MAL，JCG，和黄所支持的的NIA（R00AG033176和R01AG040081）的实验室指导研究和发展，美国能源部能源合同＃DE-AC02-05CH11231。

1。组织处理和消化<ol><li>获取作为外科手术的报废材料从人体乳腺组织。可以提供减少mammoplasties正常或良性细胞;纤维腺瘤和gynecomastias提供良性细胞;，非肿瘤乳房切除组织（外设或对侧的肿瘤，或皮下）提供细胞，范围可从正常良性含有microtumors。确保适当的IRB批准之前获得的废弃材料存在。所有材料应被视为下血源性病原体的规定。 </li><li>将材料在无菌容器中含有缓冲或媒体（ 例如 1:1 Dulbecco改良的Eagle的中和联队的F-12）中补充100 U / ml青霉素，100μg/ ml链霉素，5微克/毫升两性霉素B，多粘菌素B 50U/ml，和10％的胎牛血清，并运到实验室，在4°C. 72小时，没有显着影响的su还原隆乳组织可以在4℃下储存或运bsequent细胞活力。非减少隆乳组织的小件可能需要更迅速的处理。 </li><li>分离的上皮区域从基质使用无菌手术刀，钳子，剪刀的组合矩阵的脂肪组织，结缔组织和血管。裁片用剪刀和一个大的玻璃在无菌盘的组织（ 例如 ，150毫米）。轻轻解剖上皮区域，显示为白色链嵌入在变黄基质矩阵，使用的钳子持有的材料和手术刀刮走严重的脂肪物质。为了促进消化，削减上皮组织成小块，约3-4毫米，使用相对的手术刀，并放入50毫升的试管。如果正在处理大量的组织，可能需要新鲜的手术刀片。删除脂肪物质从盘处理，根据机构的规定。 <ol class="lalpha"><li>在大量的纤维组织（ 如皮下MAStectomies严重的纤维囊性疾病），将会有更多的白色固体，非上皮性材料。这可能是困难的解剖的上皮细胞中的这样的纤维状基质。大块纤维材料可以被切割成更小的部分，〜1-3平方毫米的面积分别消化。 </li></ol></li><li>解剖的上皮组织放置成一个圆锥形离心管中（50毫升或15毫升）与组织包含不大于管的体积的三分之一。使管到最大音量，只留下一个小的空气空间，以允许用于混合在旋转过程中，使用的组织消化混合物（DME/F-12或等效，10μg/ ml胰岛素，与上述的抗生素，和最终浓度达到10 ％FCS，200 U / ml的粗制胶原酶和100 U / ml的透明质酸酶。 </li><li>将管上HulaMixer试管旋转器和旋转360°，在37℃下在8转过夜</li><li>离心管，在600×g离心5分钟。弃上清脂肪和介质处理符合ING机构规定。 </li><li>检查完成消化稀释分装在介质中的颗粒。消化是完整的，镜检可见细胞团块，（组织体）与平滑出现的导管，腺泡或导管肺泡结构，从连接的基质（ 图1A）。减少隆乳组织通常会仍然显示附加基质过夜消化后，将需要额外的消化时间。 <ol class="lalpha"><li>不完全消化的沉淀重悬于新鲜组织消化混合物中，并再与旋转孵育在37℃下4-12小时的另一个。重复步骤1.6和1.7。如果仍然是不完整的消化，再次添加消化混合物与旋转的沉淀和重新孵育在37℃培养过夜。酶的浓度可以降低，以防止过度消化过夜。 </li></ol></li><li>消化完成后，离心管，600×g离心5分钟，吸取精华离子混合，重悬沉淀在约15 ml/50 ml管中，5 ml/15 ml管在中加用抗生素。 </li></ol> 2。过滤和冷冻吸附材料<ol><li>再悬浮的颗粒的等分试样转移到一个无菌的100微米过滤器，在一个无菌的50毫升试管中。让中漏入管，然后rewash类器官2-3毫升培养基上1-2X倍。重复以上步骤，直到所有的再悬浮颗粒已被转移。如果在过滤器上有太多的类器官和介质不再水渠容易，使用一个新的过滤器（）的其它材料。小心地翻转过滤器（S）另一个无菌管，洗管的组织体多介质。这是的100微米类器官池。 </li><li>取的材料，排出到原来的50毫升的试管，并重复这个过程，为2.1，使用一个40微米的过滤器，以获得40μm的组织体的池，其中包含主要是肺泡结构。的材料排到第Ë管构成的过滤池，其中包含单/小丛的血管间质细胞和上皮细胞和小件。 </li><li>颗粒100微米，40微米，和滤液池在600×g离心5分钟。 </li><li>吸取上清，在CPM II（DME/F-12或相当于44％FCS和6％的DMSO）重建每个管使用约1毫升的CPMII中的每0.1毫升的包装的颗粒。保持在4℃下</li><li>种子菜为2类器官池的再悬浮物质的0.1毫升下降到35毫米的组织培养塑料盘下降3.2测试。