Name: isolation and culture expansion of tumor-specific endothelial cells
Uploaded: 2015-10-14T15:00:00.000+00:00
Duration: 10 min 15 s
Description: Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

ACD is supported by a grant from the National Institute of Health (R01-CA177875). LX is a fellow in the HHMI-funded translational medicine program at UNC Chapel Hill. JVM is supported by a T32 pre-doctoral fellowship from the Integrative Vascular Biology Program at UNC Chapel Hill. We thank Clayton Davis for assistance with confocal microscopy.

以下协议进行根据实验动物医学系在北卡罗莱纳大学教堂山分校制定的准则。 1.准备以下材料和试剂开始之前<ol><li>通过补充400毫升低葡萄糖制备乳油媒体（1克/升D-葡萄糖或LG）的Dulbecco氏改良Eagle氏用50ml热灭活的胎牛血清，将50ml的Nu-血清IV，加入5ml抗菌 - 抗霉菌培养基（DMEM），和hFGF，血管内皮生长因子，表皮生长因子，R3-IGF-1，和肝素部件从商业试剂盒。 </li><li>制成500毫升之FACS缓冲液（0.5％BSA和2mM EDTA的PBS）;过滤通过无菌0.22微米的过滤杯。 </li><li>消毒或消毒解剖板。 </li><li>消毒清扫针，手术剪，和解剖。 </li></ol> 2. EC隔离（第1天，约5小时） <ol><li>安乐死小鼠与二氧化碳或其它方法兼容机智小时机构动物护理和使用委员会（IACUC）政策。 注:在大小不同的多个乳腺肿瘤（5 - 直径15mm），在一个单一的转基因小鼠可能发展，如C3标签，一定要收获所有的人都对TEC隔离。如果使用的是肿瘤的原位移植的小鼠，池二点五十八1厘米3的肿瘤为一个单一的EC隔离。在这里，我们使用乳腺肿瘤作为示范，但协议可以被修改为其它类型的肿瘤。 </li><li>通过喷洒或擦拭鼠标腹侧与足量的75％V / V乙醇消毒鼠标。 </li><li>切除肿瘤的一对使用无菌技术在无菌罩或超净工作台剪刀和解剖的。 <ol><li>伸出和引脚小鼠的四肢在解剖板。让中线腹侧切开用剪刀无需打开腹膜。皮肤并朝向乳腺肿瘤，其中位于腹膜之间解剖横向。不要打开腹膜。 </li><li>消费税只能从乳腺肿瘤组织，离开了正常乳腺的利润。仔细修剪过的非肿瘤组织如皮肤和肌肉，然后将解剖的肿瘤在锥形管中，冰上静置30 ml的LG-DMEM中。 </li></ol></li><li>把肿瘤样本，以组织培养罩; 2次 - 用无菌LG-DMEM 1洗净组织。 </li><li>从锥形管到无菌组织培养培养皿转移的肿瘤，在盘中加2ml LG-DMEM中，并剁碎有一对无菌剪刀成片&lt;5毫米。 </li><li>添加5胶原酶毫升（股票= 2毫克/毫升在Hank氏平衡盐溶液，在下文HBSS）中，1毫升分散酶（股份=在HBSS 2.5 U / ml）和75微升脱氧核糖核酸酶（股份= 1mg / ml的在PBS中）放入培养皿。总成交量为现在〜10毫升</li><li>从培养皿转移胶原酶/组织混合于组织离解器管和在组织离解器60秒运行两次（预先设定的离解公关ogram的离解:1270总发每运行）。孵育在37℃下振荡光在振荡器上75分钟。 </li><li>过滤通过100微米的细胞过滤消化的组织在一个50ml锥形管中。冲洗过滤用5ml FACS缓冲液洗任何剩余的细胞。旋在280×g离心5分钟，小心地吸出上清液，而不干扰细胞沉淀。 </li><li>稀释1毫升库存RBC裂解缓冲液（10倍）的9ml无菌水。裂解红血细胞，用10ml的裂解缓冲液（1×），并立即自旋5分钟，在280×g下。注:此步骤可以，如果血少是可见的被跳过。 </li><li>重悬在10ml的FACS缓冲液。混合10微升细胞悬浮液与10微升台盼蓝的，并使用血球计数活细胞。 </li><li>重悬的细胞在约10 7个细胞/ 100微升。加10微升每100微升细胞悬浮液的FcR块，并在冰上孵育10分钟。 </li><li>根据添加大鼠抗小鼠PE标记的CD31抗体。 表 1冰上孵育10 - 15分钟，并轻弹管偶尔。 </li><li>加入10毫升FACS缓冲液到管和旋转，在280×g离心5分钟。小心地取出上清液，并用5毫升FACS缓冲液再清洗细胞沉淀。离心以沉淀细胞。吸上清而不会破坏细胞沉淀。 </li><li> 根据表2加入FACS缓冲液和抗PE微珠冰上孵育10 - 15分钟。弗里克管偶然。 </li><li>加入10 mL FACS缓冲液和自旋样品在280×g离心5分钟;用5ml FACS缓冲液和离心再次洗一次。吸上清而不干扰沉淀。 </li><li>把体积为300微升FACS缓冲液。通过35微米的细胞过滤皑皑管5分钟旋转280×g的。使用2管和更大的体积，如果需要的。 </li><li>设置磁性multistand和磁列在罩，一柱连接到分离器和平衡柱用2ml FACS缓冲液。 </li><li> AspiratE中的上清，重悬在0.5细胞沉淀 - 1毫升FACS缓冲液。 </li><li>通过平衡的磁列通过细胞悬液。 </li><li>用2ml FACS缓冲液洗涤柱3次，并在一个15毫升管（FT级分）收集流过（FT）。 </li><li>取柱关闭隔板和用2ml FACS缓冲液洗脱入另一个15ml试管（洗脱级分）。用柱塞，以确保所有单元都断列。重复洗脱两次用2ml FACS每次缓冲。 </li><li>旋洗脱液在280×g离心5分钟。 </li><li>去除上清，悬浮颗粒在10毫升EC媒体。 </li><li>等分洗脱液级分（〜6毫升）中成10厘米明胶包被的培养皿。对了三片1 立方厘米的肿瘤，板至少在四个板块洗脱细胞。替代地，板洗脱的细胞在不同浓度的多块板（例如，种子0.5毫升，1毫升，1.5毫升，和3ml洗脱液为四个板），以确保一t个最低一个板块稀疏接种洗脱细胞。检查附着的细胞的汇合是在〜1％，第二天（即，约1.0 - 2.0×10 5附着的细胞）。 注:细胞需要被镀稀疏以便乳油菌落可以形成而不被污染的其它细胞类型。 </li><li>平板FT分数1个10 cm培养皿，让细胞恢复O / N。检查板为80 ​​- 100％，第二天汇合。冻结向下细胞（-80℃）中在低温管250微升的细胞冷冻培养基，并将它们存储在液氮中的第二天。注意:FT部分可在以后的阶段和/或作为阴性对照为隔离EC克隆的乳油的基因表达分析可用于肿瘤细胞的分离。 </li><li>更换介质每2 - 3天。菌落开始形成后7 - 10天。小EC殖民地可以早一日3.标记上盘底部的细尖标记的菌落鉴定。 </li><li>刮去非特定的CELLS周围所识别的菌落用无菌200μl的pippet小费。 </li></ol> 3.殖民地选择使用克隆环<ol><li>更换介质每2 - 3天。 7天 - EC纯度可以通过低密度脂蛋白（DII-AC-LDL）增加约5进行检查。加入50微升的LDL每10 ml的EC平台，孵育3 - 荧光显微镜下检查细胞前4小时。 注:多个低密度脂蛋白+ EC克隆可以在〜7个一天观察。 </li><li>开始收获乳油菌落，当他们到达直径的3 - 5毫米大小。 （选择一个都挤满了小LDL +细胞最好的结果大殖民地。） </li><li>收获前乳油用克隆环，预涂几6孔板用0.5％明胶。抽吸明胶，加2ml EC培养基到每个孔中，并且直到需要保持所述板在培养箱中。 </li><li>刮去非欧共体对殖民地的边缘，以确保没有其他类型的细胞将在克隆环内被困。 </li><li>使用相差显微镜（4X或10X的目标），轮廓用细尖笔在培养皿底部的含EC的殖民地区。 </li><li>用10ml的PBS洗涤板和吸移当离开一个非常薄的层的PBS中的板。 （重要提示:少量（约0.5毫升）的PBS将保持细胞克隆环过程中存活;另外，组​​织粘合剂需要水粘合。） </li><li>选择适当大小的克隆环。用一双解剖镊子拿起一个环，并用10微升枪头均匀涂抹少量的组织粘合剂到克隆环。 注:组织粘合剂对克隆环只使用少量（〜0.2微升的小环），并确保组织粘合剂分散均匀地绕在底面，以保证良好的密封性。过多的组织粘合剂产生热量，并形成薄膜，可能会杀死细胞。 </li><li>放置克隆环比EC殖民地。轻轻按下克隆环粘上里纳克到板。确保殖民地粘合环之前不会干涸。 </li><li>立即吸管25微升酶细胞剥离溶液进入克隆环孵育约1分钟或直至细胞松散附着。 </li><li>吸管中的细胞克隆环滴加入6孔板含有预热的EC介质的一个孔中。 （重要提示:不要摇晃板分散细胞; EC喜欢在紧张的集群发展。）50洗净的克隆环 - 100微升EC媒体收集尽可能多的细胞成为可能，并且所有洗涤转移到相同的6孔。 </li><li>如果在10 cm培养皿一些群体过小的收获，加入10 mL新鲜的媒体，让菌落长出了几天，然后再次重复克隆环的过程。 </li><li>生长在6孔板收获菌落直到80 - 100％汇合，并转移到细胞3个孔一个6孔板，扩大它们在10厘米组织培养皿之前。刮去污染物细胞。重复一轮又一轮的克隆环过程（步骤3.5 - 3.10），如果有必要的。保持细胞相对汇合（〜60 - 70％）扩大时。欧盟可能会停止，如果镀过于稀疏增长。 </li><li>表征乳油通过FACS，染色，PCR等。注意，语-AC-LDL是荧光灯和与PE或其他荧光抗体用于FACS可能会干扰。 </li></ol>

