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Spontaneous immortalization of chicken fibroblasts generates stable, high-yield cell lines for serum-free production of cultured meat

Nature Food volume 4, pages 35–50 (2023)Cite this article

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An Author Correction to this article was published on 12 January 2023

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Abstract

Cellular agriculture could meet growing demand for animal products, but yields are typically low and regulatory bodies restrict genetic modification for cultured meat production. Here we demonstrate the spontaneous immortalization and genetic stability of fibroblasts derived from several chicken breeds. Cell lines were adapted to grow as single-cell suspensions using serum-free culture medium, reaching densities of 108 × 106 cells per ml in continuous culture, corresponding to yields of 36% w/v. We show that lecithin activates peroxisome proliferator-activated receptor gamma (PPARγ), inducing adipogenesis in immortalized fibroblasts. Blending cultured adipocyte-like cells with extruded soy protein, formed chicken strips in which texture was supported by animal and plant proteins while aroma and flavour were driven by cultured animal fat. Visual and sensory analysis graded the product 4.5/5.0, with 85% of participants extremely likely to replace their food choice with this cultured meat product. Immortalization without genetic modification and high-yield manufacturing are critical for the market realization of cultured meat.

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Fig. 1: Spontaneous immortalization of chicken embryonic fibroblasts.
Fig. 2: Anchorage-independent growth of immortalized chicken fibroblasts in single-cell suspension.
Fig. 3: Genetic stability and safety of immortalized chicken fibroblasts.
Fig. 4: Transdifferentiation of immortalized fibroblasts to fat-storing adipocyte-like cells.
Fig. 5: High-density expansion of biomass for cultured chicken meat.
Fig. 6: Manufacturing of cultured chicken.

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Data availability

Sequencing data are available at NCBI GEO. The GEO accession number is GSE169291. 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.

Code availability

Our custom Single Cell Analysis CellProfiler Pipeline is available at https://github.com/Avnere/Single-Cell-Analysis-CellProfiler-Pipeline.

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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. Further, we thank D. Petrova, R. Barak, H. Zukerman Narodizky, L. Shirony, A. Nagawkar and M. Rosenberg for technical support.

Author information

Author notes

  1. These authors contributed equally: L. Pasitka, M. Cohen.

Authors and Affiliations

  1. Grass Center for Bioengineering, Benin School of Computer Science and Engineering, The Hebrew University of Jerusalem, Jerusalem, Israel

    L. Pasitka, M. Cohen, A. Ehrlich, M. Ayyash & Y. Nahmias

  2. Department of Cell and Developmental Biology, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel

    M. Cohen & Y. Nahmias

  3. Believer Meats, Rehovot, Israel

    B. Gildor, E. Reuveni, M. Ayyash, G. Wissotsky, A. Herscovici, R. Kaminker, A. Niv, R. Bitcover, O. Dadia, A. Rudik, A. Voloschin, M. Shimoni & Y. Nahmias

  4. Institute of Animal Science, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel

    Y. Cinnamon

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Contributions

Conceptualization and funding by Y.N. Investigation by L.P., M.C., M.S., M.A., G.W. and B.G. Methodology by Y.N., M.C., L.P., A.E., G.W., A.N., A.H., A.R., R.K., R.B., O.D. and A.V. Resources by Y.C. Software by A.E. and E.R. Writing by Y.N., L.P., M.C. and A.E.

Corresponding author

Correspondence to Y. Nahmias.

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Competing interests

Y.N. is the chief scientific officer, director and shareholder in Believer Meats. M.A., G.W., B.G., E.R., A.H., A.R., R.K., R.B., O.D., A.V. and M.S. are employees of Believer Meats. M.C. is a consultant of Believer Meats. L.P., A.E., A.N. and Y.C. declare no competing interests.

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Extended data

Extended Data Fig. 1 Spontaneous immortalization of chicken embryonic fibroblasts.

(A) Doubling time of primary cells from isolates 5 and 7. (B) Phase images of spontaneously immortalized chicken embryonic fibroblasts HUN-CF-2 and HUN-CF-4 compared to established DF-1 cell line (ATCC® CRL-12203). Scale bar equals 100 μm. (C) Gene expression analysis of TP53, PTEN, and ITGB3 in DF-1 compared with HUN-CF-2 and HUN-CF-4. (n = 3) Data are presented as means plus standard error of the mean.

Source data

Extended Data Fig. 2 Characterization of suspension-adapted chicken fibroblasts.

(A) Growth kinetics of FMT-SCF-2 in FBS-supplemented medium (DMEM10), commercial serum-free medium (UltraCULTURETM) and custom-made serum-free medium. (n = 3) (B) Species identity of cultured fibroblast origin was verified by PCR analysis. (C) Species identity of cultured fibroblast origin was verified by qRT-PCR using RapidFinder™ Chicken ID Kit validated kit. (D) Immunostaining of primary, immortalized adherent and suspension-adapted embryonic fibroblasts for Lamin A/C and Lamin B1. (E) Lamin A/C to Lamin B1 ratio in anchorage-independent fibroblasts (FMT-SCF-2) compared with primary adherent grown fibroblasts (CEF-2). Scale bar equals 20 μm. Data in panels A and E are presented as means plus standard error of the mean. Student’s t test p values are labelled: * p < 0.05, ** p < 0.01, *** p < 0.001.

Extended Data Fig. 3 Sequencing analyses of immortalized and anchorage-independent chicken fibroblasts.

