Authors have nothing to disclose.

的协议来执行动物实验，其理由和研究的目的，是由南加州机构动物护理和使用委员会的大学提交和批准。 该协议建立了从去鉴定人食管癌标本原代培养批准了南加州机构审查委员会的大学。 1.获取鼠标或人类食管<ol><li> 获得鼠标食道： <ol><li>安乐死8-12天的小鼠用2％异氟烷吸入过量颈椎脱位。 </li><li>针的动物与腹部朝上向下和润湿腹侧表面用70％的乙醇。用钳子，抓住并提起皮肤前到尿道口钳。使用标准的直外科剪刀沿着从尿道开口到下巴的腹中线切开。小心切只有皮肤。&lt;/ li&gt; <li>使从尿道下方上的动物两侧的膝盖开口的切口，形成一个倒的“Y”。使在沿肋笼动物两侧另一个切口。 </li><li>小心剥离皮肤两侧，离底层腹膜和肋骨，打下向两侧。检查左肋覆盖在胸腔和腹膜覆盖腹腔。 <ol><li>抬起用钳子腹膜和切穿它到隔膜和肋骨，向上指向剪刀，以防止损坏腹腔内容。轻轻抬起肝左叶，露出底层的胃，食管胃交界处的食道的腹部部分。 </li></ol></li><li>使用高压灭菌手术剪通过胸骨和肋骨沿中线到颈椎腰带削减。点剪刀向上，以防止损坏的胸内容。充分暴露的内容胸腔拉动左肋向两侧。 </li><li>轻轻取出心脏和肺的两叶。吸收多余的血液与Q-提​​示。胸食管是一个狭长的，柔性管躺在后方气管和前向胸椎。 注：食道可能难以识别鼠新生儿。食管胃交界处的本地化，使食管识别简单。 </li><li>认真遵循食道从胃，解剖周围的血管，脂肪和肠系膜一路攀升至食管起源于宫颈腔。手术剪与钝头最适合于此目的。 </li><li>切除食道和发生在Hanks&#39;平衡盐溶液（HBSS），用于进一步的处理如下所述的整个长度。如果需要的话为取向的目的移除胃的一部分。 注：由于小规模组织，固有肌层从肌层分离是粘膜是无法实现在小鼠新生儿食道和整个食管进​​行下面的处理进行说明。 </li></ol></li><li> 获得人食道： <ol><li>用HBSS洗食管切除标本（一般为5厘米以内），并删除任何附加的结缔组织，脂肪，血管或。 </li><li>切除的样本的一部分，并放置在福尔马林中以备将来组织学检查，如果需要的。 </li><li>由锐性剥离分开底层固有肌层其余粘膜。 </li><li>切断黏膜成子厘米的片段，并受到下面描述的协议。 注：人食道切除可以存储在PBS长达6小时前处理进行说明。食管癌的标本来源将决定试样尺寸并且将实验室的依赖。 </li></ol></li></ol> 2.隔离小鼠和人的食管间质细胞<ol><li>分离食管间质细胞˚FROM鼠和人体组织中的机械和酶消化的组合。 <ol><li>通过切碎用剪刀和多次洗涤用HBSS组织机械地消化。通过温育与300U / ml胶原酶XI和0.1毫克/毫升分散酶25分钟（鼠组织），或长达6小时（人组织）的组织酶促消化。确保用于切碎仪器已经高压灭菌消毒。 注：胶原酶是分解胶原蛋白，负责保持动物组织在一起的细胞外基质蛋白的酶。胶原酶XI具有较高的胶原酶活性，并已经看到了这种隔离协议的成功。分散酶是细菌酶的轻度蛋白水解解离的活性，可以保留细胞膜的活性，并结合使用胶原酶作为辅助酶。 </li><li>放置来自小鼠或人的组织中1.7毫升微型离心机或5毫升管分别得到粘膜片段，含有HBSS中。百果组织进一步成用剪刀以开放的尖端宽度将适用于各管2-3毫米的碎​​片。留出足够的时间，以便食管粘膜碎片沉淀到试管底部。 </li><li>慢慢倒出HBSS，小心不要无意中丢弃的组织。更换新鲜HBSS继轻轻摇动。另外，除去HBSS用1毫升吸管。 </li><li>以这种方式，共8次，洗涤时间，允许食管片段每次洗涤之间滓之间轻轻摇动在洗净组织。 注：人体组织将需要更多切碎的新生小鼠组织。 </li></ol></li><li>孵育鼠组织与300U / ml胶原酶XI和0.1毫克/毫升分散酶25分钟上的摇床上设定为低速，在室温下进行。 <ol><li>孵育人体组织与300U / ml的十一胶原酶和0.1mg / ml的分散酶长达6小时，在摇床上设定为低速，在室温下进行。 注：可用性人类esopha的GUS是可变的。人类食管粘膜孵育酶长​​达6小时的结果成功代原代培养的。 </li></ol></li><li>酶消化后，肉组织进一步用剪刀和离心机在200×g离心10分钟。 </li><li>弃去上清液。