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

Research

JoVE Journal

Medicine

16.4K 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

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

加工的主要脑肿瘤组织的干细胞检测和流量排序

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

科研

医学

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

人体前庭神经鞘瘤的原代培养

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

人掌腱膜挛缩的<em&gt;离体</em&gt;文化为肌成纤维细胞的研究和细胞外基质相互作用

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

从人乳腺癌组织及细胞系微球体形成分析

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

与人体组织转化为癌症研究工作

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

人类成胶质细胞瘤器官型切片培养模型，用于研究肿瘤细胞迁移和抗创伤药物的患者特异性作用

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

痰液诱导和实验室处理方法

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...