Development of in vitro models for the assessment of drug transfer across the mammary epithelial barrier: from 2D to 3D porcine primary mammary epithelial cell culture

There is paucity of information about the safe use of drugs during lactation, as clinical studies present ethical and practical challenges. The European project ConcePTION has been working to narrow this huge gap by developing non-clinical in vivo, in vitro and in silico lactation models that can predict drug concentrations crossing the blood-milk barrier. The pig was selected as animal model to conduct lactating experimentation for the physiological affinity with humans, in particular the Göttingen minipigs breed has been preferred for the genetic stability and microbiological control. The present work aimed to develop a minipig mammary epithelial cell (mpMECs) in vitro model to determine the extent of drug transfer into milk. The results described the isolation and characterization of mpMECs. The cells were able to reproducibly form a tight barrier within the first week of culture, with a transepithelial electrical resistance (TEER) profile similar to that obtained with mammary epithelial cells of hybrid pigs. In addition, a human mammary epithelial cell model was developed to evaluate more closely the translatability of the pig/minipig model. The primary human mammary epithelial cells (hMECs) were able to form the epithelial barrier, although measurable TEER values were reached after about 30 days, unlike mpMECs. Furthermore, the mRNA drug transporters expression levels were analyzed and the energetic metabolic profile of both mpMECs and hMECs was also evaluated. Lastly, a 3D multicellular culture model was developed to faithfully recreate a fully blood-milk barrier, by co-culturing mpMECs with two vascular-wall cell populations: the aortic endothelial cells and the mesenchymal stromal cells. In conclusion, a minipig mammary epithelial cell in vitro model to study the medicine partitioning in milk has been defined; the 3D culture model will lay the foundation for a more complex and comprehensive blood-milk barrier model than 2D by improving the Replacement and Reduction.