支持这项研究是由哈特韦尔基金会，美国心脏协会（科学家发展津贴＃0830384N）和基金会Leducq：二尖瓣07CVD04。

1。制备<ol><li>孵化鹌鹑或肥沃，鸡蛋阶段14 - （约2天）或25（约4.5天），湿度60％和37 ° C </li><li>准备无菌伯爵的平衡盐溶液（EBSS） <ol><li> 18 M 600毫升水加入100 ml 10X EBSS </li><li>添加碳酸氢钠2.2克</li><li>调节溶液的pH值至7.2 </li><li>带来的解决方案，1000毫升</li><li>穿过一个0.2微米的过滤消毒解决方案</li></ol></li><li>准备无菌M199培养基。 <ol><li> M199粉加入1包700毫升18MΩ水。 </li><li>添加碳酸氢钠2.2克</li><li>调整pH值至7.2的解决方案。 </li><li>添加10毫升（1％）青霉素，链霉素</li><li>带来的解决方案，1000毫升</li><li>穿过一个0.2微米的过滤消毒解决方案</li><li>添加1 mL无菌100X补充胰岛素转硒 - G </li><li>添加10毫升（1％）无菌鸡血清</li></ol></li><li>准备三维胶原凝胶，胶原蛋白的浓度在1.5毫克/毫升。在我们实验室先前的研究表明，胶原蛋白的浓度超过1.5毫克/毫升，提供了一个矩阵，是太生物力学僵硬的细胞移动通过。胶原凝胶浓度低于1.5毫克/毫升的有这么几个胶原纤维细胞丛本地一丝一毫的纤维，产生的细胞岛。 <ol><li> 15毫升无菌离心管中，以添加以下冰冷的试剂： <ul><li> 3X M199：体积=总体积的必要/ 3 </li><li>无菌18MΩ水：体积=必要的总量（M199音量+鸡血清音量+ 0.1 M的氢氧化钠量+胶原容积） </li><li>无菌鸡血清：体积=总体积* 0.01 </li><li>鼠尾胶原我：卷=（胶原凝胶浓度*总量必要）/胶原股票浓度</li><li>无菌0.1 M的氢氧化钠：体积​​=胶原容积* 0.2 注：添加适量的水，0.1 M的氢氧化钠和胶原蛋白的胶原蛋白原液的浓度会有所不同而有所不同。 </li></ul></li><li>良好的胶原蛋白溶液混合，用无菌吸管。 </li><li>立即转移到1.9平方厘米的增长领域井的胶原溶液0.3毫升。这是足够的胶原蛋白溶液，完全覆盖以及和提供约3毫米厚的凝胶，。一个至少250微米的凝胶厚度是对细胞的侵袭研究是必要的。 </li><li>允许至少1小时的胶原蛋白溶液凝胶在37℃，5％CO 2，湿度为100％。 </li></ol></li></ol> 2。前EMT的心内膜细胞分离和培养<ol><li>胚胎隔离<ol><li>将实验室磁带1英寸以上的蛋较大的一端的长片。这是那里的空气是位于细胞。磁带稳定脆弱的鹌鹑蛋壳。 </li><li>轻轻穿刺在空气消毒弯剪刀的尖端细胞蛋录音。 </li><li>剪下一个鸡蛋，大到足以让蛋黄通过录音的部分孔。开始穿刺孔，切一个螺旋形状向外。确保穿过外膜，但应避免刺破蛋黄。 </li><li>当在shell的洞是完整的，可视化的鹌鹑胚胎。胚胎通常位于顶面的蛋黄。 </li><li>开始倒一方面通过在蛋孔切蛋黄。 ＃55镊子保持在另一方面。随着胚胎退出顶面的鸡蛋蛋黄，轻轻滑动下，胚胎＃55镊子，从蛋黄中取出。注：鸡蛋，胚胎隔离涉及打击锋利，表面光洁的鸡蛋和鸡蛋打破，到100毫米的培养皿。蛋黄顶面应位于胚胎。轻轻滑入下，胚胎＃55镊子，从蛋黄中取出。 </li><li>胚胎放入一个100毫米的Petri菜含有冰凉的，无菌的EBSS。 </li></ol></li><li>根据汉堡包和汉密尔顿1标准，第 ​​一阶段的胚胎。胚胎阶段14 -过去阶段13，但尚未在阶段15，20-21体节和尾芽。一个14阶段的主要特点-胚胎是头部的角度来看，这是略小于90 °身体更大。 </li><li>从鹌鹑胚胎阶段14中删除的心 - ＃55镊子横向切割AV运河和公平交易与胸壁的接触。在一个培养皿的一侧放置的心在一起。 </li><li>收集用无菌移液管离体心脏，放入一个新的100毫米的Petri充满无菌，冰冷的EBSS的菜。 </li><html><li>横向切OFTs和AV运河，从＃55镊子脑室。 </li><li>节中的其余的AV运河和/或流出道（OFTs）纵向。 </li><li>设置移液器体积20微升。用吸管吸出六个纵向切AV运河和OFTs。 </li><li>驱逐AV运河上的胶原蛋白凝胶OFTs。从凝胶表面吸任何的多余的EBSS。 </li><li>使用＃55镊子东方植，所以它们是平的，使管腔侧面胶原凝胶。