A single-spheroid bovine mammary co-culture model for longitudinal monitoring and preclinical safety assessment of antimicrobial alternatives against mastitis-causing pathogens

Bovine mastitis remains a major driver of antimicrobial use in dairy production, highlighting the need for physiologically relevant in vitro models for preclinical safety assessment of novel intramammary therapeutics. Here, a previously established bovine epithelial cell line co-culture mammosphere model was adapted to a single-spheroid 96-well ultra-low attachment format, improving reproducibility and longitudinal monitoring. Mammospheres maintained epithelial organization and were further characterized for secretory function through assessment of casein expression. The suitability of the model for safety assessment was subsequently evaluated using complementary fluorescence microscopy, kinetic live-cell imaging, and flow cytometry following Triton X-100-induced cytotoxicity. Using this workflow, the safety of bacteriophage-derived recombinant lysin LysMK34 and antibiotic kanamycin, alone and in combination, was evaluated, with no cytotoxic effects observed, supporting the utility of the model for future preclinical intramammary safety assessment.