我们感谢艾米迈尔斯和ESI Quayson技能和帮助。这项工作是支持部分由NSF 0216310和奇华顿公司授予（净入学率）。

1。获得人力蕈状味觉​​乳头<ol><li>取出四到八个舌头的前部分采用弧形弹簧microscissors背表面的菌状味觉乳头。 </li><li>立即进入隔离解决方案的地方（ 碳酸氢钠 26毫米，2.5毫米的NaH 2 PO 4计 ，葡萄糖20毫米，65毫米氯化钠，氯化钾20毫米，1毫米EDTA在无核酸酶水溶解和过滤消毒）。 </li></ol> 2。人菌状的味道乳头消解人蕈状味觉细胞乳头可以是分离的，使用两种不同的酶消化协议略有不同。 <ol><li>在隔离解决方案（1毫克/毫升，Sigma Aldrich公司）与蛋白酶和弹性蛋白酶（1毫克/毫升Sigma Aldrich公司），在室温30-45分钟（协议）的菌状乳头孵育。 </li><li>菌状乳头孵育与胶原酶I型（550 U / ml的，从沃辛顿）+弹性蛋白酶（10 U / ml的，从麦汁hington）+胰蛋白酶抑制剂（0.9毫克/毫升，从沃辛顿）2毫升钙免费林格液35°C间为30分钟和95％的O 2/5％CO 2的温柔氧合循环水浴（替代方法消化，协议2） </li><li>孵育后，用铃声的乳头和磨碎用火抛光玻璃巴​​斯德吸管10倍的乳头。 </li><li>离心3分钟，在室温下在2500转和删除隔离的解决方案和味觉细胞的培养基中添加1毫升。 </li><li>转移到玻璃盘的消化菌状乳头。 </li><li>菌状乳头，轻轻地用解剖手术剃刀。 </li><li>添加到克隆到鼠尾胶原蛋白1型涂层盖玻片缸250解剖乳头液。 </li><li>每孔加入1毫升的味觉细胞培养基。 </li></ol> 3。蕈状味道人类乳头细胞的培养<ol><li>在36°C孵育板在加湿孵化器含有5％CO 2。 </li><li> 2天前的第一个变化完全培养基放置在孵化器不受干扰。味觉细胞，最终将绑定到涂层的盖玻片，虽然它可能不是清晰可见，在1至3天。 </li><li>从克隆板和中缸完全，并添加到每口井的味觉细胞培养液1毫升。 </li><li>中期每6-7天更换1/3。不改变更频繁和/或完全培养基。 </li><li>隔日检查，显微镜下细胞的生长。大多数新的细胞生长细胞簇之下。通常分离培养2-3周后离开新产生的细胞的细胞团。 </li><li>一旦扩大味觉细胞覆盖40-50％的克隆缸，trypsinize细胞使用2-3分钟0.25％W / V胰蛋白酶/ EDTA在36°C。 </li><li>转入15毫升管从水井细胞，味觉细胞的培养基中添加3卷在3000转离心5分钟，在室温下的温升。 </li><li>删除1毫升口味细胞培养基上清，重悬细胞。 </li></ol> 4。传播人类的蕈状味道乳头细胞<ol><li>转入的T25板细胞和味觉细胞培养液（通道0）添加4毫升。保持在36°C在加湿孵化器的含5％CO 2的细胞。 </li><li>中期每6-7天更换1/3，直到培养味觉细胞已达到100％汇合。此时收获，使味觉细胞，用无菌PBS洗细胞一次，然后trypsinize细胞使用2-3分钟0.25％W / V胰蛋白酶/ EDTA在36°C。 </li><li>如上所述，离心后，悬浮在新鲜的T-75瓶（通道1）完整的味觉细胞培养液和转移细胞。 </li><li>中期每6-7天更换1/3。不改变更频繁和/或完全培养基。 </li><li>重复步骤4.3和4.4时，细胞已达到接近100％汇合。细胞分裂中的T75瓶不超过1:4稀释细胞保持足够的增长，随着时间的推移。 </li></ol> 5。培养人的蕈状味觉乳头细胞冻融<ol><li>冻结初级味觉细胞的股票后，胰蛋白酶（步骤4.2），加3毫升完整的味觉细胞介质和细胞转移无菌15毫升锥形离心管。在室温下5分钟在2500转离心。 </li><li>冷冻介质中含95％胎牛血清（FBS）和5％DMSO适当的音量，小心取出上清液，轻轻重悬细胞。 </li><li>转移细胞标记，无菌冷冻管，盖紧瓶盖，冷冻集装箱含有异丙醇的地方。到1 -80℃至少一天前无限期地转移到液氮冷冻的地方。 </li><li>解冻一小瓶冷冻首要人权蕈状味觉乳头细胞，地点在37℃水浴，直到刚刚解冻（约1分钟），而在层流细胞培养工作罩。 </li><li>细胞和冷冻介质转移到无菌的15毫升锥形离心管和味觉细胞的培养基中添加5毫升。 </li><li>在室温下5分钟2500转离心细胞。小心弃上清，完整的味觉细胞培养液中，并转移到无菌的T-25的组织培养瓶轻轻重悬细胞。 </li><li>根据步骤3到4，继续培养细胞。 </li></ol> 6。代表结果成长的文化品位活检的尝试是成功的90％以上的时间。活检和分解后几天，细胞开始生长和解剖蕈状组织块分离程序后仍然向外迁移。虽然单个细胞可见电镀（ 图1A）24-48小时后，细胞生长到2-3周连接的细胞簇下，偶尔会产生子细胞（ 图1B）。从活检ING 4-8人的菌状乳头，初步分离的首要人权蕈状味觉乳头细胞达到约2000 - 8000细胞在4周文化。扩张的T25细胞培养瓶，额外的3-4周（通道0），将导致在文化适应人蕈状味觉乳头细胞（ 图1C）。首要人权蕈状味觉乳头细胞可以进一步扩大在文化，通过-1在3-4周（ 图1D）至少有一百万个细胞产生向上。主要的味觉细胞会继续增长超过8通道。 文化品味培养人的蕈状乳头细胞的形态是培养化学感受细胞类似前面描述11,12 ......培养人蕈状味觉乳头细胞保持其原始细胞体小巧美观，带或不带一个或多个进程长达15-30天。当他们到达汇合培养CELLS有极化-拉长的外观。成熟的味觉细胞组成几个不同的组织学亚型和展览味觉细胞的特定功能。在这里，我们表明，在这些文化中的细胞显示许多分子和生理功能，成熟的味觉细胞的特征。 Gustducin和磷脂酶C - β2（PLC-β2）mRNA的逆转录-聚合酶链反应和测序（ 图2）确认产品检测。 Gustducin和PLC-β2表达检测免疫细胞化学显示II型样细胞（ 图3，B和D）的存在。 Gustducin和的PLC-β2反映细胞信号通路和细胞类型的不准确地反映体内的患病率和相对表达模式。不同类型的细胞内的味蕾相对分布的因素是未知的，在培养条件，不得复制。因此，B<html>之间培养的细胞，其在体内的同行提供了一个机会，在分子水平上研究这些特点的监管，可以操纵和监控的条件下，超视距异同。 一个模型系统的一个重要的标准是存在相关的功能特性。培养的细胞还表现在几个味觉刺激（ 图4）适当浓度细胞内钙的强劲增长。在这些研究中，甜和苦的刺激，引起细胞内钙的增加可能对应到去极化。像这些味觉细胞，培养细胞的性质贷款支持的说法，我们在这里描述的模型系统将是一个宝贵的资产为进一步研究转导通路和人的味觉细胞的再生能力。 fig1.jpg“/&gt; 图1。附件和培养人的菌状的味道乳头细胞形态。成像后2天（A）的首要人权蕈状味觉乳头细胞生长在胶原涂层板-1型文化。人类蕈状的味道乳头细胞生长最多4个星期在连接的细胞团，这似乎给子细胞产生。细胞通常增长汇合四个星期内（二），（C和D）收获后，补种分别代表天2周和4周。细胞培养至少8个月持续增长。比例尺= 50纳米。 图1部分还介绍了在Ozdener 等。 10。 <img alt="图2" src="/files/ftp_upload/3730/3730fig2.jpg" /> 图2 RT-PCR结果显示在特定的味觉细胞的生物标志物的mRNA（Gustducin和PLC-β2）的存在。从培养人的蕈状细胞的总RNA反转录ribed用于RT-PCR（Invitrogen公司）使用上标第一链合成系统和PLC-β2Gustducin和检测设计的特异引物扩增的PCR用。引物（ 表1）被选为跨越一个或多个内含子。每个基因检测在体外培养的人蕈状的味道，从乳头细胞通过4或5的预期大小的扩增产物，经测序证实。没有反向酶表示基因组DNA污染，没有特定的基因控制实验检测。 PCR产物经2％琼脂糖凝胶电泳分离和凝胶的扩增产物用剃刀切除。提取DNA，使用凝胶QIAquick凝胶提取试剂盒（Qiagen）。在宾夕法尼亚大学测序设施PCR产物进行测序。的C-PCR和C-RT PCR和逆转录的对照实验，分别。 图2部分还介绍了在Ozdener 等。等。 <sup&gt; 10。 <img alt="图3" src="/files/ftp_upload/3730/3730fig3.jpg" /> 图3。培养的人类味道蕈状乳头细胞通过4个或5个免疫组织化学染色表明味觉细胞的生物标志物的存在。培养人的蕈状味觉乳头细胞（5天），用4％多聚甲醛固定，培养小学抗体（见表2）在4°C过夜图像被收购的Leica TCS SP2激光共聚焦扫描显微镜。大约60％的培养菌状的味道乳头细胞标记gustducin（B）和（四）与PLC-β2的20-30％。相应的细胞传输图像显示（A和C）。对于控件，与抗体的特异性免疫球蛋白免疫证明的非特异性免疫反应（数据未显示）的情况下。比例尺= 50纳米。部分图3中还介绍了在Ozdener 等。 10。 <img alt="图4" src="/files/ftp_upload/3730/3730fig4.jpg" /> 图4。培养人的蕈状的味道，从第4代或5乳头细胞不同刺激作出反应。培养人的蕈状味觉乳头细胞（4-5日龄）被载入1MM的Fura-2 AM（分子探针）和10毫克/毫升聚醚F127的（分子探针）。使用标准手册成像技术在体外培养的人蕈状味觉乳头细胞的细胞内钙离子水平（内[Ca 2 +] i）的变化进行测量。细胞可视化使用倒置荧光显微镜在340 nm和380 nm的激发波长和发射波长在510 nm带通滤波器中心设置。刺激的沐浴液，然后溶解在pH值和渗透压的调整，如果需要的话。培养人的蕈状味觉乳头细胞，甜和苦的刺激。 Ğ微晶说明代表反应的[Ca +2]我接触（一）Denatonium（2毫米），（二）蔗糖（1毫米）在单个细胞水平。每个跟踪代表单个体细胞。 <table border="1"><tbody><tr><td> 基因 </td><td> 序列 </td><td colspan="2"> PCR反应条件 </td><td> 预计规模 （BP） </td><td> 参考 </td></tr><tr><td> β-肌动蛋白</td><td> GGACTTCGAGCAAGAGATGG AGCACTGTGTTGGCGTACAG </td><td> 7分钟 45秒 45秒 45秒 7分钟</td><td> 95 94 53 72 72 </td><td> 234 </td><td> NM_001101.3 </td></tr><tr><td> gustducin </td><td> TCTGGGTATGTGCCAAATGA GGCCCAGTGTATTCTGGAAA </td><td> 7分钟 45秒 45秒 45秒 7分钟</td><td> 95 94 53 72 72 </td><td> 386 </td><td> NM_001102386 </td></tr><tr><td> PLC-β2 </td><td> GTCACCTGAAGGCATGGTCT TTAAAGGCGCTTTCTGCAAT </td><td> 3分钟 30秒 30秒 60秒 7分钟</td><td> 95 94 53 72 72 </td><td> 333 </td><td> NM_004573 </td></tr></tbody></table> 表1。品尝特定分子检测引物和使用条件。 <table border="1"><tbody><tr><td> 初级抗体 </td><td> 源 </td><td> 主持人 </td><td> 稀释 </td><td> 二级抗体 </td><td> 源 </td><td> 主持人 </tD&gt; <td> 稀释 </td></tr><tr><td> gustducin </td><td>圣克鲁斯</td><td>兔</td><td> 1:500 </td><td>抗兔IgG 633网站</td><td>分子探针</td><td>山羊</td><td> 1:500 </td></tr><tr><td> PLCβ-2 </td><td>圣克鲁斯</td><td>兔</td><td> 1:500 </td><td>抗兔IgG 633网站</td><td>分子探针</td><td>山羊</td><td> 1:500 </td></tr></tbody></table> 表2。抗体用于检测特定分子的表达。

