笔者想感谢她的技术专长和安德鲁·拉森Hjelm布鲁克为他的蛋白质分析。这项工作是由部分替代品的研究发展基金会（MMHK）资助和NIH NIAID性病的感染和局部杀菌剂合作研究中心IU19 AI062150-01（MMHK）。我们感谢数字重用的生殖生物学。

层流罩BSL-2的条件下，应执行的所有步骤。 1。准备STLV生物反应器<ol><li>组装STLV生物反应器，根据制造商的协议和执行戒毒协议，以确保生物反应器的无菌。覆盖带Luer帽的开放口岸，并填充为24小时95％的乙醇STLV。 </li><li>除去乙醇，用无菌蒸馏水24小时，并填写的STLV。 </li><li>重复步骤1.2只用无菌蒸馏水。 </li><li>由供应商提供的工具，松开的STLV所有螺丝帽和中心插头，反应釜，在110℃20分钟灭菌袋。 </li><li> STLV一旦被冷却，拧紧螺丝，并重复步骤1.1-1.4，以确保无菌和完整的排毒。 </li><li>冷却STLV和预消毒单向活塞的每个端口上的螺丝拧紧螺丝。 </li><li>打开活塞旋钮垂直定位。 </li><li>从10毫升和5毫升露儿的锁注射器和重新套筒活塞在包装保持无菌的柱塞。拧到活塞的注射器（鲁尔锁尖注射器这一步）。 </li><li>加入Dulbecco的磷酸盐缓冲液（DPBS）10毫升注射器，直到STLV是满满的，并开始填补了5毫升注射器。 </li><li>替换成每个注射器无菌活塞和清除残留气泡发生在生物反应器，通过推动注射器活塞之间交替。 </li><li>离开后，取出所有气泡附着生物反应器的注射器。关闭旋塞和附加STLV旋转的垂直平台和旋转最低为24小时监测泄漏。 </li></ol> 2。准备微载体珠<ol><li> STLV（1.4）相同的条件下，加入15毫升的DPBS〜250毫克cytodex微载体珠在50 mL锥形管和高压。 </li><li>冷却后，取出DPBS，添加增长epitheli的媒体人细胞的兴趣，旋流管悬浮珠。 </li><li>重复用媒体两次以上的步骤。最后洗净后加15毫升培养基珠。 </li></ol> 3。播种的STLV生物反应器中的上皮细胞和珠<ol><li>生长在组织培养瓶中单层上皮细胞的利息，直到你获得≥1×10 7细胞。从烧瓶（即trypinsization，EDTA）中取出细胞，计数细胞建立细胞浓度，细胞活力，并确定由台盼蓝染色1。 </li><li>准备珠（见2.3）的锥形管，添加所需的细胞浓度（2X10 5-1×10 7细胞）2，8，根据你的意图上皮细胞型（图1A）。确保所有细胞冲洗锥形管转移。 </li><li>删除从STLV DPBS和注射器。 </li><li>从中心端口的插头，并添加上皮ÇELL /到STLV珠悬浮液10毫升血清吸管。冲洗锥形管，以确保所有细胞/珠转移到STLV和更换中心端口插头。 </li><li> 5和10毫升的注射器和重新套回封套柱塞从柱塞保持无菌。拧入活塞开放的端口注射器。填写与媒体的STLV，更换柱塞，并关闭活塞。 </li><li>新种子在37℃培养箱STLV放置30分钟，没有旋转。 </li><li>打开旋塞，除去气泡，使用柱塞，然后关闭旋塞。 </li><li>放置在37℃，5％ 二氧化碳培养箱孵化器旋转20转（图1B）STLV。文化必须不断旋转了24公顷的天，除采样时，改变媒体，或采伐总量（见下一节）。 </li></ol> 4。