The authors want to thank Ms. Cornelia Heis for her excellent technical assistance in immunohistochemistry and preparation of chicken embryos. This work was supported by the Austrian Science Fund (FWF Grant No. P19552) and the European Union (EU FP7 project Optatio No: 278570).

根据奥地利法律，美国公共卫生服务禽流胚胎实验室动物福利办公室不被视为活的脊椎动物，直到实验动物福利hatching.The NIH办公室在这方面（HTTP提供了书面指导：// www.grants.nih.gov/grants/olaw/references/ilar91.htm和NIH出版号：06-4515）。 1.细胞培养和慢病毒转染<ol><li>培养的MM细胞系OPM-2，RPMI-8226，并从骨髓在RPMI1640培养基中的人类间质干细胞，补充有在存在10％牛胎儿小牛血清和100IU / ml青霉素，100微克/毫升链霉素和2mM谷氨酰胺的5％的CO 2在37℃。 </li><li>转染5×10 6的HEK 293FT细胞病毒包装结构（9微克）的DNA和3微克pLenti6 / V5 dest中eGFP的向量通过使用30微升脂质体转染试剂转染培养基（10毫升）中。 12小时后取出转染中，加入10 mL的DMEM培养基含10％小牛血清和1％非必需氨基酸（NEAA）。 </li><li> 5天后，游泳细胞（1000 XG，5分钟），离心后收集HEK293FT细胞的上清液。通过实时PCR测定病毒滴度为28别处描述。 </li><li>转染1×10 6个MM细胞与eGFP的慢病毒颗粒（1×10 5个颗粒）在24孔板中的完全生长培养基。 3天后，加入2微克/毫升瘟到培养基开始选择过程。对于市售的eGFP慢病毒，使用500微克/ ml新霉素的。 </li><li> 2周选择后，eGFP的簇表达MM细胞会出现;通过离心（1,000×g离心，5分钟）收集细胞，并扩大其作为OPM-2 的eGFP和RPMI-8226 eGFP的亚系进行实验（第2和3）。 </li></ol> 2. 3D-多发性骨髓瘤球体模型<ol><li>寒意胶原I型解决方案和10×DMEMØñ冰。 </li><li>混合1/10体积10​​×DMEM培养基进入胶原基质;添加NaOH水溶液（0.2 N），以中和酸性的胶原溶液以7.4的pH值;店胶原/冰介质溶液。 </li><li>转基因混合MM细胞系（OPM-2或的eGFP RPMI-8226 绿色荧光蛋白 ; 250,000球体，）与人类间质细胞（50,000个细胞/球体， 即 30微升滴）。 </li><li>在15ml试管（1000×g离心，5分钟）离心细胞混合物，冷制备胶原混合物（1mL）溶液加入到细胞沉淀拌匀（1,000微升尖）。 </li><li>立即吸取30微升胶原/细胞混合物（用100μl尖端）在一个24孔板上无菌石蜡薄膜，并允许细胞/胶原混合物在37℃（ 见图1A）聚合30分钟。 </li><li>覆盖的MM球体用含有1,10和100nM的硼替佐米1mL中培养基（参见图1B）。 </li><li>之后，在37℃72小时温育的，文件球状体由荧光立体显微镜（ 见图1C）。 </li><li>通过使用镊子具有宽扁钳口在反应管转移的每个球体的GFP的测量（ 见图1D）。 </li></ol> 3. 3D多发性骨髓瘤异种移植模型中的CAM <ol><li>孵化鸡蛋中为鸟卵的特别培养箱中在37℃和70％湿度下三天。 </li><li>此后，打开蛋和转移胚胎灭菌用乙醇，方形，10厘米的塑料称量船与细胞培养板盖和孵化“ 前卵 ”用于进一步六天，使得CAM是能够开发（参见图2A）。 </li><li>寒意胶原I型溶液和10×DMEM在冰上，混合1/10体积10​​×DMEM培养基到胶原基质，添加氢氧化钠（0.2 N），以中和酸性的胶原溶液以7.4的pH值;店胶原/冰介质溶液。 </li><li>混合转基因MM细胞系（OPM-2 的eGFP或RPMI-8226 绿色荧光蛋白 ; 250,000球体，）与人类间质细胞（50,000个细胞/球体）。 </li><li>离心细胞在15ml试管（1000×g离心，5分钟;对于每个试验化合物1小瓶），药物添加1mL中冷制备胶原混合物（在所需的工作浓度）的细胞沉淀并充分混合（1,000微升尖）。 </li><li>放置胶原滴在6孔颈部30分钟上的封口膜（各30微升），以允许细胞外基质的聚合在37℃。 </li><li>转移“onplants”来自步骤3.6与使用镊子到CAM的未处理表面（2厘米的距离胚胎）的9日龄鸡胚（4- onplants每个鸡胚，参见图2B） </li><li>经过5天的体内生长在蛋孵化器在37℃和70％的湿度，文件异种移植物通过荧光立体显微镜（ 见图2C）。受低温的冰箱5小时安乐死鸡胚胎在4℃。 </li><li>由眼科剪刀取出移植与下层组织CAM和镊子与宽阔平坦的下颌。它们用于GFP的测量（第4节， 见图2D）或血管和/或侵入的肿瘤细胞（第5）免疫组化分析。 </li></ol>通过ELISA 4.绿色荧光蛋白定量的蛋白质<ol><li>传送每个MM球体或切除异种移植入0.5毫升RIPA缓冲液含有200微克/毫升蛋白酶抑制剂。 </li><li>均质球体/异种移植物在冰上组织匀浆器。 </li><li>执行三个冷冻/解冻周期在液氮和37℃水浴中。 </li><li>离心机匀浆在4℃下20分钟（12,000克）和存储上清液。 </li><li>稀释样品中的ELISA试剂盒的测定缓冲液（200微升）1:20。测量GFP的水平通过使用生物素化抗GFP抗体商业GFP ELISA试剂盒，根据制造商的协议。 </li></ol> 5. Immunohi对血管和入侵肿瘤细胞stochemical分析<ol><li>切除修复与移植CAM面积在4％多聚甲醛O / N在4℃。 </li><li>放置固定异种移植到嵌入盒，并将它们与增加的梯度醇系（50％，70％，80％，95％的乙醇，二甲苯和石蜡;每个步骤60分钟）转移至组织包埋站。 </li><li>部异种移植物（5微米）通过使用台式旋转切片机。烘烤在56℃下在载玻片O / N石蜡切片。 </li><li> Deparaffinize部分由递减梯度酒精串联双蒸水（混合二甲苯，95％，80％，70％，50％的乙醇，双蒸水;每个步骤10分钟）。 </li><li>在水浴（95℃，20分钟）与抗原修复液进行抗原修复（柠檬酸盐缓冲液; pH值7.0;体积100微升）。 </li><li>阻断内源性过氧化物酶活性用100μl3％的H 2 O 2 /甲醇30分钟。 </li><li>在含PBS块段10％胎牛血清45分钟（体积100微升）。 </li><li>染色1小时，用100μl初级抗体（1微克/毫升）稀释在含有1％胎牛血清在RT PBS中。 </li><li>洗涤3次在PBS中后，孵育1与生物素化第二抗体（0.1微克/毫升）在含有1％胎牛血清在RT PBS中。 </li><li>洗涤3次在PBS中后由抗生物素蛋白/生物素复合物（ABC）和二氨基联苯胺（DAB）底物溶液，根据制造商的说明进行显色反应。 </li><li> 5.11。停止反应通过转移部分，以双蒸水，染液用苏木精和安装部分用合成封固剂。 </li></ol>

