Primary Culture of Flow Cytometry-Sorted Rat Mammary Epithelial Cell (RMEC) Subpopulations in a Reconstituted Basement Membrane, Matrigel

Abstract

We studied the growth of rat mammary epithelial cell (RMEC) subpopulations cultured in Matrigel. Mammary glands were removed, minced, and enzymatically dispersed. Mammary organoids (ductal and endbud fragments) were collected, enzymatically monodispersed, filtered, and labeled with fluorescein isothiocyanate-peanut agglutinin (PNA) and phycoerythrin-anti-Thy-1.1 monoclonal antibody. By flow cytometry, we separated four RMEC subpopulations: cells negative to both reagents (B-), PNA-positive cells (PNA+), Thy-1.1-positive cells (Thy-1.1+), and cells positive to both reagents (B+). The ultrastructural characteristics of sorted cells were examined by transmission electron microscopy. Thy-1.1+ cells appeared to be myoepithelial cells with actin filaments. PNA+, B-, and B+ cells were morphologically similar. Sorted RMEC subpopulations, unsorted mixed cells, and undispersed organoids were seeded in Matrigel and cultured in either a complete hormone medium (CHM) with 10% fetal bovine serum, progesterone, estradiol, hydrocortisone, insulin, and prolactin or a serum-free medium containing EGF, insulin, hydrocortisone, and human transferrin. Several types of colonies were observed: stellate, ductal, webbed, squamous, and lobuloductal colonies. At the immunocytochemical and electron micrograph levels, casein proteins were predominantly localized near the apical surfaces of the cells or in the lumina of ductal or lobuloductal colonies. Laminin and collagen IV were detected only in the basal lamina of ductal structures cultured in CHM. RMEC and subpopulations from immature virgin rats developed the capacity to synthesize and secrete lipid and casein when grown in Matrigel in serum medium or under defined serum-free conditions.