文化的培养，取样，照片文档<ol><li>经过最初的96-120小时的双向培养细胞oreactor，改变媒体倾斜STLV，使细胞/珠定居。取下注射器，同时打开活塞，倒出〜75％的媒体从侧面端口（ 图1C）。基于对媒体的细胞代谢，改变媒​​体后最初的播种和96-120 h培养期间每天或隔日。 </li><li>螺杆5和10毫升的注射器活塞无效，并按照协议，如上所述复食（1.7-1.11），然后拧紧封闭的STLV回旋转平台到位。 </li><li>监测后，每隔5-7天仔细的聚集少量（约200μL），分为两个1.5 mL管中心端口从播种到STLV总量的增长和活力。为了避免总剪切，使用所有总转让1000μL枪头，已被切割点〜2厘米和消毒。 </li><li>更换中心插头，用新注射器交换，添加新的媒体如上所述STLV（1.7-1.11），并将其放置在STLV回旋转的孵化器（见3.8）。 </li><li>用于监测细胞活力，使用两个步骤4.3 1.5 mL管分装总量，并从细胞上皮细胞组织培养瓶（如胰蛋白酶）删除珠以同样的方式。台盼蓝排斥染色1监测的可行性。 </li><li>成像细胞聚集，加上媒体的0.5-1毫升1.5毫升的第二个总等份，并转移到一个小培养皿中使用的截止1000μL枪头。图片总量，用倒置显微镜（图1D）。 </li></ol> 5。转移和收获团聚<ol><li>对于一些上皮细胞模型，它是必要的，一次性增加文化曝气2 HARV转移总量。大约一个星期前，收获总量进行分析，取出针筒和活塞从STLV倒〜50％，从侧面端口的媒体。 </li><li>雷姆奥雅纳STLV的中心插头，并仔细倒入所有内容，从中央端口（图1E）的50 mL锥形管。冲洗用5 mL媒体STLV两次，并结合冲洗聚集休息。 </li><li>继续转移，如5.2中所述的聚集，但转移总量从50 mL锥形管HARV。 </li><li>仔细从HARV〜1周后，或从STLV收获总量（根据细胞类型）进行上述（5.2）。看到下面的格式潜力分析，检测和分析后，总收成。 </li></ol> 6。潜力分析，应用与骨料进行检测<ol><li> 各种实验的格式播种总量。转入50 mL锥形管所需的聚集量1.5 mL管，24孔板，96孔板使用消毒削减了1000μL枪头（图1F）。 </li><li> 洗涤和preparING总量进行分析。删除媒体纷纷落户聚集之后。免除DPBS直接管中心或聚集激起了。一旦总量已落户的底部，删除的DPBS并视需要多次重复。 </li><li> 航运总量为异地实验和分析。加入所需的聚集量50毫升管和洗总量在6.2。完全填补围绕第50毫升，以避免漏管媒体，帽，封口膜。安全运送聚集到所需的位置，在常温下过夜。 </li><li> 固定电子显微镜的集合体（图2）。可进行扫描，传输或免疫电镜150-300微升聚集转移到1.5 mL管和洗衣机总量DPBS（6.2）的3倍。加入200μL的具体应用（透射电镜，扫描电镜，免疫EM等）所需的潜伏期电子显微镜固定液。删除修复，洗骨料ES的2倍DPBS，并进行概述Hjelm 等。为2010年2扫描和透射电子显微镜。 </li><li> 荧光显微镜成像（图3）。转让〜1.5 mL管和洗总量（6.2）100μL的聚合。修复和标签聚合与抗体与膜类似的条件下，除在1.5 mL管。装入，放在显微镜玻片上安装媒体1滴，转让的截止1000微升枪头，总量超过地方盖玻片，密封盖玻片指甲油，干通宵幻灯片2安装媒体上的标记聚合。 </li><li> 测量细胞活力/增殖采用MTT法（图4）。