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(85), (2014).</li><li>Schuler, J., Ewerth, D., Waldschmidt, J., Wasch, R., Engelhardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+multiple+myeloma:+a+critical+appraisal.">Preclinical models of multiple myeloma: a critical appraisal.</a> Expert.Opin.Biol.Ther. 13, S111-S123 (2013).</li><li>Kirshner, 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=A+unique+three-dimensional+model+for+evaluating+the+impact+of+therapy+on+multiple+myeloma.">A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma.</a> Blood. 112 (7), 2935-2945 (2008).</li><li>Jilani, S. 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=Selective+binding+of+lectins+to+embryonic+chicken+vasculature.">Selective binding of lectins to embryonic chicken vasculature.</a> J.Histochem.Cytochem. 51 (5), 597-604 (2003).</li></ol>

作者有没有竞争经济利益

的新的治疗剂用于难治性MM的发展需要较少耗时且昂贵的体内系统评估人类MM细胞的对药物的敏感性。迄今为止，只有少数的体内系统可用于新的抗骨髓瘤治疗的临床前评价。他们都有自己的化合物库29的大规模筛选的限制。 目前最好的模型为人类MM细胞是高度免疫缺陷小鼠7,13,30和火鸡胚胎29。既SCID小鼠和禽流胚胎异种移植模型可用于研究的MM的生物学和?...

