Name: transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes
Uploaded: 2017-06-03T14:00:00.000+00:00
Duration: 9 min 59 s
Description: Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

这项工作得到了国家卫生研究机构（05A1-CSPP16-014，HJL）和科技部（MOST 103-2320-B-400-015-MY3，HJL）的资助。

所有涉及动物的研究均符合动物保护机构委员会批准的方案。 细胞外囊泡/外来体分离和鉴定<ol><li>培养乳腺上皮基底细胞NAMEC4，用500mL MCDB 170，pH 7.4 + 500mL DMEM / F12与碳酸氢钠（0.2438％）制备的新鲜无血清培养基; EGF（5 ng / mL）;氢可替康（0.5μg/ mL）;胰岛素（5μg/ mL）;牛垂体提取物（BPE;35μg/ mL）;和GW627368X（1μg/ mL）。 </li><li>用血细胞计数器计数细胞后，在第0天将1.2×10 6个细胞在每15cm培养皿中的12mL培养基中种植4天4 。 </li><li>培养4天后，使用台式离心机将培养基以300×g离心5分钟。将上清液转移到锥形管（ 图1 ）。 </li><li>离心上清液在台式离心机中以2,000 x g进行20分钟。将上清液转移到超速离心管中，留下死细胞和细胞碎片（ 图1 ）。 </li><li>在4℃下以10,000xg离心上清30分钟。将上清液转移到新的超速离心管（ 图1 ）。 </li><li>在4℃下将上清液以110,000xg离心60分钟。去除上清液并将EV /外来物质沉淀重悬于PBS中（ 图1 ）。 </li><li>在4℃下将上清液以110,000xg离心60分钟。去除上清液。重悬于PBS中的EV /外来体颗粒（ 图1 ）。将从240-480 mL NAMEC条件培养基中分离的沉淀物重悬于100μL。 </li><li>用BCA蛋白测定法测定EV悬浮液的蛋白质浓度。确保浓度在20-40μg/ 100μL左右。储存于-20°C进一步分析。 </li><li>通过纳米粒子跟踪分析（NTA）测量EVs /外来体的浓度和大小，如Gardiner 等人先前所述。 11 。用PBS稀释EVs /外来体（20μg/100μL）至NTA分析10,000倍。 注意:NTA分析的结果反映了分析的囊泡的数量和大小。 </li><li>用透射电子显微镜（TEM）对EV /外来体进行成像，如Lin 等人 4所述。 </li></ol> 2.使用密度梯度的外来体纯化<ol><li>将来自步骤1.7的110,000g沉淀物重悬于40％（w / v）碘克沙醇的PBS（2mL）中。按照在PBS（每次2mL）中的30％，20％，10％和5％（w / v）碘克沙醇的等分试样依次覆盖混合物，以在超速离心管中形成密度梯度。 </li><li>将混合物在4℃下以200,000xg离心8小时。 </li><li>收集每个梯度分数（10分）附件; 1 mL /分），从管顶部移液。 </li><li>通过SDS-PAGE 12和Western印迹分析每个级分中exosome标记物（ 如 CD81，CD9，CD63和Tsg101）的存在。将每个级分的50μL悬浮液加到含有0.1％（w / v）SDS的10％凝胶上，并用凝胶电泳分离各部分的蛋白质。 </li><li>将蛋白质从凝胶转移到PVDF膜，并在4℃下将膜与抗外来体标记物（ 例如 CD81，CD9，CD63和Tsg101）和内脏蛋白GAPDH孵育过夜（ 表材料 ）。 注意:结果确定含有外来体的部分。 </li></ol>细胞外囊泡/外来体标签<ol><li>将20μl胞外蛋白/100μL的步骤1.7获得的EVs /外来体悬浮于10μM羧基荧光素琥珀酰亚胺基二乙酸酯（CFSE）中。