将滤液池可以直接接种到组织培养塑料与成纤维细胞的培养基（DME/F-12或相当于10微克/毫升胰岛素和10％FBS）。 </li><li>等分试样的剩余的再悬浮到Nunc公司类型冻结安瓿（1毫升/ 2毫升安瓿）的材料。在-80℃冷冻过夜，然后，及时转移储存在液氮中。我们没有发现任何重大损失的可行性在20世纪70年代末以来，我们原来的安瓿冷冻保存。 </li></ol> 3。播种冷冻类器官和原代培养的继代培养<ol><li>快速解冻冷冻安瓿瓶在37℃水浴中含有的类器官。种子类器官成（通常为2至10个〜6）60毫米培养皿，或1-3 100毫米培养皿，在安瓿类器官的数目取决于视觉估计。约20-40每60 mm培养皿接种的组织体是最佳的。 </li><li>解冻类器官被小心地安置，一滴一滴，盘面上1毫升吸管或巴斯德吸管均匀分布的组织体。组织体放置在靠近一起将具有有限的空间内生长，从而导致较少的细胞进行传代培养或冻结。避免刮伤盘面（的细胞往往不增长过去划伤表面）。等待1-2分钟，以使组织体连接，然后慢慢加入生长培养基，以避免撞出类器官（ 例如 2-3 ml/60毫米盘）。我ncubate在37℃下，在湿润的CO 2培养箱中。这些是原代培养。我们目前使用的M87A +催产素（X）中的最强劲的HMEC增长（ 图2）。 </li><li>一天后，请检查连接的组织体。添加额外的介质（ 例如 2-3 ml/60毫米盘）。从类器官的细胞迁移应该是可见的24-48小时，播种后48〜72小时（ 图1B，C）和有丝分裂的产物。有时，附件是差在24小时后，特别是在电镀overdigested的类器官或组织体从老年妇女，但大部分准备在72小时内将附加。小件的血管连接，并引起成纤维细胞产物（ 图1D）。 </li><li>饲料培养至少每周3次。 M87A型介质中生长的细胞长到附近汇合在5-8天之内，根据密度播种。 </li><li>如果存在显着的成纤维细胞生长，微分方程的胰蛋白酶消化分离（DT）的基础上，是快速支队从表面塑料的成纤维细胞，成纤维细胞需要删除。当上皮细胞的修补程序变大，抽吸的介质，洗菜STE（生理盐水，0.05％胰蛋白酶，0.02％EDTA），吸（ 如1 ml/60毫米菜），增添新的0.5毫升STE和在室温下离开1分钟左右，连续显微镜观察。成纤维细胞的分离，但上皮细胞仍贴壁，轻轻地，但大幅的菜敲侧对着硬表面撞出成纤维细胞，然后迅速吸。一次，PBS清洗，吸一次。严重污染的成纤维细胞的培养，可能需要额外的DT。 </li><li>继代培养原代培养时，大上皮细胞的修补程序，但在此之前汇合。密度的类器官播种和附件将影响所需的时间。要保留原代培养，并生成多个辅助文化，间隔时间的推移，我们执行部分Trypsinizations的（PT）。 </li><li>抽吸介质，洗1-2毫升，STE，60 mm培养皿中加入0.5毫升新鲜STE抛出。时间为1-5分钟，在室温下，在显微镜下观察细胞的脱落，与温柔爆震的菜，以促进细胞脱离。胰蛋白酶消化，应停止时，〜50％的细胞脱离。早期的PT通常有快速的细胞脱落。为以后的PT，细胞可以放置在37°C为快支队，仔细监测，因为所有的细胞迅速脱落。 </li><li>含血清培养基中加入2毫升的菜，repipette洗，并转移到无菌的15 mL管中。重复2倍与另一〜1-2毫升介质，加入洗管。再供给的主盘和返回孵化器。管中的细胞计数与血球计。 PT可以重复4〜8倍左右，具​​有良好的细胞再生，传代培养或冷冻二级（第二代细胞被称为二次，一次传代培养，细胞是没有LO的主要菜肴和等效长期增长NGER初选）。一旦类器官的材料不再存在于原代培养，传代培养的次级长期人口倍增潜力的下降。 </li><li><ol class="lalpha"><li>种子细胞传代培养至二级，直接从管成菜肴。播种1-2×10 5 cells/100 mm培养皿中一个强大的媒介，如M87A + X，会导致汇合4-7天。我们霍乱毒素添加到培养基中，在第二通道以增加增殖潜能;霍乱毒素的原代培养中被省略，因为它会导致多层细胞生长的组织体。 </li><li>是次要的，颗粒为冻结管在600×g离心5分钟，并重新悬浮在CPMII与10 6细胞/ ml的最终密度。等分试样到Nunc公司类型冻结安瓿重悬细胞，冷冻过夜，在-80℃，然后将其转移到存储迅速在液氮中。 </li></ol></li><li>建议冻结细胞从第一PT保管，使用从第二PT传代培养的细胞。附加PT可以冷冻储存，以备将来使用。 </li></ol>

