Name: modeling ovarian cancer multicellular spheroid behavior in a dynamic 3d peritoneal microdevice
Uploaded: 2017-02-18T15:00:00.000+00:00
Duration: 11 min 34 s
Description: Watch this Scientific Journal Video about Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice at JoVE.com

这项工作是由香港研究资助局的支持（赠款17122014，C1013-15G，719813E和17304514）。 AST皇裘槎高级研究奖学金的获得者。

1.微流体器件设计和制造<ol><li>微流控大师设计<ol><li>设计并绘制与任何计算机辅助设计（CAD）软件的微流体通道模式。 注意:通常情况下，该CAD绘图可以被发送到光掩模公司生产的光掩模。微流体设计包括三个相同的平行通道，每个通道具有以下尺寸为4mm×25毫米×250微米（宽度×长度×高度），并设置2mm的。两个信道两端分别设计有127°的角度，以促进液体的入口和出口（ 图1B）。在这个协议中使用的通道被报道在以前的出版物13。 </li><li>洗用丙酮，异丙醇超声波（500瓦/ 42千赫），和去离子水的硅晶片，每次15分钟。干燥用氮气压的晶片。加热在250℃的热板上的硅片15分钟完全干燥它。 </li><li>旋涂SU-8 2075光致抗蚀剂（125微米厚）上以1800 rpm的纺丝速度在硅晶片的层。直接烘烤光致抗蚀剂涂覆的晶片在热板上在65℃，然后在95℃下，分别以除去过量的溶剂，为5和25分钟（根据SU-8的产品手册）。 </li><li>重复步骤3和旋涂SU-8 2075光致抗蚀剂的另一层，得到250μm的最终厚度。烘烤直接在热板上在65℃下7分钟的光致抗蚀剂涂覆的晶片，然后在95℃下30分钟。慢慢冷却该晶片至室温。不要用高压空气冷却。 </li><li>对准并直接把光掩模在晶片的顶部。其暴露于UV光20秒以交联的图案。 </li><li>烘烤在65℃下用光刻胶晶片和5和12分钟95℃的加热板上，分别以除去溶剂用于聚合（根据SU-8的产品手册）。随后，冷却晶片室内温度。 </li><li>通过浸渍在SU-8显影剂将晶片25分钟除去非交联光致抗蚀剂。冲洗用异丙醇晶圆和压缩空气吹干。 注:如果用异丙醇漂洗时，表明发育不完全观察到乳状液，浸泡晶圆回SU-8开发者更长的开发时间。 </li><li>放置在180℃的热板上的晶片10分钟，以治愈潜在裂纹图案。关掉加热板和由留在热板上慢慢冷却晶片至室温。 </li><li>在小瓶帽添加三氯几滴（1H，1H，2H，2H-全氟辛基）硅烷。放入真空干燥器瓶盖。旁边放置到瓶盖晶片silanize晶片10分钟。 </li></ol></li><li> PDMS芯片制造<ol><li>裹片用铝箔创建持有者。 </li><li>混合聚二甲基硅氧烷（PDMS）基和固化剂以10:1的质量比下混合和脱气功能的离心混合器。慢慢倒入PDMS混合物到晶片至约5毫米的高度。脱气在真空下与PDMS 40分钟。固化在烘箱中的PDMS在65℃下2小时。 </li><li>仔细剥PDMS厚板关闭主并修剪沿通道的PDMS，直到它是载玻片的大小。用1毫米活检穿孔创建设备的入口和出口冲的PDMS。 </li><li>请用压缩空气和胶带PDMS表面去除灰尘和杂物PDMS。 </li><li>放置的PDMS板，与通道侧朝上，成等离子体清洁器。放置一个干净的玻璃滑动到等离子体清洁器沿着PDMS厚板。 </li><li>对待空气等离子体下的PDMS和载玻片在高RF级别1分钟。绑定PDMS到载玻片以创建一个不可逆共价键。 </li><li>放置在150℃的热板上的PDMS芯片1小时，以提高粘接强度，并返回hydrop使用前，PDMS的hobicity。 </li></ol></li></ol> 2.播种微流控芯片原发性腹膜间皮细胞（间皮细胞） <ol><li>微流体通道的纤连蛋白涂层<ol><li>用75％乙醇的30微升灭菌的微流体通道。除去乙醇并用一个枪头灭菌磷酸盐缓冲盐水的30微升（PBS）冲洗两次。在通道中充分吸PBS中。 注意:为尽量减少污染，微流体细胞培养物应仔细充分无菌条件下通过使用层流罩下进行。 </li><li>制备在无血清的M199 10微克/毫升纤连蛋白溶液:MCDB105（1:1）培养基。吸取的溶液上下，特别小心，以避免形成气泡。不要旋涡。 </li><li>慢慢地填满每个通道与纤维连接蛋白溶液30微升。密封用胶带的频道，并在4℃过夜孵育芯片。 </li></ol></li><li&gt;播种到间皮细胞微流体通道<ol><li>在10％胎牛血清（FBS）的培养基中培养的HPMC（M199:MCDB105补充有10％FBS和100U / mL青霉素/链霉素）下，5％CO 2在37℃直至80％汇合。 </li><li>冲洗用3mL的PBS的间皮细胞，并与/ 2毫升0.05％胰蛋白酶的30秒0.01％EDTA（TE）溶液trypsinize。用4mL 10％FBS的培养基中和在1000 xg离心降速细胞5分钟。 </li><li>重悬在间皮细胞2毫升10％FBS培养基。计数与血球细胞数和调节细胞浓度为3.5×10 6 /毫升。 </li><li>暖在37℃培养箱中纤连蛋白涂布的微流控芯片使用前5分钟。完全吸出纤维连接蛋白的解决方案。 </li><li>慢慢地吸取HPMC悬挂的30微升到每个通道。密封用胶带渠道和放置设备中的CO 2培养箱中过夜。 </li></ol></li></ol><ol><li>卵巢癌的球体形成<ol><li>保持人上皮卵巢癌细胞系SKOV-3中的5％FBS的培养基（M199:有5％FBS和100U / mL青霉素/链霉素MCDB105）下，5％CO 2在37℃的实验之前。 </li><li>通过煮沸溶解0.5％琼脂糖在无菌水中。分配琼脂糖溶液的50微升到96孔板各孔中。留在机罩板20分钟，以使琼脂糖固化;固化琼脂糖涂层的孔可以防止细胞粘附和指示板是可以使用了。 </li><li>冲洗SKOV-3细胞用3mL的PBS。用2ml的TE溶液3分钟Trypsinize。用4mL 5％FBS的培养基中和在1000 xg离心降速细胞5分钟。 </li><li>计数与血球SKOV-3细胞数和重悬在5％FBS的培养基中的细胞以5,000个细胞/ mL的密度。 TRansfer 200细胞悬液到每一μL的琼脂糖包被的孔，并培养，5％CO 2下在37℃下48小时。 </li></ol></li><li>癌症球体装载<ol><li>转移癌症球体到一个50毫升的离心管中预先润湿用1mL无血清培养基中并降速，在750×g离心5分钟。 注:预湿吸头和离心管可以防止意外球体附着到离心管。 </li><li>制备2.5微克/毫升的5-氯甲基二乙酸酯（CMFDA）溶液在无血清的M199:MCDB105培养基。悬浮癌症球体在1mL的CMFDA溶液孵育在37℃下30分钟。离心在750×g离心5分钟。 </li><li>冲洗癌症球体用2mL无血清培养基和离心机在750×g离心5分钟。重复此步骤两次，以从培养基中除去剩余的荧光染料。 注意:荧光信号可在球体保留时间超过96小时。 </li><li>悬浮癌症球状体在无血清的M199:MCDB105培养基和计数与血球球体数量。浓度调整到2,000球体/毫升无血清培养基。 </li><li>前球体装载，缓缓注入100微升无血清的M199中:MCDB105培养基洗去通道内的非粘附间皮细胞。 注:快速注射，吸入或气泡的形成会导致皮单层的脱离。 </li><li>负载30微升荧光标记的球体悬浮液到每个信道的。微流体芯片放置到CO 2的培养箱中。 </li></ol></li><li>灌注装配平台和联合培养<ol><li>附装用新鲜的无血清的M199注射器:MCDB105培养基为1米长的管。 </li><li>放置在注射泵，注射器和固定柱塞与注射器的主体。在相同的高度培养箱内的微流控芯片安装在水平位置的注射器泵。 ñOTE:数量和注射器的大小取决于使用的信道数和在实验的持续时间。 </li><li>按住快进键，直到媒体溢出管迅速注入与介质全管。 </li><li>运行在所需的流速注射。使用以下等式估算壁剪切应力: <img alt="式（1）" src="/files/ftp_upload/55337/eq1.jpg" /> 其中τ代表腔室，Ǫ=流速内部的壁剪应力，μ=粘度（0.01克厘米-1）; H =流动室的高度，以及w =流动室的宽度。 </li><li>管连接到该信道的入口。放置过量入口管路在孵化器，以确保在介质被加热之前注入通道。为了便于管子和通道之间的连接，相应的连接器可以选择。注意:谨防不引入任何泡到syrin通用电气，管道和连接。 </li><li>通道的出口连接到一个50毫升锥形管，收集用短输出管流出介质。 注:完整的动态共培养体系如图1D所示。 </li></ol></li></ol> 4.灌注后观察间皮细胞与癌症的球体<ol><li>观察间皮细胞和癌症球体与​​明场或荧光显微镜和灌注1小时后拍照。 </li></ol>