准备一个平行样本公司仅以相同的方式处理CFSE和PBS，作为后续EV /外来物吸收测定的阴性对照。将混合物在37℃下放置30分钟。 </li><li>将EVs /外来体悬挂在50倍体积的PBS中，并在4℃下以110,000xg离心悬浮液60分钟。去除上清液并将EV /外来物质沉淀重悬于PBS中。重复步骤3.2一次。 </li><li>将EVs /外来体悬浮在PBS中，浓度为20μg的外来蛋白/100μL，然后在将EV /外来体添加到细胞之前通过0.22μm膜过滤EV /外来体。 </li></ol>细胞外囊泡/外来体摄取测定<ol><li>为了制备培养基，将500mL的MCDB 170，pH 7.4 + 500mL的DMEM / F12与碳酸氢钠（0.2438％）混合; EGF（5 ng / mL）;氢可替康（0.5μg/ mL）;胰岛素（5μg/ mL）;和BPE（35μg/ mL） 4 。将人乳腺上皮HMLE细胞置于6孔培养皿中（1×10 6个/ sup&gt;细胞/孔），EV / exosome处理前一天。将2μg/ mL CFSE荧光标记的EVs / exosome（在步骤3.3中获得）添加到HMLE细胞的培养基中2-6小时。用平行制剂处理阴性对照组的HMLE细胞，如步骤3.1所述。 </li><li>孵育2至6小时后，在室温下用4mL PBS洗涤细胞两次。 </li><li>用0.25％胰蛋白酶分离细胞10分钟，并将细胞重新悬浮在含有0.2％FBS的PBS中。使用荧光细胞分析仪测量细胞中荧光强度的EV /外来体摄取。使用共聚焦显微镜对用EVs /外来体处理的细胞或阴性对照进行成像。 注意:细胞中的绿色荧光是由EV /外来体吸收引起的。有关显微镜设置， 请参见图6的图例。 </li></ol> 5.分离原代小鼠乳腺上皮细胞<ol><li&gt;通过向10cm的培养皿中加入12mL的0.1％明胶溶液来制备明胶包衣的菜肴。将板放在37℃的培养箱中30分钟。取出明胶溶液，并将盖子从层流罩中离开盘子4小时直到明胶涂层干燥。 </li><li>使用剪刀从12周龄的女性C57BL / 6小鼠解剖数目2,3,4和5个乳腺（ 图2 ），并使用剃须刀将腺切成小块（2mm）。 </li><li>进一步解离乳腺浆液（10只小鼠）在37℃，60分钟搅拌120毫升DMEM / F12含0.2％胶原酶IV，0.2％胰蛋白酶，5％FBS，5微克/毫升庆大霉素和1x pen-strep。 </li><li>通过在350×g离心10分钟从混合物中沉淀上皮组织。 </li><li>将上清组织悬浮于4mL含有0.1mg / mL DNA酶I的DMEM / F12中，室温5分钟。加入6mL DMEM / F12至最终体积10 mL。 </li><li>将悬浮液在室温下以400xg离心10分钟，弃去上清液。 </li><li>将沉淀重悬于10mL DMEM / F12中。离心机悬挂，当速度达到400 x g时，刹车。丢弃上清液。 注意:通过击打制动器，上皮组织被迅速沉淀下来，而成纤维细胞作为单细胞仍然停留在上清液中。 </li><li>重复5.6和5.7 5-7次。将一滴悬浮液添加到血细胞计数器中，然后在每次离心后在显微镜下检查来自有机体混合物的成纤维细胞的清除（ 图3 ）。 </li><li>将含有1x ITS，5％FBS，50μg/ mL庆大霉素，10ng / mL EGF和1×pen-strep的DMEM / F12，在步骤5.1中获得的明胶包被的培养皿中的组织样品放置48小时。 </li><li> 48小时后，用不含FBS的新鲜培养基替换培养基，去除培养物中的浮游细胞（含有ITS，50μg/ mL庆大霉素，10ng / mL EGF和1x pen-strep的DMEM / F12）。 </li><li>确认单层乳腺上皮细胞在3天内迁移并从附着的上皮组织中生长出来。 </li></ol>原代小鼠基底/乳腺上皮细胞分离<ol><li> 3天培养后，用具有蛋白水解和胶原酶活性的天然酶混合物分离小鼠原代乳腺细胞20分钟。用PBS中的5％FBS中和酶活性。 </li><li>在室温下将悬浮液以450×g离心10分钟，弃去上清液。将细胞沉淀重悬于5mg / mL分散液中20分钟。用PBS中的5％FBS中和酶活性。 </li><li>在室温下将悬浮液以450×g离心10分钟，弃去上清液。 </li><li>用浓度为10 7个细胞/ mL的稀释的0.2％FBS将细胞重新悬浮于100μLPBS中抗CD49f和抗EpCAM抗体在黑暗中1小时。 </li><li>用PBS洗涤细胞，然后在黑暗中将细胞与荧光团缀合的第二抗体在冰上孵育30分钟。 </li><li>用0.2％FBS洗涤并重新悬浮在PBS中的细胞。 </li><li>将细胞分选器4上的EpCAM hi / CD49f细胞乳腺上皮细胞分选。 </li></ol>细胞外囊泡/外来体治疗<ol><li>将来自步骤6.7的明胶包被的6孔培养皿（2×10 5个细胞/孔）上的分选的EpCAM hi / CD49f细胞乳腺上皮细胞种子，然后用PBS或2μg/ mL NAMEC衍生的EVs /外来体在步骤1.7中获得。 </li><li>为了确保EVs /外来体在长期培养处理中的生物学功效，每隔两天用含有PBS或2μg/ mL NAMEC衍生的EVs /外来体的新鲜培养基代替细胞培养基。不要分开鼠标prima在10天的治疗期间的腔细胞。 </li></ol> 8.脂肪垫注射乳腺上皮细胞<ol><li>麻醉3周龄雌性C57BL / 6小鼠与异氟烷2-3％吸入。 </li><li>把麻醉的老鼠放在背上。用剃刀/发乳去除中腹部的毛皮，并用70％酒精和聚维酮碘的三个交替周期清洁手术部位。 </li><li>用剪刀沿腹侧胸腹部区域穿过皮肤进行1.5厘米的垂直切口，然后交替暴露右侧和左侧第 4 个乳房脂肪垫。 </li><li>用剪刀去除腺体实质，清除每个脂肪垫。找到脂肪垫中的淋巴结，然后取出淋巴结下方的整个腺体实质。 注意:三分之二的脂肪垫应该保持在原位。 </li><li>用天然酶混合物（参见材料表 ）与蛋白水解a分离从步骤7.2获得的细胞nd胶原酶活性10分钟。用PBS中的5％FBS中和酶活性。 </li><li>在室温下将悬浮液以450×g离心10分钟，弃去上清液。用血细胞计数器计数细胞，并将细胞悬浮在PBS中，浓度使得15μL含有所需的细胞剂量（10 4 -10 2细胞/垫）。 </li><li>使用连接到27G针的100μL玻璃注射器将15μL乳腺上皮细胞悬液注入脂肪垫。 </li><li>对另一侧的脂肪垫重复步骤8.4-8.7。 </li><li>用伤口夹闭合皮肤。 </li></ol>乳腺全身<ol><li>细胞注射后8周用CO 2加颈椎脱位来安乐死小鼠（步骤8.7）。 </li><li>使用剪刀从胸部区域到腹股沟区域的皮肤层进行垂直切口，然后暴露右侧和左侧4 号乳房脂肪垫去除第4 个乳腺（ 图2 ）。 </li><li>在玻璃显微镜载玻片上涂抹脂肪垫，并在室温下用Kahle固定剂（4％甲醛，30％EtOH和2％冰乙酸）固定脂肪垫。 注意:Kahle的固定剂是刺激性的。在化学品罩中执行此步骤。 </li><li>在250mL 70％EtOH中洗涤脂肪垫15分钟，然后在250mL的dH 2 O中洗涤5分钟。在室温下用胭脂红明矾（1g胭脂红和2.5g硫酸铝钾）在500mL dH 2 O中染上脂肪垫。 </li><li>用250mL 70％EtOH洗涤脂肪垫15分钟，250mL 95％EtOH洗涤15分钟，加入250mL 100％EtOH 15分钟。 </li><li>清洁二甲苯中的脂肪垫数天，并在脂肪垫变透明时停止二甲苯培养。 注意:二甲苯是一种刺激性。在化学品罩中执行此步骤。 </li><li>装载幻灯片wi使用数字幻灯片扫描仪拍摄图像（2,400 dpi）的脂肪垫。 </li></ol>