传送颗粒，它由混合细胞悬浮液以5毫升管洗5次中的Dulbecco改进的Eagle培养基（DMEM），补充有2％的山梨糖醇，以消除不能存活的细胞和碎片。 </li><li>种子细胞在6孔板并培养在DMEM，用10％胎牛血清（FBS），10毫克/毫升的胰岛素，10μg/ ml的转铁蛋白，10微克/毫升庆大霉素，和2纳克/毫升的表皮生长因子（EGF） 。真空过滤器（0.22微米），它可以在过滤后加入除EGF的所有组件。分发从人切除标本获得的细胞多6孔板中，根据不同的切除大小。 </li><li>一旦井80％融合，通过贴壁细胞T25烧瓶我们荷兰国际集团0.05％胰蛋白酶/乙二胺四乙酸（EDTA）。中和与媒体和旋以400×g的。 <ol><li>对于6孔通路到T25培养瓶中，传代细胞以1：1的比例。 2或1：3的比例汇合的T25烧瓶可以在1可以传代。从T25培养瓶中于T75烧瓶中通过，使用1：1的比例。改变媒体不管容器的每2-3天。 </li><li>生长在潮湿的5％CO 2培养箱培养并在上述成肌纤维细胞的培养基，在37ºC。 注：上皮细胞没有这些文化和通道的条件下生存。用于表征，并在下面描述的研究细胞通路5和15之间。 </li><li>通过加入10％二甲亚砜的肌成纤维细胞培养基冷冻保存的细胞在液体N 2。 </li></ol></li></ol> 3.定性小鼠和人的食管间质细胞<ol><li>检查细胞的形态与倒置显微镜。观察纺锤形贴壁细胞的形态（ 图1）。 </li><li> α-SMA，波形蛋白，结蛋白，和细胞角蛋白：通过下述骨架标记物评价表征培养细胞。基质细胞与肌成纤维细胞样表型表达的细胞骨架肌成纤维细胞标记物α-SMA和波形蛋白（ 图2）。 <ol><li>为了进一步从肌肉细胞，免疫染色结蛋白区分培养基质细胞。执行免疫染色上培养的细胞，板1.5×10 4个细胞在4孔腔室玻片并生长在肌成纤维细胞培养基24小时。间质细胞，肌成纤维细胞样表型将有弱或缺乏结蛋白的表达。基质细胞缺乏上皮标记的泛细胞角蛋白的表达（数据未示出）。 注：文化的评估，包括对细胞骨架标记免疫细胞化学和细胞表面标志物用流式细胞仪评价。免疫细胞化学用作表征的主要方法为前细胞骨架蛋白PRESSION轮廓是独特的成纤维细胞，肌成纤维细胞或肌细胞。流式细胞仪通过评估上皮，内皮，细胞和造血细胞标志物的细胞表面表达建立培养纯度。 CD90是有帮助识别人类基质细胞，因为它是表达对人类成纤维细胞和肌成纤维细胞的辅助方法。 CD90是不是在小鼠基质细胞的表征有帮助，因为它也通过鼠的T细胞表达的。 </li></ol></li><li>后的细胞达到60％汇合时，冲洗载玻片用磷酸盐缓冲盐水（PBS）中，用甲醇固定。在-20℃下孵育细胞在甲醇中10分钟，然后在PBS中储存于4℃直至使用。 </li><li>按照标准的染色协议。 </li><li>简单地说，前施加第一抗体，阻断固定的细胞用PBS中的5％goatserum。稀释在5％山羊血清初级和次级抗体以及。 <ol><li>为了检测α-SMA和波形，请使用下列抗体和浓度：小鼠mAb到α-SMA，在1：250的稀释和兔多克隆到波形蛋白在1：500的稀释。 </li><li>在室温下，初级抗体孵育过夜，在4℃，冲洗3次，用PBS中并在一稀释的1添加二级抗体罗丹明缀合的山羊抗小鼠：200和的Cy2偶联的山羊抗兔的稀释度为1：1000 1小时在室温下。 </li><li>对细胞核用4&#39;，6-二脒基2Phenylindole吲哚（DAPI）以1μg/ ml的1分钟的浓度，并然后用PBS洗涤。分析幻灯片倒置显微镜。 </li></ol></li><li>通过检查鼠和人食管癌细胞的造血（CD45）和内皮（CD31）的细胞表面通过流式细胞术标记（ 图3）建立培养物的纯度。由基质细胞表面标记物CD90的表达进一步表征人类细胞。 注：CD90不能用于鉴定小鼠基质细胞如小鼠T CELLS也表达CD90。 <ol><li>通过用非酶细胞解离溶液处理分离从培养瓶中培养的小鼠食管基质细胞，在37℃下进行10分钟，用FACS缓冲液两次，计数并用FITC标记的CD45和APC-CD31与相关同种型对照，根据标准的染色程序。 </li><li>收集利用流式细胞仪刀魂细胞分析与软件的FlowJo数据。前染色细胞，优化抗体和滴定使用同种型对照，正常小鼠脾和骨髓细胞的浓度。 </li></ol></li></ol>

