Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Textured soy protein scaffolds enable the generation of three-dimensional bovine skeletal muscle tissue for cell-based meat

Nature Food volume 1, pages 210–220 (2020)Cite this article

Subjects

Abstract

Cell-based meat (CBM) production is a promising technology that could generate meat without the need of animal agriculture. The generation of tissue requires a three-dimensional (3D) scaffold to provide support to the cells and mimic the extracellular matrix (ECM). For CBM, the scaffold needs to be edible and have suitable nutritional value and texture. Here, we demonstrate the use of textured soy protein—an edible porous protein-based biomaterial—as a novel CBM scaffold that can support cell attachment and proliferation to create a 3D engineered bovine muscle tissue. The media composition was optimized for 3D bovine satellite cell (BSC) proliferation and differentiation by adding myogenic-related growth factors. Myogenesis of several cell combinations was compared, and elevated myogenesis and ECM deposition were shown in co-culture of BSCs with bovine smooth muscle cells and tri-cultures of BSCs, bovine smooth muscle cells and bovine endothelial cells. The expression of proteins associated with ECM gene sets was increased in the co-culture compared with BSC monoculture. Volunteers tasted the product after cooking and noted its meaty flavour and sensorial attributes, achieving the goal of replicating the sensation and texture of a meat bite. This approach represents a step forward for the applied production of CBM as a food product.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: TSP scaffold characterization.
Fig. 2: BSC proliferation and differentiation on TSP scaffolds.
Fig. 3: Myogenesis of multicellular cultures on TSP scaffolds.
Fig. 4: ECM deposition of multicellular cultures on TSP scaffolds after BSC differentiation.
Fig. 5: Proteomic analysis of BSC/BSMC co-culture compared with BSC monoculture on PLLA/PLGA scaffolds after differentiation.
Fig. 6: Distribution of differentially expressed proteins representing selected Gene Ontology terms.
Fig. 7: Mechanical and texture properties of TSP scaffolds.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

References

  1. Post, M. & Weele, C. Principles of Tissue Engineering for Food (Elsevier, 2014).

  2. Slade, P. If you build it, will they eat it? Consumer preferences for plant-based and cultured meat burgers. Appetite 125, 428–437 (2018).

    Article  Google Scholar 

  3. Ben-Arye, T. & Levenberg, S. Tissue engineering for clean meat production. Front. Sustain. Food Syst. 3, 46 (2019).

    Article  Google Scholar 

  4. Specht, E. A., Welch, D. R., Rees Clayton, E. M. & Lagally, C. D. Opportunities for applying biomedical production and manufacturing methods to the development of the clean meat industry. Biochem. Eng. J. 132, 161–168 (2018).

    Article  Google Scholar 

  5. Edelman, P. D., McFarland, D. C., Mironov, V. A. & Matheny, J. G. Commentary: in vitro-cultured meat production. Tissue Eng. 11, 659–662 (2005).

    Article  CAS  Google Scholar 

  6. Egozi, D. et al. Engineered vascularized muscle flap. J. Vis. Exp. 107, 52984 (2016).

    Google Scholar 

  7. Gholobova, D. et al. Endothelial network formation within human tissue-engineered skeletal muscle. Tissue Eng. Part A 21, 2548–2558 (2015).

    Article  CAS  Google Scholar 

  8. Levenberg, S. Engineering blood vessels from stem cells: recent advances and applications. Curr. Opin. Biotechnol. 16, 516–523 (2005).

    Article  CAS  Google Scholar 

  9. Shandalov, Y. et al. An engineered muscle flap for reconstruction of large soft tissue defects. Proc. Natl Acad. Sci. USA 111, 6010–6015 (2014).

    Article  ADS  CAS  Google Scholar 

  10. Listrat, A. et al. How muscle structure and composition influence meat and flesh quality. Sci. World J. 2016, 3182746 (2016).

    Article  Google Scholar 

  11. Vitello, L. et al. Enhancing myoblast proliferation by using myogenic factors: a promising approach for improving fiber regeneration in sport medicine and skeletal muscle diseases. Basic Appl. Myol. 14, 45–51 (2004).

