Three-dimensional cultures of mouse mammary epithelial cells

Abstract

The mammary gland is an ideal "model organism" for studying tissue specificity and gene expression in mammals: it is one of the few organs that develop after birth and it undergoes multiple cycles of growth, differentiation and regression during the animal's lifetime in preparation for the important function of lactation. The basic "functional differentiation" unit in the gland is the mammary acinus made up of a layer of polarized epithelial cells specialized for milk production surrounded by myoepithelial contractile cells, and the two-layered structure is surrounded by basement membrane. Much knowledge about the regulation of mammary gland development has been acquired from studying the physiology of the gland and of lactation in rodents. Culture studies, however, were hampered by the inability to maintain functional differentiation on conventional tissue culture plastic. We now know that the microenvironment, including the extracellular matrix and tissue architecture, plays a crucial role in directing functional differentiation of organs. Thus, in order for culture systems to be effective experimental models, they need to recapitulate the basic unit of differentiated function in the tissue or organ and to maintain its three-dimensional (3D) structure. Mouse mammary culture models evolved from basic monolayers of cells to an array of complex 3D systems that observe the importance of the microenvironment in dictating proper tissue function and structure. In this chapter, we focus on how 3D mouse mammary epithelial cultures have enabled investigators to gain a better understanding of the organization, development and function of the acinus, and to identify key molecular, structural, and mechanical cues important for maintaining mammary function and architecture. The accompanying chapter of Vidi et al. describes 3D models developed for human cells. Here, we describe how mouse primary epithelial cells and cell lines--essentially those we use in our laboratory--are cultured in relevant 3D microenvironments. We focus on the design of functional assays that enable us to understand the intricate signaling events underlying mammary gland biology, and address the advantages and limitations of the different culture settings. Finally we also discuss how advances in bioengineering tools may help towards the ultimate goal of building tissues and organs in culture for basic research and clinical studies.

Fig. 1

Representative schematics of the different stages of mammary gland development and of the mammary acinus. (a) Schematic showing the five mammary buds in the embryo along the milk line which will later develop into mammary glands. (b) Representative drawings of the different stages of mammary gland development, starting from birth when the mammary anlage is present, to puberty where TEB’s extend through the fat pad and the mature nulliparous gland filled with epithelial ducts. Pregnancy and lactation are modeled where the gland fills with alveoli which regress in involution such that the gland remodels to a pre-pregnant like state. (c) Representative drawing of the mammary acinus: Cuboidal luminal epithelial cells (light blue) are surrounded by contractile spindle shaped myoepithelial cells (pink) and facing a lumen on their apical side where milk droplets are deposited during lactation on their way to the ducts that terminate in the nipple. The entire structure is surrounded by a basement membrane with stromal cells including fibroblasts (yellow) and adipose cells (clear round cells).

Fig. 2

Primary mammary organoids in 3D. (a) primary mammary organoids isolated from the mammary glands of 14-week-old nulliparous mice are grown inside/embedded in lrECM (left) and on top of lrECM (right) and imaged by bright-field light microscopy on day 4 of culture, size bar, 50 µm. (b) Primary organoids isolated from the mammary glands of 8–12-week-old mice are embedded in lrECM for 4 days in additive-free medium as control (left), and stimulated with TGFα to induce alveolar growth (right). (c) Primary organoids isolated from the mammary glands of 8–12-week-old mice are embedded in collagen I (1 mg/mL) for 4 days as control (left), and stimulated with FGF2 to induce branch formation (right), size bar, 50 µm.