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肠和大肠，肺癌，膀胱癌，肝癌，和扁桃体上皮细胞的上皮细胞类型形成三维器官模型1，7-13，18。此外，该系统已用于文化类型的细胞，上皮细胞以外，目前发展的复杂，multicellular器官培养模型正在进行2，18。 协议概述，这里是用来作为指导，并可能需要进行修改，根据细胞类型的利益。最常见的修改协议培养在RWV系统的上皮细胞，包括细胞培养中STLV时间的长短，决定，并从STLV的HARV的总传输时间，为初始细胞/珠比播种中的STLV。一个更专门的修改，可能被认为是成功的聚合形成微载体珠或支架的特性，如表面积，直径，形状，表面涂层，密度，电荷，核材料，孔隙度，。此外，调整的基础培养基，配方，或补充剂，可能是必要的优化培养条件，最大限度地聚合形成。也可以进行调整，以生物反应器系统及其部件，cluding生物反应器容器的大小和转速。灌流培养系统生物反应器内的微环境，增强评估，也可以允许内聚集形成一个多产的环境，文化，确保船只的pH值，氧气和葡萄糖水平的连续监测。优化培养条件下，聚集形成，细胞分化和表征每一个新的模型系统，以及生物反应器系统的初始费用，潜在的冗长时间是值得一提的这个系统的缺点。然而，任何新的文化系统，在实验室中实现，将有类似的缺点。 我们和其他人已经表明，利用人体细胞的RWV的衍生车型，可能是一个有价值的疫苗，杀菌剂，生物制剂和一种时尚，是有关人类1，2，18药品的疗效，毒性和药代动力学预测工具。与日成功生物反应器培养系统生产正宗的人类反应，与它的灵活性相结合，RWV生物反应器系统的应用，目前正在扩大等领域作为肿瘤模型，再生医学，组织工程2，18-23。为了推进我们的RWV生成粘膜模型，我们正在努力创造一个更复杂的多细胞复制人体的黏膜组织的结构和功能的系统。然而，作者承认利用或整合多模型系统，重现药物或疫苗与人体生理系统有复杂的相互作用，它可能是必要的。总的来说，我们的目标是使用RWV生物反应器及其实验应用，以更好地了解粘膜组织生物学和细胞反应在人体器官模型环境的侮辱。

<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

Nanyang Technological University

Research

JoVE Journal

Biology

26.3K 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

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

科研

生物工程

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

专访：干细胞研究的生物反应器和三维支架材料表面改性

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

生物学

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

中空纤维生物反应器的<em&gt;在体内</em&gt;般的哺乳动物组织培养

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

功能性与极化胆囊肿的生成与定量特征

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

将3D多细胞乳房球体直接生物打印到内皮网络

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

牙齿和呼吸细胞及器官在微重力下的繁殖

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

<meta charset="utf8"></head><body>用于上皮细胞和成纤维细胞的先进双层 3D 共培养系统

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(&#949;-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

<meta charset="utf8"></head><body>作者聚焦:使用 PVA-PCL 纳米纤维膜推进 3D 共培养系统

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

使用纳米纤维膜建立上皮细胞的三维共培养模块

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...