This work was supported by funding from American Heart Association 12BGIA12040477, NC State University Chancellor&#8217;s Faculty Excellence Program, and National Natural Science Foundation of China H020381370216.

下面的协议是专为心肌细胞的新生幼鼠（10-14幼仔）一窝隔离。如果产仔显著不同，该过程可能需要进行调整以补偿。小狗应该在48±6小时岁。所有的程序在此已批准北卡罗莱纳州立大学的机构动物护理和使用委员会（IACUC）。 1.细胞分离制备注:隔离之前执行以下步骤的一天。 <ol><li>高温高压灭菌以下:大剪刀，小剪刀，小尖镊子，镊子大。使用重力周期为60分钟，在最小的121℃，30分钟的干燥时间。 </li><li>将加入40ml 0.1％胰蛋​​白酶溶液在冰箱中解冻O / N。冷藏汉克的平衡盐溶液（HBSS）如果尚未感冒。制备一个20％的胎牛血清（FBS）的股票介质溶液。 <ol><li> 400毫升的短路电流OVE氏改良的Dulbecco氏培养基（IMDM）加入100 ml的FBS，加入5ml L-谷氨酰胺（200毫米），2.5 ml庆大霉素（10毫克毫升-1），和0.9毫升2-巯基乙醇。使用消毒真空过滤。商店媒体在4℃长达3周，这是可伸缩的较小或较大的体积。稀释20％FBS的库存媒体1:1的IMDM，以产生10％FBS的NRCM介质。在小批量只会使防止反复加热和冷却介质。 </li></ol></li></ol> 2.细胞分离第1天<ol><li>在培养罩执行以下工作，以防止样品污染。为了获得最佳的时机启动这项工作在下午，因为有一个O / N孵化。 </li><li>无菌培养罩内做好准备工作区。将板凳垫在培养罩。填充两个250毫升烧杯，用70％的乙醇，并放置手术工具在其中。将小桶冰块的引擎盖。确保它是大到足以容纳数管和150毫米的菜。紫外线消毒罩FOR 10分钟。置于冰上40毫升0.1％胰蛋​​白酶。 </li><li>检索从母亲的乳鼠幼仔。为了使动物保暖，直到使用，将它们放置在一个绝缘容器，如一个冰桶包装在板凳垫，让他们温暖。 </li><li>填写150mm的培养皿中，用25ml HBSS并放置冰上。展开2-3纱布垫和放置在工作区域。通过在脖子后面的皮肤保持小狗，用70％的乙醇，并装在罩喷洒它。注:为了更好地保持无菌，这是可以由另一个人谁再放置清洗小狗在引擎盖完成。 </li><li>通过在脖子后面的皮肤保持小狗。然后使用大型剪刀，在一个快速，强大的切割断头安乐死。吸干血的纱布。注:由于在IACUC要求差异，这可能是更合适的清洗用乙醇小狗擦拭而不是喷洒它。一定要检查与大学IACUC为自己的这一步的指引。 </li><li>继续削减声道的前侧通过胸腔共青。挤在脖子上的皮肤，帮助打开胸腔，直至心脏是可见的。用小剪刀，取出心脏和地方冰上HBSS培养皿。重复其他幼仔。 </li><li>取下样品任何非心脏组织，摇动HBSS轻轻洗心。转移的心脏组织，以含有新鲜HBSS另一个培养皿中，并进一步清洗组织。切割组织进一步直到作品是1-3毫米3，然后转移到胰蛋白酶溶液。广场上的摇杆在4℃冰箱中O / N胰蛋白酶溶液。 </li></ol>细胞分离第2天注意:当吸在下面的步骤中，使用移液管，而不是一个真空管线。组织容易移动，并且真空管线将堵塞或仍然去除组织含有细胞。最好是用吸管吸出用于代替。如果组织进入吸管，吸管背出将其删除。 <ol><li>使吨10％NRCM媒体的重稀土等分:一是用25毫升和两个15毫升。将一份25毫升管媒体在37℃水浴。添加4毫升HBSS 5个不同的15毫升锥形管，并把所有的冰。标签每次15毫升管＃1-5。 </li><li>加40毫升的HBSS到50ml锥形管中。加入10 mL HBSS的胶原酶，吸管暂停，并结合解决方案，以创造50毫升1毫克毫升-1胶原酶溶液。消毒用0.22微米真空过滤器，并储存在室温。 </li><li>检索心脏组织/胰蛋白酶管。保证胰蛋白酶清晰，组织显得蓬松。允许组织沉降到管和抽吸的角落尽可能多的液体尽可能。 </li><li>加25毫升温10％FBS的NRCM媒体到管和旋流的手。帽和旋转在37℃水浴中2分钟。吸液了。组织应该浮动在这个步骤;如果是这样，从该管的底部吸出。 </li><li>加入10 mL胶原酶。旋转水浴2分钟，或直至溶液浑浊。曲ickly吸出液体。加入10 mL新鲜胶原酶含有该组织管和旋转水浴2分钟。移液器混合，转移溶液到管＃1中，并放置在冰上。它可能无法除去所有的液体。重复步骤3.2-3.6为其他三个的15毫升锥形管中。传送所有其余的液体到管＃5。 </li><li>离心15ml试管在410 RCF 8分钟。而离心，放置15毫升10％FBS NRCM媒体管水浴。放置在50ml锥形管中的顶部40微米的细胞过滤，并用2毫升冷HBSS中湿。 </li><li>吸取上清液降速细胞。加入6毫升冷HBSS中，以每管重悬。通过过滤到50ml锥形移液器细胞悬液，以消除任何组织。冲洗各15毫升管的两侧用3ml冷HBSS中，并加入到过滤器。离心细胞，在410 RCF离心10分钟。吸上清。重悬细胞沉淀在10ml 10％FBS的NRCM媒体和传输至T-75烧瓶中。冲洗锥形用5ml媒体，并加入到FL问。 </li><li>孵育在37℃下1小时和5％的CO 2。将过去的15毫升10％FBS NRCM媒体管进入水浴。转让T-75到一个新的T-175瓶内容物，冲洗T-75用15毫升的媒体，并加入到T-175。孵育1小时。 </li></ol> 4.冷冻保存<ol><li>广场上冰冻结介质。从T-175到50毫升锥形管转移细胞媒体，并收集10微升细胞悬液用台盼蓝细胞计数。离心在410 RCF 5分钟。而离心，计数使用细胞计数器细胞。 </li><li>吸文化传媒，并在每毫升2-4万个细胞浓度冷冻介质悬浮细胞。将1毫升细胞悬液在低温小瓶，然后把试管放入一个控制冷却速度冷冻集装箱，并将其放置在-80℃冷冻O / N。在1-2天内，在液氮中传送小瓶。 </li></ol> 5.细胞解冻<ol><li>大衣盘中纤维连接蛋白溶解250微升纤维连接蛋白（股票浓度为1毫克毫升-1）到9.75毫升水（终浓度为0.025毫克毫升-1）的; 60。加入足够的溶液以覆盖培养板，孵育在37℃下至少30分钟。 <ol><li>四的15毫升锥形管，使DMSO /媒体混合如下加入DMSO至20％媒体。添加（5％）9.5毫升介质用500μlDMSO中。加（2.5％）9.75毫升介质用250μlDMSO中。加入10 mL媒体（你两个人这些）。 </li></ol></li><li>加热在37℃水浴中上述介质的混合物。从液氮中取出细胞，并放在干冰。当媒体的混合物是温暖的，解冻的细胞在37℃水浴，直到刚刚解冻。不要让细胞在水浴中太久，因为这可能会损坏电池。 </li><li>传送低温小瓶的内容对5％的DMSO /介质管。离心在410 RCF 8分钟。吸上清留下细胞沉淀。加2.5％的DMSO /介质管内容物至细胞沉淀且resuspend。离心在410 RCF 8分钟。吸上清留下细胞沉淀。 </li><li>添加一个0％DMSO /媒体管内容细胞沉淀重悬。离心在410 RCF 8分钟。吸上清留下细胞沉淀。 </li><li>添加第二个0％DMSO /媒体的内容对细胞沉淀重悬。就拿细胞计数小样本。离心在410 RCF 8分钟。而离心细胞计数在血球使用台盼蓝标记死细胞。记录细胞量，以及生存力。 </li><li>吸上清留下细胞沉淀。悬浮在10％NRCM媒体（可选:加100μM溴脱氧尿苷（尿嘧啶））和板细胞纤维连接蛋白涂层的菜肴。 </li></ol> 6.细胞培养<ol><li>板细胞在每平方厘米10万左右的细胞。调整的基础上的预定用途培养物的接种密度。通过在1:10的比例稀释的20％中的介质的IMDM制备2％NRCM介质。下面是一个典型的培养物的轮廓。&lt;/ li&gt; <li>第1天，冲洗死细胞客场以热烈的IMDM，加入10％NRCM（可选100μM的BrdU）。第2天，冲洗死细胞客场以热烈的IMDM，加入10％NRCM。第3天，加2％NRCM。第4天加2％NRCM。在第5天保证在细胞融合和跳动。保持培养中的细胞对周围一周。在这一点上的成纤维细胞可能开始显著长大了新农合。 </li></ol>

