我们感谢工作人员AniCan（CRCL，里昂），动物保健技术援助。这项工作是协会倾LA RECHERCHE SUR LE癌症研究所杜国家癌症研究所国家德拉健康等德拉RECHERCHEMédicale（INSERM艾文莉计划），国家法甲抗癌症，支持，并通过奖学金从国家法甲抗癌症（JG），从研究所国家杜癌症（JG），部DE L&#39;ENSEIGNEMENT高等等德拉RECHERCHE法国（RMP和DFV）协会倾LA RECHERCHE SUR LE癌症（ DFV）。

伦理委员会批准，所有程序监管的政府机关（“委员会评估COMMUN坳中心列昂·贝拉尔，A L&#39;Animalerie中转DE L&#39;ENS，AU PBES等AU LABORATOIRE P4”（CECCAPP））。小鼠保持在无特定病原体动物设施在的“PLATEFORME AniCan中心莱昂BERARD”（法国里昂），并符合机构的指引处理。 图1中所示的程序的示意图。 1。胰腺的解剖和的弯曲齿（0天） 一个非常迅速的夹层是一个最佳的提取收率的关键，并确保良好的生存能力培养的细胞。为了减少胰腺隔离所需的时间，所有的仪器和设备必须准备好之前，鼠标安乐死。 <ol><li><html>安乐死鼠标CO 2窒息或颈椎脱位。 </li></ol>这一步，​​所有的程序都必须与无菌剥离设备无菌的气氛下进行的（生物安全柜，II级）。 <ol start="2"><li>修正了鼠标和鼠标腹部喷用70％的乙醇。与任何解剖剪刀和镊子，作出一个V形的切口，并继续在生殖器区域隔膜完全打开腹腔。 </li><li>位置对隔膜的肝叶如果身体腔是开放远远不够，他们应该留在那里。拉你的左边腹腔外，肠道和结肠，发现直肠。随着一对弯曲的镊子和解剖剪刀，抓斗和部分直肠。 </li><li>在相同的双钳，仔细展开完全从直肠的肠道胃在你的左边拉肠道。 </li></ol>耳鼻喉科“&gt;在这一步骤中，胰腺，胃和肠的开头之间的一小条出类拔萃，其结扎脾保持不变。 <ol start="5"><li>诺伊斯剪刀和一双镊子，小心地切沿着肠和胰腺具有健脾从其余的消化道中解脱出来。 </li><li>抓斗的脾脏和胰腺部分连接（ 图2）。 </li></ol>在该步骤中，确保没有肠系膜脂肪组织和/或其他邻近组织（脾，肠等），可以收集与胰腺，以避免细胞污染。 如果在该程序的其余部分，来制备所有的缓冲区必须无钙离子Ca 2 +的螯合剂，以避免在单腺泡细胞外分泌胰腺组织的完整的解离。 <ol start="7"><li>在汉克的平衡盐溶液（HBSS）1X两次冲洗胰腺。 </li></ol>在这一步，因为脂肪组织会浮，胰腺会下沉，能够容易地可视化和迅速去除污染物的白色脂肪组织仍连接到胰腺。 如果胰腺需要被运送到细胞培养设备，它必须保持在冰上在HBSS中1个。 <ol start="8"><li>在无菌培养皿中含有5毫升的HBSS 1X胰腺转移。使用诺伊斯用剪刀和解剖刀，胰腺切片在1至3毫米3（ 图3A）的小件。 </li></ol> 2。胰腺酶法和机械解离（0天） <ol><li>将它们转移到无菌的50毫升的聚丙烯管中。 </li><li>离心2分钟，在450×g离心4℃。吸弃上清，以去除细胞碎片和血细胞。 </li><li> IA型胶原酶溶液中加入10毫升（含HBSS，1X 10毫米HEPES，200 U / ml的collagena本身IA和0.25毫克/毫升的胰蛋白酶抑制剂），胰腺部分。