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	<li>Simons, M., Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes--vesicular+carriers+for+intercellular+communication.">Exosomes--vesicular carriers for intercellular communication.</a> Curr Opin Cell Biol. 21 (4), 575-581 (2009).</li><li>Th&#233;ry, C., Ostrowski, M., Segura, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles+as+conveyors+of+immune+responses.">Membrane vesicles as conveyors of immune responses.</a> Nat Rev Immunol. 9 (8), 581-593 (2009).</li><li>Boelens, 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=Exosome+Transfer+from+Stromal+to+Breast+Cancer+Cells+Regulates+Therapy+Resistance+Pathways.">Exosome Transfer from Stromal to Breast Cancer Cells Regulates Therapy Resistance Pathways.</a> Cell. 159 (3), 499-507 (2014).</li><li>Lin, M. 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=PGE2+/EP4+Signaling+Controls+the+Transfer+of+the+Mammary+Stem+Cell+State+by+Lipid+Rafts+in+Extracellular+Vesicles.">PGE2 /EP4 Signaling Controls the Transfer of the Mammary Stem Cell State by Lipid Rafts in Extracellular Vesicles.</a> Stem Cells. , (2016).</li><li>Th&#233;ry, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Curr Protoc Cell Biol. , (2006).</li><li>Gy&#246;rgy, B., 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=Membrane+vesicles,+current+state-of-the-art:+emerging+role+of+extracellular+vesicles.">Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.</a> Cell Mol Life Sci. 68 (16), 2667-2688 (2011).</li><li>Olver, C., Vidal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+secreted+exosomes.">Proteomic analysis of secreted exosomes.</a> Subcell Biochem. 43, 99-131 (2007).</li><li>Shackleton, 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=Generation+of+a+functional+mammary+gland+from+a+single+stem+cell.">Generation of a functional mammary gland from a single stem cell.</a> Nature. 439 (7072), 84-88 (2006).</li><li>Prater, M. 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=Mammary+stem+cells+have+myoepithelial+cell+properties.">Mammary stem cells have myoepithelial cell properties.</a> Nat Cell Biol. 16 (10), 942-950 (2014).</li><li>Stingl, 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=Purification+and+unique+properties+of+mammary+epithelial+stem+cells.">Purification and unique properties of mammary epithelial stem cells.</a> Nature. 439 (7079), 993-997 (2006).</li><li>Gardiner, C., Ferreira, Y. J., Dragovic, R. A., Redman, C. W., Sargent, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+sizing+and+enumeration+by+nanoparticle+tracking+analysis.">Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis.</a> J Extracell Vesicles. 2, (2013).</li><li>Shapiro, A. L., Vi&#241;uela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem Biophys Res Commun. 28 (5), 815-820 (1967).</li><li>Riches, A., Campbell, E., Borger, E., Powis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+exosome+release+from+mammary+epithelial+and+breast+cancer+cells+-+a+new+regulatory+pathway.">Regulation of exosome release from mammary epithelial and breast cancer cells - a new regulatory pathway.</a> Eur J Cancer. 50 (5), 1025-1034 (2014).</li><li>Witwer, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+sample+collection,+isolation+and+analysis+methods+in+extracellular+vesicle+research.">Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.</a> J Extracell Vesicles. 2, (2013).</li><li>van der Vlist, E. J., Nolte-'t Hoen, E. N., Stoorvogel, W., Arkesteijn, G. J., Wauben, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labeling+of+nano-sized+vesicles+released+by+cells+and+subsequent+quantitative+and+qualitative+analysis+by+high-resolution+flow+cytometry.">Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry.</a> Nat Protoc. 7 (7), 1311-1326 (2012).</li><li>Kowal, 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=Proteomic+comparison+defines+novel+markers+to+characterize+heterogeneous+populations+of+extracellular+vesicle+subtypes.">Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.</a> Proc Natl Acad Sci U S A. 113 (8), E968-E977 (2016).</li><li>Li, 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=ADVANCED+IMAGING.+Extended-resolution+structured+illumination+imaging+of+endocytic+and+cytoskeletal+dynamics.">ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.</a> Science. 349 (6251), (2015).</li><li>Outzen, H. C., Custer, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+human+normal+and+neoplastic+mammary+tissues+in+the+cleared+mammary+fat+pad+of+the+nude+mouse.">Growth of human normal and neoplastic mammary tissues in the cleared mammary fat pad of the nude mouse.</a> J Natl Cancer Inst. 55 (6), 1461-1466 (1975).</li><li>Sheffield, L. G., Welsch, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+breast+epithelia+to+mammary-gland-free+fat-pads+of+athymic+nude+mice:+influence+of+mammotrophic+hormones+on+growth+of+breast+epithelia.">Transplantation of human breast epithelia to mammary-gland-free fat-pads of athymic nude mice: influence of mammotrophic hormones on growth of breast epithelia.</a> Int J Cancer. 41 (5), 713-719 (1988).</li></ol>

