We thank all residents and attending physicians, notably Dr. A.F. Haney (the University of Chicago, Department of Obstetrics and Gynecology) for collecting omental biopsies. Also, we thank Stacey Tobin and Gail Isenberg for carefully editing this manuscript. This work was supported by Bears Care, the charitable beneficiary of the Chicago Bears Football Club, the National Institute of Neurological Disorders and Stroke (NINDS) R21 NS075702, and the National Cancer Institute grant R01 CA111882 to E.L.

描述的所有研究方案已经由芝加哥机构审查委员会的大学（IRB）审查。知情同意每位患者的术前和研究经芝加哥IRB的大学。生物安全柜型和手套应处理为保护人体组织时，减少污染细胞的危险中。 1.隔离小学未转化的基质细胞与文化<ol><li>人体组织收集和准备。 <ol><li>获得人网膜，2厘米3，腹部手术过程中移除的样本，并立即浸入组织在RT中的磷酸盐缓冲盐水（PBS）（图2A）。一块网膜3厘米×2cm的一般得1万小学人类间皮细胞（HPMC）和20万人类纤维原细胞或正常网膜成纤维细胞（NOF）。 </li><li>降速的PBS将组织浸渍于0.5×g离心3分钟尽快后收集（&lt;2小时的PBS），并在50ml锥形管中的组织转移到新鲜的20个ml的PBS中。通过在一个直径为15厘米，无菌培养皿（图2B）手术刀切碎切碎组织成5毫米3块 。 </li></ol></li><li>原代人腹膜间皮细胞分离8 <ol><li>以分离的HPMC，转移组织糜到50ml锥形管中，并等待1分钟为固体片浮到顶部 （图2C＆D）。 HPMC和红血细胞（RBC）将存在于底部的液体。使用移液管从管的底部，并进入一个新的锥形管中除去液体。旋液向下0.5 XG的3分钟，并吸从HPMC /红细胞沉淀上清。 <ol><li>重复这个过程三次：加20毫升的PBS到组织糜，让组织上升，从一个吸管，进入HPMC / RBC管底部去除液体，降速，并取出上清。毕竟自旋完成并且将上清液吸出，将丸粒将包含HPMC和RBC。 </li></ol></li><li>板的所有单元从沉淀成75厘米2烧瓶中15充满生长培养基毫升（含10％胎牛血清[FBS]，1％的MEM维生素[93毫克/升]，1％的MEM非必需氨基酸[81.4毫克/升]，1％青霉素链霉素[青霉素 - 链霉素，100U / ml青霉素和100μg/ ml链霉素]）。 </li><li>执行二次PBS洗分离另外的间皮细胞。 <ol><li>对于二次PBS洗涤，摇动剩余的固体组织在200rpm，37℃下在20毫升PBS中10分钟，然后等待1分钟为组织浮到顶部。除去从管的底部的液体进新管中，离心，在0.5×g离心3分钟，吸出上清液。 </li><li>板上的所有的HPMC / RBC从二级PBS洗涤，并在单独的75cm 2的烧瓶在15毫升完全培养基中。 </li></ol></li><li>要排除任何ř通过体积的PBS溶液：emaining HPMC，摇动组织糜在200rpm，37℃度10分钟在20毫升1：1的0.25％胰蛋白酶的25mM EDTA中。允许组织上升，收集液体在试管的底部到一个新的50ml管中，和自旋向下在0.5 GX 3分钟。 <ol><li>吸出上清液和板块全部HPMC / RBC在一个单独的75cm 2的烧瓶在15毫升完全培养基中。 </li></ol></li><li>培养在37℃和5％CO 2在加湿环境中铺板的细胞。