使用25毫升血清吸管，将他们转移到一个25厘米2烧瓶。孵育20-30分钟，在37°C。 （每5分钟），在这段时间内进行机械分离大力的往复移动胰腺十倍左右的片段，在减小尺寸的无菌移液管（25，10，和5ml血清移液管）。 </li></ol>在此步骤中，有必要频繁地监测胰腺部分酶解的程度。 <ol start="4"><li>当胰腺组织似乎是游离（根据胰腺片段的消失和该溶液的混浊度增加）（ 图3B），停止酶反应通过加入10毫升冷的洗涤缓冲液（HBSS，1X含有5 ％胎牛血清（FBS）和10毫米的HEPES）。 </li><li>转移到无菌的50毫升聚丙烯管和离心泵ifuge 2分钟，以450×g，4℃下小心吸弃上清，除去胶原酶IA解决方案。 </li><li>重悬缓冲洗涤液用10毫升洗涤沉淀。离心3分钟，在450×g离心4℃。小心吸弃上清。重复此步骤两次。 </li></ol> 3。过滤和播种分散腺泡（0天） <ol><li>重悬细胞沉淀在7ml的Waymouth培养基含2.5％FBS，1％青霉素 - 链霉素混合物（PS），0.25 mg / ml的胰蛋白酶抑制剂，和25 ng / ml的重组人表皮生长因子（EGF）。 </li><li>滤液中的细胞混合物中，允许它通过一个100微米的过滤器保留的非消化的片段（导管，血管，和胰岛）。胰腺腺泡结构（10-15细胞腺泡）通过。 </li><li>用6ml的Waymouth培养基含FBS，PS，胰蛋白酶INH冲洗过滤器ibitor，和表皮生长因子。 </li></ol>在这一步之后，细胞必须是非常仔细地处理，以避免任何腺泡解离。 <ol start="4"><li>种子隔离腺泡在6孔培养皿中，每孔（2毫升）（ 图3C）。在37℃下，5％（体积/体积）的CO 2气氛中进行培养。 </li></ol>在这一步之后，腺泡细胞的悬浮培养。 4。腺泡细胞培养（1至10天） <ol><li>二十四小时后，转移腺泡（悬浮液中）到一个新的6孔培养皿中，以消除污染物的细胞和细胞残留物附着过夜（ 图4）。 </li></ol>如果细胞培养数天或数本实验条件下，如果需要在单层培养的细胞，它是建议基质支架上，以传输和种子腺泡需要被扩展。 <ol start="2"><li>看到前一天鼎矩阵支持（0天），大衣型6孔培养皿I型胶原（5微克/厘米2）。 I型胶原蛋白的溶液中加入1毫升（50微克/毫升的0.02M的醋酸，0.2微米的过滤）在各孔中，在1小时，在37℃（或4过夜，使其被动地吸附于塑料℃下）。 </li><li>吸I型胶原蛋白溶液和，冲洗两次磷酸盐缓冲液1X涂好。 </li><li>允许涂好干（下微生物安全柜）使用前至少12小时。 </li><li>隔离的主腺泡（在步骤4.1中得到的）转移到不同的I型胶原涂层的6孔培养皿中，并对其进行培养如先前所述，在相同的条件下（在37℃下在5％（体积/体积）的CO 2气氛中） 。细胞附着到I型胶原基板2天。 </li><li>第3天，改变培养基中没有遵守，以消除非活菌。随着培养时间的增加，细胞会逐步SP阅读的含有胶原的支持。改变培养基每3天（ 图5）。 </li></ol>孤立的腺泡细胞后，可以算作一个完整的机械分离由使用托马细胞的计数室。注意隔离腺泡细胞不能被随后保持在培养。 可以得到腺泡文化的质量控制检查腺泡细胞的特异性标记物（如胰蛋白酶 ， 胰转录因子1亚基α，或羧肽酶A1（由免疫细胞化学或免疫荧光实验））的表达。

