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>

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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><tbody><tr><td colspan="4">Tissue culture reagents</td></tr><tr><td>HBSS</td><td>Sigma Aldrich</td><td>H6648</td><td></td></tr><tr><td>Dispase</td><td>GIBCO, Invitrogen</td><td>17105-041</td><td></td></tr><tr><td>Collagenase XI</td><td>Sigma-Aldrich</td><td>C9407</td><td></td></tr><tr><td>DMEM</td><td>GIBCO, Invitrogen</td><td>11965-092</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma-Aldrich</td><td>S1876</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F42442</td><td></td></tr><tr><td>Transferrin</td><td>Roche</td><td>10-652-202-001</td><td></td></tr><tr><td>trypsin/EDTA</td><td>Corning</td><td>25-052-Cl</td><td></td></tr><tr><td>Epidermal growth factor</td><td>Sigma-Aldrich</td><td>E9644</td><td></td></tr><tr><td colspan="4">Reagents for immunostaining</td></tr><tr><td>Goat serum</td><td>Sigma Aldrich</td><td>G9023</td><td></td></tr><tr><td>Mouse mAB to &amp;alpha;-SMA&amp;nbsp;</td><td>abcam</td><td>ab7817</td><td></td></tr><tr><td>Rabbit pAB to vimentin</td><td>abcam</td><td>ab45939</td><td></td></tr><tr><td>Cy2 conjugated Goat anti Rabbit</td><td>Jackson ImmunoResearch</td><td>111-225-144</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D8417</td><td></td></tr><tr><td>CD31 conjugated to eFluor 450</td><td>eBioscience</td><td>48-0319-41</td><td></td></tr><tr><td>CD90 conjugated to APC</td><td>eBioscience</td><td>17-0909-41</td><td></td></tr><tr><td>Annexin V and 7AAD</td><td>BD Pharmigen</td><td>559763</td><td></td></tr><tr><td>Mouse Fc block for CD16/CD32</td><td>BD Pharmigen</td><td>2136662</td><td></td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>5 ml tube</td><td>Eppendorf</td><td>30108310</td><td></td></tr><tr><td>Inverted microscope</td><td>Motic</td><td>AE31</td><td></td></tr><tr><td>Biosafety cabinet and incubators</td><td>Nuaire</td><td></td><td>&lt;a href=&quot;http://www.nuaire.com/products/&quot; target=&quot;_blank&quot;&gt;http://www.nuaire.com/products/&lt;/a&gt;</td></tr><tr><td>4-well chamber slides</td><td>Thermo Scientific</td><td>177437</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Olympus Vacuum-Driven Filter System</td><td>Genesee Scientific</td><td>25-227</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Nikon</td><td>Eclipse TE300</td><td></td></tr><tr><td>6-well plates</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>T25 Flasks</td><td>TRP</td><td>90026</td><td></td></tr><tr><td>T75 Flasks</td><td>Corning</td><td>43064</td><td></td></tr><tr><td>Dissection scissors</td><td></td><td></td><td></td></tr><tr><td>Dissection forceps</td><td></td><td></td><td></td></tr><tr><td>Single tipped Q-Tips</td><td>Kendall</td><td>540500</td><td></td></tr><tr><td colspan="4">Software</td></tr><tr><td>Metamorph software (Molecular Devices)</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>FACSCAlibur</td><td>BD Bioscience</td><td></td><td></td></tr><tr><td>FACSVerse</td><td>BD Bioscience</td><td></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 CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

从人类冷冻胃肠标本分离CD 90+成纤维细胞/肌成纤维细胞


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

科研

发育生物学

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

生物工程

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 ...

生物学

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

超声波增强原成人成纤维细胞隔离

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

医学

人食管类器官和气液界面培养方案

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

作者聚焦:研究嗜酸性粒细胞性食管炎的病理生理学

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.

三维细胞培养模型研究嗜酸性粒细胞性食管炎中上皮屏障 

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and ...

免疫与感染

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

小鼠肠间充质分离导致端细胞高产量

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

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 ...

从小鼠和人类食管隔离肌纤维母细胞

摘要

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此视频中的章节

从人类冷冻胃肠标本分离CD 90+成纤维细胞/肌成纤维细胞

三维组织工程人食管黏膜型号生产，表征和潜在用途

从小鼠和人类结肠组织初级肌成纤维细胞的分离

超声波增强原成人成纤维细胞隔离

三维细胞培养模型研究嗜酸性粒细胞性食管炎中上皮屏障

三维细胞培养模型研究嗜酸性粒细胞性食管炎中上皮屏障

三维细胞培养模型研究嗜酸性粒细胞性食管炎中上皮屏障

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

小鼠肠间充质分离导致端细胞高产量

成人心脏纤维细胞和肌纤维细胞的分离与表征