尽量不要用镊子穿刺胶原凝胶。 </li><li>经过2个小时，在37 ° C和5％ 的 CO 2，删除＃55镊子植心肌。心内膜细胞胶原凝胶表面上留下。另外，心肌可左外植体。随着心肌删除，不内膜细胞进行EMT的，不加干预。与目前心肌心内膜细胞的一个子集会转化成间质细胞，但转型的程度，可与外部因素调制。注：心肌即使将离开，植体必须允许前至少2小时附加的凝胶介质添加。 </li><li>加入0.4 mL的M199培养基，每孔含有植。 </li><li>培养细胞在37℃，5％CO 2，湿度为100％。 </li></ol> 3。后EMT的影音靠垫间质细胞的分离和培养<ol><li>破解尖锐，干净的表面上的鸡蛋和鸡蛋打入100毫米培养皿。蛋黄顶面应位于胚胎。拿起胚胎＃5镊子抓住周围的胚胎膜，然后将胚胎充满了冰冷，无菌EBSS到100毫米的Petri菜。 </li><li>根据汉堡包和汉密尔顿1标准，第 ​​一阶段的胚胎。一个阶段25胚胎的特性，包括独特的肘部和膝关节和轻微的眼部色素沉着。 </li><li>隔离所有HH25胚胎的心，一起放在培养皿一侧的心。 </li><li>收集用无菌移液管离体心脏，放入一个新的100毫米的Petri充满无菌，冰冷的EBSS的菜。 </li><li>一次心灵的AV地区隔离。培养皿一侧放置了AVS。轻轻搅动AV地区消除所有的血液，从而影响细胞活力。 </li><li>用无菌移液管收集隔离AVS和它们放入一个新的100毫米的Petri充满无菌，冰冷的EBSS的菜。 </li><li>顺序从AV隔离的垫子。确保有心肌目前没有在AV垫子。 </li><li>收集隔离垫，并用无菌移液管放入无菌的15 mL离心管。 </li><li> 3分钟，在15 XG旋转的离心管底部的垫子。 </li><li>使用无菌1000毫升的提示删除，而不会干扰坐垫尽可能EBSS上清。 </li><li>添加1毫升的0.25％胰蛋白酶EDTA已预热至37 ° C。允许坐垫中的胰蛋白酶孵育，直到他们完全溶解，这是一般约3-5分钟。 </li><li>添加鸡血清100μL的离心管中淬火的胰蛋白酶。 </li><li> 5分钟沉淀细胞在160 XG。取出上清液，用无菌，1000μL的提示。 </li><li>在2毫升的M199培养基和混合吹打重悬细胞。取10μL的细胞悬液对血球计数，并确定已分离出的细胞总数</li><li>确定的胶原凝胶分离的细胞数量的基础上，将。细胞需要在一个2 × 10 5细胞/毫升与250μL凝胶量的密度镀。 </li><li> 5分钟沉淀细胞在160 XG。取出上清液，用无菌，1000μL的提示。 </li><li>使用上面列出的协议，在该命令添加3X M199，水，鸡血清，胶原蛋白，和0.1M的氢氧化钠，细胞沉淀。混合轻柔吹打。 </li><li>加入250μL，每1.9厘米2的增长领域以及。 </li><li>允许至少1小时的胶原蛋白溶液凝胶在37℃，5％CO 2，湿度为100％。 </li><li>加入0.4毫升培养基孵育过​​夜。 </li><li>要继续在无压力的条件下培养凝胶，从双方的文化中解放出来的凝胶，以及使用无菌200μL枪头。机械约束的文化是保持连接双方的文化以及凝胶。 </li></ol> 4。代表性的成果<img src="/files/ftp_upload/2159/2159fig1.jpg" alt="图1" /> 图1：这里显示。范例 （一）HH14鹌鹑胚胎， 胚胎及（B）HH25鸡胚。 fig2.jpg“ALT =”图2“/&gt; 图2：HH14阀心内膜植后2个小时的文化，或（B）（A）与心肌目前与心肌删除 。 <img src="/files/ftp_upload/2159/2159fig3.jpg" alt="图3" /> 图3：HH14阀心内膜植 （A）与心肌目前，许多细胞进行心内膜间质转型和后48小时的文化有一个纺锤形，间质表型。 （二）当心肌细胞中删除所有维持一个鹅卵石形，文化在48小时后的内膜表型。 <img src="/files/ftp_upload/2159/2159fig4.jpg" alt="图4"/> 图4：HH25阀在胶原凝胶中的间质细胞的细胞四舍五入后立即播种。 <img src="/files/ftp_upload/2159/2159fig5.jpg" alt="图5"/> 图5：HH25间质细胞培养 （一）经过48小时的文化，在约束的胶原蛋白凝胶HH25间质细胞开始蔓延。 （二）HH25在播种和解放以后的6天7天之后无压力的胶原蛋白凝胶细胞压缩到原来的面积约50％的凝胶。