饲料铺板的细胞中加入15ml充满生长培养基上天3和5不除去用过的培养基。 HPMC可分裂前5-7天培养。 <ol><li>使用在所有的实验低传代HPMC（最多2代），以尽量减少去分化原来的表型和修改。最好是使用宽视场显微镜用塑料微分干涉对比功能，或相差capabilitie 4-20x物镜20倍的目标秒（在显微镜相衬滤波器）用于取细胞的所有图像，和10x物镜（）在明场来分析免疫组织化学3,3&#39;-二氨基联苯胺（DAB）染色。 </li></ol></li><li>确认HPMC是立方形，并进行免疫组化8（ 图3A，C，E）表达细胞角蛋白8和波形。 </li></ol></li><li> NOF隔离8 <ol><li>制备出10毫升胶原酶III型溶液（10倍）证券的通过组合1：1：100×青霉素 - 链霉素的1:1混合物：胶原酶III型的1500个活性单位/毫升：透明质酸酶在PBS 714单位活性/毫升。 </li><li>在10ml的10倍胶原酶III型溶液稀释于无血清培养基（DMEM，用1％的MEM维生素[93毫克/升]摇动剁碎网膜组织后保留的HPMC隔离在200rpm，37℃进行6小时， 1％的MEM非必需氨基酸[81.4毫克/升]，1％青霉素链霉素[青霉素 - 链霉素，100U / ml青霉素和100μg/ ml链霉素]）。消化的组织的外观不透明，并且可以具有一些纤维碎片（图2E）。 </li><li>离心溶液含有NOF细胞 在悬浮液中，在0.5×g离心3分钟，在室温，吸出上清液和板在75厘米2烧瓶中的沉淀在13ml充分生长培养基。 </li><li>删除旧的媒体24小时后，用15毫升新鲜完全培养基更换。 NOF可以培养分裂前1-3天。注意NOF的扩散将停止，当细胞达到汇合。 </li><li>确认原代人成纤维细胞是平的，细长的细胞和表达波形蛋白，但不通过进行免疫组化8角蛋白8（图3B，D，F）。使用低传代诺夫的实验（最好3代之前），以尽量减少去分化 与原始表型的修饰。使用宽视场显微镜用塑料DIC能力20倍的目标采取细胞的所有阶段图像，而在明场一个10倍的目标，分析免疫组化DAB染色。 </li></ol></li></ol> 2.电镀的器官文化8 <ol><li>从75厘米培养瓶中通过用10ml的PBS，接着毫升0.25％胰蛋白酶3/25毫摩尔EDTA不超过5分钟，漂洗释放NOF。中和胰蛋白酶具有完全生长介质中的至少3倍的体积。 </li><li>转移胰蛋白酶的细胞到50ml锥形管中，离心，在0.5 GX 3分钟。除去上清液，并在5毫升完全培养基带来电池备份。使用细胞计数器计数从培养烧瓶中回收细胞。 </li><li>稀释细胞在全生长培养基的适当体积到板每100微升2,000-4,000 NOF到黑色，透明底的96孔板。电镀前加入0.5微克/ 100微升鼠尾I型胶原的细胞混合物。让细胞在37℃，坐在5％CO 2在加湿环境中至少4小时，或直至细胞附着在板表面上。 </li><li>从利用在步骤2.1和2.2中描述的相同的方法培养瓶中释放的HPMC。稀释细胞在全生长培养基的合适量为板材每50μl10,000-20,000的HPMC在NOF的顶部已经镀有Ⅰ型胶原蛋白在96孔板中。让细胞在37℃，开始实验前坐在5％CO 2在加湿环境至少18小时。 </li><li>培养绿色荧光蛋白（GFP）在整个范围内生长培养基-expressing HeyA8卵巢癌细胞。 HeyA8卵巢癌细胞通过表达桡足类cGFP（PCDH-CMV-MCS-EF1）的慢病毒载体表达的荧光标记。 HeyA8卵巢癌细胞应该是在使用的当天（对数生长期）80-90％汇合。从培养板释放细胞和利用在步骤2.1-2.2为原代细胞所描述的相同的方法计数。 </li><li>允许卵巢癌细胞完全生长培养基以回收15-20分钟，在37℃下在一锥形管与盖子松松地密封。 </li></ol> 3，附着力测定8 <ol><li>恢复后，通过在0.5×g离心纺丝3分钟沉淀卵巢癌细胞，然后除去上清液和稀释细胞在无血清培养基的适当体积达到50000个细胞/100μl的的浓度。 </li><li>倒置96孔板用HPMC / NOF器官型培养以除去用过的培养基，并在无血清培养基中添加100微升癌细胞悬浮液的每个孔中。孵育板在37℃在5％CO 2在加湿环境进行30分钟至4小时。 </li><li>倒置96孔板以除去介质和非粘附细胞。使用多管移液器在低功耗仔细，轻轻吸管100微升PBS到每口井，并再次翻转板。重复PBS洗涤一次。 </li><li>倒置以除去PBS中，并加入100微升4％多聚甲醛至每个孔中。允许板坐20英里n至固定细胞。 20分钟后，反转板，并添加100微升PBS中。 </li><li>需要注意的是井的总荧光可以报告或细胞的数量进行量化。对于定量，一式两份使用HeyA8细胞以1百万个细胞/ ml的浓度的第一板的标准曲线。 <ol><li>通过停转1百万个细胞，除去培养基，并允许他们坐在在1ml的4％多聚甲醛20分钟使标准曲线。 </li><li>自旋细胞再次调出1毫升的PBS（重新计票的细胞，以确认100万/ ml浓度）。板50000，40000，30000，20000，万，5000，1000，和0细胞到每个孔中一式两份，并加入PBS中的50微升每孔的最终总体积。 </li></ol></li><li>底部读取荧光酶标仪（激发= 488纳米，发射= 528纳米）的培养板，比例高的井（标准曲线上50,000）。使用标准曲线计算有多少卵巢癌细胞粘附于organotyp在每个孔中（图4A-C）的集成电路培养。 </li></ol> 4.增殖试验10 <ol><li>后的卵巢癌细胞恢复（参见步骤2.5和2.6以上），并在0.5×g离心纺丝3分钟沉淀细胞，然后稀释所述细胞中的1％FBS的培养基的适当体积（DMEM有1％FBS，1％的MEM维生素[93毫克/升]，1％的MEM非必需氨基酸[81.4毫克/升]，1％青霉素链霉素[青霉素 - 链霉素，100U / ml青霉素和100μg/ ml链霉素]）来实现4000个细胞的浓度/ 150微升。 </li><li>用HPMC / NOF器官型培养倒置96孔板以除去用过的培养基，并在1％的添加150微升癌细胞悬浮液的FBS的介质到每个孔中。孵育板在37℃在5％CO 2在72小时的潮湿环境。 </li><li>底上读取天（激发= 488纳米，发射= 528纳米）的荧光读板器一旦培养板，评估相对增殖的细胞。继续测定96小时后，改变培养基在48小时（图5A-C）。要小心，不要改变介质时（反相板为首选），扰乱文化。 </li></ol> 5.侵袭试验8 <ol><li>电镀前的3D培养，在200μlPBS中添加7.5微克大鼠尾胶原I到一个24孔培养板插入件（8微米孔径）。让平板孵蛋O / N，然后小心翼翼地用吸管直接电镀HPMC / NOF器官文化到胶原蛋白涂层刀片（步骤2，上文）之前删除的PBS。 </li><li>制备卵巢癌细胞如在步骤3.1。到板的卵巢癌细胞上的刀片的器官型培养，小心使用吸管从刀片的顶部除去过量的介质。在无血清培养基中加入100μl卵巢癌细胞悬浮液的每个孔中。 </li><li>添加750微升全生长培养基到远低于插入物以诱导癌细胞vasion到器官文化。孵育板在37℃在5％CO 2在24小时的潮湿环境。 注意：卵巢癌细胞侵入器官培养将跨越所述插入并保持在插入件的底侧。 </li><li> 24小时后，用抓钳插入并简要冲洗在PBS的烧杯中，然后将插入到一个空的好。擦洗每个插入用棉签的两侧的顶部为总共1分钟。迅速将插入到含有每750微升4％多聚甲醛以及一盘。允许每个插入到传送插入到孔中填充有750微升的PBS之前坐在低聚甲醛10分钟。 </li><li>查看侵入细胞（粘附并固定到插入件的底部），用显微镜在2.5倍的放大倍率。当整个井是在视野内，调整放大倍数10倍，并采取5张图片，一个靠近中心和1中的井的每个象限，避免采取这一过于接近的边缘图像。按照同样的模式为每口井。 </li><li>使用制造商的方案来计算在每个图像单元的数量根据其协议来获得细胞每场，每孔（图6A-C）的侵入的平均数。 </li></ol>

