Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Hillary A. Hahm, Margot M. Ip, Kathleen Darcy, Jennifer D Black, Wendy K. Shea, Suzanne Forczek, Masami Yoshimura, Takami Oka

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)803-814
Number of pages12
JournalIn Vitro Cellular &Developmental Biology
Volume26
Issue number8
DOIs
StatePublished - Aug 1 1990

Fingerprint

Caseins
Basement Membrane
Rats
Breast
Epithelial Cells
Serum
Lipids
Assays
Immunosorbents
Electrons
Experimental Sarcomas
Enzyme-Linked Immunosorbent Assay
Eagles
Serum-Free Culture Media
Secretory Vesicles
Micelles
Enzymes
Human Mammary Glands
Transferrin
Bovine Serum Albumin

Keywords

  • differentiation
  • extracellular matrix
  • mammary epithelial cells
  • model system
  • primary culture
  • rat

ASJC Scopus subject areas

  • Developmental Biology
  • Clinical Biochemistry
  • Cell Biology

Cite this

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions. / Hahm, Hillary A.; Ip, Margot M.; Darcy, Kathleen; Black, Jennifer D; Shea, Wendy K.; Forczek, Suzanne; Yoshimura, Masami; Oka, Takami.

In: In Vitro Cellular &Developmental Biology, Vol. 26, No. 8, 01.08.1990, p. 803-814.

Research output: Contribution to journalArticle

Hahm, Hillary A. ; Ip, Margot M. ; Darcy, Kathleen ; Black, Jennifer D ; Shea, Wendy K. ; Forczek, Suzanne ; Yoshimura, Masami ; Oka, Takami. / Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions. In: In Vitro Cellular &Developmental Biology. 1990 ; Vol. 26, No. 8. pp. 803-814.
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