<ol>
	<li>Stappenbeck, T. S., Miyoshi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+stromal+stem+cells+in+tissue+regeneration+and+wound+repair.">The role of stromal stem cells in tissue regeneration and wound repair.</a> Science. 324 (5935), 1666-1669 (2009).</li><li>Powell, D. W., Pinchuk, I. V., Saada, J. I., Chen, X., Mifflin, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+cells+of+the+intestinal+lamina+propria.">Mesenchymal cells of the intestinal lamina propria.</a> Annu Rev Physiol. 73, 213-237 (2011).</li><li>Rieder, 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=T-Helper+2+Cytokines,+Transforming+Growth+Factor+beta1,+and+Eosinophil+Products+Induce+Fibrogenesis+and+Alter+Muscle+Motility+in+Patients+With+Eosinophilic+Esophagitis.">T-Helper 2 Cytokines, Transforming Growth Factor beta1, and Eosinophil Products Induce Fibrogenesis and Alter Muscle Motility in Patients With Eosinophilic Esophagitis.</a> Gastroenterology. 146 (5), 1266-1277 (2014).</li><li>Powell, D. W., 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=Paracrine+cells+important+in+health+and+disease.">Paracrine cells important in health and disease.</a> The American Journal of Physiology. 277 (1 Pt. 1), C1-9 (1999).</li><li>Powell, D. W., 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=Intestinal+subepithelial+myofibroblasts.">Intestinal subepithelial myofibroblasts.</a> The American Journal of Physiology. 277 (2 Pt. 1), C183-201 (1999).</li><li>Shaker, 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=Stromal+cells+participate+in+the+murine+esophageal+mucosal+injury+response.">Stromal cells participate in the murine esophageal mucosal injury response.</a> American Journal of Physiology Gastrointestinal and Liver Physiology. 304 (7), 662-672 (2013).</li><li>Shaker, 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=Epimorphin+deletion+protects+mice+from+inflammation-induced+colon+carcinogenesis+and+alters+stem+cell+niche+myofibroblast+secretion.">Epimorphin deletion protects mice from inflammation-induced colon carcinogenesis and alters stem cell niche myofibroblast secretion.</a> The Journal of Clinical Investigation. 120 (6), 2081-2093 (2010).</li><li>Kalabis, 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=Isolation+and+characterization+of+mouse+and+human+esophageal+epithelial+cells+in+3D+organotypic+culture.">Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.</a> Nature Protocols. 7 (2), 235-246 (2012).</li><li>Khalil, H., Nie, W., Edwards, R. A., Yoo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+primary+myofibroblasts+from+mouse+and+human+colon+tissue.">Isolation of primary myofibroblasts from mouse and human colon tissue.</a> Journal of Visualized Experiments. (80), (2013).</li><li>Rieder, 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=Gastroesophageal+reflux+disease-associated+esophagitis+induces+endogenous+cytokine+production+leading+to+motor+abnormalities.">Gastroesophageal reflux disease-associated esophagitis induces endogenous cytokine production leading to motor abnormalities.</a> Gastroenterology. 132 (1), 154-165 (2007).</li></ol>

