Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions |
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Authors: | Hillary A Hahm Margot M Ip Kathleen Darcy Jennifer D Black Wendy K Shea Suzanne Forczek Masami Yoshimura Takami Oka |
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Institution: | (1) Grace Cancer Drug Center, Roswell Park Memorial Institute, New York State Department of Health, 666 Elm Street, 14263 Buffalo, New York;(2) Laboratory of Molecular and Cellular Biology, National Institutes of Health, 20892 Bethesda, Maryland |
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Abstract: | Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within
a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and
secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within
an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting
of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor,
bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic
level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins.
Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was
observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of
casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot
analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein
mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels.
Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike
colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the
RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs
from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid
when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve
as an excellent model in which the regulation of mammary development and gene expression can be investigated.
This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health,
Bethesda, MD. |
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Keywords: | mammary epithelial cells differentiation extracellular matrix model system rat primary culture |
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