<ol>
	<li>Jemal, 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=Global+cancer+statistics.">Global cancer statistics.</a> CA: Cancer J. Clin. 61, 69-90 (2011).</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>Chau, W. K., Ip, C. K., Mak, A. S., Lai, H. C., Wong, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-Kit+mediates+chemoresistance+and+tumor-initiating+capacity+of+ovarian+cancer+cells+through+activation+of+Wnt/beta-catenin-ATP-binding+cassette+G2+signaling.">c-Kit mediates chemoresistance and tumor-initiating capacity of ovarian cancer cells through activation of Wnt/beta-catenin-ATP-binding cassette G2 signaling.</a> Oncogene. 32, 2767-2781 (2013).</li><li>Zhang, 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=Identification+and+characterization+of+ovarian+cancer-initiating+cells+from+primary+human+tumors.">Identification and characterization of ovarian cancer-initiating cells from primary human tumors.</a> Cancer Res. 68, 4311-4320 (2008).</li><li>Tang, M. K. S., Zhou, H. Y., Yam, J. W. P., Wong, A. S. T. <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+contributes+to+the+acquired+apoptotic+resistance+of+nonadherent+ovarian+cancer+cells+through+a+cross+talk+mediated+by+phosphatidylinositol+3-kinase+and+extracellular+signal-regulated+kinase+1/2.">c-Met overexpression contributes to the acquired apoptotic resistance of nonadherent ovarian cancer cells through a cross talk mediated by phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2.</a> Neoplasia. 12, 128-144 (2010).</li><li>Ksiazek, 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=Senescent+peritoneal+mesothelial+cells+promote+ovarian+cancer+cell+adhesion:+the+role+of+oxidative+stress-induced+fibronectin.">Senescent peritoneal mesothelial cells promote ovarian cancer cell adhesion: the role of oxidative stress-induced fibronectin.</a> Am. J. Pathol. 174, 1230-1240 (2009).</li><li>Hafter, R., Klaubert, W., Gollwitzer, R., Vonhugo, R., Graeff, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinked+Fibrin+Derivatives+and+Fibronectin+in+Ascitic+Fluid+from+Patients+with+Ovarian-Cancer+Compared+to+Ascitic+Fluid+in+Liver-Cirrhosis.">Crosslinked Fibrin Derivatives and Fibronectin in Ascitic Fluid from Patients with Ovarian-Cancer Compared to Ascitic Fluid in Liver-Cirrhosis.</a> Thromb Res. 35, 53-64 (1984).</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=Mesothelial+cells+promote+early+ovarian+cancer+metastasis+through+fibronectin+secretion.">Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion.</a> J. Clin. Invest. 124, 4614-4628 (2014).</li><li>Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+tumor+vasculature:+An+emerging+concept+in+antiangiogenic+therapy.">Normalization of tumor vasculature: An emerging concept in antiangiogenic therapy.</a> Science. 307, 58-62 (2005).</li><li>Chang, S. 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=Tumor+cell+cycle+arrest+induced+by+shear+stress:+Roles+of+integrins+and+Smad.">Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad.</a> Proc. Natl. Acad. Sci. USA. 105, 3927-3932 (2008).</li><li>Rutkowski, J. M., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+driving+force+for+change:+interstitial+flow+as+a+morphoregulator.">A driving force for change: interstitial flow as a morphoregulator.</a> Trends Cell Biol. 17, 44-50 (2007).</li><li>Rizvi, I., 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=Flow+induces+epithelial-mesenchymal+transition,+cellular+heterogeneity+and+biomarker+modulation+in+3D+ovarian+cancer+nodules.">Flow induces epithelial-mesenchymal transition, cellular heterogeneity and biomarker modulation in 3D ovarian cancer nodules.</a> Proc. Natl. Acad. Sci. USA. 110, E1974-E1983 (2013).</li><li>Ip, C. 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=Stemness+and+chemoresistance+in+epithelial+ovarian+carcinoma+cells+under+shear+stress.">Stemness and chemoresistance in epithelial ovarian carcinoma cells under shear stress.</a> Sci. Rep. 6, 26788 (2016).</li><li>Avraham-Chakim, L., 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=Fluid-flow+induced+wall+shear+stress+and+epithelial+ovarian+cancer+peritoneal+spreading.">Fluid-flow induced wall shear stress and epithelial ovarian cancer peritoneal spreading.</a> PloS one. 8, e60965 (2013).</li><li>Burkhalter, R. J., 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=Peritoneal+mechanobiology+and+metastatic+success+in+epithelial+ovarian+cancer.">Peritoneal mechanobiology and metastatic success in epithelial ovarian cancer.</a> Faseb Journal. 26, (2012).</li><li>Lane, W. O., 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=Parallel-plate+flow+chamber+and+continuous+flow+circuit+to+evaluate+endothelial+progenitor+cells+under+laminar+flow+shear+stress.">Parallel-plate flow chamber and continuous flow circuit to evaluate endothelial progenitor cells under laminar flow shear stress.</a> Journal of visualized experiments : JoVE. , (2012).</li><li>Botta, G. P., Manley, P., Miller, S., Lelkes, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+assessment+of+three-dimensional+cell+aggregation+in+rotating+wall+vessel+bioreactors+in+vitro.">Real-time assessment of three-dimensional cell aggregation in rotating wall vessel bioreactors in vitro.</a> Nat. Protoc. 1, 2116-2127 (2006).</li><li>Ismadi, M. Z., 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=Flow+characterization+of+a+spinner+flask+for+induced+pluripotent+stem+cell+culture+application.">Flow characterization of a spinner flask for induced pluripotent stem cell culture application.</a> PloS one. 9, e106493 (2014).</li><li>Yu, 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=A+microfluidic-based+multi-shear+device+for+investigating+the+effects+of+low+fluid-induced+stresses+on+osteoblasts.">A microfluidic-based multi-shear device for investigating the effects of low fluid-induced stresses on osteoblasts.</a> PloS one. 9. 9, e89966 (2014).</li></ol>

