Optimizing enzymatic digestion for efficient isolation and culture of primary buffalo mammary epithelial cells

Buffalo mammary epithelial cell (BuMEC) culture plays a crucial role in understanding lactation biology, milk synthesis, and mammary gland function. This comprehensive study explores the isolation, characterization, and maintenance of BuMECs under in-vitro conditions. The objective of this study was to establish a reliable in vitro culture system for BuMECs that can be used for future functional analysis of the buffalo mammary gland. We evaluated different time, doses of collagenase and its combinations for enzymatic digestion methods to obtain the optimal cell yield and viability. Cell viability was assessed using trypan blue dye after 2 and 3 hours of enzymatic digestion with varying concentrations of collagenase-II, collagenase-IV, and a mixture of both. Two-hour digestion resulted in higher cell viability and reduced contamination risk compared to 3-hour digestion. Seeded cells reached 70-80% confluency after 7 days. Morphological assessments and milk associated gene expression analysis confirmed the epithelial identity and functionality of cultured cells. This study has refined the techniques for establishing and sustaining primary BuMEC cultures, providing a robust platform for in-vitro functional analysis of mammary glands.