从SW1353软骨肉瘤细胞系细胞恳请教授的PCW Hogendoorn博士，荷兰莱顿大学教授J.博维。我们感谢的J. Mestach和G. Wagemans优秀的技术援助，G.德Bruyne专业绘图概述我们的协议。

参见图1的概述肿瘤材料 1。肿瘤样本的获取和准备的伦理委员会的批准，如果在病人的材料的使用是必要的，从病人取得的知情同意书。 <ol><li>嘉实代表材料（最小为1厘米3）干预的时间，无论是活检或切除的肉瘤。活检定义适当的地点使用动态对比度MRI。 </li><li>立即将材料在无菌小瓶含1000 U青霉素和1毫克/毫升链霉素DME​​M培养基。 </li><li>运输样品瓶立即（30 - 90分钟）到实验室。 </li></ol>所有下面的程序进行，在层流。 <ol start="4"><li>将瓶中内容物倾入无菌的陪替氏培养皿中。 </li><li>将肿瘤最大限度地1米的一小部分样品放入3用消毒的手术刀。删除所有钙化的部分，因为他们往往​​会削弱进一步的组织处理。 </li><li>随机取10个肿瘤移植应用CAM。 </li></ol> 2。肿瘤来源的单细胞悬浮液的制备持续时间：约3小时<ol><li>称取样品的其它组织。 </li><li>取约2 - 4克组织离解器管中，可能需要一个以上的管消化所有其它的肿瘤组织。 </li><li> 2 - 4克组织的消化，加2.5毫升胶原酶溶液（500 U / ml的RPMI 1640）和2.5毫升DNA酶溶液（22 KU / ml的RPMI 1640）。 </li><li>根据离解h_tumor协议处理该组织。这涉及到在CO 2培养箱中在37℃下保温2个循环的，而管轻轻摇动30分钟。 </li><li>通过150微米的细胞过滤过滤剩余的悬浮液。 </li><li>裂解液以1000rpm离心5英里名词</li><li>去除上清。 </li><li>将沉淀重悬于10ml的红细胞裂解缓冲液（ELB），允许与常规悬浮液温育10分钟。 </li></ol>注意：红细胞裂解液如下制造：加入0.037克EDTA，0.99克K 2 HPO 4，8.29克氯化铵到千毫升水产，使用10N的氢氧化钠将pH调节至7.3，过滤器在压力下通过0.22μm的过滤。贮存于4℃ <ol start="9"><li>加入25毫升的培养基（补充有10％胎牛血清，100 U / ml青霉素和100μg/ ml的链霉素的DMEM培养基），使反应停止。 </li><li>离心悬浮液，以1,000 rpm 5分钟。颗粒的颜色应该是白色。 </li><li>去除上清。 </li><li>加入5毫升的介质颗粒。通过70微米的细胞过滤，过滤悬浮液。 </li><li>通过自动细胞计数器计数活细胞/ ml的数量。 </li></ol> 3。 Ç的相关关系及细胞系电SAOS2骨肉瘤细胞（ATCC编号：HTB-85）表达增强型绿色荧光蛋白表达载体pEGFP-C1载体，使用的细胞株Nucleofector试剂盒V根据制造商的协议。建立稳定的细胞系，转染的细胞中选择G418（1毫克/毫升），连续4周线与先前建立的一种协议20。此外根据制造商的说明，通过荧光激活细胞分选（FACS）进行排序。 SW1353软骨肉瘤细胞株（ATCC编号：HTB-94）或新鲜制备肿瘤单细胞悬液的细胞标记，用红色荧光亲脂膜染色。 <ol><li>在经典的方法，用胰蛋白酶（0.5％重量/体积）乙二胺四乙酸（EDTA，0.2％重量/体积）溶液中除去细胞从培养瓶中。 </li><li>离心细胞悬浮液，以1,000 rpm 5分钟。 </li><li>删除supernatant。 </li><li>加入1 ml无血清培养基和5μl的DiI荧光（Vybrant红）/ 10 6个细胞。 </li><li>铝箔包装小瓶，并把它在10分钟，在37℃的CO 2培养箱</li><li>以1,000 rpm 5分钟，再次离心，去除上清。 </li></ol>绒毛尿囊膜试验 1。卵孵化<ol><li>保证95％的受精鸡蛋从当地的商业孵化的受精卵是必需的。 </li><li>受精卵在RT下，可以存储多达10天。 </li></ol>注：开始孵化前，仔细去除灰尘，羽毛和粪便是从蛋壳机械用纸巾，柔软的表面结构，而不是一个有一个粗略的干擦。用70％变性乙醇或其它任何清洁试剂擦拭鸡蛋的小鸡胚胎的存活率显着降低。 <ol start="3"><li>把鸡蛋放进incubATOR的一侧上，标记的上表面。在这一天的鸡蛋都放在孵化器被指定胚胎发育的第0天。 </li><li>设定的培养温度37.8℃，湿度控制在47％。确保“自动旋转”选项关闭。 </li></ol> 2。鸡蛋开幕时间：±60分钟，25个鸡蛋。 在发展的第3天，鸡蛋打开层流气流下。打开鸡蛋损害到CAM进一步发展阶段性成果，膜往往坚持到shell。红外线灯在操作过程中用于保持蛋温暖。为了提高不育，我们建议使用手套。 <ol><li>将鸡蛋的蛋杯，填划的一面朝上。 </li><li>通过去除两个毫升的蛋白水平降低的CAM通过18G的针的尖端处的蛋。如果针头钝射孔后几个蛋壳，更换针头。如果不是，用力过猛，也有需要充分发挥，造成损坏蛋黄或断裂的外壳。