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
Summary
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>在体内</em>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。
Abstract
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。
Protocol
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。 除去乙醇，用无菌蒸馏水24小时，并填写的STLV。 重复步骤1.2只用无菌蒸馏水。 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…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Alexa Fluor 488 | Invitrogen | A21131 | Used at 1:500 dilution |
| FACSDiva | BD | Flow cytometer | |
| β-tublin antibody | Calbiochem | 654162 | Used at 1:5000 dilution |
| Bio-Plex 2000 | BioRad | 171-000205 | v5 software |
| Bioreactor and components | Synthecon | RCCS-4 | |
| Cell strainer | BD Falcon | 352340 | 40μm pore size |
| Conical tube (50mL) | Corning | 5-538-60 | |
| Coverslips | VWR | 48366067 | |
| Cytokine bead array kits | BioRad | Custom human kit | |
| Cytodex beads | Sigma | C3275 | |
| DPBS | Gibco | 14190 | |
| EDTA | Sigma | ED-500G | Ethylenediaminetetraacetic acid |
| Epithelial specific antibody (ESA) | Chemicon | CBL251 | Used at 1:50 dilution |
| Fetal Bovine Serum (FBS) | Gibco | 10438 | Heat inactivated |
| HARV (Disposable) | Synthecon | D-405 | |
| Hydrochloric acid | Sigma | 258148 | 37% |
| Involucrin antibody | Sigma | I 9018 | |
| Microscope slides | VWR | 16004-368 | |
| MTT reagent | MP Biomedicals, LLC | 194592 | 3-(4,5-Dimethylthiazolyl 1-2)-2,5-Diphenyl Tetrazolium Bromide |
| MUC1 antibody (microscopy) | Santa Cruz | Sc-7313 | Used at 1:50 dilution |
| MUC1 antibody (flow cytometry) | BD Pharmingen | 559774 | Also called CD227, use 20μL per test |
| Paraformaldehyde | Electron Microscopy Sciences | 15710 | Diluted to 4% in DPBS |
| Petri dish (small) | BD Falcon | 353002 | |
| Polystyrene tube with filter | BD Falcon | 352235 | |
| Polystyrene flow tube | BD Falcon | 352058 | |
| PR antibody | DAKO | M3569 | Used at 1:100 dilution |
| ProLong Gold | Invitrogen | P36931 | Mounting media with DAPI |
| RNeasy Mini Kit | Qiagen | 74903 | |
| Sodium dodecyl sulfate | Sigma | 71725 | |
| Sterilization pouch | VWR | 11213-035 | |
| Stopcocks (one-way) | Medex | MX5061L | |
| Syringe (10mL) | BD | 309604 | Luer-lock tip |
| Syringe (5mL) | BD | 309603 | Luer-lock tip |
| Trypan Blue | Invitrogen | T10282 | |
| Vp5 antibody | Santa Cruz | sc-13525 | HSV-2 antibody Clone 6F10; used at 1:5000 dilution |
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Tags
Rotating Wall Vessel Bioreactor3D Epithelial Cell ModelsIn Vitro Cell CultureGrowth EnvironmentConventional Cell Culture SystemsPhysiologically Relevant MicroenvironmentRotating Wall Vessel (RWV) Bioreactor TechnologyOrganotypic ModelsExternal StimuliSuspension Culture SystemLow Physiological Fluid Shear ConditionsHigh-aspect Rotating Vessel (HARV)Slow-turning Lateral Vessel (STLV)Aeration Source
Cite This Article
Radtke, A. L., Herbst-Kralovetz, M. M. Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models. J. Vis. Exp. (62), e3868, doi:10.3791/3868 (2012).