A Robust Mammary Organoid System to Model Lactation and Involution-like Processes

Background

The primary function of the mammary gland is to provide nutrition to newborns via milk production. The development of the mammary gland is a highly dynamic process that occurs mainly after birth and is regulated by several factors including hormones and growth factors (Brisken and Rajaram, 2006; Sternlicht, 2006). During puberty, hormones and growth factors regulate ductal morphogenesis from a rudimentary embryonic ductal tree (Brisken and O'Malley, 2010). During each pregnancy, the mammary gland begins a new morphogenetic step initiated by hormonal stimulation, which is characterized by massive proliferation for epithelial expansion and alveolar development accompanied by adipocyte regression (Brisken and O'Malley, 2010). Importantly, prolactin signaling plays a crucial role in the terminal differentiation of luminal cells to enable milk production (Ormandy et al., 1997). At the end of lactation after weaning of the progeny, the mammary gland enters the involution stage characterized by programmed cell death, tissue remodeling, and redifferentiation of adipocytes (Hughes and Watson, 2012; Macias and Hinck, 2012; Zwick et al., 2018; Jena et al., 2019).

Histologically, the mammary gland is composed of a bilayered epithelium consisting of an inner layer of luminal cells (keratin 8+) and an outer layer of contractile basal cells (keratin 5+). Luminal cells are responsible for milk production during lactation, while basal cells aid milk ejection. The epithelium is surrounded by a stromal fat pad that comprises fibroblasts, nerves, vasculature, lymphatics, immune cells, adipocytes, and extracellular matrix (ECM) (Richert et al., 2000).

Over the past decade, organoids of various tissues, such as stomach, colon, lung, and pancreas, have been developed (Huch and Koo, 2015), offering many exciting possibilities for the study of tissue development and disease. The organoid system is a powerful tool that combines the advantages of a 2D culture (easy manipulation, precise control of cell composition and microenvironment, live imaging) with the opportunity to study complex cell–cell and cell–ECM interactions in a more controlled ex vivo manner (Huch and Koo, 2015; Shamir and Ewald, 2015; Koledova, 2017; Artegiani and Clevers, 2018).

Several models have been developed to study the mechanisms of mammary branching morphogenesis in primary mammary epithelium using different protocols (Ewald et al., 2008; Huebner et al., 2016; Neumann et al., 2018), cell lines (Xian et al., 2005), sorted cells (Jamieson et al., 2017; Linnemann et al., 2015), or induced pluripotent stem cells (Qu et al., 2017). However, an organoid system modeling key aspects of the late postnatal developmental stages of the mammary gland has remained challenging to establish.

Previously, there have been several attempts to model lactation in 3D culture: spheroids of a breast adenoma cell line were used to study copper secretion into milk (Freestone et al., 2014); organoids of primary epithelium were shown to produce milk following the administration of a lactogenic stimulus (Mroue et al., 2015; Jamieson et al., 2017); and co-culture of breast epithelium and pre-adipocyte cell lines was shown to initiate an involution-like process (Campbell et al., 2014). However, in-depth characterization of milk production and involution or the proper bilayered architecture of mammary epithelium remained to be carried out.

Recently, we developed a model of lactation and involution of mammary epithelium based on organoids of primary mammary gland tissue cultured in 3D Matrigel^® (Sumbal et al., 2020b). Under lactogenic stimuli, primary organoids maintain long-term milk production, retain the contractile myoepithelial layer, and enter involution following hormone withdrawal. Moreover, after involution, the organoids remain hormonally sensitive and are able to enter another round of lactation (Sumbal et al., 2020b). Here, we present a methodological guideline to establish the primary mammary organoid-based ex vivo model of lactation and involution, with detailed procedures for obtaining tissue, isolating organoids, establishing and maintaining 3D culture, and preparing organoid samples for subsequent RNA or protein expression analysis or histological examination. This model can be used for studies on lactation biology, mammary stem cell plasticity, regulatory mechanisms of mammary epithelial cell differentiation and death, or other interesting biological phenomena. We believe that this model will initiate the further development of organoid technology, including creative applications in biotechnology and regenerative medicine (Sumbal et al., 2020a).