A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation (original) (raw)

Data availability

RNA-seq data has been deposited to the GEO (accession number GSE173199). Source data are provided with this paper. Further data supporting the findings of this study are available from the authors upon request.

Code availability

Analysis code is available from the authors upon request.

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Acknowledgements

We thank K. Derks and D. Tserpelis (Genome Services Maastricht UMC+) for their support in the acquisition and analysis of RNA-seq data, R. Mohren (Imaging Mass Spectrometry, M4I, Maastricht University) for proteomics data, and H. Duimel and C. López Iglesia (Microscopy CORE Lab, M4I, Maastricht University) for SEM. We also thank J. Melke and D. Remmers (Mosa Meat BV) for their assistance with confocal microscopy.

Author information

Authors and Affiliations

  1. Mosa Meat BV, Maastricht, the Netherlands
    Tobias Messmer, Iva Klevernic, Carolina Furquim, Ekaterina Ovchinnikova, Arin Dogan, Helder Cruz, Mark J. Post & Joshua E. Flack
  2. Department of Physiology, Maastricht University, Maastricht, the Netherlands
    Tobias Messmer & Mark J. Post

Authors

  1. Tobias Messmer
  2. Iva Klevernic
  3. Carolina Furquim
  4. Ekaterina Ovchinnikova
  5. Arin Dogan
  6. Helder Cruz
  7. Mark J. Post
  8. Joshua E. Flack

Contributions

T.M., I.K., C.F., E.O., A.D. and J.E.F. performed experiments and analysis. A.D., H.C., M.J.P. and J.E.F. supervised the study. T.M., M.J.P. and J.E.F. wrote the manuscript with input from all authors.

Corresponding author

Correspondence toJoshua E. Flack.

Ethics declarations

Competing interests

T.M., I.K., C.F., E.O., A.D., H.C. and J.E.F. are employees of Mosa Meat BV. M.J.P. is co-founder and stakeholder of Mosa Meat BV. The study was funded by Mosa Meat BV. Mosa Meat BV has patents pending on serum-free proliferation medium (PCT/P125933PC00) and serum-free differentiation medium (JBB/P126144NL00). All authors declare no other competing interests.

Additional information

Peer review information Nature Food thanks Deepak Choudhury, Laura Domigan and Min Du for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

a, RNA-seq-derived median fold changes of selected muscle-related genes during serum starvation compared to 0 h. b, Mean fold changes of genes shown in a), determined via RT–qPCR; error bars indicate SD, n = 3. c, Median fold changes of selected strongly upregulated myogenic genes compared to 0 h, determined via RNA-seq. d, Median fold changes of selected downregulated genes compared to 0 h, determined via RNA-seq; boxes indicate IQR, whiskers show 1.5 × IQR.

a, Volcano plot showing differentially expressed genes between 0 h (yellow) and 96 h (red) of serum starvation. Selected differentially expressed muscle, stem cell or cell cycle-related genes are indicated. b, Scatter plot showing correlation of log2-fold changes of overlapping genes from bovine (x-axis) with C2C12 (y-axis) with indicated Pearson correlation coefficient (R). Colours indicate upregulation (red) or downregulation (yellow) in bovine gene expression, shapes indicate whether differentially expressed genes are simultaneously up/downregulated in both species (squares) or significantly up/downregulated in one species while inversely regulated in the other (triangles). c, Median fold changes of muscle-related protein levels from 0 h to 72 h post serum starvation, normalised against 0 h; boxes indicate IQR, whiskers show 1.5 × IQR. d, Scatter plot showing the Pearson correlation of log2-fold changes of genes from RNA-seq (y-axis) and corresponding proteins from mass spectrometry (x-axis) upon serum starvation with indicated correlation coefficient (R). Colours indicate upregulation (red) or downregulation (yellow) while shapes indicate significantly regulated proteins (points), genes (triangles), or both (squares). e, Mean fold gene expression changes of differentially regulated surface receptors, determined by RT–qPCR; error bars indicate SD, n = 3.

a, Normalised nuclei counts of SCs differentiating on indicated coatings after 72 h in SFB, SFDM and serum starvation as percentage against SFB; statistical significance is indicated for each media against respective Matrigel control, error bars indicate SD, n = 3. b, Normalised nuclei counts of SCs after 72 h of SFB, SFDM or serum starvation at early (left), medium (centre), and late (right) passages with indicated PDs, as percentage of low PDs in SFB; asterisks directly above bars indicate statistical significance against SFB; error bars indicate SD, n = 4. c, Representative fluorescence images of differentiating SCs at early (top), medium (middle) or late (bottom row) passages after 72 h in SFB (left), SFDM (centre) or serum starvation (right), corresponding to Fig. 4e, Extended Data Fig. 3b; green, desmin; blue, Hoechst. Scale bar, 500 µm. *P < 0.05, **P < 0.005, ***P < 0.001.

a, Normalised nuclei counts of SCs from different donor animals after 72 h of myogenic differentiation as percentage of SFB with statistical significance indicated between SFDM and serum starvation respectively for each donor; error bars indicate SD, n = 4. b, Mean fusion indices of SCs from different donor animals after 72 h of serum-free or serum starvation induced differentiation, normalised against respective SFB condition. Statistical significance is indicated between SFDM and serum starvation respectively for each donor; error bars indicate SD, n = 4. c, Representative fluorescence images of myogenic differentiation of SCs from different donor animals after 72 h in SFB (top), SFDM (middle) and serum starvation (bottom row); green, desmin; blue, Hoechst. Scale bar, 500 µm. *P < 0.05, **P < 0.005, ***P < 0.001.

a, Representative fluorescence images of SCs after 72 h in SFB, SFDM with DMEM/F-12 and DMEM base, and upon serum starvation; green, desmin; blue, Hoechst. Scale bar, 500 µM. b, Normalised nuclei counts from a) as percentage of SFB with statistical significance indicated against SFDM (DMEM/F-12); error bars indicate SD, n = 4. c, Mean fusion indices derived from a) with statistical significance performed against SFDM (DMEM/F-12); error bars indicate SD, n = 4. d, Scatter plot indicating correlation of log2-fold changes between SFGM and DMEM/F-12-based SFDM (x-axis) against log2-fold changes between SFGM and DMEM-based SFDM (y-axis) with Pearson correlation coefficient indicated. *P < 0.05, **P < 0.005, ***P < 0.001.

a, Ultrastructure of BAMs after 192 h in SFB or SFDM (with DMEM/F-12 or DMEM basal media) or serum starvation. Scale bar, 100 µm. b, Representative fluorescence images of BAMs after 192 h in DMEM-based SFDM without (left) and with (right) 10 µM acetylcholine (ACh); pink, desmin; red, 𝛂-actin; green, myosinHC; blue, Hoechst. Scale bars, 100 µm. c, Ultrastructure (top), wide (middle) and close-up (bottom) scanning electron microscopy images of BAMs after 192 h in DMEM-based SFDM with (right) and without (left) 10 µM acetylcholine. Scale bars, 100 µm.

Extended Data Table 1 Media Formulations

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Extended Data Table 2 RT–qPCR primers

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Messmer, T., Klevernic, I., Furquim, C. et al. A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation.Nat Food 3, 74–85 (2022). https://doi.org/10.1038/s43016-021-00419-1

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