Multiple myeloma (MM) is characterized by proliferation of malignant plasma cells in the bone marrow, bone lesions and immunodeficiency 1. Although new treatment options such as proteasome inhibitors (bortezomib) and immune modulatory drugs (pomalidomide and lenalidomide) are available, MM still remains an incurable malignancy with a grim prognosis 2. The bad prognosis might be explained by the extraordinary heterogeneity of MM cell clones that contributes to variable responses to therapy, in particular under long time treatment and selection pressure of MM clones 3.
Preclinical testing of new drugs and their combinations in vitro and in vivo is a critical and time-consuming step for future drug development. Thus, useful in-vivo models of MM are required to gain a better understanding of the biology of the disease and to enable the discovery of new drugs. Actually, the best xenotransplantation models for hematological malignancies and therapeutics are immune-deficient mice, such as the severe-combined immunodeficient (SCID) mice 4-7, the non-obese diabetic/SCID (NOD/SCID) mice 8,9 or the &#946;-microglobulin-knockout NOD/SCID mice 10,11.
Although murine models of human MM in some aspects can resemble the phenotype of human disease, immune-deficient mice are inbred, therefore simulate only one individual response to a drug and costs are very high. Due to immunosuppression animals require special maintenance conditions and the engraftment of human MM in mice requires 6 weeks to 2 months 9,12, unless cells are grafted directly to the bone marrow using a technically demanding procedure with lower rates of animal survival 7,13. Therefore, new methods using stem-cell based organoid models 14, tissue engineering 15 or sophisticated 3D cell culture models 16 have been established. They will compete in the near future with classical animal experiments for preclinical drug testing, but cannot replace systemic toxicity tests in living organisms.
The chicken embryo has been demonstrated before to be a suitable organism for xenotransplantation of human cells and tissues due to lack of adaptive immune response until hatching 17-19. Moreover, each chicken embryo reflects an individual reaction to applied drugs or tumor cells due to genetic diversity within the chicken population. The chorioallantoic membrane (CAM) is a well-established system to study tumor-dependent angiogenesis 20-22. When solid tumors are grafted to the CAM, they display many characteristics of cancers in vivo, including proliferation, invasion, angiogenesis and metastasis 23-27. 
Based on the previous experience of our group with CAM xenograft models20,26,27, a human MM model was established that combines the advantage of a human 3D culture system with the model of ex ovo developing chicken embryos. This MM model system allows real time monitoring of MM growth progression, quantification of cell mass and preclinical drug testing.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RPMI-8226 cells</td>
			<td>DSMZ</td>
			<td>ACC 9</td>
			<td>STR profiled</td>
		</tr>
		<tr>
			<td>OPM-2 cells</td>
			<td>DSMZ</td>
			<td>ACC 50</td>
			<td>STR profiled</td>
		</tr>
		<tr>
			<td>Human mesenchymal stem cells&#160;</td>
			<td>PromoCell</td>
			<td>PC-C-12974</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293FT cells&#160;</td>
			<td>Invitrogen</td>
			<td>R700-07</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 Medium</td>
			<td>Sigma Aldrich</td>
			<td>R0883</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160; HyClone</td>
			<td>ThermoScientific</td>
			<td>SH30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glut- Pen- Strep solution</td>
			<td>Sigma</td>
			<td>G6784</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Medium</td>
			<td>Gibco</td>
			<td>31966</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Sigma Life Sciences</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection Medium/Opti-MEM&#160;</td>
			<td>Gibco</td>
			<td>51985</td>
			<td></td>
		</tr>
		<tr>
			<td>eGFP lentiviral particles</td>
			<td>GeneCopoeia</td>
			<td>LPP-EGFP-LV105</td>
			<td>Ready to use viral particles</td>
		</tr>
		<tr>
			<td>pLenti6/V5Dest6 eGFP vector</td>
			<td>Invitrogen</td>
			<td>PN 35-1271</td>
			<td>from authors</td>
		</tr>
		<tr>
			<td>ViralpowerTM packaging mix&#160;</td>
			<td>Invitrogen</td>
			<td>P/N 35-1275</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection reagent/ Lipofectamin 2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Invitrogen</td>