转移聚集到一个24孔板和625μL酚红的自由媒体取代媒体。每孔加入62.5μL5毫克/毫升噻唑蓝四氮唑盐（MTT）和4小时于37°C。加入625μL100毫克/毫升SDS-0.01M盐酸每口井和孵育过夜。阅读在570 nm处的吸光度，并获得％的细胞活力，用公式： </li></ol><img alt="式（1）" src="/files/ftp_upload/3868/3868eq1.jpg" /><ol start="7"><li> 毒理学研究 。转移至24孔板的聚集和执行台盼蓝排斥≥2井的初始细胞存活率/浓度。添加浓度范围复制井测试化合物。对于细胞活力（图5A），洗总量和执行处理后的台盼蓝排斥。为TC 50（图5B），每个浓度随着时间的推移重复井细胞活力，并用芦苇Muench方法，以确定中毒浓度50％，15。 </li><li> 式微珠阵列（CBA）或酶联免疫吸附试验 。细胞上清可以采取播种后在细胞实验绘图格式。刺激细胞成长为种子聚合单层，收集分析酶联免疫吸附试验或CBA（图6）上清（≥120μL），并储存于-80°C。 </li><li> RNA分析。转移1.5毫升管与DPBS洗总量≥500μL的骨料。继续提取，使用动物细胞裂解使用20号针头的同质化Qiagen公司RNAeasy套件和协议。确保让珠管底部在解决之前，转移上清，不转移空珠离心柱（珠将堵塞列膜造成RNA产量低）（图7）。 </li><li> 蛋白质分析。根据相同的方法与使用1.5 mL管或更大的实验格式的单层收获总量。然而，溶解后的聚集，使珠定居和运行前的一种蛋白质凝胶或其他形式的蛋白质分析（图8）转移到一个单独的管裂解。 </li><li> 感染研究。种子聚合成所需的实验格式。从珠子trypsinizing细胞枚举细胞数量和细胞活力量化的使用至少有两个井/样品。感染与利益的病原体聚集在选定的多重感染与细胞生长为单层（图9）执行。 6.2洗涤步骤必须执行。 </li><li> 流式细胞仪检测：种子总量在50毫升管，洗总量（6.2），2MM EDTA的添加为5-10分钟，在37°C。加入5-10毫升媒体细胞，通过细胞一个细胞过滤器。等分1×10 6细胞在寒冷的封闭液（在PBS FBS的1％），聚丙烯管和洗涤细胞。根据制造商的抗体和孵育重悬细胞。洗，悬浮在PBS，离心细胞和转移细胞过滤管进行分析（图10）。 </li></ol> 7。代表结果一个例子图2可以看出，在有区别的人类上皮细胞在的STLV生物反应器系统的总种植。 SEM和TEM图像采集从人阴道中的STLV成长为39天的细胞，microridges，内陷，细胞分泌囊泡，并在根尖表面的微绒毛可以观察。 体内生物反应器中生长的上皮细胞样特性的进一步论证，可以观察到免疫荧光共聚焦显微镜图像（ 图3）粘蛋白（MUC1基因）表达的3-D阴道细胞和上皮细胞的特定标志物（ESA）的终末分化（外皮;兴业）。 的3-D总量所示，显示类似组织的超微结构特点，但他们更多的有关生理表型也很好的平台，为评估化合物的毒性。 MTT法，常用的检测，以测量细胞活力，新陈代谢或苔TION，可以用来测量各种化学品和化合物（ 图4）的毒性。的Triton X-100，洗涤剂，破坏细胞膜，被用来作为阳性对照，MTT法验证。一个额外的方法来衡量毒性测试化合物治疗后，台盼蓝排斥（ 图5）。壬苯醇醚9（N）的治疗之后，阴道总量表现出宫颈外植体模型的毒性反应类似，但不同的配置文件相比，16单层2。 