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作者宣称，他们有没有竞争经济利益。

在这个协议中，我们描述了一个程序隔离胰腺腺泡细胞。在小于1小时，这种方法使得有可能超过20×10 6个腺泡细胞，每只动物隔离。由于其快速和简单的实现（多达10个胰腺可以被独立处理每个实验并行），该协议将出现一个很好的妥协之间的隔离方法3-5,9-12,17。 关键步骤/故障排除 这种方法的效率依赖在几个关键点上采取的预防?...

一个经常遇到的问题为研究实验室工作外分泌胰腺组织腺泡细胞在体外培养一段时间足够长，以允许长期实验的难度。 的一个因素阻碍这种培养系统的发展是由于在糖酵解，蛋白水解和脂解酶，字面消化的胰腺组织胰腺细胞的分离过程中，当它们被释放到高含量的实验操作胰腺组织所固有的灵敏度。 第二个因素是显着的体外可塑性腺泡细胞，这往往会失去它们的分泌特性和转分化其他成熟细胞，如胰腺导管细胞或肝细胞样细胞1。 在体外 ，这种细胞的可塑性变化与实验条件（如培养基成分）2 </燮&gt;和适当的培养条件下，胰腺外分泌细胞的1到设计中引入了一定程度的复杂性。 已经开发了几种方法的腺泡细胞的分离和培养，从豚鼠胰腺3-5的第一。最初，这些协议涉及消化胰腺组织胶原酶糜蛋白酶和蛋白酶鸡尾酒的，剧烈的机械分离与最终的隔离。以这种方式分离的胰腺细胞显示异常的结构和功能特征，特别是根尖的结构和显着损害其膜受体的损失。分离出的细胞仍然是可行的只有1或2天。 制备分散，腺泡维护其内和细胞间的建筑，保护细胞膜，限制损坏表面受体，从而改善外分泌泌6-8。作为一个RESU，LT，这种方法提供延长7-10 天的体外腺泡细胞活力的主要优点。此外，这种方法目前优选的腺泡细胞分离9-12，因为维持细胞间的接触，包括通过间隙连接的细胞耦合，是一个重要的决定因素外分泌胰腺腺泡细胞表型的13。 由于去分化，腺泡细胞和导管上皮细胞转分化的起源积极的胰腺外分泌癌14建议的机制之一，分散腺泡模式也是足够的系统来研究胰腺癌的可塑性和其随后的分子机制。此外，在结合使用转基因动物的15,16和转基因技术的发展（2腺病毒或慢病毒转导，使用的纳米颗粒等），这可以在体外原腺泡细胞模型在确定各种遗传功能障碍如何影响腺泡细胞分化或去分化和调控，应该提供更好的了解胰腺炎的发作，癌前病变，细胞的可塑性变化的分子事件负责是非常有用的。 隔离分散腺泡是我们使用的方法，在我们的实验室中培养胰腺腺泡细胞。在这里，我们描述和讨论所采用的方法。它涉及酶解耦合无腺泡细胞的分离机械破碎胰腺组织（细菌胶原酶）。虽然大多数协议涉及培养腺泡，暂停或经过特殊处理的塑料基板，我们的成长他们只是简单地悬浮（24小时），播种之后如果需要长时间的细胞培养基质支架上。 此协议允许快速分离（少于1小时）分散胰交流INI，持续一个多星期的文化。它允许超过20×10 6每小鼠胰腺腺泡细胞的隔离。它的简单性使得能够处理多达10个独立的胰腺平行。保持腺泡内和细胞间的架构，从而腺泡分离的原代细胞的表型，这种模式构成了系统的首选转分化机制的研究，胰腺外分泌功能的机型，目前市面上的所有其他来自显示许多遗传性胰腺肿瘤改变导致细胞转化。

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>0.2 &mu;m filter</td>
			<td>Dutscher</td>
			<td>146560</td>
			<td></td>
		 </tr>
		 <tr>
			<td>10 ml serological pipettes</td>
			<td>Beckton Dickinson</td>
			<td>357551</td>
			<td></td>
		 </tr>
		 <tr>
			<td>100 &mu;m filter</td>
			<td>Beckton Dickinson</td>
			<td>352360</td>
			<td></td>
		 </tr>
		 <tr>
			<td>100 mm Petri dish</td>
			<td>Beckton Dickinson</td>
			<td>353003</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1000 &mu;l filter tips</td>
			<td>Starlab</td>
			<td>S1122-1830</td>
			<td></td>
		 </tr>
		 <tr>
			<td>20 &mu;l filter tips</td>
			<td>Starlab</td>
			<td>S1120-1810</td>
			<td></td>
		 </tr>
		 <tr>
			<td>200 &mu;l filter tips</td>
			<td>Starlab</td>
			<td>S1120-8810</td>
			<td></td>
		 </tr>
		 <tr>
			<td>25 ml serological pipettes</td>
			<td>Beckton Dickinson</td>
			<td>357535</td>
			<td></td>
		 </tr>
		 <tr>
			<td>5 ml serological pipettes</td>
			<td>Beckton Dickinson</td>
			<td>357543</td>
			<td></td>
		 </tr>
		 <tr>
			<td>50 ml polypropylene tube</td>
			<td>Beckton Dickinson</td>
			<td>352070</td>
			<td></td>
		 </tr>
		 <tr>
			<td>6-well plate</td>
			<td>Beckton Dickinson</td>
			<td>353046</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Acetic acid 100%</td>
			<td>VWR BDH Prolabo</td>
			<td>20104.298</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagenase IA</td>
			<td>Sigma-Aldrich</td>
			<td>C2676</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Curved forceps, Dumont #7</td>
			<td>World Precision Instruments</td>
			<td>14188</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Dissecting scissors, straight</td>
			<td>World Precision Instruments</td>
			<td>14393</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Epidermal Growth Factor, human</td>
			<td>Promokine</td>
			<td>C-60180</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethanol absolute (AnalaR Normapur)</td>
			<td>VWR BDH Prolabo</td>
			<td>20821.310</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-801F</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Forceps, Dumont #5</td>
			<td>World Precision Instruments</td>
			<td>14098</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Hank's Balanced Salt Solution 1x </td>
			<td>Gibco</td>
			<td>14025050</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HEPES 1 M (pH 6.98-7.30)</td>
			<td>Lonza</td>
			<td>17-737F</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Incubator O2/CO2</td>
			<td>Sanyo</td>
			<td>MCO-19M</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Matrigel</td>
			<td>Beckton Dickinson</td>
			<td>356234</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Microbiological Safety Cabinet, level II</td>
			<td>Faster</td>
			<td>SafeFast Elite 212 S</td>
		 </tr>
		 <tr>
			<td>Noyes scissors, sharp/sharp tips, German</td>
			<td>World Precision Instruments</td>
			<td>500228-G</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Penicillin-Streptomycin mixture</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phosphate Buffer Saline 10x </td>
			<td>Gibco</td>
			<td>14200067</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipet-Aid</td>
			<td>Drummond Scientific Company</td>
			<td>Pipet-Aid XP</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipetman P1000</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipetman P20</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipetman P200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scalpel</td>
			<td>Paramount Surgimed Ltd.</td>
			<td>Disposable Scalpel Size 23</td>
		 </tr>
		 <tr>
			<td>T25 flask, 25 cm2</td>
			<td>Sigma-Aldrich</td>
			<td>Z707481</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin inhibitor, from Glycine Max</td>
			<td>Sigma-Aldrich</td>
			<td>T6522</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Type I collagen</td>
			<td>Beckton Dickinson</td>
			<td>354236</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Waymouth's medium</td>
			<td>Gibco</td>
			<td>31220-023</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

