A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids
A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids
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DOI：
10.3791/2720
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发表时间：
2011-05-01
影响因子：
1.2
通讯作者：
Foty, Ramsey
中科院分区：
文献类型：
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作者：
Foty, Ramsey
细胞-细胞凝聚力和细胞-基质粘附的研究历来是在粘附于刚性基质的单层培养物上进行的。然而，组织内的细胞通常被包裹在紧密堆积的组织块内，其中细胞与许多近邻和细胞外基质组分建立紧密连接。因此，3D组织内的细胞所经历的化学环境和物理力与单层培养中生长的细胞所经历的化学环境和物理力根本不同。这已被证明会显著影响细胞形态和信号传导。已经设计了几种方法来产生3D细胞培养物，包括将细胞包封在胶原凝胶（1）或生物材料支架（2）中。这些方法虽然有用，但不能概括在正常组织中发现的紧密的直接细胞-细胞粘附结构。相反，它们更接近培养系统，其中单细胞松散地分散在ECM产物的3D网状结构中。在这里，我们描述了一种简单的方法，其中细胞被放置在悬滴培养和在生理条件下孵育，直到它们形成真正的三维球体，其中细胞直接接触彼此和细胞外基质成分。该方法不需要专门的设备，并且可以适于包括添加非常少量的任何生物试剂，其可能对阐明对细胞-细胞或细胞-ECM相互作用的影响感兴趣。该方法还可用于共培养两种（或更多种）不同的细胞群，以阐明细胞-细胞或细胞-ECM相互作用在指定细胞之间的空间关系中的作用。细胞-细胞凝聚和细胞-ECM粘附是研究胚胎发育、恶性侵袭中的肿瘤-基质细胞相互作用、伤口愈合以及应用于组织工程的基础。这种简单的方法将提供一种产生组织样细胞聚集体的方法，用于测量生物力学特性或用于生理相关模型中的分子和生物化学分析。
Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels(1)or in biomaterial scaffolds(2). Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.