这项工作是在肠道生理健康（AS）和赖特基金会试点奖（AS）支持NIH / NCI K08CA153036-01A1（AS），生AGA，通用磨坊贝尔研究所和营养学研究学者奖。

该技术主要培养一代大体相似使用具有适于组织大小切碎和消化时间鼠或人体组织时。我们用小鼠组织的经验表明，用上述的方法始终导致成功建立原代培养物的。在协议中的关键步骤包括手术设备，消毒和下面的组织培养标准的无菌技术。小鼠的年龄也是一个关键步骤。 8 - 19日龄新生儿持续取得成功的主要文化。基质细胞从年轻的新生儿隔离尚未尚未成功生成。试图从老年小鼠建立肌成纤维?...

上皮-间质相互作用中涉及的各种胃肠道功能，包括黏膜再生，修复，纤维化和癌变1,2的调节。这些相互作用已被研究最多在小肠和结肠，并且可以类似地发挥在食管粘膜紊乱3的作用。肠和结肠的基质细胞被称为成肌纤维细胞的一个亚群已被证实参与介导组织损伤，炎症和修复4,5。在远端胃肠道，这些梭形细胞位于邻近所述基底膜在上皮和粘膜固有层之间的界面，并被定义为α-SMA和波形蛋白阳性，泛细胞角蛋白阴性，弱阳性或阴性结蛋白5。 食管基质未被严格特征在于在细胞或分子水平。我们在鼠食管工作脱monstratedα-SMA和波形蛋白的细胞在食管基质，偶尔下层的鳞状上皮6。上皮-间质相互作用已牵涉食管粘膜障碍如胃食管损伤介导6和嗜酸细胞性食管3。纤维化狭窄也食管损伤和间质细胞的公知的并发症有牵连胃肠纤维化的发病机制。这些细胞的分离将帮助完成必要的研究，调查疯狂的信号通路。 这次提交提供必要建立α-SMA阳性，波形积极肌纤维母细胞原代培养，使得知识有关的信号通路介导这些相互作​​用存在的差距可以解决的技术。所描述的技术已成功地用于由作者建立原代鼠结肠肌成纤维细胞7和furthe适合建立小鼠6和人类肌成纤维细胞样食管间质细胞的河在此我们描述建立和表征小鼠或将来的功能研究，使用前人类食道建立这些文化需要的条件。培养物可以生长并用于向上以15的通道。隔离并建立原代培养物的通过的方法概述如下生成的基质细胞与成肌纤维细胞表型; α-SMA，波形蛋白阳性以及弱阳性或阴性结蛋白，和细胞角蛋白阴性。这种表型是从食道成纤维细胞的表型主要是波形蛋白阳性，α-SMA的负3或粘膜肌层6的α-SMA阳性，波形蛋白阴性表型明显。