isolation and culture of mouse primary pancreatic acinar cells

此协议允许快速分离小鼠胰腺腺泡（少于1小时），使得有可能保持它们在培养一周以上。可以从一个单一的小鼠胰腺获得超过20×10 6个腺泡细胞。此协议可提供在独立处理多达10个并行胰腺。因为它保留腺泡结构，这种模式非常适合从胰腺肿瘤，这显示许多基因的改变，造成部分或者全部丧失腺泡分化的细胞系的建立在体外胰腺外分泌的生理研究。

图1图式“分散”腺泡主要腺泡细胞的分离方法。的关键步骤，这在协议过程中都必须严格遵守的讨论部分中描述的。 以方便其剥除，胰腺从腹部与附加的脾脏（ 图2）一起被收集。两个机构需要削减分开，仍然可以连接到胰腺残余的脂肪组织必须被删除（步骤1.6）。 宏观和微观图片， 如图3所示，代表每一个步骤的...

Watch this Scientific Journal Video about Isolation and Culture of Mouse Primary Pancreatic Acinar Cells at JoVE.com

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

在本出版物中，我们描述了一个快速，便捷的程序，用于隔离和培养主要从小鼠胰腺腺泡细胞。这种方法构成一个有价值的方法，以新鲜的正常/未转化的胰腺外分泌细胞的生理研究。

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

isolation-and-culture-of-mouse-primary-pancreatic-acinar-cells

Research

JoVE Journal

Biology

35.4K 次观看.  Centre de Recherche en Cancérologie de Lyon. 在本出版物中，我们描述了一个快速，便捷的程序，用于隔离和培养主要从小鼠胰腺腺泡细胞。这种方法构成一个有价值的方法，以新鲜的正常/未转化的胰腺外分泌细胞的生理研究。 

视频: Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

分离小鼠原代滋养细胞及滋养细胞侵袭实验

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

科研

生物学

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 ...

Assessing the Secretory Capacity of Pancreatic Acinar Cells

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex&#160;vivo culture will represent the in&#160;vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.

Isolated pancreatic acini retain their in&#160;vivo morphology and activity and offer powerful ways for monitoring and manipulating secretion. This work demonstrates how acini can be isolated from the mouse pancreas, and how their secretory capacities can be assessed.

评估胰腺腺泡细胞的分泌能力

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

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 Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

微型猪原主主动脉内皮细胞的隔离与培养

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

从成人小鼠腭中分离和培养原代口腔角质形成细胞

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

小鼠原发性肝正弦内皮细胞的分离与表征

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

小鼠原代视网膜色素上皮细胞的分离

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...