Development and Hormonal Control of a Primary Culture System of Epithelial Cells Isolated from Rat Mammary Gland

Abstract

Techniques for the growth of rat mammary epithelium in vitro have been developed. Epithelial elements were dissociated by collagenase digestion of the mammary fat pad and separated from contaminating connective tissue elements by a ficoll gradient centrifugation procedure. Monolayer cultures with characteristic epithelial morphology have been established and maintained for prolonged periods of time. Comparison of morphological characteristics and hormonal growth requirements of these cells with fibroblasts isolated from the same tissue has confirmed their epithelial nature. The labeling index and DNA synthesis rate have been determined for cells grown in medium supplemented with various concentrations and combinations of hormones in an attempt to define those hormonal requirements which support cell growth and simulate physiological levels. On the basis of these experiments, the following standard medium supplements have been established: insulin (0.05 to 0.5 pg per ml), aldosterone (0.005 to 0.05 pg per ml), prolactin (0.05 pg per ml), estradio1-17β (0.00003 pg per ml) and progesterone (0.003 to 0.03 pg per m1). Specific hormonal responsiveness of the mammary epithelium to estrogen has been studied in vitro. Following a 1 hour treatment of mammary epithelial cell cultures with estradio1-17β, the synthesis of an induced protein (IP) characteristic of estrogen action in other estrogen responsive tissues can be detected. This system provides a baseline for future studies of hormone-target cell interactions including those concerning alterations in macromolecular synthesis and in vitro neoplastic transformation.