    Google Scholar 

  12. Yin, H., Price, F. & Rudnicki, M. A. Satellite cells and the muscle stem cell niche. Physiol. Rev. 93, 23–67 (2013).

    Article  CAS  Google Scholar 

  13. Purslow, P. P. Muscle fascia and force transmission. J. Bodyw. Mov. Ther. 14, 411–417 (2010).

    Article  Google Scholar 

  14. Jockenhoevel, S. et al. Fibrin gel—advantages of a new scaffold in cardiovascular tissue engineering. Eur. J. Cardiothorac. Surg. 19, 424–430 (2001).

    Article  CAS  Google Scholar 

  15. Guo, B. et al. Transcriptome analysis of cattle muscle identifies potential markers for skeletal muscle growth rate and major cell types. BMC Genomics 16, 177 (2015).

    Article  Google Scholar 

  16. Jain, R. K., Au, P., Tam, J., Duda, D. G. & Fukumura, D. Engineering vascularized tissue. Nat. Biotechnol. 23, 821–823 (2005).

    Article  CAS  Google Scholar 

  17. Christov, C. et al. Muscle satellite cells and endothelial cells: close neighbors and privileged partners. Mol. Biol. Cell 18, 1397–1409 (2007).

    Article  CAS  Google Scholar 

  18. Butler, J. M., Kobayashi, H. & Rafii, S. Instructive role of the vascular niche in promoting tumour growth and tissue repair by angiocrine factors. Nat. Rev. Cancer 10, 138–146 (2010).

    Article  CAS  Google Scholar 

  19. Rafii, S., Butler, J. M. & Ding, B.-S. Angiocrine functions of organ-specific endothelial cells. Nature 529, 316–325 (2016).

    Article  ADS  CAS  Google Scholar 

  20. Kyriakopoulou, K., Dekkers, B. & van der Goot, A. J. in Sustainable Meat Production and Processing (ed. Galanakis, C. M.) 103–126 (Academic Press, 2019).

  21. Day, L. Proteins from land plants—potential resources for human nutrition and food security. Trends Food Sci. Technol. 32, 25–42 (2013).

    Article  CAS  Google Scholar 

  22. Zeltinger, J., Sherwood, J. K., Graham, D. A., Müeller, R. & Griffith, L. G. Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition. Tissue Eng. 7, 557–572 (2001).

    Article  CAS  Google Scholar 

  23. Hayes, J. S., Czekanska, E. M. & Richards, R. G. in Tissue Engineering III: Cell-Surface Interactions for Tissue Culture (eds Kasper, C., Witte, F. & Pörtner, R.) 1–31 (Springer, 2012).

  24. Rodriguez, B. L. & Larkin, L. M. in Functional 3D Tissue Engineering Scaffolds (eds Deng, Y. & Kuiper, J.) 279–304 (Woodhead Publishing, 2018).

  25. Choi, J. S., Lee, S. J., Christ, G. J., Atala, A. & Yoo, J. J. The influence of electrospun aligned poly(epsilon-caprolactone)/collagen nanofiber meshes on the formation of self-aligned skeletal muscle myotubes. Biomaterials 29, 2899–2906 (2008).

    Article  CAS  Google Scholar 

  26. Aviss, K. J., Gough, J. E. & Downes, S. Aligned electrospun polymer fibres for skeletal muscle regeneration. Eur. Cells Mater. 19, 193–204 (2010).

    Article  CAS  Google Scholar 

  27. Levenberg, S. et al. Engineering vascularized skeletal muscle tissue. Nat. Biotechnol. 23, 879–884 (2005).

    Article  CAS  Google Scholar 

  28. Perry, L., Landau, S., Flugelman, M. Y. & Levenberg, S. Genetically engineered human muscle transplant enhances murine host neovascularization and myogenesis. Commun. Biol. 1, 161 (2018).

    Article  Google Scholar 

  29. Specht, L. An Analysis of Culture Medium Costs and Production Volumes for Cell-Based Meat (The Good Food Institute, 2019).

  30. Loh, Q. L. & Choong, C. Three-dimensional scaffolds for tissue engineering applications: role of porosity and pore size. Tissue Eng. Part B Rev. 19, 485–502 (2013).

    Article  CAS  Google Scholar 

  31. Du, M., Wang, B., Fu, X., Yang, Q. & Zhu, M.-J. Fetal programming in meat production. Meat Sci. 109, 40–47 (2015).

    Article  Google Scholar 

  32. Ding, S. et al. Maintaining bovine satellite cells stemness through p38 pathway. Sci. Rep. 8, 10808 (2018).

    Article  ADS  Google Scholar 

  33. Verbruggen, S., Luining, D., van Essen, A. & Post, M. J. Bovine myoblast cell production in a microcarriers-based system. Cytotechnology 70, 503–512 (2018).

