Establishment and Characterization of Primary Culture of Mammary Epithelial Cells from Bakarwali Sheep

Abstract

Aims: This study aimed to establish a primary mammary epithelial cell culture from the Bakarwali sheep of Jammu. Place and Duration of Study: All experimental procedures of this study were performed at Cell culture lab, Division of livestock production management, F.V.SC&A.H, Sher-e-kashmir university of agricultural sciences and technology of Jammu, India. Methodology: Mammary gland tissue obtained from slaughter house was cleaned and transported to laboratory. Whole mammary gland was rinsed with phosphate buffer saline (PBS) containing antibiotics and then small tissue was taken for mincing. Minced tissue was treated with collagenase 0.05% for 2 hours at 37˚C. The minced tissue was further dissociated with trypsin- EDTA for 30 minutes at 37˚C and then filtered through a cell strainer. The ovine mammary epithelial cell preparation was suspended in growth medium and antibiotics. The mixed population of cells were subjected to selective trypsinization to remove the fibroblast cells from the primary culture of mammary epithelial cells. After establishment of cell culture, cells were characterized by partial amplification of Keratin 18 (Krt18), β-casein (Csn2), and Vimentin (Vim). Results: The heterogeneous population of cells was obtained at day six which consists of epithelial and mesenchymal cells (fibroblast-like) by enzymatic digestion method. Further passaging and selective trypsinization of cells resulted in removal of fibroblast-like cells and formed a homogenous layer of epithelial cells. To differentiate epithelial cells from other cells in the heterogenous population partial amplification of Keratin 18 (Krt18), β-casein (Csn2), and Vimentin (Vim) were done. The amplification products yielded 196 bp, 177 bp and 151 bp product size respectively for Vim, Krt18 and Csn2 genes. Conclusion: In the present study we have established primary culture of mammary epithelial cells of Bakarwali sheep of Jammu region. The primary cell culture can be used for further cell lines development, for studying the mammary gland biology, transfection and expression studies, as mastitis model, immune response study involving infection and host response replacing experiments.