进一步的研究表明上皮细胞的能力，在生物反应器中生长，函数和信号同样在体内的组织，在图6中可以观察到。由Toll样受体（TLR）识别微生物的分子刺激后，聚合反应和分泌促炎症细胞因子，包括IL-6 1。表达孕激素受体 ​​（PR），响应激素刺激的受体，也可在阴道细胞在RNA和蛋白水平（ 图7和图8），分别观察。的3-D聚集不仅有能力应对病原体和激素分子，但也能功能支持疱疹性病毒2型（HSV-2）感染。共聚焦显微镜图像的HSV-2感染的3-D（ 图9）阴道聚集演示感染。不显示并行2ð阴道上皮细胞培养细胞HSV-2感染引起的严重破坏的结果。 RWV衍生的3-D总量也已用于量化，通过细胞生长曲线和斑块检测细菌和病毒的复制，分别为8，9。最后，单个细胞内聚集的物理和功能特性，也可以通过流式细胞仪进行量化。对于examp乐，我们采用了流式细胞仪检测，以测量MUC1的个人（ 图10）阴道细胞表面表达。阴道的3-D总量表示的MUC1的比例较低（27.7％）比单层（67.2％）。 MUC1的比例相比，以单层的3-D骨料表面上可能是一个MUC1的分泌细胞，观察阴道道17，18多数的结果。 <img alt="图1" src="/files/ftp_upload/3868/3868fig1.jpg" /> 图1。人类上皮细胞的培养，在RWV和使用RWV派生聚集的潜在应用示意图。）上皮细胞开始生长在组织培养瓶中单层。一旦融合，细胞将被删除从烧瓶结合STLV生物反应器微载体珠。比例尺：80微米）STLV旋转在一个平台上创造低f恒定的自由落体环境LUID剪切，使细胞附着和生长从而形成可见细胞聚集，C珠）96小时后，在STLV媒体必须改变，以适应细胞的新陈代谢。媒体倒入一个侧面端口，新媒体是通过一个附加的10毫升注射器（柱塞删除）取代，更换注射器活塞，用于挤出泡沫。 四）（四）〜5天之后开始聚集形成。采样总量每7天，并通过光镜成像监测其发展的进展（3-D人力阴道上皮细胞的20倍放大）E），一旦发生完整的团聚体的形成和细胞分化，聚合生物反应器和收获转移到50 mL锥形管F）骨料，可接种到1.5毫升试管或一个多孔板格式进行了实验分析和检测。G）的大纲，POTE<html>总量上可以进行的。107检测和分析。这种分析代表名单并不意味着是一个对所有潜在的下游应用的详尽目录。 <img alt="图2" src="/files/ftp_upload/3868/3868fig2.jpg" /> 图2。检查使用电子显微镜的3-D上皮细胞聚集体的形态和结构特点。（一）扫描电子显微镜（SEM）图像的3-D人体阴道上皮细胞聚集。比例尺：200微米（100倍），100微米（200X），50微米（500X）。透射电子显微镜（TEM）影像（二）细胞分泌囊泡和微绒毛或（C）“突起”的箭头指向一个3-D的人类上皮阴道细胞聚集。比例尺：2微米（Hjelm 等2010年修改）2。 g3.jpg“/&gt; 图3。共焦荧光显微镜，用来确定在3-D的上皮细胞聚集交界的分化和蛋白质标记。人阴道细胞的3-D收获后32天，从生物反应器，固定，染色（一）蛋白MUC1粘蛋白抗体，（B）抗体特定上皮细胞，欧空局，或（三）反外皮（INV）承认终末分化的上皮细胞的抗体。间接标记聚合网站488中学抗体（绿色）。比例尺：60微米（Hjelm 等2010年修改）2。 <img alt="图4" src="/files/ftp_upload/3868/3868fig4.jpg" /> 图4。 MTT法是用来测量细胞活力。