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Res. 99 (8), 861-869 (2006).</li><li>Wylie-Sears, J., Aikawa, E., Levine, R. A., Yang, J. -. H., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitral+valve+endothelial+cells+with+osteogenic+differentiation+potential.">Mitral valve endothelial cells with osteogenic differentiation potential.</a> Arterioscler. Thromb. Vasc. Biol. 31 (3), 598-607 (2011).</li><li>Ginsberg, 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=Efficient+direct+reprogramming+of+mature+amniotic+cells+into+endothelial+cells+by+ETS+factors+and+TGF&#946;+suppression.">Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGF&#946; suppression.</a> Cell. 151 (3), 559-575 (2012).</li><li>Sapino, 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=Expression+of+CD31+by+cells+of+extensive+ductal+in+situ+and+invasive+carcinomas+of+the+breast.">Expression of CD31 by cells of extensive ductal in situ and invasive carcinomas of the breast.</a> J. Path. 194 (2), 254-261 (2001).</li><li>Maddaluno, 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=EndMT+contributes+to+the+onset+and+progression+of+cerebral+cavernous+malformations.">EndMT contributes to the onset and progression of cerebral cavernous malformations.</a> Nature. 498 (7455), 492-496 (2013).</li><li>Cooley, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+signaling+mediates+endothelial-to-mesenchymal+transition+(EndMT)+during+vein+graft+remodeling.">TGF-&#946; signaling mediates endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling.</a> Sci. Transl. Med. 6 (227), 227ra34-227ra34 (2014).</li><li>Garcia, 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=Tie1+deficiency+induces+endothelial-mesenchymal+transition.">Tie1 deficiency induces endothelial-mesenchymal transition.</a> EMBO Rep. 13 (5), 431-439 (2012).</li><li>Xiao, 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=Tumor+endothelial+cells+with+distinct+patterns+of+TGF&#946;-driven+endothelial-to-mesenchymal+transition.">Tumor endothelial cells with distinct patterns of TGF&#946;-driven endothelial-to-mesenchymal transition.</a> Cancer Res. 75 (7), 1244-1254 (2015).</li><li>Kusumbe, A. P., Ramasamy, S. K., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+angiogenesis+and+osteogenesis+by+a+specific+vessel+subtype+in+bone.">Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone.</a> Nature. 507 (7492), 323-328 (2014).</li><li>Wang, 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=Identification+of+a+clonally+expanding+haematopoietic+compartment+in+bone+marrow.">Identification of a clonally expanding haematopoietic compartment in bone marrow.</a> EMBO J. 32 (2), 219-230 (2012).</li><li>Sawamiphak, 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=Ephrin-B2+regulates+VEGFR2+function+in+developmental+and+tumour+angiogenesis.">Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis.</a> Nature. 465 (7297), 487-491 (2010).</li><li>Chi, J. -. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+diversity+revealed+by+global+expression+profiling.">Endothelial cell diversity revealed by global expression profiling.</a> Proc. Natl. Acad. Sci. 100 (19), 10623-10628 (2003).</li><li>Nolan, D. 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=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev. Cell. 26 (2), 204-219 (2013).</li><li>Ingram, D. 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=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105 (7), 2783-2786 (2005).</li></ol>

