Fibroblast-Epithelium Co-culture Methods Using Epithelial Organoids and Cell Line–Derived Spheroids

Fibroblasts are an integral cell type of mammary gland stroma, which plays crucial roles in development, homeostasis, and tumorigenesis of mammary epithelium. Fibroblasts produce and remodel extracellular matrix proteins and secrete a plethora of paracrine signals, which instruct both epithelial and other stromal cells of the mammary gland through mechanisms, which have not been fully understood. To enable deciphering of the intricate fibroblast-epithelial interactions, we developed several 3D co-culture methods. In this chapter, we describe methods for establishment of various types of embedded 3D co-cultures of mammary fibroblasts with mammary epithelial organoids, mammary tumor organoids, or breast cancer spheroids to investigate the role of fibroblasts in mammary epithelial development, morphogenesis, and tumorigenesis. The co-culture types include dispersed, aggregated, and transwell cultures.