In vitro mammary gland model: establishment and characterization of a caprine mammary epithelial cell line | In vitro model mlecne zleze: vzpostavitev in dolocitev znacilnosti epitelne celicne linije iz kozje mlecne zleze
2009
Ogorevc, J., Ljubljana Univ. (Slovenia). Biotechnical Fac., Dept. of Animal Science | Prpar, S. | Dovc, P.
Demanding transcriptomic studies in combination with challenging experiments in livestock animal species could be replaced by good in vitro models mimicking the function of ruminant mammary gland. The objective of our study was to establish epithelial cell line obtained from primary cell culture of lactating goat mammary gland. Mammary tissue from lactating Saanen goat (Capra hircus) was digested in collagenase and hyaluronidase solution and plated on plastic flasks. When growing on plastic, typical cobblestone morphology of epithelial cells and larger irregularly shaped cells, corresponding to myoepithelial cells were observed. When growth medium was supplemented with lactogenic hormones (insulin, hydrocortisone, and prolactin) and cells were cultured on plastic for extended period of time at high density, dome-like structures appeared as a result of cell to cell contact induced differentiation. Immunofluorescence staining using antibodies against smooth muscle á-actin, vimentin and various cytokeratins were used to distinguish between different cell types. Cell types of epithelial and myoepithelial cells were confirmed. Complete differentiation of cells was achieved when growing them on a commercial basal membrane matrix preparation which contains laminin, collagen IV, and various growth factors. Cells grown on basal membrane matrix in growth medium supplemented with lactogenic hormones differentiated morphologically and functionally. Spherical structures that resembled the alveoli of lactating mammary gland were observed. Reverse transcription PCR (RT-PCR) was performed on the total RNA extracted from the cultured cells in order to detect the potentially present milk protein mRNAs.
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