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
Summary
传统的，二维细胞培养技术，往往导致与分化标志物，细胞因子和生长因子的改变的特点。旋转细胞培养系统的三维细胞培养（农村信用社）重新建立许多因素的表达，绒毛外滋养细胞系。
Abstract
人类滋养细胞研究领域的艾滋病在认识复杂的环境中建立在胎盘。由于这些研究的性质， 人体内实验是不可能的。一个小学文化，外植体培养和滋养层细胞系的组合，支持我们的绒毛外滋养细胞（EVTs），这是怀孕的成功建立所必需的入侵的子宫壁和子宫螺旋动脉重塑3,4的理解。尽管从这种模型中收集的知识财富，它是接受，使用类似的EVT -细胞株在体外细胞培养模型显示改变细胞特性相比，其在体内的同行5,6时。培养细胞在旋转细胞培养系统（农村信用社）显示的EVT样细胞株的形态，表型和功能特性，更加紧密地模仿u的区别人Tero EVTs，调解入侵（如基质金属蛋白酶（MMPs））和滋养层细胞分化7,8,9的基因表达增加。圣乔治医院的胎盘细胞株- 4（SGHPL 4）（请盖伊惠特利博士和博士朱迪思卡特赖特捐赠）是一个EVT样细胞系，用于在农村信用社的测试。
农村信用社培养容器的设计是基于对器官和组织的原则，即在一个三维（3 – D）环境功能。由于在船只动态的文化条件，包括有关生理剪切条件，细胞生长在三个方面形成聚集基于自然细胞亲和力和分化成器官组织的集会10,11,12。维护流体轨道提供了一个低剪切，低动荡的环境相似，在体内发现的条件。培养细胞沉淀是通过调整旋转反驳加快农村信用社，以确保细胞恒定的自由落体。气体交换发生通过透水疏水膜生物反应器的背面。像他们的父母在体内的组织，能够应对在三个层面（即在其顶端，基底和横向表面），因为他们培养的多孔微载体珠表面上化学和分子梯度力场的细胞生长。像塑料防渗表面的二维膜生长时，细胞被剥夺了这一重要的沟通，在其基底表面。因此，空间限制的环境所施加深刻影响细胞如何从周围的微环境意识和解码信号， 从而暗示13的3 – D环境的重要作用。
我们已经使用了农村信用社工程师7,14,15,16各种人类上皮组织的生物学意义的3 – D模型。事实上，许多以前的报告中有DEMonstrated农村信用社培养细胞可以承担有关生理尚未10,17-21其他机型可能表型。总之，在农村信用社的文化代表了一种简单，重现性好，高通量平台，提供大量分化的细胞，适合各种实验操作。在下面的协议，作为一个例子使用EVTs，我们清楚地说明需要三个维度文化的贴壁细胞在农村信用社的步骤。
Protocol
1。胶原珠的制备在此之前的3 – D细胞培养加载EVTs，需要准备的Cytodex – 3微载体珠： 称取适量的实验所需的Cytodex – 3珠。该协议是适应农村信用社的10毫升船只，其中珠0.05克需要。对于一个50毫升农村信用社的船只，相应的规模。在一个50毫升的高压灭菌的锥形管，混合磷酸12mL贝科250毫克Cytodex – 3珠缓冲液（DPBS）。这一数额是足够5个农村信用社的船只。 确保有足够的量?…
Discussion
这里介绍的文化技术提供具有高度侵袭性的EVT样细胞的调查。如今，它已被公认，由于抑制细胞反应，化学和分子线索的三个层面（心尖，基底和横向细胞表面 ）10,13在单层发生分化的损失。这种技术反映在子宫内指出入侵的EVT细胞的特点。由于程序模仿传统的单层组织培养的时间动力学，但提供了差异表达的细胞，比较分析可能被雇用。在实验中使用的细胞团可收获，在任?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生授予NIH / NICHD的HD051998（CAM）的机构的支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Cytodex microcarrier beads | Sigma-Aldrich | C3275 | |
| Rotating Cell Culture System (RCCS) | Synthecon | RCCS-D | Includes rotor base, power supply, 4 disposable RCCS units |
| RCCS Disposable Units | Synthecon | Contact Synthecon | |
| 3ml Luer-Lock tip syringe | BD | 309585 | |
| 10ml wide-tip serological pipette | BD | 357504 | |
| MEM Alpha | Invitrogen | 12561-072 | |
| Leibovitz’s L-15 medium, powder | Invitrogen | 41300-039 | |
| H2O, Endotoxin free | Fisher | MT-25-055-CM | |
| Sodium Bicarbonate | Sigma-Aldrich | S-7795 | |
| Peptone | Fisher Scientific | BP1420-100 | |
| Fructose | Sigma-Aldrich | F3510-100 | |
| Galactose | Sigma-Aldrich | G5388-100 | |
| Glucose | Sigma-Aldrich | G7528-250 | |
| HEPES | Invitrogen | 15630-080 | |
| L-Glutamine | Invitrogen | 25030 | |
| Insulin-Transferrin-Sodium Selenite (ITS) | Sigma-Aldrich | I1884 | |
| FBS | Invitrogen | 10437 | |
| Penicillin-Streptomycin | Invitrogen | 15140 |
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Tags
Rotating Cell Culture SystemsHuman Cell CultureHuman Trophoblast CellsModelPrimary CulturesExplant CulturesTrophoblast Cell LinesInvasion Of Uterine WallRemodeling Of Uterine Spiral ArteriesExtravillous Trophoblast CellsSuccessful Establishment Of PregnancyIn Vitro Cell Culture ModelsRotating Cell Culture System (RCCS)Morphological PropertiesPhenotypic PropertiesFunctional PropertiesEVT-like Cell LinesDifferentiation Of EVTsGene ExpressionMatrix Metalloproteinases (MMPs)Trophoblast DifferentiationSaint Georges Hospital Placental Cell Line-4 (SGHPL-4)RCCS Culture VesselThree-dimensional Environment
Citar este artigo
Zwezdaryk, K. J., Warner, J. A., Machado, H. L., Morris, C. A., Höner zu Bentrup, K. Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model. J. Vis. Exp. (59), e3367, doi:10.3791/3367 (2012).