作者什么都没有透露。

该测定提供了一个灵活的和生理学相关模型，可以与各种生物化学和基于细胞的测定，包括被结合，但不限于，粘附测定，间皮间隙测定法，和药物筛选。它可以应用到的腹腔微环境对癌症发展的效果的评估。然而，一些实验条件可能需要进行优化，这取决于计划的目标（ 例如，每信道，共培养时间等接种的HPMC和癌症球体的数量）。在腹膜内微环境的其它细胞类型，诸如成纤维细胞，内皮细胞，或免疫细胞，也可以为了研究卵巢癌球体和附近小区之间的重要的相互作用被包括在系统中。其它ECM成分，如层粘连蛋白，玻连蛋白或胶原，也可以使用。该协议可能需要考虑的一个弱点是，虽然营养补充和剪切应力的耦合使人想起在体内看出，在我们目前的系统营养补充和剪切应力的没有单独的控制。此外，将需要的技术，如片上胰蛋白酶消化，用荧光激活细胞分选组合以分别收集卵巢癌细胞和间皮细胞有关不同类型的细胞的进一步的分子研究。 当与传统方法相比，该技术具有几个优点。首先，三维共培养更好地模拟卵巢癌细胞和间皮细胞的体内细胞间通讯。第二，细胞是在流体流动培养的，但不是在静止状态下，产生有潜在代表的体内情况12,13细胞应答。此外，流体流动可以精确控制，模仿hydrodynami条件C在肿瘤进展的不同阶段。 总之，动态3D卵巢癌间皮共培养这里提出的模型提供了用于在腹膜腔建模肿瘤行为的灵活框架。用该模型，动态水动力条件下的癌细胞和间皮细胞间的相互作用可全面分析。它可以极大地推进我们的转移进程的基本机制的理解，也可能有助于治疗的发展。

<html>卵巢癌是最致命的妇科肿瘤，其特征是广泛腹膜扩散和恶性腹水1的形成。这种广泛的腹膜转移是一项重大的临床挑战，并与较差的临床结果有关。不同于通过血液转移最坚实癌，卵巢癌主要是腹腔内传播。肿瘤细胞转移2的过程中，存在作为多细胞聚集体/球体。即悬浮培养能丰富卵巢癌干/肿瘤起始细胞，这一事实进一步表明，这些球体可能与两个肿瘤侵袭性相关联，并增强化疗3,4。有在2D和3D培养，这大概有不同的分子机制5之间药物的反应差异。 _content"&gt;与间皮的基本相互作用构造为卵巢肿瘤进展的主要微环境，这些皮细胞躺在的胞外基质（ECM），其中纤连蛋白是一种普遍存在的组分。间皮细胞衍生纤连蛋白的表达和增加之间的链接肿瘤进展已经表明，纤维粘连蛋白是在恶性腹水6,7大量存在。卵巢癌细胞也能够诱导从间皮细胞纤连蛋白的分泌，以促进早期卵巢癌转移8。 新出现的证据表明，机械性刺激，包括剪切应力，可以调节细胞形态，基因表达，并且因此，肿瘤细胞9，10，11的表型。由于恶性腹水 开发和t内积聚umor进展，卵巢肿瘤细胞暴露于流体流，将所得的剪切应力。若干组，我们包括，已显示出剪切应力对卵巢癌的进展的影响，包括细胞骨架修饰，上皮至间质转变，和癌症干性12，13，14，15。因此，生理相关微环境是肿瘤腹膜转移的调查重要。然而，目前的体外流体力学培养系统对模拟和控制常数，低，生理学相关的剪切应力16，17，18，19限制。 常规体外方法侧重于无论是蜂窝或机械环境依然有限模仿适当生理相关的腹腔微环境的复杂性。 这里，为了工程师腹膜一个新的模型，以克服常规的策略的限制，以推进在癌转移腹膜内室的研究中，以控制流体流动的基于3D微流体平台的设计。在这个模型中，卵巢癌球体分别与在微流控芯片在连续的流体流动（ 图 1A）的原代人腹膜间皮细胞共培养。铺板于纤连蛋白的间皮细胞。非粘附性卵巢癌球状体接种到用由注射泵灌注连续流动介质的微流体通道。两个3D环境和动态机械力转移级联的非常重要的因素。此平台可用于在复杂的CEL方面调查腹膜内微细胞性和共培养的相互作用，以及关于动态力学线索。