<table border="0" cellpadding="0" cellspacing="0" width="1647">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:549px;">Name</td>
			<td style="width:549px;"></td>
			<td style="width:549px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture reagents</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS&#160;</td>
			<td>Sigma Aldrich</td>
			<td>H6648</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dispase&#160;</td>
			<td>GIBCO, Invitrogen</td>
			<td>&#160;17105-041</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">collagenase XI&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C9407&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM&#160;</td>
			<td>GIBCO, Invitrogen&#160;</td>
			<td>11965-092</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sorbitol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S1876</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Sigma)</td>
			<td>F42442</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">transferrin&#160;</td>
			<td>Roche</td>
			<td>10-652-202-001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trypsin/EDTA&#160;</td>
			<td>Corning</td>
			<td>25-052-Cl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">epidermal growth factor&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E9644</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trypsin/EDTA&#160;</td>
			<td>Corning</td>
			<td>&#160;25-052-Cl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reagents for immunostaining</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">goat serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse mAB to &#945;-SMA&#160;</td>
			<td>abcam</td>
			<td>ab7817</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit pAB to vimentin&#160;</td>
			<td>abcam</td>
			<td>ab45939</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cy2 conjugated Goat anti Rabbit&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-225-144</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI&#160;</td>
			<td>sigma-aldrich</td>
			<td>D8417</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD31 conjugated to eFluor 450&#160;</td>
			<td>ebiosciences</td>
			<td>48-0319-410</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD90 conjugated to APC&#160;</td>
			<td>ebiosciences&#160;</td>
			<td>17-0909-41&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Annexin V and 7AAD&#160;</td>
			<td>BD Pharmigen</td>
			<td>559763</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mouse Fc block for CD16/CD32&#160;</td>
			<td>BD Pharmigen</td>
			<td>2136662</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 ml tube&#160;</td>
			<td>eppendorf</td>
			<td>30108310</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Motic AE31 inverted microscope</td>
			<td>Motic AE31 inverted microscope</td>
			<td>Motic AE31 inverted microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuaire Biosafety Cabinet and Incubators</td>
			<td>Nuaire Biosafety Cabinet and Incubators</td>
			<td>Nuaire Biosafety Cabinet and Incubators</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-well chamber slides&#160;</td>
			<td>Thermo Scientific</td>
			<td>177437</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf Centrifuge 5810R</td>
			<td>Eppendorf Centrifuge 5810R</td>
			<td>Eppendorf Centrifuge 5810R</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olympus Vacuum-Driven Filter System&#160;</td>
			<td>Genesee Scientific&#160;</td>
			<td>25-227</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon Eclipse TE300 Fluorescent Microscope&#160;</td>
			<td>Nikon Eclipse TE300 Fluorescent Microscope (Tokyo, Japan)&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 well plates&#160;</td>
			<td>Corning</td>
			<td>3516</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T25 Flasks&#160;</td>
			<td>TRP&#160;</td>
			<td>90026</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T75 Flasks&#160;</td>
			<td>Corning</td>
			<td>43064</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissection Scissors</td>
			<td>Dissection Scissors</td>
			<td>Dissection Scissors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection Forceps</td>
			<td>Dissection Forceps</td>
			<td>Dissection Forceps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Single Tipped Q-Tips&#160;</td>
			<td>Kendall</td>
			<td>540500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T75 Flasks</td>
			<td>Corning</td>
			<td>43064</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Metamorph software (Molecular Devices)</td>
			<td>Molecular Devices</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACSCAlibur&#160;</td>
			<td>BD Bioscience&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACSVerse</td>
			<td>BD Bioscience</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of myofibroblasts from mouse and human esophagus

通过酶消化产生小鼠和人的食管肌成纤维细胞。新生儿（8-12天龄）鼠食管收获，切碎，清洗，并进行酶促消化用胶原酶和分散酶25分钟。人食管切除标本被剥离固有肌层和外膜的，剩余的粘膜被切碎，并经受酶促消化用胶原酶和分散酶长达6小时。培养的细胞表达α-SMA和波形和快递结蛋白弱或根本没有。培养条件不利于上皮，造血，或内皮细胞的生长。培养纯度通过潜在污染造血细胞和内皮细胞的细胞表面标志物表达的流式细胞仪评估进一步证实。所描述的技术很简单，导致一致的新一代非hematopoieitc，非内皮细胞间质中。这种技术的局限性是固有的使用分子生物学的研究， 也就是小学文化，不可避免的遇到变异在不同老鼠或人类建立文化之间。然而，小学文化是在体内状态的比较有代表性的反映相比，细胞系。这些方法还提供了调查以分离的能力，并从不同的临床和实验条件培养基质细胞，使组间的比较。其特征食管基质细胞还可以在功能性研究调查食管疾病上皮 - 基质相互作用使用。

食管基质细胞使用机械和酶消化分离的最初铺板并在6孔板中培养。检查细胞以电镀后数小时内倒置显微镜的演示了一个混合细胞悬浮液松散粘连到板的底部（ 图1A）。在接下来的24小时，纺锤形细胞牢固地粘附到板底观察从混合细胞悬浮液（ 图1B）。这些发芽细胞覆盖的整个面积的5天内良好，可以成功地传代，保持形态的拍摄至少15代。纺锤形贴壁细胞以低密度（ ...

Watch this Scientific Journal Video about Isolation of Myofibroblasts from Mouse and Human Esophagus at JoVE.com

Isolation of Myofibroblasts from Mouse and Human Esophagus

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

从小鼠和人类食管隔离肌纤维母细胞

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

isolation-of-myofibroblasts-from-mouse-and-human-esophagus

Research

JoVE Journal

Biology

10.9K 次观看.  Keck School of Medicine, University of Southern California. We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express α-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions. 

视频: Isolation of Myofibroblasts from Mouse and Human Esophagus

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

科研

生物学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

从人类Lipoaspirates脂肪来源干细胞的手动隔离

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

从鼠标阻力动脉分离微血管内皮管

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

小非编码RNA从人血清中分离

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...