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The authors have nothing to disclose.

该协议允许新农合被孤立，冷冻，解冻。解冻的细胞是该过程的一个重要部分。一系列的DMSO稀释液被用来慢慢地从单元14除去DMSO中。重要的是DMSO中提取被快速地执​​行作为所述细胞是特别敏感解冻后立即死亡。如果需要更多或更少的细胞解冻，根据需要的DMSO溶液的体积可以按比例。 一个挑战NRCM隔离和文化是成纤维细胞的快速增殖。该协议使用预镀已显示减少的成纤维细胞15的数量。通过使用未涂覆烧瓶到的成纤维细胞附着比心肌快得多预镀作品。媒体的变化顺序可以改变，以减少成纤维细胞生长。成纤维细胞将会超过10％NRCM媒体新农合，但成纤维细胞生长显著慢在2％NRCM MEDIA。有电镀16,17或添加的BrdU缓慢成纤维细胞生长3之前的其他方法，例如珀梯度以除去成纤维细胞。过度的成纤维细胞生长可以是硬质相用显微镜来检测，但使用NRCM特异性染色，如α-SA（ 图3）很容易显示。如果仍然有过大的成纤维细胞生长，FBS在媒体的量可以降低到2％FBS的上2.如果需要一天，电镀可在5％FBS的介质进行，尽管这可能导致增加的细胞死亡。 此过程是有限的，并且不再有益的，如果使用的是需要大量的小区18,19的细胞培养物。的情况下所需要的细胞的量接近一半量的细胞可以从一个垫料收获的，此过程将需要更多的动物，因为大约一半的细胞被因冷冻保存丢失。冷冻保存的好处使用较少的大公时，有更大LS用于测定诸如免疫染色。 此协议说明了方法的分离，冷冻保存，和新农合的后续。很少有替代品，比如买冷冻细胞厂商，使用这种方法，但买盘细胞可能成本过高，一些实验室。我们的方法只需要在细胞培养中通常使用的材料和试剂。此外，利用这些方法可以大大提高实验室工作的效率和灵活性，同时减少使用实验室动物。所描述的方法产生可用于随后的体外研究活培养物。

