Spontaneous immortalization of bovine fibroblasts following long-term expansion offers a non-transformed cell source for cultivated beef

Spontaneously immortalized cell lines provide an essential, non-transformed resource for cultivated meat production. Although chicken fibroblasts readily immortalize in culture, bovine fibroblasts have not been shown to immortalize without genetic manipulation of TP53 or TERT. Here we demonstrate the spontaneous immortalization of fibroblast lines from Simmental and Holstein cows. We track the molecular basis of the immortalization process over 500 days of culture, corresponding to 240 population doublings. Cells entered senescence at population doubling 60, showing γH2AX foci, telomere shortening and an active senescence-associated secretory phenotype profile. Breakthroughs occurred following 400 days in culture, resulting in stable fibroblast lines. Telomerase and PGC1A activation during senescence resolve telomere shortening and mitochondrial dysfunction without activating P53, driving spontaneous immortalization. We explored the economic potential of cultivated beef production using spontaneously immortalized bovine fibroblasts, showing that price parity could be theoretically reached using continuous manufacturing.