**3. Mammary tissue–derived primary cells as an** *in vitro* **model**

Mammary gland development occurs in stages; proliferation and differentiation of mammary cells are dependent on sexual development and reproduction, which are under control of endocrine system [12]. The gland is primarily composed of mesenchymal and epithelial tissue and the latter is subjected to significant remodeling during lactation cycles. The tissue remodeling involves proliferation and differentiation of the epithelial cells forming functional glandular tissue, followed by regression of the tissue due to apoptosis and loss of glandular structure and function (involution). The functional part of mammary gland is glandular tissue connected by a branched system of secondary ducts, combining into larger primary ducts, which end in the gland cistern. The milk is synthesized by secretory luminal cells arranged in spherical structures called alveoli, which form larger structures called lobules. Alveoli and ducts of lactating mammary gland are composed of different types of epithelial cells important for milk synthesis and secretion, while connective and fat tissue surround and support the epithelial structures. An alveolus is comprised of a single layer of milk secreting luminal epithelium, surrounded by a single layer of contractile myoepithelial cells, which lie adjacent to the basal membrane, where mammary stem/progenitor cells also reside [10].

In agreement with the "Replacement" of the three Rs principle (3Rs: Replacement, Reduction, and Refinement) adequate *in vitro* model, mimicking the function of the mammary gland allows the study of physiological, biochemical, and immune functions of the mammary gland, substituting *in vivo* experiments. In addition to the ethical issues, cell lines enable use of many technical replicas, better control of the environment, and surmount the problem of variation introduced by animal's individuality [13] and the problem of systemic effects, which makes elucidation of a contribution of a particular cell type of interest difficult [14]. A main limitation of primary cell models is a finite life span, and a limiting number of available biological replicas, as a derived cell culture, represent a single genotype, while establishment and characterization of a large number of cell cultures/lines are quite a challenging and laborious process. Additionally, cell cultures do not always properly model *in vivo* conditions; therefore, limitations should be considered for each individual purpose.

Several ruminant immortalized mammary epithelial cell lines as MAC-T [15] and BME-UV [16] were generated by genomic integration of Simian virus large T-antigen (SV40LTA). However, transformed mammary cells are genetically and (usually) phenotypically changed. Transient mammary lines show low responsiveness to lactogenic hormones [17] and are not proper approximation of *in vivo* lactation. Genetic modifications and adaptations to growth in cell cultures alter metabolic pathways in continuous cell lines; therefore, the use of primary cells is a much better approximation of the *in vivo* system [18].