<ol>
	<li>Mbiene, J. P., Maccallum, D. K., Mistretta, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+cultures+of+embryonic+rat+tongue+support+tongue+and+gustatory+papilla+morphogenesis+in+vitro+without+intact+sensory+ganglia.">Organ cultures of embryonic rat tongue support tongue and gustatory papilla morphogenesis in vitro without intact sensory ganglia.</a> J. Comp. Neurol. 377, 324-340 (1997).</li><li>Miyamoto, T., Fujiyama, R., Okada, Y., Sato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+difference+in+amiloride-sensitivity+of+salt-induced+responses+in+mouse+non-dissociated+taste+cells.">Strain difference in amiloride-sensitivity of salt-induced responses in mouse non-dissociated taste cells.</a> Neurosci. Lett. 277, 13-16 (1999).</li><li>Caicedo, A., Jafri, M. S., Roper, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+Ca2++imaging+reveals+neurotransmitter+receptors+for+glutamate+in+taste+receptor+cells.">In situ Ca2+ imaging reveals neurotransmitter receptors for glutamate in taste receptor cells.</a> J. Neurosci. 20, 7978-7985 (2000).</li><li>Qin, Y. M., Shi, J. Q., Zhang, G. H., Deng, S. P., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reliable+method+to+obtain+cells+of+taste+buds+from+fungiform+papillae+of+mice.">A reliable method to obtain cells of taste buds from fungiform papillae of mice.</a> Acta Histochem. 112, 107-112 (2010).</li><li>Spielman, A. I., Mody, I., Brand, J. G., Whitney, G., MacDonald, J. F., Salter, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+isolating+and+patch-clamping+single+mammalian+taste+receptor+cells.">A method for isolating and patch-clamping single mammalian taste receptor cells.</a> Brain Res. 503, 326-329 (1989).</li><li>Kishi, M., Emori, Y., Tsukamoto, Y., Abe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+rat+taste+bud+cells+that+retain+molecular+markers+for+taste+buds+and+permit+functional+expression+of+foreign+genes.">Primary culture of rat taste bud cells that retain molecular markers for taste buds and permit functional expression of foreign genes.</a> Neuroscience. 106, 217-225 (2001).</li><li>Ruiz, C. J., Stone, L. M., McPheeters, M., Ogura, T., Bottger, B., Lasher, R. S., Finger, T. E., Kinnamon, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+rat+taste+buds+in+primary+culture.">Maintenance of rat taste buds in primary culture.</a> Chem. Senses. 26, 861-873 (2001).</li><li>Stone, L. M., Tan, S. S., Tam, P. P., Finger, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell+lineage+relationships+in+taste+buds.">Analysis of cell lineage relationships in taste buds.</a> J. Neurosci. 22, 4522-4529 (2002).</li><li>Ozdener, H., Yee, K. K., Cao, J., Brand, J. G., Teeter, J. H., Rawson, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+long-term+maintenance+of+rat+taste+cells+in+culture.">Characterization and long-term maintenance of rat taste cells in culture.</a> Chem. Senses. 31, 279-290 (2006).</li><li>Ozdener, M. H., Brand, J. G., Spielman, A. I., Lischka, F. W., Teeter, J. H., Breslin, P. A., Rawson, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Human+Fungiform+Papillae+Cells+in+Culture.">Characterization of Human Fungiform Papillae Cells in Culture.</a> Chem. Senses. 36, 601-612 (2011).</li><li>Gomez, G., Rawson, N. E., Hahn, C. G., Michaels, R., Restrepo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+odorant+elicited+calcium+changes+in+cultured+human+olfactory+neurons.">Characteristics of odorant elicited calcium changes in cultured human olfactory neurons.</a> J. Neurosci. Res. 62, 737-749 (2000).</li><li>Wolozin, B., Sunderland, T., Zheng, B. B., Resau, J., Dufy, B., Barker, J., Swerdlow, R., Coon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+culture+of+neuronal+cells+from+adult+human+olfactory+epithelium.">Continuous culture of neuronal cells from adult human olfactory epithelium.</a> J. Mol. Neurosci. 3, 137-146 (1992).</li><li>Wick, N., Saharinen, P., Saharinen, J., Gurnhofer, E., Steiner, C. W., Raab, I., Stokic, D., Giovanoli, P., Buchsbaum, S., Burchard, A., Thurner, S., Alitalo, K., Kerjaschki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomal+comparison+of+human+dermal+lymphatic+endothelial+cells+ex+vivo+and+in+vitro.">Transcriptomal comparison of human dermal lymphatic endothelial cells ex vivo and in vitro.</a> Physiol. Genomics. 17, 179-192 (2007).</li><li>Fu, L., Zhu, L., Huang, Y., Lee, T. D., Forman, S. J., Shih, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+neural+stem+cells+from+mesenchymal+stemcells:+evidence+for+a+bipotential+stem+cell+population.">Derivation of neural stem cells from mesenchymal stemcells: evidence for a bipotential stem cell population.</a> Stem Cells Dev. 17, 1109-1121 (2008).</li><li>Sandow, S. L., Grayson, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limits+of+isolation+and+culture:+intact+vascular+endothelium+and+BKCa.">Limits of isolation and culture: intact vascular endothelium and BKCa.</a> Am. J. Physiol. Heart Circ. Physiol. 297, H1-H7 (2009).</li></ol>