<table><tbody><tr><td>Silicon wafer</td><td>University wafer</td><td>#1196</td><td>100 mm</td></tr><tr><td>SU-8 2075 photoresist&amp;nbsp;</td><td>Microchem</td><td></td><td></td></tr><tr><td>SU-8 developer&amp;nbsp;</td><td>Microchem</td><td>108-65-6</td><td></td></tr><tr><td>Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane</td><td>Sigma</td><td>448931</td><td></td></tr><tr><td>Sylgard 184</td><td>Dow Corning</td><td>1673921</td><td>Polydimethylsiloxane (PDMS) + curing agent kit</td></tr><tr><td>Biopsy punch&amp;nbsp;</td><td>Miltex</td><td>33-31AA</td><td>1 mm diameter</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-002</td><td></td></tr><tr><td>Polyethylene tubing</td><td>SCI</td><td>BB31695-PE/5</td><td>0.86 mm (inner diameter)</td></tr><tr><td>Syringe</td><td>Terumo</td><td></td><td></td></tr><tr><td>Syringe pump</td><td>Longer precision pump&amp;nbsp;</td><td>&amp;nbsp;LSP01-2A</td><td></td></tr><tr><td>Medium 199</td><td>Invitrogen</td><td>31100-035</td><td>Add 2.2 g/L sodium bicarbonate</td></tr><tr><td>MCDB 105 Medium</td><td>Sigma</td><td>M6395</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone</td><td>SH30068.02</td><td></td></tr><tr><td>Penicillin/streptomycin&amp;nbsp;</td><td>Invitrogen</td><td>15070-063</td><td></td></tr><tr><td>Trypsin EDTA solution&amp;nbsp;</td><td>Gibco</td><td>25300-054</td><td>0.05% Trypsin - 0.01% EDTA, phenol red</td></tr><tr><td>Fibronectin human</td><td>BD</td><td>354008</td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Invitrogen</td><td>15510-027</td><td></td></tr><tr><td>5-chloromethylfluorescein diacetate</td><td>Life technologies</td><td>C7025</td><td>Green CMFDA</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>SANYO</td><td>MCO-18AIC</td><td></td></tr><tr><td>Centrifuge</td><td>Hitachi</td><td>CT15RE</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Nikon</td><td></td><td>Model: 80i or ECLIPSE Ti; software: SPOT</td></tr><tr><td>SKOV-3&amp;nbsp;</td><td></td><td></td><td>Gift from Dr. N Auersperg (University of British Columbia)</td></tr></tbody></table>

modeling ovarian cancer multicellular spheroid behavior in a dynamic 3d peritoneal microdevice

卵巢癌的特征在于丰富的腹膜转移，与恶性腹水通常发现肿瘤球。这与不良临床预后相关，目前缺乏有效的治疗方法。两个三维（3D）环境和动态机械力在此转移级联非常重要的因素。然而，传统的细胞培养不能概括这个自然肿瘤微环境。因此， 在体内样，可以效仿腹腔内环境模型的重要性显而易见。在这项研究中，腹膜的一个新的微流体平台成立转移期间以模仿卵巢癌的球体的情况在腹膜腔。一个非粘附的条件下生成的卵巢癌的球体均涂覆有经受生理学相关的剪切应力腹膜间皮细胞微流体通道中培养。总之，这个动态三维卵巢癌，间皮麦克风rofluidic平台可以在基本的癌症生物学提供了新的知识，并作为潜在的药物筛选和开发平台。