The authors have nothing to disclose.

由于获 ​​得纯TEC初级培养物，许多体外研究替代TEC与市售的EC系或原乳油例如人脐静脉EC（HUVEC）13的困难。然而，从正常组织这些EC人群可能只能作为TEC从正常的同行明显不同的代理。例如，TEC表型上和功能异常在体内和一些这些异常可能是可透射体外 14-18。 TEC有异常生长，迁移和分化的能力，并与CD31 +肿瘤细胞可能聚结形成血管样结构16,19,20。...

内皮细胞（EC）是实体肿瘤的发展过程中是至关重要的。从休眠中的肿瘤血管生成开关来传播和远处转移的播种开始，欧共体形成提供血液，氧气管道，和营养，以维持肿瘤生长1。作为最近提出，EC也有灌注无关的功能，并形成一个支持癌症干细胞和其他肿瘤基质细胞2-5的生长利基。因此，高纯度的肿瘤特异性EC（TEC） 进行体外培养允许常规功能研究，将揭示介导肿瘤血管生成和肿瘤细胞串扰新型的分子机制的光。 EC是高度专业化的视原产6的组织。由于不同的肿瘤类型的异质性和肿瘤微环境，TEC也可以显示反映肿瘤特异性专业化ö独特的功能f显示血管。例如，有在TEC中的基因表达签名来自不同类型或肿瘤7,8-牌号分离引人注目变性。然而，频繁的共纯化非欧共体，特别是肿瘤相关的成纤维细胞和肿瘤细胞，与TEC的可混淆的全基因组表达分析。这些不需要的细胞类型是依赖于长期的 TEC培养体外扩增研究，特别是有问题的。 这里介绍的是，始终从生产肿瘤和其他组织的纯EC文化高保真方法。继欧盟分数和去除共纯化的非欧共体的免疫柱富集，一个额外的克隆环一步捕捉到纯粹的欧共体殖民地使用9。各菌落可以在培养物中多次传代而不会污染非EC中的出现进行扩展。这种方法也产生多重EC克隆从单一孤立的过程，这是理想的内切的研究thelial异质性。另外，它表明，CDH5 CRE:ZsGreen升/秒/升报告小鼠是一种有价值的工具，用于产生"命运映射"和擦掉标记EC其中保持ZsGreen荧光培养10。有了小幅调整方案，这种方法应该适应不同的肿瘤类型或正常组织。

