Reconstituting Breast Tissue with Organotypic Three-dimensional Co-culture of Epithelial and Stromal Cells in Discontinuous Extracellular Matrices

Abstract

Co-culture systems utilizing reconstituted or synthetic extracellular matrix (ECM) and micropatterning techniques have enabled the reconstruction of surface epithelial tissues. This technique has been utilized in the regeneration, disease modeling and drug screening of the surface epithelia, such as the skin and esophagus. On the other hand, the reconstruction of glandular epithelia would require more intricate ECM organizations. Here we describe a protocol for a novel three-dimensional organotypic co-culture system for the reconstruction of mammary glands that utilizes the discontinuous ECM. In this technique, primary mammary fibroblasts first establish a layer of the connective tissue rich in collagen I. Then, mammary epithelial cells form acinar structures, the functional glandular units, within the laminin-rich basement membrane embedded in the connective tissue. This method allows for the regeneration of the in vivo-like architecture of mammary glands and could be utilized for monitoring the real-time response of mammary glands to drug treatment.

Loss of Nitric Oxide Induces Fibrogenic Response in Organotypic 3D Co-Culture of Mammary Epithelia and Fibroblasts—An Indicator for Breast Carcinogenesis

“…#2301) supplemented with 1% Fibroblast Growth Supplement (FGS, ScienCell Research Laboratories, Carlsbad, CA, USA, Cat. #2352) and 1% penicillin/streptomycin as described [ 24 ].…”

“…Cultures were maintained for 10–21 days with addition of fresh medium on alternate days [ 33 ]. For 3D mono-culture of mammary fibroblasts (MFs), cells were grown in Matrigel: collagen I (1:1) mixture in the same manner as 3D co-culture (see below) [ 24 ]. To determine the effects of the inhibition of NO production, 3D cultures were kept under the treatment of 2.5 mM L-NAME (NOS inhibitor, N ω -Nitro-L-arginine methyl ester hydrochloride, Sigma-Aldrich, St. Louis, MO, USA), vehicle control (PBS) or NO synthase agonist L-arginine (2.5 mM) for 2–3 weeks with medium change every other day [ 34 ].…”

“…These lesions were characterized by desmoplastic stroma and intraductal papillomas that highly expressed TGF-β and HER2 oncogene [ 23 ]. In the present report, we tested whether such fibrogenic and carcinogenic effects of NO deprivation could be recapitulated in our novel organotypic 3D co-cultures [ 24 ]. Through this study, we examined whether this in vitro co-culture system could exhibit desmoplastic responses toward a known fibrogenic and carcinogenic factor to test its utility in the deep studies of the causal linkage between fibrosis and cancer.…”

“…This new organotypic 3D co-culture system utilizes the discontinuous ECM to model the complexity of glandular tissues, such as mammary glands [ 24 ]. The mammary gland is the arboreal structure extended from the nipple towards the terminal lobes composed of mammary acini—the milk producing units.…”

“…In our 3D co-culture, mammary epithelial cells and fibroblasts are separately seeded in physiologically relevant matrices: laminin-rich BM for epithelia vs. collagen I-rich membrane for fibroblasts. Both cell types interact through paracrine signaling, but without direct physical interactions [ 24 ]. This system is different from the original 3D organotypic co-culture system developed for surface epithelia (such as skin and respiratory tracts) which utilizes the continuous ECM that resembles the collagen-rich interstitial matrix ( Figure 2 A,B) [ 27 , 28 , 29 ].…”