In vitro culture of Boer goat mammary epithelial cells to form a monolayer constituting a tight barrier to drug movement

Abstract

In rural areas of developing countries like South Africa, people typically depend on goat farming for both meat and milk production due to the shortage of grazing and the higher maintenance cost of cattle. An understanding of the functionality of the mammary gland and subsequent drug transport into milk are important factors in determining milk withdrawal periods and drug residues in milk intended for human consumption. Tight cellular monolayers, cultured to resemble the in vivo blood-milk-barrier, are used to evaluate the transepithelial transport of drugs into milk in vitro. The aim of this study was to culture and maintain tight monolayers of primary Boer goat mammary epithelial cells that would be a barrier to paracellular drug movement. Cells were cultured and maintained similarly to the method described by Pantschenko and colleagues (2000), with some adaptations and with MCF10a as growth medium. The formation of tight barriers was evaluated by measuring transepithelial electrical resistance (TEER) and the paracellular movement of dextran-FITC. An aggregated monolayer was established which had the characteristic cobblestone appearance, typical of epithelial cells, with no fibroblasts seen microscopically. On day 11 the monolayers appeared to be confluent under microscopic examination, they presented a significant barrier to the movement of FD70 dextran (Papp = 0.001), and the transepithelial electrical resistance (TEER) was greater than 200 ?.cm2. At day 18 of culture, macroscopically the cells started to stack and cell debris formed, presumably due to overgrowing and cell differentiation, and the monolayers were no longer appropriate for use. Furthermore, cryopreservation techniques were performed on the cells and these cells were frozen, stored, and regrown as viable epithelial cells. Primary Boer goat mammary epithelial cells, cultured and maintained using the methods described in this dissertation, form tight monolayers that are a significant barrier to the paracellular movement of relatively large molecules like dextran70, with TEER values appropriate for xenobiotic transcellular flux studies between day 11 and 18 of culture. This timeframe corresponds with the time in which drug transfer studies are typically done in cell cultures from other species. Viable cryopreservation of Boer goat mammary epithelial cells is a useful tool that can be used to enhance these studies.