作者没有什么可以披露的。

外来体通常携带释放它们的细胞的特征，并且释放的外来体的量可以由刺激诱导4 。可以收集细胞的培养基并进行差异超速离心用于EV /外来物质收集（ 图1 ）。目前对于分离EV /外来体的理想方法目前尚无普遍的共识。这里使用的最佳方法已经由下游应用14确定 。超速离心是分离EV /外来体的相对快速的方法，可以保护EV /外来体的生物活性?...

外来体可以通过在细胞之间转移膜和细胞质蛋白，脂质和RNA来介导细胞交流1 。已经证明外来体介导的通信涉及许多生理和病理过程（ 即抗原呈递，耐受性发展2和肿瘤进展3 ）。外来体通常具有与释放它们的源细胞相似的内容。因此，外来体可以从源细胞携带特定的细胞特性，并将这些性质转移到摄取它们的细胞中。 外来体是50至150纳米的双层膜囊泡，并呈现特异性标记（ 例如 CD9，CD81，CD63，HSP70，Alix和TSG101）。因此，外来体必须通过不同方面的各种方法来表征。透射电子显微镜可用于观察膜囊泡如外来体4,5 。纳米粒子跟踪分析（NTA）和动态光散射分析（DLS）用于测量纯化外来体的大小和数量4 。可以通过密度梯度验证外来体的脂质膜含量。染色体标志物，例如CD9，CD81，CD63，HSP70，Alix和TSG101,6,7可以通过Western印迹法测定。 乳腺基底细胞在植入脂肪垫时具有产生乳腺的能力，而腔细胞不能8,9,10 。因此，乳腺基底细胞也称为乳腺再生单位。通过使用乳腺基底细胞和腔细胞模型，可以检测EVs /外来体在不同细胞群体之间转运细胞特征的能力。这个工作证明了通过使用源自乳腺基底上皮细胞的EVs /外来体将腺体形成能力从乳腺基底上皮细胞转移到乳腺腔上皮细胞的方法。乳腺上皮细胞摄取基底细胞分泌的EV /外来体后获得基础细胞特性，然后形成乳腺。