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Metastasis. 5, 181-197 (1987).</li><li>Kanemoto, T., Martin, G. R., Hamilton, T. C., Fridman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synthetic+peptides+and+protease+inhibitors+on+the+interaction+of+a+human+ovarian+cancer+cell+line+(NIH:OVCAR-3)+with+a+reconstituted+basement+membrane+(matrigel).">Effects of synthetic peptides and protease inhibitors on the interaction of a human ovarian cancer cell line (NIH:OVCAR-3) with a reconstituted basement membrane (matrigel).</a> Invasion Metastasis. 11, 84-92 (1991).</li><li>Rieppi, 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=Mesothelial+cells+induce+the+motility+of+human+ovarian+carcinoma+cells.">Mesothelial cells induce the motility of human ovarian carcinoma cells.</a> Int. J. Cancer. 80, 303-307 (1999).</li><li>Barbolina, M. V., Adley, B. P., Ariztia, E. V., Liu, Y., Stack, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+membrane+type+1+matrix+metalloproteinase+activity+in+ovarian+carcinoma+cells+via+collagen-induced+EGR1+expression.">Microenvironmental regulation of membrane type 1 matrix metalloproteinase activity in ovarian carcinoma cells via collagen-induced EGR1 expression.</a> J. Biol. Chem. 282, 4924-4931 (2007).</li><li>Burleson, K. 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=Ovarian+carcinoma+ascites+spheroids+adhere+to+extracellular+matrix+components+and+mesothelial+cell+monolayers.">Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers.</a> Gynecol. Oncol. 93, 170-181 (2004).</li><li>Suzuki, N., 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=HMOCC-1,+a+human+monoclonal+antibody+that+inhibits+adhesion+of+ovarian+cancer+cells+to+human+mesothelial+cells.">HMOCC-1, a human monoclonal antibody that inhibits adhesion of ovarian cancer cells to human mesothelial cells.</a> Gynecol. Oncol. 95, 290-298 (2004).</li><li>Kishikawa, 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=Two+distinct+pattern+of+peritoneal+involvement+shown+by+in+vitro+and+in+vivo+ovarian+cancer+dissemination+models.">Two distinct pattern of peritoneal involvement shown by in vitro and in vivo ovarian cancer dissemination models.</a> Invas. Metast. 15, 11-21 (1995).</li><li>Casey, R. 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=Establishment+of+an+in+vitro+assay+to+measure+the+invasion+of+ovarian+carcinoma+cells+through+mesothelial+cell+monolayers.">Establishment of an in vitro assay to measure the invasion of ovarian carcinoma cells through mesothelial cell monolayers.</a> Clin. Exp. Metastasis. 20, 343-356 (2003).</li><li>Niedbala, M. J., Crickard, K., Bernacki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+human+ovarian+tumor+cells+with+human+mesothelial+cells+grown+on+extracellular+matrix.">Interactions of human ovarian tumor cells with human mesothelial cells grown on extracellular matrix.</a> Exp. Cell Res. 160, 499-513 (1985).</li><li>White, E. A., Kenny, H. A., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+modeling+of+ovarian+cancer.">Three-Dimensional modeling of ovarian cancer.</a> Adv. Drug Deliv. Rev. 79-80, 184-192 (2014).</li><li>Kenny, H. A., 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=Quantitative+high+throughput+screening+using+a+primary+human+three-dimensional+organotypic+culture+predicts+in+vivo+efficacy.">Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy.</a> Nature Comm. , (2015).</li><li>Sawada, K., 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=c-Met+overexpression+is+a+prognostic+factor+in+ovarian+cancer+and+an+effective+target+for+inhibition+of+peritoneal+dissemination+and+invasion.">c-Met overexpression is a prognostic factor in ovarian cancer and an effective target for inhibition of peritoneal dissemination and invasion.</a> Cancer Res. 67, 1670-1680 (2007).</li><li>Kenny, H. A., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP-2+functions+as+an+early+response+protein+in+ovarian+cancer+metastasis.">MMP-2 functions as an early response protein in ovarian cancer metastasis.</a> Cell cycle. 8, 683-688 (2009).</li><li>Sawada, K., 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=Loss+of+E-cadherin+promotes+ovarian+cancer+metastasis+via+alpha+5-integrin,+which+is+a+therapeutic+target.">Loss of E-cadherin promotes ovarian cancer metastasis via alpha 5-integrin, which is a therapeutic target.</a> Cancer Res. 68, 2329-2339 (2008).</li><li>Mitra, A. K., 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=Ligand-independent+activation+of+c-Met+by+fibronectin+and+&#945;(5)&#946;(1)-integrin+regulates+ovarian+cancer+invasion+and+metastasis.">Ligand-independent activation of c-Met by fibronectin and &#945;(5)&#946;(1)-integrin regulates ovarian cancer invasion and metastasis.</a> Oncogene. 30, 1566-1576 (2011).</li></ol>

