Three-dimensional culture models of normal and malignant breast epithelial cells
Three-dimensional culture models of normal and malignant breast epithelial cells
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DOI：
10.1038/nmeth1015
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发表时间：
2007-04-01
期刊：
影响因子：
48
通讯作者：
Bissell, Mina J.
中科院分区：
文献类型：
--
作者：
Lee, Genee Y.;Kenny, Paraic A.;Bissell, Mina J.
细胞外基质是正常稳态和组织表型的关键调节因子（1）。当细胞在二维塑料基质上离体培养时，重要的信号丢失。许多这些关键的微环境线索可以使用富含层粘连蛋白的细胞外基质（IrECM）的三维（3D）培养物来恢复（2）。这些3D培养测定允许非恶性和恶性乳腺细胞之间的表型区分，因为前者在3D环境中生长形成极化的、生长停滞的腺泡样集落，而后者形成无序的、增殖的和非极性的集落（3）。当细胞暴露于基底膜样凝胶时，在塑料上培养的细胞中平行发挥作用的信号传导途径变得不可整合（4-7）。因此，适当的3D培养提供了离体分析基因功能和细胞表型的更生理相关的方法。我们在这里描述了一个强大的和通用的方法，用于培养各种人类乳腺细胞系的三维和描述的准备从这些文化的分子分析的细胞提取物。以下程序描述了3D“包埋”测定，其中细胞包埋在lrECM凝胶中进行培养（8）（图1）。lrECM是指源自Engelbreth-Holm-Swarm小鼠肉瘤细胞的溶解提取物（9）。有关3D矩阵的用户选项的讨论，请参见方框1。或者，可以如框2中所述使用3D“在顶部”测定，其中细胞在覆盖有lrECM稀溶液的薄lrECM凝胶的顶部上培养（图1和图2）。
Extracellular matrix is a key regulator of normal homeostasis and tissue phenotype(1). Important signals are lost when cells are cultured ex vivo on two-dimensional plastic substrata. Many of these crucial microenvironmental cues may be restored using three-dimensional (3D) cultures of laminin-rich extracellular matrix (lrECM)(2). These 3D culture assays allow phenotypic discrimination between nonmalignant and malignant mammary cells, as the former grown in a 3D context form polarized, growth-arrested acinus-like colonies whereas the latter form disorganized, proliferative and nonpolar colonies(3). Signaling pathways that function in parallel in cells cultured on plastic become reciprocally integrated when the cells are exposed to basement membrane-like gels(4-7). Appropriate 3D culture thus provides a more physiologically relevant approach to the analysis of gene function and cell phenotype ex vivo. We describe here a robust and generalized method for the culturing of various human breast cell lines in three dimensions and describe the preparation of cellular extracts from these cultures for molecular analyses. The procedure below describes the 3D 'embedded' assay, in which cells are cultured embedded in an lrECM gel(8) ( Fig. 1). By lrECM, we refer to the solubilized extract derived from the Engelbreth-Holm-Swarm mouse sarcoma cells(9). For a discussion of user options regarding 3D matrices, see Box 1. Alternatively, the 3D 'on-top' assay, in which cells are cultured on top of a thin lrECM gel overlaid with a dilute solution of lrECM, may be used as described in Box 2 ( Fig. 1 and Fig. 2).