<table><tbody><tr><td>MCDB 170&amp;nbsp;</td><td>USBiological</td><td>M2162</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermo</td><td>1250062</td><td></td></tr><tr><td>Optima L-100K ultracentrifuge</td><td>Beckman</td><td>393253</td><td></td></tr><tr><td>SW28 Ti Rotor</td><td>Beckman</td><td>342204</td><td></td></tr><tr><td>SW41 Rotor</td><td>Beckman</td><td>331306</td><td></td></tr><tr><td>NANOSIGHT LM10</td><td>Malvern</td><td>NANOSIGHT LM10</td><td>for nanoparticle tracking analysis (NTA)</td></tr><tr><td>Optiprep&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D1556</td><td>60% (w/v) solution of iodixanol in water (sterile).</td></tr><tr><td>CD81 antibody</td><td>GeneTex</td><td>GTX101766</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CD9 antibody</td><td>GeneTex</td><td>GTX100912</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CD63 antibody</td><td>Abcam</td><td>Ab59479</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>TSG101 antibody</td><td>GeneTex</td><td>GTX118736</td><td>1:1,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>GAPDH</td><td>GeneTex</td><td>GTX100118</td><td>1:6,000 in 5% w/v nonfat dry milk, 1x TBS, 0.1% Tween 20 at 4 &amp;deg;C, overnight&amp;nbsp;</td></tr><tr><td>CFSE (carboxyfluorescein succinimidyl diacetate ester)</td><td>Thermo</td><td>V12883</td><td></td></tr><tr><td>FACSCalibur</td><td>BD Biosciences</td><td></td><td>fluorescence cell analyzer</td></tr><tr><td>collagenase Type IV&amp;nbsp;</td><td>Thermo</td><td>17104019</td><td></td></tr><tr><td>trypsin</td><td>Thermo</td><td>27250018</td><td></td></tr><tr><td>&amp;nbsp;ITS</td><td>Sigma-Aldrich</td><td>I3146</td><td>a mixture of recombinant human insulin, human transferrin, and sodium selenite</td></tr><tr><td>accutase</td><td>ebioscience</td><td>00-4555-56</td><td>a natural enzyme mixture with proteolytic and collagenolytic enzyme activity</td></tr><tr><td>dispase&amp;nbsp;</td><td>STEMCELL</td><td>7913</td><td>5 mg/mL = 5 U/mL</td></tr><tr><td>anti-CD49f antibody</td><td>Biolegend</td><td>313611</td><td>1:50</td></tr><tr><td>anti-EpCAM antibody</td><td>Biolegend</td><td>118213</td><td>1:200</td></tr><tr><td>FACSAria</td><td>BD Biosciences</td><td></td><td>cell sorter</td></tr><tr><td>carmine alum</td><td>Sigma-Aldrich</td><td>C1022</td><td></td></tr><tr><td>human mammary epithelial cells (HMLE cells, NAMECs)</td><td></td><td></td><td>gifts from Dr. Robert Weinberg</td></tr><tr><td>permount</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>SP15-500</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Zymeset&amp;nbsp;</td><td>BSB101</td><td></td></tr><tr><td>EGF</td><td>Peprotech</td><td>AF-100-015</td><td></td></tr><tr><td>Hydrocoritisone</td><td>Sigma-Aldrich</td><td>SI-H0888</td><td></td></tr><tr><td>Insulin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SI-I9278</td><td></td></tr><tr><td>BPE (bovine pituitary extract)</td><td>Hammod Cell Tech&amp;nbsp;</td><td>1078-NZ</td><td></td></tr><tr><td>GW627368X&amp;nbsp;</td><td>Cayman</td><td>10009162</td><td></td></tr><tr><td>15 cm culture dish</td><td>Falcon&amp;nbsp;</td><td>353025</td><td></td></tr><tr><td>table-top centrifuge</td><td>Eppendrof&amp;nbsp;</td><td>Centrifuge 3415R</td><td></td></tr><tr><td>ultracentrifuge tube</td><td>Beckman</td><td>344058</td><td></td></tr><tr><td>PBS (Phosphate-buffered saline)&amp;nbsp;</td><td>Corning</td><td>46-013-CM</td><td></td></tr><tr><td>BCA Protein Assay</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>23228</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>Hitachi</td><td>HT7700</td><td></td></tr><tr><td>gelatin&amp;nbsp;</td><td>STEMCELL</td><td>7903</td><td></td></tr><tr><td>10 cm culture dish</td><td>Falcon&amp;nbsp;</td><td>353003</td><td></td></tr><tr><td>6-well culture dish</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>female C57BL/6 mice</td><td>NLAC (National Laboratory Animal Center</td><td></td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>BioWest&amp;nbsp;</td><td>S01520</td><td></td></tr><tr><td>gentamycin</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>15710072</td><td></td></tr><tr><td>Pen/Strep</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>DNase I</td><td>5PRIMER</td><td>2500120</td><td></td></tr><tr><td>isofluorane&amp;nbsp;</td><td>Halocarbon</td><td>NPC12164-002-25</td><td></td></tr><tr><td>formaldehyde</td><td>MACRON</td><td>H121-08</td><td></td></tr><tr><td>EtOH (Ethanol)</td><td>J.T. Baker</td><td>800605</td><td></td></tr><tr><td>glacial acetic acid</td><td>Panreac</td><td>131008.1611</td><td></td></tr><tr><td>aluminum potassium sulfate</td><td>Sigma-Aldrich</td><td>12625</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Leica</td><td>3803665</td><td></td></tr><tr><td>0.22 &amp;mu;m membranes</td><td>Merck Millipore</td><td>Millex-GP</td><td></td></tr><tr><td>AUTOCLIP Wound Clips, 9 mm</td><td>BD Biosciences</td><td>427631</td><td></td></tr><tr><td>AUTOCLIP Wound Clip Applier</td><td>BD Biosciences</td><td>427630</td><td></td></tr><tr><td>CellMask&amp;trade; Deep Red</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>C10046</td><td>plasma membrane stain</td></tr></tbody></table>

transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes

细胞可以通过外来体，含有蛋白质，脂质和核酸的〜100-nm胞外囊泡（EVs）进行通信。非贴壁/间充质乳腺上皮细胞（NAMEC）衍生的细胞外囊泡可以通过差异超速离心从NAMEC培养基中分离。基于它们的密度，EV可以通过110,000×g的超速离心纯化。可以使用连续密度梯度进一步分离来自超速离心的EV制剂，以防止可溶性蛋白质的污染。然后可以使用纳米颗粒跟踪分析进一步评估纯化的EV，其测量制剂中囊泡的大小和数量。尺寸范围为50至150nm的细胞外囊泡是外来体。 NAMEC衍生的EVs /外来体可以通过乳腺上皮细胞摄取，可以通过流式细胞术和共焦显微镜检测。一些乳腺干细胞性质（ 如乳腺形成能力）可以通过来自NAMEC的EVs /外来体从茎状NAMEC转移到乳腺上皮细胞。分离的原代EpCAM hi / CD49f细胞乳腺上皮细胞在移植到小鼠脂肪垫后不能形成乳腺，而EpCAM lo / CD49f基底乳腺上皮细胞在移植后形成乳腺。通过EpCAM hi / CD49f细胞乳腺上皮细胞摄取NAMEC衍生的EVs /外来体允许它们在移植到脂肪垫中之后产生乳腺。衍生自干样乳腺上皮细胞的EVs /外来体转移到EpCAM hi / CD49f细胞乳腺上皮细胞的乳腺形成能力。

由于已经显示阻断PGE 2 / EP 4信号传导从乳腺基底样干细胞4引发EV /外来体释放，这项工作提出了从乳腺上皮基底细胞（NAMEC）培养物中分离诱导的EVs /外来体的方法。由于NAMECs在无血清培养基中培养，所以不存在来自血清13的预先存在的EV /外来体。对于在含有血清的培养基中培养的细胞，培养基中预先存在的外来体必须通...

Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

该方案描述了从非贴壁/间充质乳腺上皮细胞中纯化，定量和表征细胞外囊泡（EVs）/外来体的方法，并使用它们将乳腺形成能力转移到乳腺上皮细胞。衍生自茎状乳腺上皮细胞的EVs /外来体可以将该细胞特性转移到摄入EV /外来体的细胞中。

通过细胞外囊泡/外来体转移乳腺基质上皮细胞和乳腺腔细胞之间的乳腺形成能力

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

transfer-mammary-gland-forming-ability-between-mammary-basal

Research

JoVE Journal

Developmental Biology

11.6K 次观看.  National Health Research Institutes. 该方案描述了从非贴壁/间充质乳腺上皮细胞中纯化，定量和表征细胞外囊泡（EVs）/外来体的方法，并使用它们将乳腺形成能力转移到乳腺上皮细胞。衍生自茎状乳腺上皮细胞的EVs /外来体可以将该细胞特性转移到摄入EV /外来体的细胞中。 

视频: Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

C57BL/6J小鼠乳腺间质细胞和癌细胞的移植

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

科研

医学

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

大鼠乳腺内皮细胞移植到间膜白脂肪垫

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

癌症研究

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

生物学

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

乳腺上皮移植手术

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

发育生物学

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

乳腺的发育和功能在小鼠模型的评价

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

非酶中，预浸润性乳腺癌病变的无血清组织培养的微球体的自然发生

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

对小鼠乳腺冰冻切片间接免疫荧光

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

完整、全小鼠乳腺的分离及细胞外基质表达及腺体形态学分析

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

通过细胞外囊泡/外来体转移乳腺基质上皮细胞和乳腺腔细胞之间的乳腺形成能力

摘要

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C57BL/6J小鼠乳腺间质细胞和癌细胞的移植

大鼠乳腺内皮细胞移植到间膜白脂肪垫

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型

乳腺上皮移植手术

通过微分试穿素生成马赛克乳腺有机体

乳腺的发育和功能在小鼠模型的评价

非酶中，预浸润性乳腺癌病变的无血清组织培养的微球体的自然发生

对小鼠乳腺冰冻切片间接免疫荧光

完整、全小鼠乳腺的分离及细胞外基质表达及腺体形态学分析

通过细胞外囊泡/外来体转移乳腺基质上皮细胞和乳腺腔细胞之间的乳腺形成能力

摘要

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此视频中的章节

C57BL/6J小鼠乳腺间质细胞和癌细胞的移植

大鼠乳腺内皮细胞移植到间膜白脂肪垫

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

乳腺上皮移植手术

通过微分试穿素生成马赛克乳腺有机体

乳腺的发育和功能在小鼠模型的评价

非酶中，预浸润性乳腺癌病变的无血清组织培养的微球体的自然发生

对小鼠乳腺冰冻切片间接免疫荧光

完整、全小鼠乳腺的分离及细胞外基质表达及腺体形态学分析

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型