<table><tbody><tr><td>Antibiotic-Antimycotic&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s medium (1 g/L D-glucose) (LG-DMEM)</td><td>Gibco</td><td>11885-084</td><td></td></tr><tr><td>EGM-2 Bullet Kit&amp;nbsp;</td><td>Lonza</td><td>CC4176</td><td>Not all components used</td></tr><tr><td>Fetal bovine serum (Hyclone)</td><td>Thermo Scientific</td><td>SH30071.03</td><td>Heat inactivated at 56 &amp;deg;C for 30 min</td></tr><tr><td>Nu-Serum IV</td><td>Corning</td><td>CB-51004</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBBS)</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>FACS buffer&amp;nbsp;</td><td></td><td></td><td>0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 &amp;mu;m filter</td></tr><tr><td>75% v/v ethanol for disinfection</td><td></td><td></td><td></td></tr><tr><td>Anti-PE microbeads&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-048-801</td><td></td></tr><tr><td>Bovine serum albumin (BSA) fraction V, 7.5%</td><td>Gibco</td><td>15260-37</td><td></td></tr><tr><td>Cell freezing media (Bambanker)</td><td>Wako Chemicals</td><td>302-14681</td><td></td></tr><tr><td>Collagenase type II&amp;nbsp;&amp;nbsp;</td><td>Worthington Biochemical</td><td>LS004176</td><td>Make stock concentration 2 mg/ml in HBSS</td></tr><tr><td>Deoxyribonuclease I (DNase)</td><td>Worthington Biochemical</td><td>LS002004</td><td>Make stock concentration 1 mg/ml in PBS</td></tr><tr><td>Dil-Ac-LDL</td><td>Biomedical Technologies</td><td>BT-902</td><td></td></tr><tr><td>EDTA, 0.5M, pH 8.0</td><td>Cellgro</td><td>46-034-CL</td><td></td></tr><tr><td>Enzymatic cell detachment solution (Accutase)</td><td>Sigma-Aldrich</td><td>A6964-100ML</td><td></td></tr><tr><td>Gelatin, 2% in water, tissue culture grade</td><td>Sigma-Aldrich</td><td>G1393-100ML</td><td>Dilute in PBS to make 0.5% gelatin solution</td></tr><tr><td>Mouse FcR Blocking Reagent&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-092-575</td><td></td></tr><tr><td>Neutral protease (Dispase)</td><td>Worthington Biochemical</td><td>LS02104</td><td>Make stock concentration 2.5 U/ml in HBSS</td></tr><tr><td>PE-rat anti-mouse CD31 antibody</td><td>BD Pharmingen</td><td>553373</td><td></td></tr><tr><td>RBC lysis buffer (BD Pharm Lyse)</td><td>BD Pharmingen</td><td>555899</td><td></td></tr><tr><td>Sterile water</td><td></td><td></td><td></td></tr><tr><td>Trypan blue, 0.4 %&amp;nbsp;</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>10 mm tissue culture dishes</td><td>Corning</td><td></td><td></td></tr><tr><td>15 ml conical tubes (sterile)</td><td>Corning</td><td></td><td></td></tr><tr><td>50 ml conical tubes (sterile)&amp;nbsp;</td><td>Corning</td><td></td><td></td></tr><tr><td>6-well tissue culture plates</td><td>Corning</td><td></td><td></td></tr><tr><td>Tissue-dissociator tubes (gentleMACS) C tubes)&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-093-237</td><td></td></tr><tr><td>Cell Separator&amp;nbsp; (MidiMACS)</td><td>Miltenyi Biotech</td><td>130-042-302</td><td></td></tr><tr><td>Cell strainer 100 &amp;mu;m&amp;nbsp;</td><td>Corning</td><td>352360</td><td></td></tr><tr><td>Cloning rings (assorted sizes)</td><td>Bel-Art Products</td><td>378470000</td><td></td></tr><tr><td>Cryotubes</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Dissecting board</td><td></td><td></td><td>Sterilize or disinfect with 75% v/v ethanol before use&amp;nbsp;</td></tr><tr><td>Dissecting forceps and scissors</td><td></td><td></td><td>Sterilize before use&amp;nbsp;</td></tr><tr><td>Dissecting pins 2&quot;</td><td></td><td></td><td>Sterilize before use&amp;nbsp;</td></tr><tr><td>FACS tubes with 35 &amp;mu;m filter cap</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>Filter cup (Stericup, 0.22 &amp;mu;m)</td><td>Millipore</td><td>SCGPU05RE</td><td></td></tr><tr><td>Fine-tip marker</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotech</td><td>130-042-401</td><td></td></tr><tr><td>Magnetic Multistand</td><td>Miltenyi Biotech</td><td>130-042-303</td><td></td></tr><tr><td>Tissue adhesive (Vetbond)</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>Or a centrifuge with similar capacity for 15 ml and 50 ml conical tube centrifugation</td></tr><tr><td>Tissue culture hood</td><td></td><td></td><td></td></tr><tr><td>Tissue dissociator (gentleMACS)</td><td>Miltenyi Biotech</td><td>130-093-235</td><td>Preset program &quot;m_impTumor_01&quot; used for tissue dissociation&amp;nbsp;</td></tr><tr><td>Liquid nitrogen freezer</td><td></td><td></td><td></td></tr><tr><td>Microplate or rotary shaker</td><td></td><td></td><td></td></tr><tr><td>Phase contrast light microscope</td><td></td><td></td><td></td></tr></tbody></table>