人力阴道聚集的3-D，接种于24孔板，并与媒体单独（未处理）或0.01％，0.1％或1.0％的Triton X-100为1小时的清洁剂，治疗37° C。跟随着<html> Ğ治疗，媒体被删除，以MTT法测量细胞活力。 **表示P &lt;0.001;单尾学生的t检验比较的Triton X-100处理的细胞，未经处理的控制。 <img alt="图5" src="/files/ftp_upload/3868/3868fig5.jpg" /> 图5。利用屏幕化合物毒性的3-D的上皮细胞聚集（A）壬苯醇醚-9（N）剂量依赖性的可行性曲线相比，24小时后治疗的3-D人阴道上皮细胞模型（红线/条）相同的细胞类型成长为合流膜（黑线/条），（B）的N-9 TC 50（一）为1.5小时，4小时，8小时和24小时的治疗后阴道上皮细胞培养水平。 *表示P &lt;0.05;单尾学生的t检验比较，单层3-D细胞在每次曝光时间。 （修改Hjelm 等 2010）2。 / files/ftp_upload/3868/3868fig6.jpg“/&gt; 图6。 FSL-1（TLR2的/ 6），PIC（TLR3的），旗（TLR5的刺激生产的3-D上皮细胞的细胞因子在分析三D人力阴道细胞Toll样受体（TLR）激动剂刺激的反应。） ，CL097（TLR7 / 8）为24小时和分泌细胞因子式微珠阵列法测定。 IL-6所示为代表的炎症细胞因子的产生和测量。 *表示P &lt;0.05;单尾学生的t检验比较刺激PBS组样品。 （修改Hjelm 等 2010）2。 <img alt="图7" src="/files/ftp_upload/3868/3868fig7.jpg" /> 图7。监测的3-D上皮细胞基因表达的半定量RT-PCR分析孕激素受体 ​​（PR）的表达在单层（毫升）或3-D人阴道上皮细胞。GAPDH的负荷控制。扩增产物显示30个PCR循环后发生的谈话表达的结果。 箭头头部点公关带只发生在3-D采样。 <img alt="图8" src="/files/ftp_upload/3868/3868fig8.jpg" /> 图8。与抗孕激素受体 ​​（PR）抗体的3-D上皮细胞的蛋白质表达分析。单层的3-D人阴道上皮细胞的全细胞裂解液（30μg）Western blot分析探讨。 β-微管蛋白作为探针加载控制。 <img alt="图9" src="/files/ftp_upload/3868/3868fig9.jpg" /> 图9。立体人类上皮细胞模型，支持生产的病毒感染。共焦荧光显微镜图像的3-D人阴道上皮细胞感染HSV-2总。细胞感染了两个小时，洗净，固定24 h后感染（4％PFA）的，在MOI 1.0沾满了HSV-2的具体VP5基因Çapsid抗体（绿色）和核染色的DAPI（蓝色）。比例尺：50微米。 <img alt="图10" src="/files/ftp_upload/3868/3868fig10.jpg" /> 图10。在单个细胞的3-D的人类上皮细胞模型，流式细胞仪分析。 （一）单层或（乙）的3-D人阴道上皮细胞聚集与EDTA分离，标有1 FITC标记蛋白MUC1抗体（蓝色），同型对照（绿色），或PBS（红色），FITC标记的表达量化流式细胞仪。数字表示，染色门后保持了背景荧光染色细胞的同型对照的粘蛋白抗体阳性的细胞百分比。非均匀峰是众多glycosolation这些细胞表面的图案和MUC1的不同程度的结果。

