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> 2。组织收集<ol><li>在手术时从宏观上由病理学家鉴定为癌症领域收集卵巢癌（〜5克大小）的固体试样。临床标本是去确定，并根据机构的协议注册。 </li><li>将样品在一个无菌，SC珠螺杆盖，聚丙烯试样容器填充用30ml冰冷的PBS。从外科病理学实验室运送标本的研究实验室进行处理在冰上，并在从标本收集30分钟（ 图1）。 </li></ol> 3。组织处理<ol><li>在无菌条件下工作的，传送含有10ml新鲜的冰冷PBS中，利用无菌的刀片，进一步切成可能（2毫米或更小）的最小片样品上的培养皿（60毫米×60毫米）（ 图2A和2 B）。 </li><li>转移剁碎组织成包含（30分钟，37℃）分散酶II（2.4 U / ml）的DMEM中加入10ml预热的15毫升锥形管和孵化在5％CO 2和37℃下30分钟。为确保标本的最佳消化，手动搅拌的浆细胞，每隔5分钟。 </li><li>经过30分钟的培养，转让（使用10ml的血清吸液管）的料浆细胞上的细胞过滤器（70微米网孔）放置在50ml锥形管中的顶部和应用对使用注射器柱塞上的网状物的温和的压力。丢弃任何未解离的组织（残留在网格的顶部），并收集所得到的细胞悬浮在50ml的无菌锥形管中。离心机在320×g离心7分钟，在4℃（ 图3）。 </li><li>弃去上清，重悬细胞沉淀在含有10％FBS的10ml DMEM。 </li><li>培养在培养皿中的细胞悬浮液在5％CO 2和37℃（ 图4）。 </li><li>从最初的镀24小时后更换培养基。这样就可以去除细胞碎片和目前大多数文化中的红细胞。 </li><li>每三天在随后的两个星期之后，主平机会的文化准备好下游应用改变介质。 </li></ol>

<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>

作者什么都没有透露。

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分钟的临床标本。 因为主细胞从固体样品与腹水液中分离，它具有该疾病的早期阶段之前，当腹水形成发生的时间分离细胞的优点。这种技术还具有副词antage来自特定站点的隔离原发性卵巢癌细胞。在使用这种协议获得可行的原发性卵巢癌细胞的成功在很大程度上依赖于如何迅速样品可以在手术之后进行处理。为了达到最佳效果，标本应在30分钟内从手术接收。如果这是不可能的，试样应保持在4℃（在PBS中）中过夜，在以下的天。根据我们的经验，样本下过夜存放处理降低了产量。暴露于分散酶II的时间超过30分钟，也降低细胞活力和不应该执行。另一种方法来分散酶II的治疗可以用胶原酶和透明质酸酶治疗。然而，这导致成品率下降8。因为在临床标本中的巨大可变性，相比于其他一些可能有较大的红血细胞污染。如果是这样的话，我们建议"洗涤"的剁碎的组织（步骤3.1）用PBS之前将它们暴露酶消化。这将减少的红细胞制剂中的数量，并最终促进初级上皮细胞的粘附。 由于临床标本不育（他们只要他们在手术病理实验室抵达失去无菌），关键是要严格无菌条件下执行所有的步骤。而培养的污染是可能的，通常发生在的情况下，小于10％。上述协议可以用于临床标本的任何一致性。 这个协议的主要限制是细胞从各样品中回收的数量是高度依赖于所提供的试样的尺寸。一个典型的标本的大小约3厘米×3厘米。另一个限制是由以下事实表示，虽然原发性卵巢癌细胞成倍增长达10次传代体外 （因此普罗维鼎的机会，使可行的股票），他们最终衰老和死亡，从而限制了细胞从单一供体的可用性。虽然我们不执行从成纤维细胞中的肿瘤细胞的任何分离，我们推测血清的相对高的（10％）的浓度在整个过程中可以得到卵巢癌的细胞生存优势比成纤维细胞。它也可能是细胞是不容易从基质隔室（成纤维细胞）分离可以不分解，并将分离过程中能够保持在过滤器上。 与此协议获得的细胞是高度适合于未来的应用包括调查的目的是了解导致卵巢癌的发生和发展的过程。未来的应用还包括使用这些原代细胞在体外和在卵巢癌以及生物标记物的早期检测的新的化疗剂的体内测试。