使用该协议，一个微流体平台的成立，卵巢癌的球体水动力条件下的间皮细胞模型。原代人腹膜间皮细胞在微型装置中培养16小时，明视场显微镜下观察。 如图2A所示，通道底部被成功覆盖着的HPMC的单层。要注意的纤连蛋白或HPMC图案形成过程中气泡的形成会导致信道涂层失败是重要的。通过非粘附悬浮培养，直径大约为100微米，这是在大小中的那些患者的腹水中发现类似的多细胞球状体，产生与绿色荧光染料，CMFDA标记。然后将该球体转移至HPMC包衣的微流体通道和保持在悬浮液中。在显微镜下观察细胞形态和图像被捕获（ 图2B）。荧光标记癌症球体可以由荧光显微镜（ 图2C）下的间皮细胞很容易地鉴别。 <img alt="图1" src="/files/ftp_upload/55337/55337fig1.jpg" /> 图1.动态3D腹膜微型建模卵巢癌多细胞球体行为。 （A）中使用的共同培养的卵巢癌的球体与流条件下的HPMC的微流体芯片的示意图。 （B）中示出了微流体通道的设计原理图。在协议中使用的微流控芯片（C）照片，其渠道与清晰的可视化染料溶液注入。栏= 1厘米。 （D）的照片示出的实验装置。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55337/55337fig1large.jpg" target="_blank">请点击此处查看大版本O</a>F该数字。 <img alt="图2" src="/files/ftp_upload/55337/55337fig2.jpg" /> 图2.卵巢癌球体，间皮细胞动态共培养模型的图像。 （一）在微流体通道种植的HPMC单层的形象。 （B）在涂覆有根据相差显微镜（左图）或（C）的荧光显微镜（绿色，CMFDA;右图）腹膜间皮细胞微流体通道卵巢癌球体图像栏= 100微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55337/55337fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice at JoVE.com

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

为了研究卵巢肿瘤进展的生理相关的模型，多细胞球体下模拟流体流动的微型培养。这种动态3D模型模拟与在卵巢癌转移发生的细胞和机械部件的腹膜内环境。

模拟卵巢癌多细胞球体行为动态三维微型腹膜

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

modeling-ovarian-cancer-multicellular-spheroid-behavior-dynamic-3d

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.6K 次观看.  University of Hong Kong. 为了研究卵巢肿瘤进展的生理相关的模型，多细胞球体下模拟流体流动的微型培养。这种动态3D模型模拟与在卵巢癌转移发生的细胞和机械部件的腹膜内环境。 

视频: Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

科研

生物工程

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 ...

医学

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 ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

癌症研究

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

一种三维球体模型研究肝细胞癌肿瘤-基质相互作用

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

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 ...

建立卵巢癌-网膜相互作用的 3D 离体模型

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

作者聚焦:用于研究癌症-脂肪微环境相互作用的高级离体模型 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

基于人网膜的卵巢癌腹膜转移离 体 模型

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

模拟卵巢癌多细胞球体行为动态三维微型腹膜

摘要

探索更多视频

此视频中的章节

微流控装置重新创建一个肿瘤微环境

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

异型三维

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

一种三维球体模型研究肝细胞癌肿瘤-基质相互作用

在实时的间皮细胞卵巢癌球体附件和入侵评估

基于人网膜的卵巢癌腹膜转移离体模型

基于人网膜的卵巢癌腹膜转移离体模型

基于人网膜的卵巢癌腹膜转移离体模型

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

模拟卵巢癌多细胞球体行为动态三维微型腹膜

摘要

探索更多视频

此视频中的章节

微流控装置重新创建一个肿瘤微环境

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

异型三维

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

一种三维球体模型研究肝细胞癌肿瘤-基质相互作用

在实时的间皮细胞卵巢癌球体附件和入侵评估

基于人网膜的卵巢癌腹膜转移离 体 模型

基于人网膜的卵巢癌腹膜转移离 体 模型

基于人网膜的卵巢癌腹膜转移离 体 模型

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

基于人网膜的卵巢癌腹膜转移离体模型

基于人网膜的卵巢癌腹膜转移离体模型

基于人网膜的卵巢癌腹膜转移离体模型