心肌细胞培养是现代心脏研究的一个重要工具。乳鼠心肌细胞（新农合），因为隔离常用和文化是比成年大鼠心肌1更容易。该NRCM方法仍然有一些限制，包括长期分离过程和有限的细胞增殖的菜。有对新农合的大多数一般需要4-48小时工作2-6的隔离无数协议。此外，将细胞分离经常由1至2天龄的幼鼠2,4-7;诞生时间是不可预知和冲突，在实验室的其他工作。如果需要对实验只有少量细胞的隔离可能是低效和浪费的。最努力改善工作流程专注于减少隔离时间，但是这并没有解决定时幼崽诞生的问题。 作为替代，许多实验室利用从胚胎干细胞（ESC）或诱导的多能干细胞（iPS细胞）衍生的心肌细胞。然而，重新编程和/或分化的过程可能是非常耗时和昂贵的为好。使用这些细胞作为体外肌细胞模型时，可以有其他的问题。既ESC-和iPSC-衍生的心肌细胞已显示出表现从初级心肌8-10在电生理学差异。 解离合管能够被存储为使用冷冻11几天，然而这并允许长期存储。液氮通常用于保存细胞的时间较长期间，但是需要冷冻保护剂，如二甲基亚砜（DMSO）。此前有研究表明，在冻结媒体5-10％DMSO的理想浓度允许新农合的冷冻保存，但即使是这样的生存能力仍然很低12。虽然DMSO有助于释放过程中保护细胞诚，它可以是对细胞有毒的浓度高于1.5％的13。以前的研究已经表明，慢慢地从细胞中除去DMSO，可提高细胞活力14。 我们试图提高NRCM基于细胞的测定通过冷冻保存的细胞下述分离的效率。这允许了细胞被解冻，并且在必要时，减小隔离和动物消费的频率使用。使用这种方法，我们表明，它是可能的冷冻保存新农合和解冻它们用于在以后的时间。解冻后细胞维持可接受的可行性，并产生NRCM文化是积极的α-横纹肌辅肌动蛋白（α-SA）和合同油然而生。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>IMDM (+25mM HEPES +L-glutamine)</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td>With added 25mM HEPES and L-glutamine</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (+Ca +Mg)</td>
			<td>Corning</td>
			<td>21-020-CV</td>
			<td>With added calcium and magnesium, pH 7.1-7.4</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%</td>
			<td>Gibco</td>
			<td>25300-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS5</td>
			<td>BioLife Solutions</td>
			<td>205102</td>
			<td>Freezing media</td>
		</tr>
		<tr>
			<td>Cryogenic Vial</td>
			<td>Corning</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C1889-50MG</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m Cell Strainer</td>
			<td>Greiner Bio-One</td>
			<td>542040</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizing Vacuum Filter (0.22&#956;m)</td>
			<td>Corning</td>
			<td>431118</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Conical</td>
			<td>Corning</td>
			<td>430790</td>
			<td></td>
		</tr>
		<tr>
			<td>trypan blue</td>
			<td>Cellgro</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr Frosty Freezing Container</td>
			<td>ThermoScientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell EZ&#160;SLIDES</td>
			<td>Millipore</td>
			<td>PEZGS0416</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-sarcomeric actinin antibody</td>
			<td>Sigma</td>
			<td>A7811</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine</td>
			<td>BD Biosciences</td>
			<td>51-7581KZ</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cryopreservation of neonatal rat cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

以下的新生大鼠心肌细胞的分离，将细胞冷冻向下液氮，并且可以贮存至少几个月。通常在解冻时，用台盼蓝分析测定的生存能力将介于40-60％。虽然这是比其他类型的细胞时，用适当的接种和培养的细胞将增殖（ 图1）。为了验证收缩，细胞可以用相差显微镜进行成像。细胞应开始于约三天（ 图2）自发收缩。对于免疫细胞化学（ICC）分析，将细胞接种在4孔培养载玻片用每孔200,000个细胞。为了表明该细胞在培养是合管的细胞用α-SA和使用荧光显微镜成像。有无数α-SA的阳性细胞表示在培养新农合（ 图4）。其他的实验已经进行包括小路图灵合管与人心脏干细胞衍生的外来体以测试外来体（ 图5）的再生性能。 <img alt="图1" src="/files/ftp_upload/52726/52726fig1.jpg" /> 图1. NRCM 体外增殖。NRCM的荧光显微镜图像与染色α-SA（绿色）和Ki67（红色），和DAPI（蓝色），两个（A）的新鲜分离合管和（B）的冷冻保存，然后解冻合管。箭头表示增殖细胞（Ki67的阳性）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52726/52726fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52726/52726fig2.jpg" /> 图2.收缩冻存和解冻NRCM的体外。新农合spontaneously承包体外 。与新鲜分离的新农合，这可能需要几天时间收缩恢复前。视频记录解冻后五天。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52726/Figure2.m4v" target="_blank">请点击此处观看该视频。</a> <img alt="图3" src="/files/ftp_upload/52726/52726fig3.jpg" /> 图3.收缩新鲜分离NRCM的体外。新鲜分离的新农合的自发收缩体外 。这些细胞就会开始更快呈现收缩。视频记录隔离后3天。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52726/figure_3_rgb.m4v" target="_blank">请点击此处观看该视频。</a> <img alt="图4" src="/files/ftp_upload/52726/52726fig4.jpg" /> 图4.纯度NRCM文化。&lt;/ STRONG&gt;无论（A）的荧光显微镜图像经过5天的文化新鲜分离的文化和（B）新农合冷冻解冻和新农合。新农合显示阳性染色α-SA（绿色），而纤维母细胞中可以看出，其中的细胞核（蓝色）不是由α-SA包围。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52726/52726fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52726/52726fig5.jpg" /> 图5.使用解冻NRCM的基于细胞的分析，新农合的共聚焦显微镜图像，通过α-SA（绿色）强调，培养与心脏干细胞的DiI标记的外来体（红色）。标记用DAPI（蓝色）的细胞的细胞核。为了创造一个氧化应激模型中，合管分别与100μM的过氧化氢处理18小时，孵育用exoso之前MES 4小时扭转氧化应激。箭头指示切体高浓度的某些细胞的地区。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52726/52726fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