有时针阻塞的系带，其中的2个螺旋带，暂停的蛋黄蛋清。这个问题可以通过轻轻推入柱塞回落，直至该块被解决。如果被吸入到注射器蛋黄，更换的针和注射器。有时胚胎死亡后，此事件。如果除去蛋白量不足，则CAM将被损坏，当外壳被除去。 </li><li>在标记的上侧的外壳，以防止溢出壳粒子到CAM切割窗口的同时应用的半透性粘接薄膜。 </li><li>用小锥子在蛋的表面作一介绍孔。 </li><li>切为1cm 2：在外壳的窗口，使用一对无菌尖头手术剪。 </li><li>胚胎的跳动的心脏和毗邻船只可以观察到日E面的蛋黄。删除未受精的蛋或死胚。一个被中断的血管方面的特征死胚。 </li><li>密封窗口的半透性粘接薄膜。这是没有必要覆盖穿刺部位，在插入针的过程中，除非壳破裂。 </li></ol> 3。接种程序时间：±1小时，每细胞系在小鸡胚胎发育的第9天，卵层流气流下接种。 遍布的CAM的肿瘤细胞评价需要的细胞在接种过程中的有限表面的遏制。基底膜是一种细胞外基质（ECM）凝胶中经常使用的实验细胞培养条件从Engelbreth-Holm的群小鼠肉瘤。在冷却时，它是一种流体，但将进行热激活的聚合时，到第20 - 40℃，从而形成一个稳定的凝胶。中实验中，基底膜保持在一个盒子里含有融化的冰。 注：我们坚持不使用穿孔盖玻片。我们观察到接枝的癌细胞的粘附和生长到塑料圆盘，从而偏置膜本身上的细胞的生长潜力。 <ol><li>重悬细胞沉淀在冷却的ECM凝胶的浓度为10 6 cells/100微升ECM凝胶。保持这个解决方案上的冰融化。 </li><li>海关部分的半透膜，根据层流无菌手术的一对。如果粘接膜完全除去，这将导致胚胎死亡，由于污染的比例较大。 </li><li>触摸shell窗口的CAM正下方用无菌玻璃棒轻轻地，为了除去上皮细胞层。触摸CAM用无菌玻璃棒被证明是比使用手术刀的编号为11，这需要更多的轻便和经验的创伤小。可能会出现小出血。 </li><li>一肿瘤材料dding。 </li><li>对于肿瘤移植：轻轻放下一对无菌镊子的CAM组织到一块，不挤。 </li><li> ECM凝胶过程：4倍的25微升的细胞-ECM凝胶悬浮液加入到CAM，在彼此的顶部，使ECM凝胶聚合在应用程序之间的。以这种方式，包含癌细胞粘附斑块被创建。 </li><li>再次用一个半透胶粘剂膜密封外壳窗口。 </li></ol> 4。在CAM的成像和收获时间：2小时25个鸡蛋。 在小鸡胚胎发育的第16天，凸轮收获。下工作的层流是没有必要的。 <ol><li>一对手术剪，放大该窗口。确保不削减太低，否则将被损坏的CAM，导致受伤的血管出血。 </li><li>添加300 - 500微升的PBS D到CAM教派用吸管含有接种物的离子。如果没有荧光探针的使用，甲醛缓冲液可以用作使膜更硬，促进膜的切断。 </li><li>通过立体声荧光显微镜，配备一个数字彩色摄像机，使用GFP或的DSR滤波器，没有过滤器，使照片的CAM蛋。对于肿瘤移植，所有照片都没有过滤器。强烈建议从上述LED灯照明，在拍摄过程中（ 图2）。 </li><li>记录肿瘤细胞标记细胞迁移。肿瘤的边界可能是不规则磨损。分散的细胞可能是肿瘤边界附近。在一些样品中，可以观察到肿瘤细胞的行（ 图3）。 </li><li>无菌剪刀的一对切膜对壳躺在上面的边界。 </li><li>将收获的CAM在无菌培养皿中充满与PBS D或缓冲甲醛。 </li><li>的上侧和下侧的凸轮，与不带过滤器的照片。 </li><li>将膜与缓冲甲醛中至少72小时，在有标签的容器。 </li><li>嵌入凸轮的石蜡和准备作病理检查。小心切肿瘤移植在结节的中心，确保切割表面是平行于磁带盒的底部。如果没有，CAM和肿瘤移植物之间的相互作用将不可见。相同的策略适用于对ECM凝胶斑块：相对的面的切割刀片应垂直于斑块。 </li></ol> 5。组织学评价苏木精 - 伊红染色和Ki-67免疫组化进行了进一步澄清，如果需要的话。免疫组化法对福尔马林固定，石蜡包埋的组织切片厚度为3.5μm，使用自动幻灯片染色机根据制造商的指示。鼠标主单克隆抗体（克隆MIB-1，稀释1/100），Ki-67的使用。热诱导抗原活化检索使用细胞，书桌1，可视化，实现与通用的DAB检测试剂盒，根据制造商的说明。组织切片进行了Dehydratation使用自动盖片。 的的SAOS2和SW1353细胞系，Ki67的阳性和Ki67阴性细胞nuclei/100μm2的数量进行计数在三个区域：一个比较接近的CAM的边界，一个关闭的表面和一个在中心的ECM凝胶牌匾。这个过程重复每个Ki-67的染色后的载玻片的6倍。指数增殖细胞计数的总数除以Ki67的阳性细胞的数量为每个幻灯片确定。 坏死的定义是在细胞核内染色质的微细分散的损失，伴随着法迪纳克的不同单元格边距。异种移植，坏死得分完整，部分（通常在表面），或缺席。由观察到CAM的肿瘤细胞的浸润是指肿瘤细胞的CAM中胚层内，被评价为存在或不存在。 三个项目都取得了主机的行为。成纤维细胞浸润被定义为拉长的细胞，使CAM和入侵移植瘤/斑块，得分为存在或不存在。向内生长增殖的小鸡容器，其特征在于成核的小鸡红细胞的存在下，可观察到血管向内生长。

