During lactation a tight blood-milk barrier (BMB) is formed by mammary epithelial cells (MECs) connected by tight junctions. These junctions prevent the paracellular flow of compounds, allowing the BMB to regulate milk composition (Nguen, 1998). The BMB therefore plays an important role in determining the quality and safety of milk.
There are still a lot of unanswered questions regarding the cellular mechanisms by which chemical compounds are transported across the mammary epithelium. In vitro cell culture models are useful to study how chemicals cross epithelial barriers such as the BMB. To mimic the in vivo barrier, the epithelial cells in such a model should form a confluent monolayer to present a tight barrier against the paracellular flow of compounds. These cells must also be well characterised with regards to drug transporter expression and activity, to ensure accurate, reliable interpretation of data.
Endogenous and exogenous compounds can cross the BMB via the transcellular route by passive or facilitated diffusion, or active transport. Breast cancer resistance protein (BCRP, decoded by ABCG2), is a transporter of the ATP-binding cassette family that plays an important role in the active accumulation of substrates in milk (Jonker et al. 2005). BCRP is strongly induced (i.e. upregulated) in the lactating mammary gland of humans, mice and dairy animals when compared to non-lactating tissue (Jonker et al. 2005; Lindner et al. 2013). In contrast, another important efflux transporter in mammals, multi-drug resistance protein, MDR1 (also known as P-glycoprotein, or P-gp, decoded as ABCB1), is downregulated in the mammary gland during lactation (Alcorn et al. 2002; Yagdiran, 2015). Both BCRP and P-gp have a broad range of substrates, that overlaps to some extent (Mao and Unadkat, 2015). The reason for this relative up- and downregulation of BCRP and P-gp respectively during lactation, remains unclear, but an important and consistent feature of this epithelial barrier in the lactating mammary gland is that BCRP is the predominant efflux transporter (Lidner et al.2013, Yagdiran, 2015).
An immortalised bovine mammary epithelial cell line frequently used by researchers studying the BMB is BME-UV (Zavision et al.1996). Several apical cationic and anionic transporters have been identified (Al-bataineh et al. 2009), but researchers have not been able to differentiate BME-UV cells to express P-gp and BCRP in a relationship that is representative of the lactating BMB. BCRP expression is low in this cell line (Yagdiran, 2015). To the authors’ knowledge there are no mammary epithelial cell lines from dairy animals that express BCRP appropriately. Transfected cell lines, for example the MDCKII-BCRP cell line, have been used as an alternative by researchers to specifically study the transport of BCRP-substrates (Wasserman et al. 2013), but is less preferred as a biologically relevant option for the BMB, since these cells does not share all the characteristics of MECs. Primary bovine mammary epithelial cells (pbMEC) could have several advantages over immortalized and transfected cell lines. Primary cells are isolated from the tissue of interest, can be cultured directly and are not genetically manipulated. They are thus expected to have more normal cell morphology and maintain many of the important markers, responses and functions seen in vivo. The data obtained from primary cell culture experiments could be more relevant and representative of the in vivo environment and include individual donor characteristics (Mather and Barnes, 1998). Since first described in 1983 (McGrath), pbMECs are most commonly harvested from udder tissue at the slaughterhouse, with some variations in methodology between researchers. These pbMECs isolated from tissue requires several trypsinisations to purify the culture from co-isolated fibroblasts. An alternative method is to isolate cells from raw milk. This method as described by Beuhring (1990) was later refined by researchers such as Sorg (2012) and Danowski (2013). Both methods of pbMEC isolation have their own advantages and disadvantages. Important to note is that milk isolated cells can be used from the first passage as fibroblast contamination is no concern, but the number of cells isolated varies greatly between isolation attempts and there is also a high risk of contamination (Beuhring, 1990).
In a study by Vachkova and colleagues (2021), pbMEC isolated from udder tissue showed the presence of moderate quantities of BCRP mRNA at 80% confluence in culture. Their study concluded that this model should be further validated as a potentially interesting in vitro model for studying active transport across the BMB. Although this study investigated the expression and function of several biotransformation enzymes, as well as the transcription of BCRP, they did not consider P-gp. The aim of the study reported in this communication was to determine the relationship between the transcription of ABCG2 and ABCB1 (encoding for BCRP and P-gp respectively) in pbMECs in culture, at 80% and 100% confluence.