从三维混合细胞球体共培养的乳腺上皮细胞的隔离
Summary
被形容为一个简单的方法，分析相关的上皮细胞组织成纤维细胞的影响。这种方法和三维组织培养相结合，可以方便从3D隔离后的细胞分析。该技术适用于不同恶性潜能的细胞，使系统的研究对肿瘤细胞的肿瘤相关基质的影响。
Abstract
虽然已经进入了巨大的努力识别信号通路和分子参与正常细胞和恶性行为1-2，使用传统的二维细胞培养模型，这项工作已完成，允许细胞操纵方便。它已成为明确的细胞内信号通路的影响外的力量，包括维和细胞的表面张力3-4。已采取多种途径，开发三维模型，更准确地反映生物组织架构3。虽然这些模型纳入多维度和建筑讲，由此产生的对细胞影响的研究是比在两维模型的局限性和困难，在随后的分析中提取细胞组织培养的简便。
重要作用，肿瘤周围的微环境在肿瘤发生的行为我之所以成为越来越认识4。肿瘤基质是由多种细胞类型和细胞外分子。在肿瘤的发展有肿瘤细胞和基质细胞之间的双向信号。虽然参与肿瘤基质协同进化的一些因素已经确定，仍然有必要制定简单的技术，系统地识别和研究这些信号6阵列。成纤维细胞是最丰富的细胞类型，在正常或肿瘤相关间质组织，促进基底膜和旁分泌生长因子7的沉积和维护。
许多团体已经使用三维培养系统研究成纤维细胞的各种细胞功能的作用，包括肿瘤反应治疗，免疫细胞招聘，信号分子，细胞增殖，凋亡，血管生成，侵袭8-15。我们已经优化了屁股的简单方法对乳腺上皮细胞，使用市售的细胞外基质的模型来创建三维混合细胞群（联合培养）16-22文化essing乳腺成纤维细胞的影响。续共培养细胞的形成与球体内部的聚类成纤维细胞和上皮细胞很大程度上取决于外部的球体，形成多细胞的预测，到矩阵。操纵改变上皮细胞的侵袭，导致成纤维细胞可以很容易量化的数量和长度23上皮预测的变化。此外，我们已经制定为隔离上皮细胞三维共培养，促进成纤维细胞的对上皮行为暴露的影响分析的方法。我们已经找到了合作文化的影响，持续数周后上皮细胞的分离，允许充裕的时间来执行多个检测。这种方法是适应细胞不同恶性潜能，不需要专门的设备。这种技术允许多个条件下，在体外细胞模型的快速评估和相应的结果，可以在动物体内组织模型以及人体组织样本。
Protocol
1。建立三维共培养建立联合培养的前一天，从4℃冰箱中冷冻和解冻冰基底膜一夜之间。 在当天的实验，准备共同的文化，它对应的上皮细胞，可用于介质（在这个例子中的三菱电梯中等介质：DME/F12介质与10％小牛血清，5微克/毫升胰岛素1微克/毫升氢化可的松，1毫微克/毫升表皮生长因子）。保持冰至少1小时前与基底膜混合介质。 冲淡了解冻的基底膜，确定总的基底膜所需…
Discussion
这里介绍的方法代表着一个简单的方法分析，包括肿瘤细胞的上皮细胞间质成纤维细胞的影响。它允许直接的细胞内细胞外基底膜基质的三维背景的互动，作进一步的分析，同时使基质细胞容易提取。这可以应用于研究肿瘤相关基质，成纤维细胞系可以被操纵，影响信号通路的选择和上皮线，可以在不同的侵入性的潜力，如在图1所示。三维共培养模型，使细胞侵袭的变化已经引起视?…
Disclosures
The authors have nothing to disclose.
Materials
| Name of reagent | Company | Catalogue number | Comments |
| BD Matrigel | BD Biosciences | 356237 | Phenol-red free |
| Hyclone Classical Liquid Medium: DMEM F12 | ThermoScientific | SH30023.01 | |
| Hyclone Bovine Calf Serum | ThermoScientific | SH3007303 | |
| Insulin | EMD Chemicals | 407709 | Dissolve in 0.005N HCl; 0.22 μM filter |
| Hydrocortisone | Sigma-Aldrich | H4001 | Dissolve in 50% EtOH; 0.22 μM filter |
| EGF | Sigma-Aldrich | E9644 | Dissolve in 10mM acetic acid; 0.22 μM filter |
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Tags
Mammary Epithelial CellsThree-dimensional Mixed-cell Spheroid Co-cultureSignaling PathwaysMoleculesNormal Cell BehaviorMalignant Cell BehaviorTwo-dimensional Cell Culture ModelsIntracellular Signaling PathwaysExtracellular ForcesDimensionalityCell Surface TensionThree-dimensional ModelsMicroenvironmentTumorsTumorigenesisTumor BehaviorTumor StromaBidirectional SignalsStromal CellsFibroblasts
Cite This Article
Xu, K., Buchsbaum, R. J. Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture. J. Vis. Exp. (62), e3760, doi:10.3791/3760 (2012).