这项工作是由卫生部授予R01 HL105734到VVPMDL国家研究院​​赞助支持，部分是由健康研究事业奖K08 HL094748国家机构。 VVP是美国心脏协会的创新研究员被授予11IRG900384支持。

1，准备必要的解决方案和设备<ol><li>首先，通过高压灭菌他们15分钟消毒手术器械（直形镊子，弯形镊子，直板造型剪刀和弯曲的形状剪）。在121℃。准备2台仪器，其中一组用于解剖出来的心和另一组中的将被用于切碎起来的组织。 </li><li>溶解MCDB153培养基1包900毫升的消毒，过滤后的水，搅拌缓慢，直到粉末完全溶解。添加1.2克碳酸氢钠，或毫升钠15.7碳酸氢盐溶液，对于每升培养基的最终体积被制备并进行搅拌直到溶解[w / v的7.5％。将pH调节至7.2，加入任一的1N HCl或1N的NaOH，然后通过在组织培养罩（生物安全柜中）在无菌条件下用0.2微米的瓶顶过滤器倾介质。然后收集媒体，并将其保存在4℃。 </li><li>准备补充添加到培养介质（材料清单）。的补充剂应该被添加到需要的只是在媒体前电镀细胞的培养基。 </li><li>制备DPBS有10％FBS加抗生素（青霉素和链霉素）。 </li><li>至100毫升DPBS加抗生素（青霉素和链霉素）中，接着，通过添加0.25克胰蛋白酶（USB牛胰腺，活动&gt; 2500 USP单位/ mg，超纯水）制备0.25％胰蛋白酶溶液。 </li><li>完成此过程所需的其他必要的设备，包括立体显微镜，培养皿（10厘米），50毫升锥形管，40微米的细胞过滤器，DPBS加用抗生素，60毫米培养皿和35毫米的培养皿。 </li></ol> 2，隔离新生乳鼠心脏<ol><li>首先，获得P0到P2新生乳鼠组（n = 6〜8）。建议使用由养殖DCT的Cre重组酶的女性纯合子雄性是杂无论是R26R产生的幼崽:EYFP的等位基因或Z / EG基因标记的黑素细胞样细胞。这两个R26R:EYFP和Z / EG小鼠可以从杰克逊实验室（股票号码006148和003920，分别）。 </li><li>其次，安乐死的新生小鼠幼崽，用酒精棉签擦拭胸前，然后将幼崽在一个10厘米的培养皿用DPBS。 </li><li>切开皮肤在胸腔和胸骨用灭菌镊子和剪刀，然后打开箱子仔细地在立体显微镜下。新生小鼠幼崽的心是非常小的，所以要小心，以确保与连接心房切除整个心脏。 </li><li>在DPBS洗心，将它们转移到一个新的10厘米的培养皿。 </li></ol> 3，酶消化<ol><li>把培养皿的切心来组织培养罩（生物安全柜），然后按照下面使用无菌技术，在通风橱中的步骤。 </li><li>首先，洗心三次在无菌的DPBS加用抗生素。 </li><li>将心中（N = 6-8）在60毫米的培养皿中，加入6-8毫升的0.5％胰蛋白酶溶液的培养皿中。 </li><li>接着，切割的心成小块（小于1毫米的大小），并在5％CO 2气氛中孵育它们在37℃下进行30分钟。 </li><li>孵育期结束后，倒入所得的心脏组织，并从盘的胰蛋白酶溶液到无菌的50ml锥形管中。 </li><li>然后，用10毫升无菌吸管，快速而轻，磨碎的50毫升锥形管30-50倍的解决方案。 </li><li>过滤通过一个新的，干净的50毫升锥形管的顶端40微米的细胞过滤网，所得溶液以收集滤液。 </li><li>接着，离心50-ml管中，在240×g下在台式离心机，5分钟，在室温下进行。然后，轻轻地吸上清，悬浮颗粒残留在6-8毫升无菌的DPBS。重复此步骤2次以上。这一步小球心房肌细胞和心脏的黑色素细胞，但不会拖垮网络由于成纤维细胞的成纤维细胞体积较小，将保留在上清液中。 </li></ol> 4，电镀黑素细胞心肌样细胞<ol><li>离心后的第三冲洗，小心排出或吸断洗涤液（DPBS），重悬在6ml MCDB培养基的沉淀到该补充剂已被添加。 </li><li>现在，绘制再悬浮液进入无菌移液管，然后将细胞在35毫米的菜，或在6孔板的好，游泳池，距离3新生小鼠离体心肌细胞在一个35毫米的菜或好。 </li><li>孵化菜过夜，在37℃，5％CO 2气氛中，吸脱培养基连同任何未附着的细胞，然后用DPBS冲洗所述板一次。然后加入新鲜培养基，以将板和更换介质每2天。细胞从新生儿或胚胎心脏分离可维持培养达3周，而细胞从成年听到分离TS可被保持在培养长达10天。 </li></ol>从成年小鼠心脏5隔离心黑素细胞样细胞<ol><li>首先，请从成熟老鼠的心（3-4周龄，N = 3）通过给予肝素100单位腹腔注射给小鼠，然后它们实施安乐死后中线胸骨。它建议用戊巴比妥安乐死的小鼠在此过程中，但这种药物是不是在所有地区提供。 </li><li>接下来，洗切心含在组​​织培养罩（生物安全柜）抗生素无菌DPBS。轻轻挤压心脏采用弧形解剖钳去除他们剩余的血液，然后冲洗心中的DPBS一次。 </li><li>从心里卸下心房和房室环（这些结构含有黑素细胞样细胞），然后将切除的组织标本中有35毫米的菜。 </li><li>用弯剪和切割力的心切成小块PS，然后添加6-8毫升的0.5％胰蛋白酶溶液的培养皿中。然后孵育的菜，在37℃，45〜60分钟。 </li><li>现在，遵循的程序上面的步骤从步骤3.6起开始。 </li></ol>