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作者没有透露。

利用这里RWV生物反应器技术，可提供与能力的研究，以促进他们的细胞培养系统，以更生理相关的器官细胞培养模型。 RWV生物反应器细胞培养系统提供了一个低剪切力的微环境，使细胞在体内类似的特征，包括紧密连接，粘蛋白生产过程（即微绒毛）外，与细胞极性形成的3-D细胞聚集。这里提出的数据和例子，大部分是利用阴道上皮细胞生长在RWV系统，但是RWV系统也被用于其他文化包括小...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>的Alexa Fluor 488 </td><td> Invitrogen公司</td><td> A21131 </td><td>在1:500稀释使用</td></tr><tr><td> FACSDiva </td><td>屋宇署</td><td></td><td>流式细胞仪</td></tr><tr><td> β-tublin抗体</td><td> Calbiochem公司</td><td> 654162 </td><td>用于在1:5000稀释</td></tr><tr><td>生物Plex的2000 </td><td> BioRad公司</td><td> 171-000205 </td><td> V5软件</td></tr><tr><td>生物反应器及零件</td><td> synthecon </td><td>信用社-4 </td><td></td></tr><tr><td>细胞过滤器</td><td>屋宇署猎鹰</td><td> 352340 </td><td> 40μm的孔大小</td></tr><tr><td>锥形管（50毫升） </td><td>康宁</td><td> 5-538-60 </td><td></td></tr><tr><td>盖玻片</td><td>厂商VWR </td><td> 48366067 </td><td></td></tr><tr><td>因子珠阵列包</td><td> BioRad公司</td><td>自定义人类套件</td><td></td></tr><tr><td> cytodex珠</td><td>西格玛</td><td> C3275 </td><td></td></tr><tr><td> DPBS </td><td> Gibco公司</td><td> 14190 </td><td></td></tr><tr><td> EDTA的</td><td>西格玛</td><td> ED-500G </td><td>乙二胺四乙酸</td></tr><tr><td>上皮细胞特异性抗体（ESA） </td><td> Chemicon公司</td><td> CBL251 </td><td>在1:50稀释使用</td></tr><tr><td>胎牛血清（FBS）， </td><td> Gibco公司</td><td> 10438 </td><td>热灭活</td></tr><tr><td> HARV（一次性） </td><td> synthecon </td><td>的D-405 </td><td></td></tr><tr><td>盐酸</td><td>西格玛</td><td> 258148 </td><td> 37％ </td></tr><tr><td>外皮抗体</td&gt; <td>西格玛</td><td>我9018 </td><td></td></tr><tr><td>显微镜载玻片</td><td>厂商VWR </td><td> 16004-368 </td><td></td></tr><tr><td> MTT法试剂</td><td> MP Biomedicals，LLC </td><td> 194592 </td><td> 3 - （4,5-Dimethylthiazolyl 1-2）-2,5 - 二苯基四氮唑溴化物</td></tr><tr><td>粘蛋白抗体（显微镜） </td><td>圣克鲁斯</td><td> SC-7313 </td><td>在1:50稀释使用</td></tr><tr><td>粘蛋白抗体（流式细胞仪） </td><td>屋宇署Pharmingen公司</td><td> 559774 </td><td>也称为CD227，使用20μL每个测试</td></tr><tr><td> 多聚甲醛 </td><td>电子显微镜科学</td><td> 15710 </td><td>在DPBS稀释到4％ </td></tr><tr><td>培养皿中（小） </td><td>屋宇署猎鹰</td><td> 353002 </td><td></td></tr><tr><td>带过滤器的聚苯乙烯管</td><td>屋宇署猎鹰</td><td> 352235 </td><td></td></tr><tr><td&gt;聚苯乙烯流管</td><td>屋宇署猎鹰</td><td> 352058 </td><td></td></tr><tr><td>公关抗体</td><td> DAKO公司</td><td> M3569 </td><td>在1:100稀释使用</td></tr><tr><td>延长黄金</td><td> Invitrogen公司</td><td> P36931 </td><td>用DAPI安装媒体</td></tr><tr><td>的RNeasy迷你套件</td><td> Qiagen公司</td><td> 74903 </td><td></td></tr><tr><td>十二烷基硫酸钠</td><td>西格玛</td><td> 71725 </td><td></td></tr><tr><td>灭菌袋</td><td>厂商VWR </td><td> 11213-035 </td><td></td></tr><tr><td>活塞（单程） </td><td> Medex公司</td><td> MX5061L </td><td></td></tr><tr><td>注射器（10ML） </td><td>屋宇署</td><td> 309604 </td><td>露儿的锁尖</td></tr><tr><td>注射器（5ML） </td><td>屋宇署</td><td> 309603 </td><td>露儿的锁尖</td></tr><tr><td>台盼蓝</td><td> Invitrogen公司</td><td>T10282 </td><td></td></tr><tr><td> VP5抗体</td><td>圣克鲁斯</td><td> SC-13525 </td><td> HSV-2抗体克隆6F10; 1:5000稀释</td></tr></tbody></table>

culturing and applications of rotating wall vessel bioreactor derived 3d epithelial cell models