没有利益冲突的声明。

我们一直保持着味道从人类的蕈状乳头细胞获得超过8通道，横跨12个月的期限。这些文化产生新的细胞，其中许多在体外成熟的阶段，他们表示，除了味觉受体细胞的关键分子和生理属性的几个指标，成熟的味蕾细胞类型。存在gustducin和PLC-β2在培养的细胞亚群的免疫反应是与这一结论相一致。最重要的是，某些细胞对甜和苦味刺激的应用与细胞内钙离子的短暂增加，这是一个功能分离的味...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td></tr><tr><td> MCDB上123 </td><td> Sigma-Aldrich公司</td></tr><tr><td> iscove的DMEM培养基</td><td>敏达</td></tr><tr><td>弹性蛋白酶</td><td> Sigma-Aldrich公司</td></tr><tr><td>蛋白酶E </td><td> Sigma-Aldrich公司</td></tr><tr><td>弹性蛋白酶</td><td>沃辛顿</td></tr><tr><td>大豆胰蛋白酶抑制剂</td><td>沃辛顿</td></tr><tr><td>型胶原酶1 </td><td>沃辛顿</td></tr><tr><td>鼠尾胶原蛋白1型</td><td>屋宇署科学</td></tr><tr><td> FURA-2 </td><td>分子探针</td></tr><tr><td>标用于RT-PCR的第一链合成系统</td><td> Invitrogen公司</td></tr><tr><td>徕卡TCS的SP2光谱共焦显微镜</td><td>莱卡微系统公司</TD&gt; </tr><tr><td>愉-1成像站和MetaMorph软件</td><td>分子器件</td></tr><tr><td>小精尖钳和额外的鸟语花香剪刀</td><td>罚款科学工具</td></tr></tbody></table>