<ol>
	<li>Hornyak, T. 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=Mitf+dosage+as+a+primary+determinant+of+melanocyte+survival+after+ultraviolet+irradiation.">Mitf dosage as a primary determinant of melanocyte survival after ultraviolet irradiation.</a> Pigment Cell Melanoma Res. 22, 307-318 (2009).</li><li>Yonetani, 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=In+vitro+expansion+of+immature+melanoblasts+and+their+ability+to+repopulate+melanocyte+stem+cells+in+the+hair+follicle.">In vitro expansion of immature melanoblasts and their ability to repopulate melanocyte stem cells in the hair follicle.</a> J. Invest. Dermatol. 128, 408-420 (2008).</li><li>Levin, M. 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=Melanocyte-like+cells+in+the+heart+and+pulmonary+veins+contribute+to+atrial+arrhythmia+triggers.">Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers.</a> J. Clin. Invest. 119, 3420-3436 (2009).</li><li>Patel, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+insights+in+the+cellular+basis+of+atrial+fibrillation.">Novel insights in the cellular basis of atrial fibrillation.</a> Expert Rev. Cardiovasc. Ther. 8, 907-915 (2010).</li><li>Sviderskaya, E. V., 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+neurons+and+melanocytes+induced+from+immortal+lines+of+postnatal+neural+crest-like+stem+cells.">Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.</a> FASEB J. 23, 3179-3192 (2009).</li><li>Sviderskaya, E. V., 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=P16Ink4a+in+melanocyte+senescence+and+differentiation.">P16Ink4a in melanocyte senescence and differentiation.</a> J. Natl. Cancer. Inst. 94, 446-454 (2002).</li><li>Cook, A. 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+melanoblasts+in+culture:+Expression+of+BRN2+and+synergistic+regulation+by+fibroblast+growth+factor-2,+stem+cell+factor,+and+endothelin-3.">Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3.</a> J. Invest. Dermatol. 121, 1150-1159 (2002).</li><li>Fang, 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=Defining+the+conditions+for+the+generation+of+melanocytes+from+human+embryonic+stem+cells.">Defining the conditions for the generation of melanocytes from human embryonic stem cells.</a> Stem Cells. 24, 1668-1677 (2006).</li><li>Brito, F. 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=Timeline+and+distribution+of+melanocyte+precursors+in+the+mouse+heart.">Timeline and distribution of melanocyte precursors in the mouse heart.</a> Pigment Cell Melanoma Res. 21, 464-470 (2008).</li><li>Yajima, I., 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+isolation+of+heart+melanocytes+is+specified+and+the+level+of+pigmentation+in+the+heart+may+correlate+with+coat+color.">The isolation of heart melanocytes is specified and the level of pigmentation in the heart may correlate with coat color.</a> Pigment Cell Melanoma Res. 21, 471-476 (2008).</li></ol>