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(33), e1620 (2009).</li><li>Balke, M., Neumann, 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=Morphologic+characterization+of+osteosarcoma+growth+on+the+chick+chorioallantoic+membrane.">Morphologic characterization of osteosarcoma growth on the chick chorioallantoic membrane.</a> BMC Research Notes. 3, 58 (2010).</li><li>Hendrix, A., Maynard, D., 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=Effect+of+the+secretory+small+GTPase+Rab27B+on+breast+cancer+growth,+invasion,+and+metastasis.">Effect of the secretory small GTPase Rab27B on breast cancer growth, invasion, and metastasis.</a> Journal of the National Cancer Institute. 102, 866-880 (2010).</li><li>Ausprunk, D., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+of+normal+and+neoplastic+tissues+grafted+to+the+chick+chorioallantois:+role+of+host+and+preexisting+graft+vessels.">Vascularization of normal and neoplastic tissues grafted to the chick chorioallantois: role of host and preexisting graft vessels.</a> American Journal of Pathology. 79, 597-618 (1975).</li><li>Lokman, N. A., Elder, A. S. F., Riciardelli, C., Oehler, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+Chorioallantoic+membrane+(CAM)+Assay+as+an+in+vivo+model+to+study+the+effect+of+newly+identified+molecules+on+ovarian+cancer+invasion+and+metastasis.">Chick Chorioallantoic membrane (CAM) Assay as an in vivo model to study the effect of newly identified molecules on ovarian cancer invasion and metastasis.</a> International Journal of Molecular Sciences. 13, 9959-9970 (2012).</li><li>Vargas, A., Zeisser-Labou&#232;be, M., Lange, N., Gurny, R., Delie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chick+embryo+and+its+chorioallantoic+membrane+(CAM)+for+the+in+vivo+evaluation+of+drug+delivery+systems.">The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems.</a> Advanced Drug Delivery Reviews. 59, 1162-1176 (2007).</li><li>Hagedorn, M., Javerzat, 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=Accessing+key+steps+of+human+tumor+progression+in+vivo+by+using+an+avian+embryo+model.">Accessing key steps of human tumor progression in vivo by using an avian embryo model.</a> Proceedings of the National Academy of Sciences U.S.A. 102, 1643-1648 (2005).</li><li>De Wever, O., Hendrix, 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=Modeling+and+quantification+of+cancer+cell+invasion+through+collagen+type+I+matrices.">Modeling and quantification of cancer cell invasion through collagen type I matrices.</a> International Journal of Developmental Biology. 54, 887-896 (2010).</li><li>Albini, A., Benelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemoinvasion+assay:+A+method+to+assess+tumor+and+endothelial+cell+invasion+and+its+modulation.">The chemoinvasion assay: A method to assess tumor and endothelial cell invasion and its modulation.</a> Nature Protocols. 2, 504-511 (2007).</li><li>Hanahan, D., Coussens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessories+to+the+crime:+functions+of+cells+recruited+to+the+tumor+microenvironment.">Accessories to the crime: functions of cells recruited to the tumor microenvironment.</a> Cancer Cell. 21, 309-322 (2012).</li></ol>