			<td>R210-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin</td>
			<td>Biochrom</td>
			<td>A2912</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen-Type1&#160; Rat Tail</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM powder</td>
			<td>Life Technologies</td>
			<td>Art.Nr. 10338582</td>
			<td></td>
		</tr>
		<tr>
			<td>plitidepsin</td>
			<td>Pharmamar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bortezomib</td>
			<td>LKT Lab., Inc.</td>
			<td>B5871</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">SPF-white hen eggs</td>
			<td rowspan="3">Charles River</td>
			<td rowspan="3"></td>
			<td rowspan="3">Fertilized&#160; white Leghorn&#160; chicken eggs</td>
		</tr>
		<tr>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Plastic weighing boats</td>
			<td rowspan="2">neoLab</td>
			<td rowspan="2">Art.Nr. 1-1125</td>
			<td rowspan="2">for ex-ovo culture</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Petridish square (Lids)</td>
			<td>Simport</td>
			<td>D210-16</td>
			<td>for ex-ovo culture</td>
		</tr>
		<tr>
			<td>RIPA Buffer (10x)</td>
			<td>Cell Signaling</td>
			<td>#9806</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Tablets</td>
			<td rowspan="2">Roche</td>
			<td rowspan="2">11 836 170 001</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Complete Mini EDTA-free</td>
		</tr>
		<tr>
			<td>GFP ELISA</td>
			<td>Cell Biolabs, Inc.</td>
			<td>AKR-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Histocette II</td>
			<td>Simport</td>
			<td>M493-6</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA&#160; 37%</td>
			<td>Roth</td>
			<td>7398.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Lonza</td>
			<td>BE17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolut</td>
			<td>Normapur</td>
			<td>20,821,321</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti-Histol</td>
			<td>Roth</td>
			<td>Art.Nr.6640.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Sigma</td>
			<td>A6330</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">SuperFrost Microscope Slides</td>
			<td>R. Langenbrinck&#160;</td>
			<td>Art.-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Labor- u. Medizintechnik</td>
			<td>03-0060</td>
		</tr>
		<tr>
			<td rowspan="2">DakoCytomation Wash Buffer 10x</td>
			<td rowspan="2">DakoCytomation</td>
			<td>Code-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>S 3006</td>
		</tr>
		<tr>
			<td rowspan="2">Target Retrieval Solution (10x)&#160; pH 6,1</td>
			<td rowspan="2">DAKO</td>
			<td>Code-Nr.</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>S 1699</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu ASMA clone 1A4</td>
			<td>DAKO</td>
			<td>M0851</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu CD138 clone MI15</td>
			<td>DAKO</td>
			<td>M7228</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Vimentin clone V9</td>
			<td>DAKO</td>
			<td>M0725</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Desmin clone D33</td>
			<td>DAKO</td>
			<td>&#160;M0760</td>
			<td></td>
		</tr>
		<tr>
			<td>m-a-hu Ki67&#160; clone MIB-1&#160;&#160;</td>
			<td>DAKO</td>
			<td>&#160;M7240</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated goat- anti-mouse IgG</td>
			<td>Vector Laboratories Inc.</td>
			<td>BA-9200</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain Elite ABC Kit</td>
			<td>Vector Laboratories Inc.</td>
			<td># PK-6100</td>
			<td></td>
		</tr>
		<tr>
			<td>FAST DAB Tablet Set.</td>
			<td>Sigma Biochemicals</td>
			<td># D4293</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#8217;s haemalaun solution</td>
			<td>Merck</td>
			<td>1,092,490,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti Histokitt</td>
			<td>Roth</td>
			<td>Art.Nr.6638.2</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bench top rotary microtome</td>
			<td>Thermo Electron, Shandon Finesse ME+</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue embedding station</td>
			<td>Leica, TP1020</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Egg-Incubator</td>
			<td>Grumbach&#160;</td>
			<td>BSS160</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo fluorescence microscope equipped with an connected with a digital camera (Olympus E410) and flexible cold light&#160;</td>
			<td>Olympus, SZX10</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Turrax&#160;</td>
			<td>IKA T10</td>
			<td></td>
			<td>Homogenizer</td>
		</tr>
	</tbody>
</table>

