Development and validation of a new bovine teat apex epithelial cell (bTAEC) line.

The teat sphincter and canal serve as the primary physical barrier against the entry of pathogens into the mammary gland, playing a crucial role in the udder's defence. While much effort has been dedicated to the study of the interaction between mastitis causing pathogens and secretory mammary epithelial cells, there are currently no studies on such interactions with mammary keratinocytes originating from the teat canal. To address this challenge, we are developing a new bovine teat apex epithelial cell line, termed bTAEC, designed to deepen our understanding of the role this unique extramammary region plays in the development of intramammary infection. Two isolation protocols, utilizing collagenase and trypsin, respectively, were investigated to compare the efficacy of the isolation of keratinocytes from dairy cows’ teat canals and assess their immunophenotype. Collagen-coated plates were used to culture tissues, and two protocols were employed: one with collagenase type III and another with trypsin 0.25%. Immunophenotyping involved the application of Cytokeratin Type I Monoclonal Antibody (AE-1) and BD Horizon™ BV421 Rat Anti-Mouse IgG1. Viability of XX was measured using 7AAD, and FlowJo analyses were conducted. The trypsin protocol demonstrated superior results, yielding 23.9-28.0% keratinocytes compared to 16.0-18.0% in the collagenase protocol. However, total viability was slightly lower in the trypsin protocol (74.1% vs. 76.6%), and keratinocyte viability was low at approximately 9.1%. Moving forward, the following steps will be taken: firstly, enhancing keratinocyte viability over time; secondly, minimizing fibroblast contamination; and finally, cultivating keratinocytes for subsequent sorting and immortalization. The resulting innovative cell line will then be used as a valuable tool for advancing our understanding of bovine mastitis pathogenesis.