isolation and culture of human fungiform taste papillae cells

味觉细胞是高度专业化的，具有独特的组织学，分子生物学和生理特性，允许一个简单的刺激和食品中含有复杂的化学分子的广泛检测。在人类，个体的菌状乳头，包含从零到多达20个味蕾。有没有人的味觉细胞培养建立的协议，尽管保持文化品位乳头细胞的多种细胞周期的能力将是相当实用，这些独特的感官细胞的特征分子，再生和功能特性。味觉细胞的早先的研究已经完成，使用新鲜细胞原代培养，外植体培养，从啮齿类动物或半完整的味蕾，在组织切片1,2,3,4。虽然这些准备工作的每个人都有优势，将有长远的文化发展提供了重要的好处，特别是对味觉细胞增殖和复合驱研究rentiation。几个团体，包括我们在内，一直在味觉细胞培养模型的建立和发展有兴趣。大多数尝试以文化品位细胞报道有限的活力，细胞通常不超过3-5ð5,6,7,8持久。我们最近报道对啮齿动物的味觉细胞9扩展文化的一个成功的方法。我们在这里报告的第一次孤立的人类蕈状味觉乳头细胞在体外培养体系的建立。活检取自人体的菌状乳头细胞成功地保持了八个多通道（12个月）没有丧失活力的文化。细胞表现出成熟的味觉细胞特征的许多分子和生理功能。 Gustducin和β2，磷脂酶C（PLC - β2）的mRNA在许多细胞，通过逆转录 - 聚合酶链反应检测和测序证实。免疫细胞化学分析表明，在场的阵风ducin和PLC-β2培养味觉细胞中的表达。体外培养的人蕈状细胞也表现出适当的浓度表明，味觉受体和信号通路至少是目前的几种口味刺激细胞内钙离子的增加。这些结果充分表明，成年人类的味觉细胞可以产生和保持为至少8个通道。许多人保留着细胞的急性分离的从成年味道上皮细胞，以支持其作为一种模式味觉系统的使用和生理生化特性。这个系统将使参与增殖，分化和功能在哺乳动物味觉受体细胞， 在体外制备过程的进一步研究。 蕈状人的味觉乳头建立人力蕈状细胞培养的捐赠研究适当的知情同意后，根据研究进行了审查协议由IRB委员会批准。舒尔曼联营机构审查委员会公司，俄亥俄州辛辛那提市批准的协议（＃0934）。书面协议下面是根据报告公布的参数由Ozdener 等 。 2011年10。

Watch this Scientific Journal Video about Isolation and Culture of Human Fungiform Taste Papillae Cells at JoVE.com

Isolation and Culture of Human Fungiform Taste Papillae Cells

我们旨在建立一个孤立和维护人类菌状的味道乳头细胞的长期文化重现协议。活检取自人体的菌状乳头细胞成功地保持了八个多通道（12个月）没有丧失活力的文化。

人力蕈状味觉​​乳头细胞的分离和培养

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

isolation-and-culture-of-human-fungiform-taste-papillae-cells

Research

JoVE Journal

Neuroscience

14.3K 次观看.  Monell Chemical Senses Center. 我们旨在建立一个孤立和维护人类菌状的味道乳头细胞的长期文化重现协议。活检取自人体的菌状乳头细胞成功地保持了八个多通道（12个月）没有丧失活力的文化。

视频: Isolation and Culture of Human Fungiform Taste Papillae Cells

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

从人类毛囊神经嵴干细胞的分离和培养的

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

科研

神经科学

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

人类胶质母细胞瘤肿瘤细胞的分离和扩大使用的神经球含量

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

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

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

从胚胎前脑内皮细胞的分离和培养

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

Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller &#38; Reedy&#8217;s 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller &#38; Reedy&#8217;s original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller &#38; Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies.

Here we present a protocol to measure fungiform papilla density from digital photographs. This method builds prioritization and objective characteristic metrics into the original descriptive work on fungiform papillae by Miller &#38; Reedy (1990).

为菌状乳头的客观分析丹佛乳头协议

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

胚胎小鼠神经干细胞的分离与培养

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

小鸡西里神经神经元的分离与培养

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...

人力蕈状味觉​​乳头细胞的分离和培养

人力蕈状味觉乳头细胞的分离和培养