Abstract
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.
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Data availability
Sequencing data are available via https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE282140. All data generated or analysed during this study are included in this published article and its Supplementary Information files. Source data are provided with this paper.
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Acknowledgements
We thank the Sam and Rina Frankel Foundation (donation; Y.N.) and Believer Meats (Y.N.) for funding this work. The authors also thank Y. Tzfati for his advice, K. Asulin, C. Zisman, L. Shirony, L. Ravid Lustig and Y. Friedmann for technical support, and Believer Meats for contributing its cell lines to this research.
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Conceptualization and funding by Y.N. Investigation by L.P., M.C., S.R., A.E., B.G., A.G. Methodology by Y.N., L.P., M.C., S.R., A.E., B.G., A.G. Software by S.R. and A.E. Writing by Y.N., L.P. and M.C.
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Y.N. is a director and shareholder in Believer Meats. B.G. and A.G. are employees of Believer Meats. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Long-term bovine dermal fibroblast propagation in vitro.
(a) Expression of fibroblast markers in primary bovine fibroblasts and bovine liver tissue (n=3). Data are presented as means plus s.e.m. (b) Doubling time of primary cells ranged between 30 and 8,200 h. Cultures entered senescence around 200 d post isolation. (c) Accumulative population doublings (PD) of primary cells. Senescence was reached at PD 40 to 60 following over 40 passages in culture.
Extended Data Fig. 2 Characterization of senescent bovine fibroblasts.
(a) Top 24 terms appearing in the enrichment analysis of overlapping differentially expressed genes between PBF-15 and Sn-BF-15, and PBF-13 and Sn-BF-13, respectively (n=3). (b) Senescent fibroblasts exhibit a fragmented nucleus as seen in transmission electron microscopy (TEM) images (arrowheads). Scale bar 10 µm. (c) TEM images showing cytoplasmic organelles and structures in primary and senescent fibroblasts. Mitochondria (M), rough- and smooth- ER (RER and SER) respectively, membrane whorls (MW), autophagosome (AP). Scale bar 2 µm.
Extended Data Fig. 3 Characterization of spontaneously immortalized bovine fibroblasts.
(a) Doubling time of bovine fibroblasts in extended in vitro cultures. Spontaneously immortalized lines are referred to as HUN-BF-15 and HUN-BF-13 derived from Holstein and Simmental cows, respectively. (b) Phase images of primary fibroblasts and emerging fibroblast colonies. Black arrows indicate senescent cells, red arrow indicates edge of emerging colony of immortalized fibroblasts. Scale bar 200 µm. (c) Accumulative population doublings of immortalized lines HUN-BF-15 and HUN-BF-13 as a function of passage number. (d) Top 24 terms appearing in the enrichment analysis of overlapping differentially expressed genes between Sn-BF-15 and HUN-BF-15, and Sn-BF-13 and HUN-BF-13, respectively (n=3).
Extended Data Fig. 4 DNA repair capability of immortalized bovine fibroblasts.
(a) Comet assay showing functional DNA damage repair in primary and immortalized fibroblasts PBF-13 and HUN-BF-13. Scale bar 500 µm.
Extended Data Fig. 5 Mitochondrial structure and function of primary, senescent and immortalized fibroblasts.
(a) Transmission electron microscopy (TEM) imaging showing changes in mitochondrial structure in primary, senescent and immortalized fibroblasts from a Holstein cow. Arrowheads indicate damaged mitochondria. Scale bar 1 µm.
Extended Data Fig. 6 Anchorage-independent growth of immortalized bovine fibroblasts in single-cell suspension.
(a) Schematic depiction of adaptation and selection of clones for single-cell suspensions. (b) Doubling times tracked during immortalized bovine fibroblast adaptation to anchorage-independent growth. (c) Phase image of bovine fibroblast single cell suspension. Scale bar 50 µm. Part a created with BioRender.com.
Extended Data Fig. 7 Cell culture validation.
(a) Mycoplasma test of primary, senescent and immortalized bovine fibroblasts. (b) Species confirming test of primary, senescent and immortalized bovine fibroblasts.
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Pasitka, L., Cohen, M., Regenbaum, S. et al. Spontaneous immortalization of bovine fibroblasts following long-term expansion offers a non-transformed cell source for cultivated beef. Nat Food 6, 1079–1094 (2025). https://doi.org/10.1038/s43016-025-01255-3
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DOI: https://doi.org/10.1038/s43016-025-01255-3