作者什么都没有透露。

接种和收获的时间时序进行SAOS2天接种ECM凝胶（36的CAM）和胚胎发育天5和10之间变化。 孵化前9天，CAM是不能持续大到足以支持我们采用ECM凝胶。收获时，肿瘤细胞有时不得不从更深的CAM检索，并在蛋白或CAM一些ECM凝胶样品躺在松动。在第5和第6天，这是很难避免把小鸡畸形或死亡，导致肿瘤细胞鸡胚。从第9天，在CAM被完全覆盖的大部分鸡蛋的窗口下面的表?...

肉瘤是一种罕见的肿瘤治疗抵抗1,2由于死亡率高的结缔组织。患者的生存率进展受阻低的年发病，其广泛的多样性，以及肉瘤细胞的事实，据报道， 在体外难以培养3,4。 使用培养细胞的临床前治疗评价已经表明，新的，显然是在体外的活性分子并不总是反映在临床上的结果。此外，揭示了基因表达阵列的基因畸变并不总是与个别病人5-7的肿瘤行为特征。为了试图解决这些问题，已经获得了个性化医疗的重要性，这也反映在增加搜索异种移植模型8-12。 在体内试验中的一种反映复杂的相互作用的优点是与c的相关关系在固体肿瘤细胞与宿主组织环境，必要的癌细胞增殖和侵袭13。目前，我们研究使用绒毛膜尿囊膜法（CAM法）作为一个可重复的异种移植物模型肉瘤14,15。本试剂盒被广泛用于肿瘤血管生成的研究16,17。然而，在文献中，我们已经发现这个实验的不同的协议，而其他的研究观察到的生长或血管生成的显着性差异，根据不同的协议18,19。 在这篇文章中，我们探讨不同的CAM检测条件对细胞行为的肿瘤移植，肿瘤来源的单细胞悬液和既定的肉瘤细胞培养的效果。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Cell Line Nucleofector Kit V</td>
			<td>Amaxa</td>
			<td>VCA-1003</td>
		</tr>
		<tr>
			<td>collagenase 2 solution (500 U/ml RPMI 1640)</td>
			<td>Sigma Aldrich</td>
			<td>C6885</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41965-039</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
		</tr>
		<tr>
			<td>Dnase solution</td>
			<td>Sigma Aldrich</td>
			<td>DN25</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Invitrogen</td>
			<td>11811031</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Sigma-Aldrich</td>
			<td>E1270</td>
		</tr>
		<tr>
			<td>mouse primary monoclonal antibody Ki67</td>
			<td>Dako Denmark</td>
			<td>MIB-1</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fluka</td>
			<td>D76240</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>20012019</td>
		</tr>
		<tr>
			<td>PBSD</td>
			<td>Invitrogen</td>
			<td>14040083</td>
		</tr>
		<tr>
			<td>peGFP-C1 vector</td>
			<td>Clontech</td>
			<td>632470</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15140163</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Invitrogen</td>
			<td>22409-015</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Invitrogen</td>
			<td>25300054</td>
		</tr>
		<tr>
			<td>Vybrant cell-labeling DiI</td>
			<td>Lifetechnologies</td>
			<td>22885</td>
		</tr>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>C10227</td>
		</tr>
		<tr>
			<td>digital color camera</td>
			<td>Leica</td>
			<td>DFC 340 FX</td>
		</tr>
		<tr>
			<td>Digital Egg Incubator</td>
			<td>Auto Elex Co</td>
			<td>R-COM 50</td>
		</tr>
		<tr>
			<td>FACS</td>
			<td>BD Biosciences</td>
			<td>FACSAriaIII</td>
		</tr>
		<tr>
			<td>Gentlemacs C-Tube</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
		</tr>
		<tr>
			<td>Gentlemacs Dissociator</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
		</tr>
		<tr>
			<td>Gentlemacs Dissociator User Manual containing h_tumor protocol</td>
			<td>Miltenyi Biotech</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>semipermeable adhesive film (Suprasorb F)</td>
			<td>Lohmann&amp;Rauscher</td>
			<td>20468</td>
		</tr>
		<tr>
			<td>stereo fluorescence microscope</td>
			<td>Leica</td>
			<td>M205 FA</td>
		</tr>
		<tr>
			<td>Tissue-Tek Film automated Coverslipper</td>
			<td>Sakura</td>
			<td>6400</td>
		</tr>
		<tr>
			<td>ultraView Universal DAB Detection Kit</td>
			<td>Ventana Medical Systems Inc</td>
			<td>760-500</td>
		</tr>
		<tr>
			<td>Ventana Automated Slide Stainer</td>
			<td>Ventana Medical Systems</td>
			<td>Benchmark XT</td>
		</tr>
	</tbody>
</table>

