Effects of cell culture techniques on gene expression and cholesterol efflux in primary bovine mammary epithelial cells derived from milk and tissue

Abstract

Primary bovine mammary epithelial cells (pbMEC) are often used in cell culture to study metabolic and inflammatory processes in the udder of dairy cows. The most common source is udder tissue from biopsy or after slaughter. However, it is also possible to culture them from milk, which is non-invasive, repeatable and yields less contamination with fibroblasts. Generally, not much is known about the influence of cell origin and cell culture techniques such as cryopreservation on pbMEC functionality. Cells were extracted from milk and udder tissue to evaluate if milk-derived pbMEC are a suitable alternative to tissue-derived pbMEC and to test what influence cryopreservation has. The cells were cultivated for three passages and stored in liquid nitrogen. The relative gene expression of the five target genes kappa-casein, lingual antimicrobial peptide (LAP), lactoferrin, lysozyme (LYZ1) and the prolactin receptor normalised with keratin 8 showed a tendency to decrease in the tissue cultures, but not in the milk-derived cultures, suggesting a greater influence of the cultivation process on tissue-derived cells, freezing lowered expression levels in both cultures. Overall expression of LAP and LYZ1 tended to be higher in milk cells. Cholesterol efflux was measured to compare passages one to seven in milk-derived cells. Passage number did not alter the efflux rate (p ≤ 0.05). We showed for the first time that the extraction of pbMEC from milk can be a suitable alternative to tissue extraction.

Temporal variation of milk fat globule diameter, fat and cholesterol content and milk epithelial cell gene expression in dairy cows

“…Also, as shown by Sorg et al . (), the obtained gene measurement results are fully comparable with the other techniques, as only living and fully intact mammary epithelial cells are extracted.…”

“…During the last years, we have optimised a technique for isolating purified bovine milk epithelial cells (pbMECs) out of fresh milk samples and postulated it as an optimal alternative to the above‐mentioned traditional methods (Sorg et al . ). This technique allows a live picture of processes in the mammary gland, and it presents three important advantages: it is non‐invasive, very economical and easy to perform, and samples can be taken as often as needed without causing any damage to the sensitive gland tissue, which is often the case with biopsies of the mammary gland.…”

Bovine mammary epithelial cell cultures for the study of mammary gland functions

“…What is more, we have also tested the mammary gland material for establishing cell cultures from three relevant bovine stages (non-lactating heifers, individuals in mid-lactation, and individuals in involution). Some studies present BMEC established from fragments of mammary parenchymal tissue obtained from pregnant individuals (Emerman et al 1977; Huynh and Pollak 1995; Rose et al 2002), but most often, the mammary cell cultures are established from lactating individuals (Pantschenko et al 2000; German and Barash 2002; Hu et al 2009; Zhao et al 2010; Lu et al 2012; Sorg et al 2012). On the other hand, little is known about the suitability of tissue fragments from heifers in establishing primary cell cultures.…”

Mammary immunity of White Park and Highland cattle compared with Brown Swiss and Red Holstein

“…It yields less contamination by fibroblasts (Buehring, 1990) and has been shown to be an applicable alternative to tissue sampling (Sorg et al, 2012). We chose to culture them from milk because it is a non-invasive method and therefore especially suited for rare and valuable animals.…”