<td>11-150-059</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen regulator</td>
			<td>Praxair</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen tanks -</td>
			<td>Praxair</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pasteur pipettes</td>
			<td>Fisher</td>
			<td>13-711-48</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher</td>
			<td>AC193780010</td>
			<td></td>
		</tr>
		<tr>
			<td>Printer Timing Belt</td>
			<td>Amazon</td>
			<td></td>
			<td><a href="https://www.amazon.com/Uxcell-a14081200ux0042-PRINTER-Precision-Timing/dp/B00R1J3KDC/" target="_blank">https://www.amazon.com/Uxcell-a14081200ux0042-PRINTER-Precision-Timing/dp/B00R1J3KDC/</a></td>
		</tr>
		<tr>
			<td>Razor rectangle blades</td>
			<td>Fisher</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF basic</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-025/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human VEGF</td>
			<td>R&#38;D Systems</td>
			<td>293-VE-010/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Retractable scalpels</td>
			<td>Fisher</td>
			<td>22-079-716</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Fisher</td>
			<td>AC217125000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher</td>
			<td>AC327300010</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating Microtome</td>
			<td>Campden Instruments</td>
			<td>7000 SMZ-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine HCl (100 mg/mL)</td>
			<td>Heartland Veterinary Supply</td>
			<td>NADA 139-236</td>
			<td></td>
		</tr>
	</tbody>
</table>

slicing and culturing pig hearts under physiological conditions

许多新药在临床研究中失败，由于心毒副作用，因为目前可用的体外测定和体内动物模型对人体心脏负债的预测不佳，给制药业带来了数十亿美元的负担。因此，全世界在进行昂贵且耗时的"人类第一"试验之前，需要更好的方法来识别药物心毒性， 在全球范围内未得到满足。目前，只有未成熟的心脏细胞（人类诱导多能干细胞衍生心肌细胞[hiPSC-CMs]）用于测试治疗效率和药物毒性，因为它们是唯一可以长期培养的人类心脏细胞需要测试药物疗效和毒性。然而，单个细胞类型不能复制由多种细胞类型组成的复杂3D心脏组织表型。重要的是，药物对成人心肌细胞的影响需要测试，与不成熟的hiPSC-CMs相比，其特征和毒性反应不同。 培养人类心脏切片是完整人体心肌的一种有前途的模式。该技术提供一个完整的多细胞系统，模拟人类心脏组织，反映人类心肌的生理或病理状况。最近，通过优化培养介质组件和培养条件，包括1.2 Hz的连续电刺激和培养介质的间歇性氧合，我们开发了一种新的培养系统设置，以保持生存能力和功能的人和猪心脏切片6天的培养。在目前的协议中，我们以研究猪心切片和培养的方法为例。同样的协议用于培养来自人类、狗、绵羊或猫心的切片。该培养系统有可能成为急性心毒性检测的强有力的预测性人类原位模型，从而缩小临床前和临床检测结果之间的差距。

我们使用一种商用细胞培养电刺激器，可以同时容纳8个6个井板，通过诱导生理频率（1.2 Hz）的电刺激来模拟成人心脏环境，并筛选基本介质成分，以延长培养13中功能性猪心片的持续时间。由于猪和人类心脏的大小和解剖量相似，我们利用猪心开发了一种生物化心脏切片培养系统，随后在人类心脏切片中验证了它。在这里，我们详细介...

Watch this Scientific Journal Video about Slicing and Culturing Pig Hearts under Physiological Conditions at JoVE.com