the in ovo cam-assay as a xenograft model for sarcoma

肉瘤是一种非常罕见的疾病，在本质上是异质的，都阻碍了发展的新疗法。肉瘤患者的理想人选后分层的个性化医学，解释目前在开发这种疾病的一个重现性好，成本低的异种移植模型的兴趣。鸡胚绒毛尿囊膜，是一种天然的免疫缺陷宿主能够维持嫁接的组织和细胞，没有种属特异性的限制。此外，它很容易访问，操纵和使用光学和荧光立体显微镜成像。组织学进一步允许异型细胞相互作用的详细分析。 该协议详细描述了OVO绒毛尿囊膜移植肉瘤派生的新鲜肿瘤组织，其单细胞悬液，和永久和瞬态荧光标记的建立肉瘤细胞株（系Saos-2和SW1353）。小鸡存活大鼠上课是高达75％。该模型用于研究移植（活力，Ki67的增殖指数，坏死，浸润）和主机（成纤维细胞浸润，血管长入）的行为。对于单细胞悬液的本地化嫁接，ECM凝胶提供了显着的优势，在惰性容器材料。 Ki67增殖指数的距离的凸轮表面的细胞和CAM上，后者确定的治疗产品的另外的时间框架中的应用的持续时间有关。

在CAM的评价肿瘤移植变得附着到CAM（ 图2A）。单细胞悬液经常从病人的材料显示干燥，微微凸起的牌匾（ 图2D）。切除后的CAM，标志着起皱发生膜（ 图2E和2F）。 对于商业细胞株的牌匾变得更加不透明，表明细胞的增殖。 ECM凝胶在不同的细胞系有明显的增长模式上的CAM。 SAOS2形成一个蔓延斑块，表面从接?...

Watch this Scientific Journal Video about The In ovo CAM-assay as a Xenograft Model for Sarcoma at JoVE.com

The In ovo CAM-assay as a Xenograft Model for Sarcoma

该<em&gt;在OVO</em&gt;绒毛尿囊膜（CAM）接枝源性肉瘤的新鲜肿瘤组织中，其单细胞悬液，并永久和瞬态荧光标记的建立肉瘤细胞株。该模型用于研究移植（活力，Ki67的增殖指数，坏死，浸润）和主机（成纤维细胞浸润，血管长入）的行为。

该<em&gt;在OVO</em&gt; CAM作为一个肉瘤Xenograftmodel的检测

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

the-in-ovo-cam-assay-as-a-xenograft-model-for-sarcoma

Research

JoVE Journal

Medicine

The In ovo CAM-assay as a Xenograft Model for Sarcoma

25.3K 次观看.  Ghent University Hospital. 该在OVO绒毛尿囊膜（CAM）接枝源性肉瘤的新鲜肿瘤组织中，其单细胞悬液，并永久和瞬态荧光标记的建立肉瘤细胞株。该模型用于研究移植（活力，Ki67的增殖指数，坏死，浸润）和主机（成纤维细胞浸润，血管长入）的行为。

视频: The In ovo CAM-assay as a Xenograft Model for Sarcoma

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

科研

医学

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

一个简单的指南螺杆颅内移植瘤小鼠的研究方法

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

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 ...

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

谷氨酸和缺氧作为离体脊椎动物视网膜应力模型

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

该<em&gt;卵内</em&gt;鸡胚尿囊膜（CAM）含量为肝癌的有效异种移植模型

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

油酸注射液在猪急性呼吸窘迫综合征模型中的研究

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...