Journal of Dairy Research



Original article

In vitro differentiation of a cloned bovine mammary epithelial cell


MICHAEL T.  ROSE  a1 c1 p1, HISASHI  ASO  a1, SHINICHI  YONEKURA  a2, TOKUSHI  KOMATSU  a1, AKIHIKO  HAGINO  a2, KYOUHEI  OZUTSUMI  a1 and YOSHIAKI  OBARA  a2
a1 Department of Animal Physiology, National Institute of Animal Industry, P.O. Box 5, Tsukuba-Norindanchi, Ibaraki 305-0901, Japan
a2 Laboratory of Animal Physiology, Tohoku University, Aoba-ku, Sendai 981-8555, Japan

Abstract

The aim of the study was to establish in vitro a bovine mammary epithelial cell (MEC) clone, able to respond to mitogenic growth factors and to lactogenic hormones. Mammary tissue from a 200-d pregnant Holstein cow was used as a source of MEC, from which a clone was established through a process of limiting dilution. When plated on plastic, the cells assumed a monolayer, cobblestone, epithelial-like morphology, with close contact between cells. Inclusion of IGF-1 and EGF in the media significantly increased the number of cells 5 d after plating. All cells stained strongly for cytokeratin and moderately for vimentin at young and old passage stages, indicating the epithelial nature of this cell clone. When the cells were plated at a high density on a thin layer of a commercial extracellular matrix preparation (Matrigel), lobular, alveoli-like structures developed within approximately 5 d, with a clearly visible lumen. When cells were plated onto Matrigel in differentiation media (containing lactogenic hormones), detectable quantities of α-casein were present in the media and particularly on the lumen side of the structures. Omission of one of the lactogenic hormones (insulin, prolactin or hydrocortisone) reduced α-casein release to the limit of detection of the assay used. Lactoferrin was also produced when the cells were plated on Matrigel, again principally on the lumen side of the lobules, though this was independent of the lactogenic hormones. By passage 40, the cells had senesced, and it was not possible to induce α-casein or lactoferrin production. This study notes the establishment of a functional bovine mammary epithelial cell clone, which is responsive to mitogenic and lactogenic hormones and an extracellular matrix.

(Received February 14 2001)
(Accepted January 21 2002)


Key Words: Casein; lactoferrin; lactogenesis; matrigel.

Correspondence:
c1 For correspondence; e-mail: mir@aber.ac.uk
p1 Present address: Institute of Rural Studies, Llanbadarn Campus, University of Wales, Aberystwyth, SY23 3AL, UK.