Name: method for obtaining primary ovarian cancer cells from solid specimens
Uploaded: 2014-02-04T15:00:00
Description: Watch this Scientific Journal Video about Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens at JoVE.com

我们要感谢明尼苏达大学的组织采购基金的工作人员与病人的组织样本收集方面的援助。这项工作是由国防部的卵巢癌研究发展计划（OCRP）OC093424到MB的支持下，由兰迪剃须刀癌症研究和社区基金，MB的，由明尼苏达卵巢癌联盟MB，并由妇科肿瘤学部门资金MB。资助者在研究设计，数据收集和分析，发布决定或准备的手稿没有作用。

操守准则 卵巢癌的固体样品从明尼苏达大学的组织采购基金（TPF）后的机构审查委员会委员会获得：人类主题委员会（IRB）的批准。 1。试剂设置<ol><li>通过补充的DMEM含10％FBS和100单位青霉素 - 链霉素准备完整的DMEM培养基中。存储介质在4℃和温暖至37℃后才能使用。 </li><li>分装分散酶II为5-10毫升单独的卷，以避免重复冷冻解冻并储存在-20°C下冻存。 </li></...

<ol>
	<li>Kurman, R. J., Visvanathan, K., Roden, R., Wu, T. C., Ie M, S. h. i. h. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+and+treatment+of+ovarian+cancer:+shifting+from+early+stage+to+minimal+volume+of+disease+based+on+a+new+model+of+carcinogenesis.">Early detection and treatment of ovarian cancer: shifting from early stage to minimal volume of disease based on a new model of carcinogenesis.</a> Am. J. Obstet. Gynecol. 198 (4), 351-356 (2008).</li><li>Kuhn, E., Kurman, R. J., Shih, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+Cancer+Is+an+Imported+Disease:+Fact+or+Fiction.">Ovarian Cancer Is an Imported Disease: Fact or Fiction.</a> Curr. Obstet. Gynecol. Rep. 1 (1), 1-9 (2012).</li><li>Sarojini, S., 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=Early+detection+biomarkers+for+ovarian.">Early detection biomarkers for ovarian.</a> J. Oncol. , (2012).</li><li>Shepherd, T. G., Theriault, B. L., Campbell, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nachtigal+M.W.+Primary+culture+of+ovarian+surface+epithelial+cells+and+ascites-derived+ovarian+cancer+cells+from+patients.">Nachtigal M.W. Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients.</a> Nat. Protoc. 1 (6), 2643-2649 (2006).</li><li>Tsao, S. 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=Characterization+of+human+ovarian+surface+epithelial+cells+immortalized+by+human+papilloma+viral+oncogenes+(HPV-E6E7+ORFs).">Characterization of human ovarian surface epithelial cells immortalized by human papilloma viral oncogenes (HPV-E6E7 ORFs).</a> Exp. Cell. Res. 218 (2), 499-507 (1995).</li><li>Auersperg, N., Maines-Bandiera, S. L., Dyck, H. G., Kruk, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cultured+human+ovarian+surface+epithelial+cells:+phenotypic+plasticity+and+premalignant+changes.">Characterization of cultured human ovarian surface epithelial cells: phenotypic plasticity and premalignant changes.</a> Lab. Invest. 71 (4), 510-518 (1994).</li><li>Brewer, M., 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=In+vitro+model+of+normal,+immortalized+ovarian+surface+epithelial+and+ovarian+cancer+cells+for+chemoprevention+of+ovarian+cancer.">In vitro model of normal, immortalized ovarian surface epithelial and ovarian cancer cells for chemoprevention of ovarian cancer.</a> Gynecol. Oncol. 98 (2), 182-192 (2005).</li><li>Sueblinvong, T., 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=Establishment,+characterization+and+downstream+application+of+primary+ovarian+cancer+cells+derived+from+solid+tumors.">Establishment, characterization and downstream application of primary ovarian cancer cells derived from solid tumors.</a> PLoS One. 7 (11), (2012).</li><li>Rockwell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo-in+vitro+tumour+cell+lines:+characteristics+and+limitations+as+models+for+human+cancer.">In vivo-in vitro tumour cell lines: characteristics and limitations as models for human cancer.</a> Br. J. Cancer Suppl. 41 (4), 118-122 (1980).</li><li>Wong, C., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development,+application+and+limitations+of+breast+cancer+cell+lines+to+study+tamoxifen+and+aromatase+inhibitor+resistance.">The development, application and limitations of breast cancer cell lines to study tamoxifen and aromatase inhibitor resistance.</a> J. Steroid. Biochem. Mol. Biol. 131 (3-5), 83-92 (2012).</li></ol>

更好地了解卵巢癌的病因和发展是至关重要的提高受到这种破坏性疾病的女性的结果。在此背景下，利用建立和“市售的”卵巢癌细胞系无疑是相当有用的。然而，今天我们知道，癌症细胞系并不能代表它们来自于许多方面，包括缺乏异质性9,10的人类肿瘤。 在这里，我们报告一个快速，可靠的方法分离和原发性卵巢癌的细胞培养直接从卵巢癌中的手术标本分离30分钟...