Slicing and Culturing Pig Hearts under Physiological Conditions

该协议描述了如何在生理条件下切片和培养心脏组织6天。该培养系统可用作测试新型心力衰竭治疗效果的平台，以及3D心脏模型中急性心毒性的可靠检测平台。

生理条件下猪心的切片和培育

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

该协议用于在生理条件下切片和培养心脏组织部分六天，可用于急性心脏毒性测试以及心力衰竭疗法的疗效测试。这种培养系统有可能成为急性心脏毒性测试的有力预测人类原位模型，缩小临床前和临床测试结果之间的差距。演示程序将是我们实验室的实验室经理曲庆辉。要获得猪心组织切片，首先在振动器的组织浴冷却夹克中加入冰，并在浴池中加入Tyrode溶液。使用一升塑料罐收集组织浴冷却夹克中融化的冰径流，将猪心转移到生物安全柜中含有一升新鲜冷心肺溶液的托盘中。使用可伸缩的无菌手术刀解剖心脏以隔离左心室，并使用矩形剃须刀刀片将左心室切成一到两个立方体厘米块。留出一个组织块进行切片，将剩余的组织块块放入 50 毫升的冷 Tyrode 溶液管中。轻轻按摩保留的组织块，并将一到两滴组织胶水添加到振动器的金属样品支架上。将一块 4% 的 agar 块粘在胶水上，表面面积约一平方厘米，并在胶中加入一到两滴组织胶水。将心脏块连接到 agar 心脏瓣膜侧向下，组织尽可能平放在支架上，将带心脏块的组织支架放入振动器的切片浴中。至关重要的是，坚持心脏块的阿加与心脏史诗侧朝下在组织胶水，并确保组织是尽可能平坦的。然后将氧气管连接到切片浴和装满 Tyrode 溶液的金属托盘上，将 40 微米细胞过滤器放在金属托盘上，以收集切片时的心脏组织部分。使用振动器操作软件调整刀片和样品的高度，使刀片靠近组织顶部，但低于心肌和液体覆盖组织和刀片。选择前进以调整开始切片组织的地方。按切片以加快速度，并使用旋钮将刀片向组织边缘移动。再次按切片以停止并前进以停用进程。然后按指示调整切割参数，然后按切片开始切片组织。当刀片到达组织端，但在到达试样支架的端部之前，再次按切片停止，然后按回，返回以返回开始位置。当切片达到全长，看起来质量良好时，使用塑料巴氏移液器，用冷Tyrode溶液轻轻地从浴池中收集组织，使用钳子和弹簧剪刀，以脱位切片从心脏时，必要时。将每个收集的切片放在含氧的 Tyrode 浴液中的单个细胞过滤器中，并使用移液器中的溶液将组织压在细胞过滤器表面。然后将金属垫圈放在组织顶部，按住它。在氧化泰洛德溶液中至少一小时后，修剪每个切片的外部以去除任何不均匀的边缘，将每片的端部粘在一块六毫米宽的消毒聚氨酯打印机正时带上，并配有嵌入的金属线。将支撑的心脏片放入六孔板中，每孔含有六毫升培养介质，并使用无菌钳子确保组织位于井的中心。注意板盖不会接触组织，将板放在培养板上，使板盖的弯曲侧与板的角面匹配，方角向上倾斜。将切片放入 37 摄氏度和 5% 的二氧化碳培养箱中。以六毫升新鲜含氧培养基代替培养超级钠，每天三次。将盖连接到细胞培养电刺激器并调整刺激器，以连续向心脏片提供 10 伏电力和 1.2 赫兹频率。插入刺激后，心脏切片将开始跳动。每天中午介质更换时，更换培养皿中的刺激板盖，防止有毒石墨颗粒释放至培养介质中。插入白色泡沫塞，其中用过的盖连接到电缆的细胞培养电刺激器，以防止水损坏电路，并将板盖放入一个浴，辅以2X抗生素/抗霉菌。第二天，在70%乙醇浴中去污染板盖5至15分钟，然后将盖转移到一个新鲜的自洗水浴中，并辅以2X抗生素/抗霉菌3分钟。冲洗板盖后，使用乙醇喷洒的清洁无绒擦拭，以去除塑料部件中残留的水，并清除白色泡沫塞。然后，盖板准备放回培养板。使用这种新的仿生培养装置作为证明，猪心脏切片的生存能力保持六天，由MTT测定评估。在头六天，类似于观察到的新鲜心脏切片的反应，在猪心脏切片心肌细胞内没有自发的钙瞬变。心肌细胞确实对外部电和β-肾上腺素刺激有反应。此外，在心脏切片培养中保持了长达六天的收缩力和对异丙醇的反应，与在新鲜心脏切片中观察到的相似。可以在心脏切片上进行多种结构和功能评估，包括 MTT 可行性测定、免疫污染、电子显微镜、钙瞬态测量、收缩功能评估、代谢评估和分析。我们目前正在使用这种培养系统来测试对猪和人类心脏切片感兴趣的药物和基因疗法的急性心脏毒性和疗效。

Research

JoVE Journal

Medicine

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

在分离的小鼠使用红桃心功能和能量学的评估 31 P核磁共振

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

科研

医学

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

NADH的荧光成像隔离双心室工作离体兔心

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

左室壁应力和改进功能衰竭心脏使用Algisyl LVR减少

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

心肌内细胞交货：观察在小鼠心脏

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

A Detailed Protocol for Physiological Parameters Acquisition and Analysis in Neurosurgical Critical Patients

神经外科危重患者生理参数采集与分析的详细方案

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ...

Cochlear Implantation in the Guinea Pig

豚鼠耳蜗植入术

Cochlear implants are highly efficient devices that can restore hearing in subjects with profound hearing loss. Due to improved speech perception ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

标准化生物力学条件下小鼠软骨骨折愈合的微创模型分析

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Functional and Physiological Methods of Evaluating Median Nerve Regeneration in the Rat

大鼠中位神经再生评价的功能与生理方法

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

前维沃·兰根多夫注入心脏的心电生理学研究

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...