Spontaneous Immortalization of Bovine Fibroblasts through Extended In Vitro Culture

Spontaneous immortalization in large animals like cattle is rare and has previously required genetic manipulation of TP53 or TERT. Here, we demonstrate the spontaneous immortalization of fibroblast lines from Holstein and Simmental cows over 500 days of continuous culture, corresponding to 240 population doublings. Initial senescence was observed at population doubling 60, marked by telomere shortening, ?H2AX foci, and an active SASP profile. After 400 days, stable immortalized lines with a doubling time of 45 hours emerged. Transcriptomic and metabolic analyses revealed a reversal of senescence-associated apoptotic processes and mitochondrial stress, with no change in telomerase or p53 activity. Our findings suggest bovine fibroblast immortalization can occur through alternative pathways, with applications in cultivated meat production and aging research. Overall design: Primary fibroblasts were isolated from Holstein (PBF-15) and Simmental (PBF-13) cows and cultured in vitro over 500 days. Senescence markers, telomere lengths, and doubling times were tracked throughout culture, with immortalization events noted after extended periods. Transcriptomic analysis (RNA-seq) compared primary, senescent, and immortalized cell stages to characterize the molecular changes associated with spontaneous immortalization. Additional metabolic and chromosomal stability assays were performed to assess telomere maintenance and mitochondrial function.