我们什么都没有透露。

心黑素细胞存在于心脏内的解剖位置中通常引起心律失常的触发器。这些区域包括肺静脉，房室瓣环，后左心房和卵圆孔（ 图4）。要理解中的作用，这些细胞在心律失常玩，我们开发的技术来隔离他们，研究他们的生理机能。但是，我们认识到，提高该技术以产生更健康，更大量的心脏黑色素细胞将允许进一​​步的研究，以探讨心脏黑色素细胞对外源性刺激的反应。此外，改进的...

我们最近发现在表达黑色素合成酶和形态上类似皮肤的黑素细胞的小鼠和人类的心脏了一种新的细胞群。我们称这些细胞的黑素心"，发现他们是存在于解剖位置，从该房性心律失常的触发往往起源于人类。我们还发现心脏黑色素细胞在小鼠生殖细胞缺失的DCT导致房性心律失常。心黑素细胞稀疏地分布在整个小鼠心房，他们包括所有心房细胞少于0.1％。调查心脏黑色素细胞的电特性和生物特性，我们开发了一种方法，从使用Cre重组酶诱导的黑素细胞特异性标记物的小鼠心脏隔离。我们的初始步骤，从那些用于分离新生鼠心肌细胞，并产生非常小的数值适用于膜片钳记录或PCR分析细胞的开发。然而，为了提高愈合日和心脏的黑色素细胞，进行更深入的分析，电生理或生化研究的可行性，我们制定了从那些旨在隔离皮肤的黑素细胞精致的过程。描述和展示了在本次审查的程序有生产，可作为镀纯人群或心肌与心脏健康的黑素细胞的优势。

<table><tbody><tr><td>MCDB 153</td><td>Sigma-Aldrich</td><td>M7403</td><td>Dissolve in water</td></tr><tr><td>DPBS, no calcium no magnesium</td><td>Invitrogen</td><td>14190136</td><td>Stock conc.: 1x&lt;br/&gt; Working conc.: 1x</td></tr><tr><td>Penicillin-streptomycin </td><td>Invitrogen</td><td>15140163</td><td>Stock conc.: 100x&lt;br/&gt; Working conc.: 1x</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technology</td><td>Certified</td><td>Working conc.: 4%</td></tr><tr><td>TPA</td><td>Sigma-Aldrich</td><td>P1585</td><td>Dissolve in DMSO&lt;br/&gt;Stock conc.: 3.2 &amp;mu;M&lt;br/&gt;Working conc.: 32 nM</td></tr><tr><td>Transferrin</td><td>Sigma-Aldrich</td><td>T8158</td><td>Dissolve in DPBS&lt;br/&gt;Stock conc.: 0.1 mg/ml&lt;br/&gt;Working conc.: 1 &amp;mu;g/ml</td></tr><tr><td>Dibutyryl cyclic AMP</td><td>Sigma-Aldrich</td><td>D0627</td><td>Dissolve in DPBS&lt;br/&gt;Stock conc.: 0.5 mM&lt;br/&gt;Working conc.: 5 &amp;mu;M</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td>Dissolve in 10 mM acetic acid&lt;br/&gt;Stock conc.: 1 mg/ml&lt;br/&gt;Working conc.: 5 &amp;mu;g/ml</td></tr><tr><td>Basic FGF</td><td>BD Biosciences</td><td>354060</td><td>Dissolve in water&lt;br/&gt;Stock conc.: 4 ng/&amp;mu;l&lt;br/&gt;Working conc.: 4 ng/ml</td></tr></tbody></table>