The authors have nothing to disclose.

成立腹膜微环境的一个器官模型来评估两者的细胞和先天在卵巢癌传播微环境的组件的单独和集体功能（多个）。提供了用于镀敷和自定义三维器官型培养来研究卵巢癌的细胞粘附，增殖和侵袭的具体协议。原代人网膜间皮细胞和成纤维细胞从患者分离并使用在早期通道保持正常形态和患者的变化。在这种模式下，正常成纤维细胞网膜和ECM（通常胶原I型）的分层与伯人腹膜间皮细胞单层。该模型...

Ovarian cancer is the deadliest gynecologic malignancy1. The majority of patients are diagnosed after the cancer has disseminated throughout the peritoneal cavity. Once the cancer has spread throughout the peritoneal cavity, cytoreductive surgery and chemotherapy are often not sufficient treatment to prevent cancer recurrence and chemoresistance, resulting in a less than 30% 5-year survival rate. Ovarian cancer metastasis is predominantly limited to the peritoneal cavity, and several other cancer types, including gastric, pancreatic, and colon cancers, metastasize to the same anatomic sites in the peritoneal cavity. In general, ovarian cancer cells detach from the in situ carcinoma in the fallopian tube or the primary ovarian tumor, travel in peritoneal fluid as single cells or spheroids, and attach to mesothelium-lined surfaces of the omentum, bowel, and abdominal wall2. 
The tumor microenvironment plays an important role in disease progression and chemoresistance in many cancers3-6. The peritoneal cavity is a unique microenvironment, with a mesothelial cell monolayer covering the majority of surfaces (Figure 1A)7. The mesothelial lining acts as a barrier that creates a low-friction surface, which tends to be protective against cancer cell adhesion8. Immediately underneath this mesothelial-lined surface is a layer made predominantly of fibroblasts and extracellular matrix (ECM), which promote cancer cell adhesion and invasion8. Ovarian cancer cells secrete factors that induce changes in the mesothelial cell lining that enhance ovarian cancer cell adhesion, invasion, and metastasis9,10. Ovarian cancer cells adhere to the mesothelial surface via integrin and CD44-mediated mechanisms (Figure 1B)11-16. 
Historically, several 3D models have been developed to investigate ovarian cancer interactions with the microenvironment. Some of the first models studied ovarian cancer-ECM interface17-21, ovarian cancer-mesothelial cell communication13,14,21-24, or both25 (reviewed by us 26). Niedbala et al. discovered that ovarian cancer cells display a quicker and firmer adhesion to ECM than to mesothelial cells or to plastic alone25. However, these models did not histologically resemble the peritoneal microenvironment. Therefore, we established a 3D organotypic model to more thoroughly replicate the ovarian cancer microenvironment. In order to better understand the role of the microenvironment and the interaction between cancer and peritoneal cells in the peritoneal dissemination of ovarian cancer, we have developed a 3D organotypic in vitro culture model of the peritoneal cavity lining (schematic in Figure 1C). The proposed model is composed of primary human fibroblasts and ECM, covered with a layer of primary human mesothelial cells-each cell type is isolated from human omentum. Histologically, this model resembles the normal peritoneal or omental lining, and provides a surface on which we can study the tumor microenvironment, the interaction between cancer cells and normal tissue, and the processes of cancer cell adhesion, invasion, and proliferation8.