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。

<table border="0" cellpadding="0" cellspacing="0" width="720">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Name of Material/ Equipment</td>
			<td style="width:125px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">REAGENTS:</td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X PBS, sterile&#160;</td>
			<td style="width:125px;">Invitrogen</td>
			<td style="width:155px;">14190-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#160;Screw lid, polypropylene specimen container, sterile (Thermoscientific)</td>
			<td style="width:125px;">Thermoscientific</td>
			<td style="width:155px;">02 1090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;DMEM medium, sterile&#160;</td>
			<td style="width:125px;">Invitrogen</td>
			<td style="width:155px;">11995-065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">&#160;Fetal bovine serum, sterile</td>
			<td style="width:125px;">Thermo Scientific Hyclone</td>
			<td style="width:155px;">SH30396.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#160;100x penicillin-streptomycin, liquid (Invitrogen)</td>
			<td style="width:125px;">Invitrogen</td>
			<td style="width:155px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Dispase II, sterile (Roche)</td>
			<td style="width:125px;">Roche</td>
			<td style="width:155px;">04942 078 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">&#160;Small fine-tip forceps and scalpel blade, sterile</td>
			<td style="width:125px;">Fisher Scientific</td>
			<td style="width:155px;">forceps: 08-953E, blades: 08-927-5D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Small dissecting scissors, sterile</td>
			<td style="width:125px;">Fisher Scientific</td>
			<td style="width:155px;">08-945</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#160;15 ml conical capped tubes, sterile (BD Falcon)</td>
			<td style="width:125px;">BD, Falcon</td>
			<td align="right" style="width:155px;">352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">50 ml conical capped tubes, sterile (BD Falcon)</td>
			<td style="width:125px;">BD, Falcon</td>
			<td align="right" style="width:155px;">352027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;10 ml serological pipette, sterile&#160;</td>
			<td style="width:125px;">Corning</td>
			<td style="width:155px;">13-678-11E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 ml syringe plunger, sterile&#160;</td>
			<td style="width:125px;">Tyco Healthcare</td>
			<td align="right" style="width:155px;">8881516911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nylon filter (70 &#956;m ) cell strainer, sterile (BD Falcon)</td>
			<td style="width:125px;">BD Falcon</td>
			<td align="right" style="width:155px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-cm Petri dish, sterile&#160;</td>
			<td style="width:125px;">Corning</td>
			<td align="right" style="width:155px;">430166</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<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>
		</tr>
	</tbody>
</table>

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。将回收的细胞悬浮液置于5％CO 2和37℃（F IGURE 4）。在此阶段的细胞培养物的形态学揭示两者EOC细胞的存在作为单细胞悬浮液，并在小团块。在这一点上，培养也揭示了PRE​​SEN<html> ce的红细胞和图5所示的小碎片。重要的是，而平机会会慢慢（历时1-3天）坚持以塑料，红细胞和细胞碎片不会和他们最终将逐步"从文化的消失"。从最初的电镀3日，平机会的文化越来越显示贴壁EOC细胞团，并逐渐减少红细胞和碎片， 如图6。由6-7天，EOC细胞形成涡流状的形状（箭头所示），并开始扩散到塑料以形成较大的多细胞聚集体，导致EOC细胞作为如图7所示的典型的鹅卵石形态。第14天，平机会细胞通常汇合（ 图8），现在已经准备好为下游测试和应用。 pload/51581/51581fig1.jpg"/&gt; 图1。收集临床标本。卵巢癌的固体样品被收集在手术时间，并放置在无菌，螺丝盖，聚丙烯试样填充用30ml冰冷的PBS的容器。 <img alt="图2a" fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig2ahighres.jpg" src="/files/ftp_upload/51581/51581fig2a.jpg" /><img alt="图2b" fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig2bhighres.jpg" src="/files/ftp_upload/51581/51581fig2b.jpg" /> 图2。处理临床标本。 A）转移到含有10ml新鲜的冰冷的1×PBS。 乙培养皿固体样品）临床标本进一步切成约2mm大小的块。 图3。从未解离的组织分离EOC细胞。以下酶消化，临床样本被转移到一个细胞过滤及温和的压力是通过使用注射器柱塞上施加的消化临床标本。这允许对平机会的结缔组织和EOC细胞的恢复机械剥离。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig4highres.jpg" src="/files/ftp_upload/51581/51581fig4.jpg" /> 图4。所得EOC细胞悬浮液的电镀。除去任何未离解的组织后，将细胞悬浮液离心并重新悬浮于10ml的DMEM接触器的癌宁10％FBS和5％CO 2和37℃下培养在培养皿中<img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig5highres.jpg" src="/files/ftp_upload/51581/51581fig5.jpg" /> EOC的细胞培养物的处理。细胞悬浮电镀后立即后立即的图5。形态示EOC作为单细胞悬浮液，并在团块（包括箭头所示），红细胞和细胞碎片仍然很多在这个阶段。原来的放大倍率，20X。 <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig6highres.jpg" src="/files/ftp_upload/51581/51581fig6.jpg" /> EOC细胞培养3天的图6。形态在第3天电镀。EOC细胞培养镀层呈半贴壁EOC℃后后埃尔簇（由箭头表示）和较少的红细胞污染。原来的放大倍率，20X。 <img alt="图7" fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig7highres.jpg" src="/files/ftp_upload/51581/51581fig7.jpg" /> 图7。一个星期后，建立了平等机会委员会文化从最初的电镀后的一个星期电镀。EOC细胞培养显示细胞在组织培养塑料传播漩涡般的集群。原来的放大倍率，20X。 <img alt="图8" fo:content-width="5in" fo:src="/files/ftp_upload/51581/51581fig8highres.jpg" src="/files/ftp_upload/51581/51581fig8.jpg" /> 图8。融合EOC文化。EOC显示细胞在培养14天，典型的上皮鹅卵石形态的融合单层。

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

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。

为获取原发性卵巢癌细胞固体样品的方法

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

method-for-obtaining-primary-ovarian-cancer-cells-from-solid-specimens

Nanyang Technological University

Research

JoVE Journal

Medicine

13.4K 次观看.  University of Minnesota. 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。 

视频: Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

科研

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

量化衰老癌症细胞的敏感方法

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

两种方法从子宫切除标本小学建立人子宫内膜基质细胞

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

在实时的间皮细胞卵巢癌球体附件和入侵评估

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

小鼠模型的非侵入性成像检测和监测卵巢癌复发

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

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

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

在卵巢癌肿瘤起始细胞体外富集

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...