<ol>
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没有利益冲突的声明。

人类乳腺组织的处理方法能够获得的异构混合类型的细胞在人乳腺癌的的纯乳腺上皮细胞。过滤通过固定大小的孔，允许不同的馏分的乳腺分离（ 例如 ，导管和导管肺泡与肺泡）从消化的基质矩阵。等基因乳腺成纤维细胞可以得到相匹配的上皮细胞。冷冻消化的物质，保留了良好的生存能力超过30年。此方法已成功地工作，但在所有的乳腺组织的纤维状，是简单的执行。其他乳腺组织处理的变化，存在不提供是可行的，清洁，或分离的上皮馏分。配售文化低应力诱导培养基，如M87A加工类器官可以长期增长的HMEC多个谱系标记。 HMEC已培养在塑料上仍然保留的能力，属TE三维结构与正常的在体内像天堂关系。这些的HMEC文化是适合广泛的实验研究，包括高吞吐量，正常的的HMEC行为和因素，可能推动或抑制衰老和改造。

<table border="1"><tbody><tr><td>玻璃培养皿15厘米</td><td> VWR </td><td> 89000-308 </td></tr><tr><td>剪刀6.5" </td><td> VWR </td><td> 82027-594 </td></tr><tr><td>钳8" </td><td> VWR </td><td> 82027-436 </td></tr><tr><td>手术刀一次性</td><td> MILTEX </td><td> 4-411 </td></tr><tr><td>胶原酶</td><td> SIGMA </td><td> C0130 </td></tr><tr><td>透明质酸酶</td><td> SIGMA </td><td> H3506 </td></tr><tr><td>胰岛素</td><td> SIGMA </td><td> I5500 </td></tr><tr><td> DMSO </td><td> SIGMA </td><td> D8418 </td></tr><tr><td>青霉素/链霉素</td><td> Life Technologies公司</td><td> 15140-122 </td></tr><tr><td>两性霉素B </td><td> Life Technologies公司</td><td> 15290-018 </td></tr><tr><td>硫酸多粘菌素B </td><td> Life Technologies公司</td><td> 21850-029 </td></tr><tr><td>胎牛血清</td><td> Life Technologies公司</td><td> 26140-087 </td></tr><tr><td>胰蛋白酶0.05％，100毫升</td><td> Life Technologies公司</td><td> 25300-054 </td></tr><tr><td> DMEM/F12 </td><td> Life Technologies公司</td><td> 11039-021 </td></tr><tr><td> HulaMixer </td><td> Life Technologies公司</td><td> 159-20D </td></tr><tr><td>细胞过滤器100微米</td><td> BD猎鹰</td><td> 352360 </td></tr><tr><td>细胞过滤40微米</td><td> BD猎鹰</td><td> 352340 </td></tr><tr><td>管15毫升</td><td>格雷纳生物一</td><td> 188-261 </td></tr><tr><td>管50毫升</td><td>格雷纳生物一</td><td> 227-261 </td></tr><tr><td> TC菜10厘米</td><td>格雷纳生物一</td><td> 664-160 </td></tr><tr><td> TC菜6厘米</td><td>格雷纳生物一</td><td> 628-160 </td></tr><tr><td>低温瓶</td><td>的Nalgene </td><td> 5000-1020 </td></tr><tr><td> Heamocytometer </td><td>豪瑟科学</td><td> 1490 </td></tr><tr><td>离心分离</td><td> Eppendorf公司模型</td><td> 5702 </td></tr></tbody></table>

processing of human reduction mammoplasty and mastectomy tissues for cell culture