身体经验的环境条件，影响他们的建筑间的通信，和整体功能的细胞和组织。 在体外细胞培养模型，以准确地模仿利益的组织，文化的生长环境是一个重要的方面考虑。常用的传统的细胞培养系统中传播的平面两维（2D）不透水表面的上皮细胞。虽然已经从传统的细胞培养系统了解到，许多研究结果都无法在人体临床试验或组织外植体的重现，可能由于缺乏一个有关生理微环境的结果。 在这里，我们描述了一个文化系统，克服许多的2-D细胞培养的培养条件的界限，通过创新的旋转壁式（RWV）生物反应器技术，。我们和其他人已经表明，器官RWV派生模型可以概括结构E，功能，和外界刺激的类似人类的外植体组织1-6正宗的人类反应。 RWV生物反应器悬浮培养体系，使上皮细胞的生理低流体剪切条件下的生长。在两个不同的格式，高方面的旋转容器（HARV）或缓慢转弯外侧的船只（STLV），他们在不同的曝气源生物反应器。添加到上皮细胞的生物反应器的选择结合多孔胶原涂层的微载体珠（图1A）。这些细胞在生物反应器在恒定的自由落体（图1B）利用作为增长脚手架的珠子。由生物反应器提供的微环境，使这些细胞形成三维（3 - D）聚集在体内类似的特征显示，往往不按标准的2-D培养条件（图1D）观察。这些特征包括紧路口，MUC我们的生产，根尖/基底取向， 在体内蛋白质的定位，和额外的上皮细胞类型的特定属性。 由单层上皮细胞发展到一个完全分化的3-D总会有所不同，根据细胞类型，7-13。从生物反应器的定期抽样允许监测上皮细胞聚集形成，细胞的分化和活力（图1D）。一旦建立细胞分化和聚合形成，收获细胞生物反应器，可以应用于2-D细胞进行类似的实验（图1E-G）的几个因素的3-D的聚集。在这项工作中，我们描述的详细步骤如何培养RWV生物反应器系统的3-D在上皮细胞聚集和各种潜在的检测和分析，可以执行的3-D的总量。这些分析包括，但不仅限于，结构/平方米orphological分析（聚焦，扫描和透射电子显微镜），细胞因子/趋化因子的分泌和细胞信号（）流式细胞小球微阵列和Western blot分析，基因表达分析（实时PCR），毒理学/药物分析和宿主 - 病原体相互作用。利用这些检测为更深入和广泛的研究，如代谢组学，转录组学，蛋白质组学和其他基于阵列的应用奠定了基础。我们的目标是培养人类上皮细胞非传统的手段来产生器官概括在人类体内组织，一个浅显的和强大的系统与多样的科学兴趣的研究人员使用的3-D模型。

Watch this Scientific Journal Video about Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models at JoVE.com

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

描述一个旋转细胞培养系统，使上皮细胞在3 - D细胞聚集形成的生理条件下成长。聚合生成显示<em&gt;在体内</em&gt;样的特点不遵守传统的文化模式，并为众多的科学调查的器官更精确的模型系统。

旋转壁式生物反应器的培养和应用推导出三维上皮细胞模型

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. ...

culturing-applications-rotating-wall-vessel-bioreactor-derived-3d

Research

JoVE Journal

Biology

26.2K 次观看.  University of Arizona College of Medicine - Phoenix. 描述一个旋转细胞培养系统，使上皮细胞在3 - D细胞聚集形成的生理条件下成长。聚合生成显示在体内样的特点不遵守传统的文化模式，并为众多的科学调查的器官更精确的模型系统。

视频: Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

科研

生物学

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

粉末样品的绝对量子产量测量

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

一个培养人关节软骨细胞的三维系统在滑液

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

精子冷冻保存和恢复使用I ·冷冻包

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

膜和细胞壁选择性荧光染料在丝状真菌活细胞成像中的应用

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

肠道维利表皮的器官3D培养进行分化

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...