<table>
	<tbody>
		<tr>
			<td>1. Isolation and culture of primary cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Scientific</td>
			<td>SH3001304</td>
			<td></td>
		</tr>
		<tr>
			<td>Single-edged razor blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm culture dishes</td>
			<td>BD Biosciences</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass flask</td>
			<td>?</td>
			<td>?</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>16000044_3616914956</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with L-Glutamine</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamins</td>
			<td>Corning</td>
			<td>25-020-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Nonessential amino acids</td>
			<td>Corning</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker&#160;</td>
			<td>Thermo-Fisher</td>
			<td>MaxQ 4450</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5702</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo-Fisher</td>
			<td>Forma Series II Water Jacketed CO2 Incubator Model 3100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA, 1x (0.25%)</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Worthington Biochemical</td>
			<td>LS002592</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 Flasks</td>
			<td>BD Biosciences</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>T-175 Flasks</td>
			<td>BD Biosciences</td>
			<td>353112</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>Rainin</td>
			<td>P2, P10, P20, P200 and P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>Corning</td>
			<td>Filtered tips P2, P10, P20, P200 and P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>2. Plating 3D culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counter</td>
			<td>Invitrogen</td>
			<td>Countess</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen</td>
			<td>C10313</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I (Rat Tail)</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate, clear bottom, black</td>
			<td>BD Biosciences</td>
			<td>353219</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>3. Adhesion assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipet</td>
			<td>Eppendorf</td>
			<td>Xplorer 300</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4% in PBS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Molecular Devices</td>
			<td>Minimax</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>5. Invasion assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Inserts (8um, 24-well)</td>
			<td>BD Biosciences</td>
			<td>353097</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Q-tips</td>
			<td>cotton swabs</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 200m</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Profiler</td>
			<td>public domain</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>BD Biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>6. Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Integrin &#945;V&#946;3 Antibody, clone LM609</td>
			<td>EMD Millipore</td>
			<td>MAB1976</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta 1&#160;</td>
			<td>Oncosynergy</td>
			<td>OS2966</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha 5 [CD49e]</td>
			<td>ID Pharmingen</td>
			<td>555615</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta 4 [CD104]</td>
			<td>EMD Millipore</td>
			<td>MAB 2058</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling the early steps of ovarian cancer dissemination in an organotypic culture of the human peritoneal cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

器官型培养物组装通过首先混合主人成纤维细胞与I型胶原蛋白，然后与间皮细胞的5倍的数量覆盖该培养。将培养物温育至少18小时，加入前卵巢癌细胞，研究粘附，浸润或增殖。每个测定重复与多个（N = 3-5），从不同的患者和许多井获得3D培养在每种条件下进​​行了测试的粘合（N = 5），增殖（N = 5）和侵入测定（n = 3时） 。卵巢癌细胞附着在三维器官型培养4小时（图4）</str...

Watch this Scientific Journal Video about Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity at JoVE.com

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

modeling-early-steps-ovarian-cancer-dissemination-an-organotypic

Research

JoVE Journal

Medicine

26.5K 次观看.  University of Chicago. Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation. 

视频: Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

科研

医学

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

异型三维<em在体外</em&gt;卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

一个器官高通量系统原发性多发性骨髓瘤细胞的药物敏感性的表征

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

腹膜内卵巢癌转移的定量

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

输卵管的管状细胞学作为卵巢癌早期检测的有前景的工具

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

小鼠视网膜微动脉的反应性测定的制备步骤前体

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...