<ol>
	<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J Morphol. 88, 49-92 (1951).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+A+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue.</a> Developmental Biology. 95, 108-108 (1983).</li><li>Butcher, J. T., Norris, R. A., Hoffman, S., Mjaatvedt, C. H., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periostin+promotes+atrioventricular+mesenchyme+matrix+invasion+and+remodeling+mediated+by+integrin+signaling+through+Rho/PI+3-kinase.">Periostin promotes atrioventricular mesenchyme matrix invasion and remodeling mediated by integrin signaling through Rho/PI 3-kinase.</a> Developmental Biology. 302, 256-256 (2007).</li></ol>

阶段HH14隔离内膜细胞的方法 -心中最初是由润和Markwald开发提供了控制，在体外环境研究的因素，启动和调节胚胎EMT的2。 HH14 -培养无心肌内膜植不会发生EMT的无生物力学或生化干预。如果心肌左外植体，它会发出信号内膜细胞的一个子集来进行间质改造，但外部因素可能会规范这一过程。胚胎祖细胞间质隔离HH25影音阀垫使用屠夫等人所描述的方法提供了一?...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Extra fine Bonn scissors, curved</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14085-08</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #55</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14099</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #5</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14098</td>
<td>&nbsp;</td>
<tr>
<td>Sterile transfer pipettes</td>
<td>&nbsp;</td>
<td>Samco Scientific </td>
<td>2041S</td>
<td>&nbsp;</td>
<tr>
<td>4-well culture plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>&nbsp;</td>
<tr>
<td>Sterile disposable filter units, PES, 0.2 &mu;m pore size membrane</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>566-0020</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
<tr>
<td>Sterile petri dishes, 100 mm x 15 mm</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>25384-342</td>
<td>&nbsp;</td>
<tr>
<td>Laboratory tape</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>89097-920</td>
<td>&nbsp;</td>
<tr>
<td>Rat-tail collagen I</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>10X Earl's Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Quality Biological, Inc.</td>
<td>119-064-131</td>
<td>&nbsp;</td>
<tr>
<td>M199 powder</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31100-035</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Insulin-Transferrin-Selenium-G supplement (100X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41400-045</td>
<td>&nbsp;</td>
<tr>
<td>Chicken serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16110-082</td>
<td>&nbsp;</td>
<tr>
<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>Sodium hydroxide solution, 1.0 N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S2770</td>
<td>&nbsp;</td>
<tr>
<td>Fertile quail eggs (Coturnix coturnix)</td>
<td>&nbsp;</td>
<td>Lake Cumberland Game Bird Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fertile chicken eggs (Gallus gallus)</td>
<td>&nbsp;</td>
<td>Cornell University Poultry Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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isolation and culture of avian embryonic valvular progenitor cells

正确的形成和胚胎心脏瓣膜的功能的发育进程的关键。早期胚胎的心脏是一个U形管的心内膜心肌包围。心肌细胞分泌的心脏果冻，一个透明质酸丰富的胶质基质，到房室交界处和流出道（OFT）流明。在第一阶段开始HH14 valvulogenesis内膜细胞的一个子集时，从心肌收到的信号，经过内膜间质转化（EMT），并侵入心脏果冻。阶段HH25瓣膜垫子mesenchymalized，这是间质细胞，最终形成的心脏瓣膜和室间隔仪器。了解启动和调节EMT和细胞分化过程中的机制是非常重要的，因为严重的先天性心脏缺陷的连接。在这项研究中，我们目前的方法来隔离前EMT的心内膜和后EMT的间质细胞，这是两个不同的细胞表型prevalvular垫。前EMT的心内膜细胞可以培养的心肌有或没有。后EMT的影音垫间质细胞可以培养的胶原凝胶内机械限制或无压力。这些在体外模型的三维模拟关键瓣膜形态发生事件和解构早期和晚期valvulogenesis的机制非常有用。

Watch this Scientific Journal Video about Isolation and Culture of Avian Embryonic Valvular Progenitor Cells at JoVE.com

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

本文将提供一个隔离的方法，培养鹌鹑鸡HH14<sup&gt; -</sup&gt;阀心内膜细胞及间质细胞HH25阀垫。

禽流感胚胎瓣膜祖细胞的分离和培养

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

isolation-and-culture-of-avian-embryonic-valvular-progenitor-cells

Research

JoVE Journal

Biology

10.1K 次观看.  Cornell University. 本文将提供一个隔离的方法，培养鹌鹑鸡HH14 -阀心内膜细胞及间质细胞HH25阀垫。

视频: Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

产后小鼠小脑颗粒神经元祖细胞和神经元的分离和培养

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

科研

生物学

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

瓣膜内皮细胞的分离

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚胎心室血管内皮细胞的分离

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...