(A) K-mean clustering using Over-Representation (OR) Analysis of CEF-2 and HUN-CF-2. Full list of pathways (1-24) is provided in Supplementary Table 1 (S1A). Green: Downregulated genes, red: Upregulated genes. (B) K-mean clustering using Gene Set Enrichment (GSE) Analysis of CEF-2 and HUN-CF-2. Full list of pathways (1-20) is provided in Supplementary Table 1 (S1B). Green: Downregulated genes, red: Upregulated genes. (C) A heat map based on copy number variations (CNVs) of four immortalized chicken fibroblast lines, with FMT-SCF-1 and −2 derived from broiler Ross 308 and FMT-SCF-3 and −4 derived from Israeli Baladi chicken, respectively. Green: normal copy of genomic sequences (2n), purple: deletions of one copy (1n), turquoise: 2-copy deletion (0n). Red: (3n), orange: (4n) and black: (> 5 copies) mark duplication events. Boundaries between chromosomes are labelled black due to the repetitive sequence of the telomeres. (D) A phylogenetic tree of the four immortal chicken fibroblast lines based on similarities and differences in CNVs. (E) PCA of primary cells from broiler Ross 308 (CEF-2), Israeli Baladi (CEF-4), immortalized adherent and immortalized suspension lines of East Lansing (DF-1). Scale bar equals 100 μm.

Extended Data Fig. 4 Characterization of transdifferentiation potential in immortalized fibroblasts.

(A) Confocal image analysis of HUN-CF-2 cultured with adipogenesis-inducing agents pristanic acid, rosiglitazone, and lecithin. Nucleus rounded morphology and localizations as well as lipid droplet accumulation are indicative of immature adipocytes1. (B) TEM images showing cell shape and accumulation of fat in cells transdifferentiated with soy lecithin and oleic acid as compared to untreated control cells. Scale bars left to right, top to bottom equal 5 μm, 500 nm, 5 μm, and 500 nm, respectively. (C) Confocal images of HUN-CF-2 and FMT-SCF-2 following 7 days of transdifferentiation in medium containing oleic acid and oleic acid combined with soy lecithin. Scale bars equal 50 μm. (D) FACS analysis of FMT-SCF-2 cultured with oleic acid or lecithin and oleic acid. (n = 10,000 cells) (E) Gene expression analysis of PPARG and its target genes in FMT-SCF-2 treated with oleic acid. Fatty acid binding protein 4 (FABP4) upregulated in oleate stimulation possibly due to increased fatty acid trafficking2. (F) Confocal images of HUN-CF-2 following 7 days of transdifferentiation in adipogenic induction medium. Scale bars equal 50 μm. (G) K-mean clustering heatmap of transdifferentiated adherent and suspension cultures with their respective input cell lines. (n = 3) Data in panels A and E are presented as means plus standard error of the mean. Student’s t test p values are labelled: * p < 0.05, ** p < 0.01, *** p < 0.001.

Extended Data Fig. 5 Characteristics of high density expanded biomass.

(A) Three independent fed-batch bioreactor runs showing cell density and viability in a culture of FMT-SCF-4. (B) Summary of the three fed-batch bioreactor outcomes. (C) Three independent continuously perfused bioreactor runs in a culture of FMT-SCF-4. (D) Summary of the three perfusion bioreactor run outcomes. (E) Confocal images of FMT-SCF-4 cells before and after bioreactor expansion immunostained for Vimentin, Integrin B1, SOX2 and SSEA-1. Scale bars equal 50 μm. (F) Gene expression analysis of fibroblast markers in FMT-SCF-4 cells before and after expansion in a bioreactor. (n = 3) Data are presented as means plus standard error of the mean. Student’s t test p values are labelled: * p < 0.05, ** p < 0.01, *** p < 0.001.

Extended Data Fig. 6 Quality assessment of bioreactor expanded biomass.

(A) Pathogen and pesticide testing of fibroblasts and adipocyte-like cells obtained from three independent bioreactor runs and commercially obtained chicken breast. (B) Nutrition profile of fibroblasts, adipocyte-like cells and commercially obtained chicken breast presented as means ± standard deviation (n = 3). * indicates value of a single sample. (C) Dry-matter based amino acid profile of three independent samples of fibroblasts, adipocyte-like cells and chicken breast. (n = 3) (D) Dry-matter based fatty acid profile of fibroblasts, adipocyte-like cells and chicken breast. (n = 3) (E) Quantification of lipid oxidation in harvested biomass following extended storage in -20 °C using TBARS assay. (F) Quantification of lipid oxidation in harvested biomass following storage at room temperature, 4 °C and -20 °C using TBARS assay3. (n = 1-3) Data in panels C, D and F are presented as means plus standard error of the mean.

Supplementary information

Supplementary Information

Supplementary Tables S1–S5, References for Extended Data Figures and Source Data for Extended Data Fig. 2b (uncropped images of gels).

Reporting Summary.

Supplementary Table 1

S1a: Enrichment over-representation analysis of immortalization pathways comparing primary chicken fibroblasts (CEF-2) and immortalized fibroblasts (HUN-CF-2). S1b: Enrichment gene set enrichment analysis of immortalization pathways comparing primary chicken fibroblasts (CEF-2) and immortalized fibroblasts (HUN-CF-2). S1c: single nucleotide variation (SNV) analysis of TP53 in primary and immortalized chicken fibroblasts.

Supplementary Video 1

Grilling of cultured chicken.

Source data

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Source Data Extended Data Fig. 1-6

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Pasitka, L., Cohen, M., Ehrlich, A. et al. Spontaneous immortalization of chicken fibroblasts generates stable, high-yield cell lines for serum-free production of cultured meat. Nat Food 4, 35–50 (2023). https://doi.org/10.1038/s43016-022-00658-w

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