Name: isolation and culture of mouse primary pancreatic acinar cells
Uploaded: 2013-08-13T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Mouse Primary Pancreatic Acinar Cells at JoVE.com

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） <p class="jove_con...

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholecystokinin+induction+of+mob-1+chemokine+expression+in+pancreatic+acinar+cells+requires+NF-kappaB+activation.">Cholecystokinin induction of mob-1 chemokine expression in pancreatic acinar cells requires NF-kappaB activation.</a> Am. J. Physiol. 277, 74-82 (1999).</li><li>Ji, B., Kopin, A. S., Logsdon, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+differences+between+rat+and+mouse+CCKA+receptors+determine+the+divergent+acinar+cell+response+to+the+cholecystokinin+analog+JMV-180.">Species differences between rat and mouse CCKA receptors determine the divergent acinar cell response to the cholecystokinin analog JMV-180.</a> J. Biol. Chem. 275, 19115-19120 (2000).</li><li>Ji, K. A., Yang, M. S., Jou, I., Shong, M. H., Joe, E. 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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=Inactivation+of+TIF1gamma+cooperates+with+Kras+to+induce+cystic+tumors+of+the+pancreas.">Inactivation of TIF1gamma cooperates with Kras to induce cystic tumors of the pancreas.</a> PLoS Genet. 5, e1000575 (2009).</li><li>Dorrell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+pancreatic+alpha,+beta,+duct+and+acinar+populations+with+cell+surface+markers.">Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.</a> Mol. Cell Endocrinol. 339, 144-150 (2011).</li><li>Case, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+intracellular+transport+and+discharge+of+exportable+proteins+in+the+pancreatic+acinar+cell+and+other+cells.">Synthesis, intracellular transport and discharge of exportable proteins in the pancreatic acinar cell and other cells.</a> Biol. Rev. Camb. Philos. Soc. 53, 211-354 (1978).</li><li>Blauer, M., Nordback, I., Sand, J., Laukkarinen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+explant+outgrowth+culture+model+for+mouse+pancreatic+acinar+cells+with+long-term+maintenance+of+secretory+phenotype.">A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype.</a> Eur. J. Cell. Biol. 90, 1052-1060 (2011).</li></ol>

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 ...

Research

JoVE Journal

Biology

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

科研

生物学

Isolation of Primary Mouse Trophoblast Cells and Trophoblast 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 ...

Isolation and Primary Culture of Rat Hepatic Cells

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 Immune Cells from Primary Tumors

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

制备胰腺腺泡细胞钙成像的目的，细胞损伤的测量，以及腺病毒感染

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Assessing the Secretory Capacity of Pancreatic Acinar Cells

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

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 ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

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 ...