实验检验的正常人类乳腺上皮细胞（HMEC）的行为，与正常细胞如何获得异常的属性，可促进体外培养系统，能够更准确地在体内的生物学模型。研究细胞分化，衰老，衰老和永生化人源性材料的使用是特别有利的，因为许多重要的分子差异，在这些属性之间的人，通常利用老鼠细胞1-2。乳腺细胞中存在一个简单的模型系统，因为正常和异常组织提供了大量的频率减少隆乳术和乳房切除术的手术。 乳腺由许多不同的细胞类型， 如上皮细胞，脂肪间质，血管内皮一个复杂的混合。上皮细胞是负责的乳腺泌乳功能的差异化，绝大多数的人类乳腺癌的起源。我们已开发的方法来处理进入纯上皮组件以及间充质细胞3乳腺手术丢弃组织。处理后的材料可以被无限期冷冻保存，或开始进入小学文化。外科丢弃材料被运送到实验室，手动解剖丰富上皮含组织。随后的解剖组织的消化利用胶原酶，透明质酸酶带基质材料的上皮细胞基底膜。将所得小片的上皮树（类器官）消化的基质是可以分开的，通过顺序固定孔径的膜过滤。取决于孔径大小，可以得到较大的导管/肺泡件，较小的牙槽簇，或基质细胞组成的馏分。我们观察到卓越的增长，当文化作为组织体发起的，而不是显示sociated单细胞。放置在低应力诱导培养基培养的组织体支持长期增长的正常HMEC标记多个系类型（肌上皮，管腔，祖）4-5。从一个个体的组织，可以得到足够数量的细胞，以允许广泛的实验检验，使用标准化的细胞批次，以及使用高通量的方式的讯问。 培养HMEC已在各种正常的生长，分化，衰老，衰老，以及如何改变这些正常的过程不朽的和恶性转化过程中4-15,16过程的研究。在细胞外基质材料，以及其他类型的细胞，和/或三维培养的存在下生长的影响，可以进行比较与生长在塑料5,15。培养HMEC，与正常细胞开始，提供了一个实验听话的系统检查的因素可能会推动或阻止人类衰老和癌变。