隔离和乳鼠心肌细胞冷冻保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

isolation-and-cryopreservation-of-neonatal-rat-cardiomyocytes

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.4K 次观看.  College of Veterinary Medicine, North Carolina State University. The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

视频: Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

科研

发育生物学

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

隔离和卫星细胞的表征鼠头Branchiomeric肌肉

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

诱发心肌细胞的使用多顺反子构建表达GATA4，MEF2C和Tbx5中的最优比率改善发电

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

新生小鼠胰腺胰岛功能的有效隔离

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

细胞周期变化的可视化和核测定心肌出生后

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

宽吻海豚 (海豚截形) 精子: 收集、 冷冻保存和外源体外受精

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

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

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

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 ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

原发性大鼠瓣膜间质细胞的分离与鉴定: 主动脉瓣钙化的新模型研究

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

对雄性欧洲鳗鲡 (安圭拉) 进行激素成熟和精子冷冻保存的处理和处理

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...

隔离和乳鼠心肌细胞冷冻保存

摘要

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此视频中的章节

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

隔离和卫星细胞的表征鼠头Branchiomeric肌肉

诱发心肌细胞的使用多顺反子构建表达GATA4，MEF2C和Tbx5中的最优比率改善发电

小鼠胚胎Hemogenic内皮细胞的分离

新生小鼠胰腺胰岛功能的有效隔离

细胞周期变化的可视化和核测定心肌出生后

宽吻海豚 (海豚截形) 精子: 收集、冷冻保存和外源体外受精

使用电刺激人干细胞衍生的心肌细胞的成熟中Biowires

原发性大鼠瓣膜间质细胞的分离与鉴定: 主动脉瓣钙化的新模型研究

对雄性欧洲鳗鲡 (安圭拉) 进行激素成熟和精子冷冻保存的处理和处理

隔离和乳鼠心肌细胞冷冻保存

摘要

探索更多视频

此视频中的章节

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

隔离和卫星细胞的表征鼠头Branchiomeric肌肉

诱发心肌细胞的使用多顺反子构建表达GATA4，MEF2C和Tbx5中的最优比率改善发电

小鼠胚胎Hemogenic内皮细胞的分离

新生小鼠胰腺胰岛功能的有效隔离

细胞周期变化的可视化和核测定心肌出生后

宽吻海豚 (海豚截形) 精子: 收集、 冷冻保存和外源体外受精

使用电刺激人干细胞衍生的心肌细胞的成熟中Biowires

原发性大鼠瓣膜间质细胞的分离与鉴定: 主动脉瓣钙化的新模型研究

对雄性欧洲鳗鲡 (安圭拉) 进行激素成熟和精子冷冻保存的处理和处理

宽吻海豚 (海豚截形) 精子: 收集、冷冻保存和外源体外受精