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. 2007 Apr;4(4):359-65.
doi: 10.1038/nmeth1015.

Three-dimensional culture models of normal and malignant breast epithelial cells

Genee Y Lee  1 , Paraic A Kenny, Eva H Lee, Mina J Bissell
Affiliations

Three-dimensional culture models of normal and malignant breast epithelial cells

Genee Y Lee et al. Nat Methods. 2007 Apr.

Abstract

Extracellular matrix is a key regulator of normal homeostasis and tissue phenotype. 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). 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. Signaling pathways that function in parallel in cells cultured on plastic become reciprocally integrated when the cells are exposed to basement membrane-like gels. 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 (Fig. 1). By lrECM, we refer to the solubilized extract derived from the Engelbreth-Holm-Swarm mouse sarcoma cells. 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).

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Figures

Figure 1
Figure 1
Breast epithelial cell morphology in different culture conditions. (a) Schematic of nonmalignant breast epithelial cells grown as a monolayer on tissue-culture plastic (left), in the 3D embedded assay (middle) and in the 3D on-top assay (right). (b) Phase-contrast images of nonmalignant HMT-3522 S1 cells grown in the three different culture conditions (top) and malignant HMT-3522 T4-2 cells grown in the same conditions (bottom). Scale bars, 50 μm.
Figure 2
Figure 2
Breast cancer cell lines in 3D culture. (a–c) Phase-contrast images (left) and confocal cross-sections of Phalloidin staining of F-actin (right) of BT-474 (a), SK-BR-3 (b) and MDA-MB-231 (c) cell lines grown for 4 d in the 3D on-top assay. In a and b, colonies were completely extracted from the gel for immunostaining; in c, colonies were immunostained in the gel. Scale bars, 100 μm (left) and 50 μm (right).
Figure 3
Figure 3
3D drug response assay. (a–c) HMT-3522 S1 (a), HMT-3522 T4-2 (b) and HMT-3522 T4-2 treated with an EGFR inhibitor, AG1478 (c) were cultured in the 3D on-top assay for 4 d. Colonies were then extracted and immunostained against α6 integrin (green) and β-catenin (red). Nuclei were counterstained with DAPI (blue). Confocal sections through the centers of the colonies are shown. Scale bar, 20 μm.
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