isolating primary melanocyte-like cells from the mouse heart

我们确定了一个新的黑色素细胞样细胞群（也称为心源性黑色素细胞），在小鼠中导致房性心律失常触发小鼠和人类的心。调查心脏黑色素细胞的电特性和生物特性，我们开发了一种程序，从小鼠心脏，我们从这些设计以分离新生鼠心肌细胞衍生的隔离。为了获得适合于更广泛的膜片钳或生化研究健康心脏的黑素细胞，我们开发了一种精制方法分离和镀基于那些最初设计以隔离皮肤黑素细胞中黑素细胞的心脏。精致的过程证明了本次审查，并产生更大的健康黑色素细胞样细胞，可作为镀纯人口或心肌细胞的数量。

在这篇文章中描述的协议是相对于我们以前出版的从小鼠心脏心脏分离黑素细胞之一的改进技术。这个过程采用了利用由黑色素合成酶特异性Cre重组酶从动标注心脏黑色素细胞样细胞的荧光标记物。使用的标记物与新鲜分离的胚胎或新生儿细胞中工作时，由于该区分心脏的黑素细胞从心房肌细胞的形态差异尚未发展（ 图1A）标记心脏的黑素细胞是重要的。 如图1A时，分离...

Watch this Scientific Journal Video about Isolating Primary Melanocyte-like Cells from the Mouse Heart at JoVE.com

Isolating Primary Melanocyte-like Cells from the Mouse Heart

在这个协议中，W E 发现了新的黑色素细胞样细胞群（也称为心源性黑色素细胞）的小鼠和人类中，有助于房性心律失常引发小鼠的心。

从小鼠心脏中分离主黑素细胞样细胞

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

isolating-primary-melanocyte-like-cells-from-the-mouse-heart

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.4K 次观看.  University of Pennsylvania. 在这个协议中，W E 发现了新的黑色素细胞样细胞群（也称为心源性黑色素细胞）的小鼠和人类中，有助于房性心律失常引发小鼠的心。 

视频: Isolating Primary Melanocyte-like Cells from the Mouse Heart

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

科研

发育生物学

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

初级毛质素黑色素细胞和纤维细胞培养的快速生成

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

癌症研究

以单细胞分辨率从小鼠心脏祖细胞中分离细胞核

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

作者聚焦:从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率下的表观基因组和基因表达分析  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

生物学

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

隔离成年小鼠心肌细胞的细胞信号传导和文化及<em&gt;在体外</em&gt;心肌肥厚

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

一种持续分离小鼠心肌细胞的简单有效的方法

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

医学

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

制备用于心肌梗死后成年小鼠心脏单细胞RNA测序的非心肌细胞悬液

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

从小鼠心脏中分离主黑素细胞样细胞

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此视频中的章节

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

初级毛质素黑色素细胞和纤维细胞培养的快速生成

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

鼠心肌细胞的分离及生理分析

隔离成年小鼠心肌细胞的细胞信号传导和文化及

一种持续分离小鼠心肌细胞的简单有效的方法

制备用于心肌梗死后成年小鼠心脏单细胞RNA测序的非心肌细胞悬液

成人小鼠心脏干细胞的分离、鉴定及分化