尽管其发病率相对较低，卵巢癌是最致命的妇科疾病和妇女1,2癌症死亡的第五大原因。这主要是由于缺乏可靠的工具和模型，忠实地概括本病3的启动和发展。我们大部分的关于卵巢癌知识今天已经可以通过使用从腹水4-7永生恢复卵巢表面上皮细胞（IOSEs），卵巢癌细胞系和原发性卵巢癌细胞。不幸的是，它们的使用也有一些局限性包括一些与永生化过程或在体外的通道相关联的遗传和表型的变化，并从腹水中制备得到的人口的异质性。 因此，从卵巢癌的身份和具体的固体样品得出原发性卵巢癌细胞代表UNIQUË工具，研究卵巢癌的进展。 参与获取这些恶性细胞的主要困难是由于基质细胞或成纤维细胞一起损失生存能力和过早缺乏这些EOC细胞的培养增殖能力的过度生长。有几种方法来从实体瘤产生单细胞悬液目前存在的，通过机械方式或酶解，但是某些技术产生了较多的首选结果8。这里，我们表明，酶消化用分散酶II的结果可行的，成纤维细胞 - 自由EOC细胞的一种有效的恢复。将如此获得的培养物EOC极易受遗传操纵和也可在药物筛选试验有用的，表明这些EOC培养物适合于许多下游应用。

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			<td height="21" style="height:21px;width:275px;">Name of Material/ Equipment</td>
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			<td height="40" style="height:40px;">&#160;Screw lid, polypropylene specimen container, sterile (Thermoscientific)</td>
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			<td height="40" style="height:40px;">50 ml conical capped tubes, sterile (BD Falcon)</td>
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			<td height="21" style="height:21px;">&#160;10 ml serological pipette, sterile&#160;</td>
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			<td height="21" style="height:21px;">6 ml syringe plunger, sterile&#160;</td>
			<td style="width:125px;">Tyco Healthcare</td>
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			<td height="40" style="height:40px;">Nylon filter (70 &#956;m ) cell strainer, sterile (BD Falcon)</td>
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			<td align="right" style="width:155px;">352350</td>
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			<td height="21" style="height:21px;">5-cm Petri dish, sterile&#160;</td>
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			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">10-cm Petri dish, sterile&#160;</td>
			<td style="width:125px;">Corning</td>
			<td align="right" style="width:155px;">430167</td>
			<td></td>
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method for obtaining primary ovarian cancer cells from solid specimens

急需研究卵巢癌的发生和发展可靠的工具。而采用卵巢癌细胞系的保持对于理解卵巢癌的一种有价值的工具，它们的使用有很大的局限性。这些包括缺乏异质性，并延长体外传代相关的遗传改变的太多了。这里我们描述了一种方法，其允许快速建立原发性卵巢癌细胞的形成在外科手术时收集的固体临床标本。该方法包括遭受临床标本酶消化30分钟的。分离的细胞悬浮液被允许生长和可用于下游应用，包括药物筛选。以上建立的卵巢癌细胞系原发性卵巢癌细胞系的优点是，它们是代表它们来自于并且可以从不同的网站是否来自原特异性临床标本的表面RY或转移性卵巢癌。

卵巢癌的新鲜临床标本手术后收集（ 图1）和切成小片（ 图2A和2B）构成细胞浆液。这允许试样的最佳暴露于酶处理。细胞浆暴露于酶消化并在37℃下进行30分钟。在孵育时间浆液变得越来越混浊（尤其是各搅拌后），这是组织分解的迹象。在温育时间结束时，将浆液转移到一个细胞过滤到EOC细胞免受任何未解离的组织（ 图3）中分离出来。将回收?...

Watch this Scientific Journal Video about Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens at JoVE.com

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

为获取原发性卵巢癌细胞固体样品的方法

这项研究描述了隔离原发性卵巢癌细胞从固体临床标本和表征了详细的方法。卵巢癌的临床标本进行酶消化，以获取可行的，成纤维细胞无上皮性卵巢癌（EOC）细胞非常适合用于下游应用。

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

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Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

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In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

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