在图1中所示的适当消化组织体，并放置在培养的组织的代表数字。不完全消化的组织会显示材料仍连接到这些结构的外侧，并且将可能有一些成纤维细胞的原代培养细胞的生长，需要DT以移除。 Overdigested组织会显示不那么顺畅的外部边界，并可能需要较长时间附着在原代培养。上皮的产物内开始48-72小时（1B，C）。小件的血管（1D）可以是一个来源的间充质细胞的产物。上皮增生表现形态异质的人口，人口与增殖含有混合物的细胞与肌上皮细胞，祖细胞和管腔系（1E，3C-E）的标记。在低应力媒体，如M87A补充与霍乱毒素和催产素的增长，支持优势长期增长的正常预瘀HMEC相比之前媒体的配方（ 图2）。 M87A型媒体也将支持贯通通道的管腔和祖细胞生长4-8（ 图3C-E，4），此后，大部分细胞谱系标记仅显示肌上皮。 HMEC培养在塑料上可以保留，它们能形成适当的三维微图案三维微孔自组织，与管腔内部的肌上皮细胞（ 图4A，B）的细胞，并以形成当在基质胶（ 图4C，D）的镀覆时的有组织的结构。 <img src="/files/ftp_upload/50011/50011fig1.jpg" alt="图1"/> 图1。 HMEC类器官和原代培养 （A）类器官消化，过滤后的导管肺泡结构。 （B，C）类器官导管和腺泡结构放置在原代培养后2天内开始注意电池outgr零增长（白色箭头）。 （D）连接的小血管和成纤维细胞相同的文化产物，从开始B，C （E）上皮细胞的产物，从初级化培养后第4天。谱系的识别与抗体染色K14（红色）和K19（绿色）;细胞核用DAPI（蓝）进行染色。管腔（K14-/K19 +，绿色），肌上皮（K14 + / K19-，红色），和祖（K14 + / K19 +黄色细胞）是可见的。未染色的细胞中观察到的组织体核心，由于不完全抗体渗透。 <img src="/files/ftp_upload/50011/50011fig2.jpg" alt="图2"/> 图2。开始从一个个体，标本184次，获得增长的HMEC文化在不同的培养基配方。类器官的原代培养，使用不同的培养基配方。我们最近的配方，M87A +催产素（X）4，获得最佳的长期增长。该媒体还支持生长的多个HMEC谱系（见图1E）。较早的媒体制定，MM提供较少的强劲增长，而一个无血清培养基，MCDB170（商业MEGM）17引线快速诱导周期素激酶抑制剂p16基因INK4A异常细胞7,11,13和选择。 <img src="/files/ftp_upload/50011/50011fig3.jpg" alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/50011/50011fig3highres.jpg"/> 图3。谱系多样性比较蒙昧的组织体和培养HMEC在4 个通道 （A，B）流式细胞仪分析的教养，酶解法分离组织体（A）表达的，EpCAM和CD49f/alpha 6整合，及（B）CD227/Muc1 CD10/CALLA。 （C，D）4 个通道前血瘀HMEC流式细胞仪分析表达（C）EpCAM和CD49f/alpha 6的整合，和（D）CD227/Muc1 CD10/CALLA。可识别populations标记为LEP（表达的管腔标志物EPCAM或CD227），MEP（CD49f或CD10的表达肌上皮标记），或PROG（丰富的CD49f + / EPCAM +人口）。请注意，在文化适应调控的EpCAM及CD49f的变化相比，没有文化的组织体。 （E）未分选的HMEC和FACS富集LEP和MEP在4 日通过染色，免疫荧光法检测角蛋白K14（MEP标记）和K19（LEP标记），以验证血统鉴定。细胞核用DAPI染色显示为蓝色。 <img src="/files/ftp_upload/50011/50011fig4.jpg" alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/50011/50011fig4highres.jpg"/> 图4。培养HMEC能够在体内的血统关系时，放置在适当的微环境，形成有组织的结构。（A）血瘀前4 个通道HMEC FACS富集我使用置身于管腔LEP和肌上皮的MEP谱系标志物CD227和CD10，K14和K19的表达验证这些谱系（图中未示出）。 （B）在微图案微孔混合在一起时，培养的细胞能够自我组织成双层MEP在外面和内部LEP [改编自香颂等 。5]。荧光标记的LEP（绿色）和MEP（红色）被成像的激光共聚焦显微镜，在0小时，24小时，和48小时后，除微孔（上部）。控制HMEC被任意带有红色或绿色的荧光标记（低级）标记。 （C）明场像FACS富集祖细胞（CKIT +）镀在3D（基底膜）文化和生长18天。由此产生的结构可以表现出肺泡的形态。 （D）结构中提取基底膜染色检测K14和K19;与DAPI复染细胞核。免疫荧光分析显示正确的管腔和基础波拉组织结构Rity的。

Watch this Scientific Journal Video about Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture at JoVE.com

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

一个方法来处理人类乳腺外科丢弃的材料。加工组织，在组织体的形式，可以存储无限期冻结或放置在文化的长期增长。此方法使正常的人类上皮细胞生物学，实验考试和外生扰动的影响。

处理人减少乳房整形和乳房切除术的组织细胞培养

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

processing-human-reduction-mammoplasty-mastectomy-tissues-for-cell

Nanyang Technological University

Research

JoVE Journal

Medicine

16.5K 次观看.  Lawrence Berkeley National Laboratory. 一个方法来处理人类乳腺外科丢弃的材料。加工组织，在组织体的形式，可以存储无限期冻结或放置在文化的长期增长。此方法使正常的人类上皮细胞生物学，实验考试和外生扰动的影响。

视频: Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

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

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

癌症研究

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

使用微生理系统模拟人体乳房组织中的乳腺癌

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

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

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

实时检测和捕获共培养物中的侵袭细胞亚群

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

从正常和肿瘤乳腺组织中生成和成像小鼠和人上皮类器官，无需传代

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

制备基于微流体的血管化人类微肿瘤模型

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

作者聚焦：创建人类血管化微肿瘤作为转化癌症研究的模型 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

建立用于癌症研究的生理性人血管化微肿瘤模型

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

处理人减少乳房整形和乳房切除术的组织细胞培养

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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

人类乳腺上皮细胞致癌转化的体外评价

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

使用微生理系统模拟人体乳房组织中的乳腺癌

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

实时检测和捕获共培养物中的侵袭细胞亚群

从正常和肿瘤乳腺组织中生成和成像小鼠和人上皮类器官，无需传代

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型