该<em>在OVO</em> CAM作为一个肉瘤Xenograftmodel的检测
Summary
该<em>在OVO</em>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。
Abstract
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。
Introduction
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。
Protocol
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。 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。 立即将材料在无菌小瓶含1000 U青霉素和1毫克/毫升?…
Representative Results
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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of Reagent | Company | Catalog Number |
| Cell Line Nucleofector Kit V | Amaxa | VCA-1003 |
| collagenase 2 solution (500 U/ml RPMI 1640) | Sigma Aldrich | C6885 |
| DMEM | Invitrogen | 41965-039 |
| DMSO | Sigma | D8418 |
| Dnase solution | Sigma Aldrich | DN25 |
| G418 | Invitrogen | 11811031 |
| Matrigel | Sigma-Aldrich | E1270 |
| mouse primary monoclonal antibody Ki67 | Dako Denmark | MIB-1 |
| Paraformaldehyde | Fluka | D76240 |
| PBS | Invitrogen | 20012019 |
| PBSD | Invitrogen | 14040083 |
| peGFP-C1 vector | Clontech | 632470 |
| Penicillin/streptomycin | Invitrogen | 15140163 |
| RPMI | Invitrogen | 22409-015 |
| Trypsin-EDTA solution | Invitrogen | 25300054 |
| Vybrant cell-labeling DiI | Lifetechnologies | 22885 |
| Name of Equipment | Company | Catalog Number |
| Countess Automated Cell Counter | Invitrogen | C10227 |
| digital color camera | Leica | DFC 340 FX |
| Digital Egg Incubator | Auto Elex Co | R-COM 50 |
| FACS | BD Biosciences | FACSAriaIII |
| Gentlemacs C-Tube | Miltenyi Biotech | 130-093-237 |
| Gentlemacs Dissociator | Miltenyi Biotech | 130-093-235 |
| Gentlemacs Dissociator User Manual containing h_tumor protocol | Miltenyi Biotech | |
| semipermeable adhesive film (Suprasorb F) | Lohmann&Rauscher | 20468 |
| stereo fluorescence microscope | Leica | M205 FA |
| Tissue-Tek Film automated Coverslipper | Sakura | 6400 |
| ultraView Universal DAB Detection Kit | Ventana Medical Systems Inc | 760-500 |
| Ventana Automated Slide Stainer | Ventana Medical Systems | Benchmark XT |
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Cite This Article
Sys, G. M., Lapeire, L., Stevens, N., Favoreel, H., Forsyth, R., Bracke, M., De Wever, O. The In ovo CAM-assay as a Xenograft Model for Sarcoma. J. Vis. Exp. (77), e50522, doi:10.3791/50522 (2013).