establishment of a human multiple myeloma xenograft model in the chicken to study tumor growth, invasion and angiogenesis

多发性骨髓瘤（MM），恶性浆细胞疾病，无法治愈的和新的药物都需要改善患者的预后。由于缺乏骨微环境和自动/旁分泌生长因子的人的MM细胞不易培养。因此，目前迫切需要建立适当的体外和体内培养系统来研究对人MM细胞新的治疗的作用。这里，我们提出一个模型，以在体外和体内生长的人类多发性骨髓瘤细胞在一个复杂的三维环境。 MM细胞系OPM-2和RPMI-8226被转染以表达该转基因GFP和培养在人类间质细胞和胶原的存在I型基质为三维球状体。此外，球状体接枝在鸡胚绒毛尿囊膜（CAM）和肿瘤生长通过立体声荧光显微镜监测。这两种型号允许新的治疗DRU研究GS在一个复杂的三维环境和肿瘤细胞块移植物在转基因特异性GFP-ELISA均化后进行量化处理。此外，主机和肿瘤细胞的侵袭进入下卧宿主组织的血管生成反应，可以每日通过立体显微镜监测和针对人肿瘤细胞（Ki-67的，CD138，波形蛋白）或宿主壁细胞覆盖血管免疫染色分析（结蛋白/ ASMA）。 总之，onplant系统允许研究在一个复杂的三维环境MM细胞生长和血管生成，使筛选靶向生存MM细胞的增殖和新的治疗的化合物。

在目标化合物的3D多发性骨髓瘤球体测定体外分析 由于在体外原代人MM细胞培养的限制，我们建立了新的三维体外培养模型为人类MM细胞系，利用细胞外生长基质和支持原代人骨髓间充质细胞从骨髓的（ 图1A，B）。 EGFP转基因MM细胞系允许可视化和MM肿瘤块的量化的三维生长球状体之后。两个MM细胞系OPM-2 的eGFP和RPMI-8...

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

人多发性骨髓瘤（MM）细胞需要间质细胞和细胞外基质成分为存活和增殖的支持性微环境。我们建立了与植入人类骨髓瘤和间质细胞体内鸡胚模型来研究癌症药物对肿瘤的生长，侵袭和血管生成作用。

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 ...

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12.9K 次观看.  Innsbruck Medical University. 人多发性骨髓瘤（MM）细胞需要间质细胞和细胞外基质成分为存活和增殖的支持性微环境。我们建立了与植入人类骨髓瘤和间质细胞体内鸡胚模型来研究癌症药物对肿瘤的生长，侵袭和血管生成作用。 