isolation and culture expansion of tumor-specific endothelial cells

新鲜分离的肿瘤特异性内皮细胞（TEC）可以用来探索肿瘤血管生成的分子机制，并用作体外模型用于开发新的血管生成抑制剂用于癌症。然而，长期的鼠内皮细胞（EC）的体外扩增是具有挑战性，因为文化的表型漂移（内皮细胞间质转化），并污染非欧共体。这是为TEC其通过在培养共纯化的成纤维细胞或肿瘤细胞容易outcompeted尤其如此。在这里，高保真的隔离方法，它利用免疫磁珠富集加上菌落筛选和体外扩增的描述。这种方法产生纯乳油级分是完全不含污染基质或肿瘤细胞。它也表明，谱系追踪CDH5 CRE:ZsGreen升/秒/升报告小鼠，与该协议使用本文中所描述，是一种宝贵的工具来验证细胞纯度从这些小鼠孤立欧盟的菌落表现出持久的文化和灿烂的ZsGreen荧光。

统代表了总细胞群体中大多数成人组织11只有轻微的一小部分。因此重要的是充分消化收获组织成单细胞悬浮液，以确保从细胞外基质（ECM）和结缔组织的最大释放乳油。根据我们的经验，CD31介导的免疫选择仅提供丰富的，但不是纯统的部分;因此，另一个关键步骤是物理去除使用克隆环（图1）EC菌落共纯化非EC和选择/扩张。例如，当CD31富集EC铺板无需进一步菌落筛选，非欧?...

Watch this Scientific Journal Video about Isolation and Culture Expansion of Tumor-specific Endothelial Cells at JoVE.com

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

isolation-and-culture-expansion-of-tumor-specific-endothelial-cells

Research

JoVE Journal

Medicine

11.1K 次观看.  University of North Carolina at Chapel Hill. We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models. 

视频: Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

前肿瘤反应性T细胞的体外扩增Bryostatin 1/Ionomycin手段和共同的γ链细胞因子配方

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

科研

免疫与感染

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

正常结肠和结直肠癌患者内皮细胞的分离--一种改进的协议

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

癌症研究

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

人脐血内皮祖细胞的分离

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

发育生物学

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

生物工程

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

生物学

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

神经科学

A Patient-Derived Xenograft Model for Venous Malformation

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7&#8210;9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue.

静脉畸形的患者衍生异种移植模型

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

分离的CD133 +肝干细胞的克隆扩增

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.