    Article  CAS  Google Scholar 

  34. Péault, B. et al. Stem and progenitor cells in skeletal muscle development, maintenance, and therapy. Mol. Ther. 15, 867–877 (2007).

    Article  Google Scholar 

  35. Du, M., Huang, Y., Das, A. K., Yang, Q. & Duarte, M. S. Manipulating mesenchymal progenitor cell differentiation to optimize performance and carcass value of beef cattle. J. Anim. Sci. 91, 1419–1427 (2013).

    Article  CAS  Google Scholar 

  36. Chapman, M. A., Meza, R. & Lieber, R. L. Skeletal muscle fibroblasts in health and disease. Differentiation 92, 108–115 (2016).

    Article  CAS  Google Scholar 

  37. Krieger, J., Park, B.-W., Lambert, C. R. & Malcuit, C. 3D skeletal muscle fascicle engineering is improved with TGF-β1 treatment of myogenic cells and their co-culture with myofibroblasts. PeerJ. 6, e4939 (2018).

    Article  Google Scholar 

  38. Bauman, T. M. et al. Characterization of fibrillar collagens and extracellular matrix of glandular benign prostatic hyperplasia nodules. PLoS ONE 9, e109102 (2014).

    Article  ADS  Google Scholar 

  39. Suvik, A. & Effendy, A. W. M. The use of modified Masson’s trichrome staining in collagen evaluation in wound healing study. Mal. J. Vet. Res. 3, 39–47 (2012).

    Google Scholar 

  40. Mehta, F., Theunissen, R. & Post, M. J. in Myogenesis: Methods and Protocols (ed. Rønning, S. B.) 111–125 (Springer, 2019).

  41. Frey, R. S., Johnson, B. J., Hathaway, M. R., White, M. E. & Dayton, W. R. Growth factor responsiveness of primary satellite cell cultures from steers implanted with trenbolone acetate and estradiol-17β. Basic Appl. Myol. 5, 71–79 (1995).

    Google Scholar 

  42. Lapin, M. R., Gonzalez, J. M. & Johnson, S. E. Substrate elasticity affects bovine satellite cell activation kinetics in vitro. J. Anim. Sci. 91, 2083–2090 (2013).

    Article  CAS  Google Scholar 

  43. Lu, R., Chen, Y.-R., Solomon, M. B. & Berry, B. W. Tensile properties and Warner–Bratzler tenderness measurement of raw and cooked beef. Trans. ASAE 41, 1431–1439 (1998).

    Article  Google Scholar 

Download references

Acknowledgements

The authors thank M. Hathaway for providing the BSCs and relevant protocols, Y. Dahan for assistance with bovine cell isolation, I. Redenski for support with micro-CT experimental design, the BCF Bioimaging Center, Faculty of Medicine, Technion, for help with micro-CT imaging and analysis, J. Zavin for assistance with cryosectioning, O. Katovitz for assistance with data quantification, I. Michael for assistance with experimental design and Y. Posen for editorial assistance during preparation of this manuscript. The authors thank Technion’s MIKA Center for support with the SEM measurement and Technion’s Smoler Center for support with proteome measurements and analysis. The research was supported by funding from Aleph Farms.

Author information

Author notes

  1. These authors contributed equally: Tom Ben-Arye, Yulia Shandalov.

Authors and Affiliations

  1. Department of Biomedical Engineering, Technion – Israel Institute of Technology, Haifa, Israel

    Tom Ben-Arye, Yulia Shandalov, Shahar Ben-Shaul, Shira Landau, Yedidya Zagury, Iris Ianovici & Shulamit Levenberg

  2. Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa, Israel

    Tom Ben-Arye & Shulamit Levenberg

  3. Aleph Farms Ltd, Ashdod, Israel

    Neta Lavon & Shulamit Levenberg

Authors
  1. Tom Ben-Arye
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yulia Shandalov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shahar Ben-Shaul
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shira Landau
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yedidya Zagury
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Iris Ianovici
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Neta Lavon
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shulamit Levenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.B.-A., Y.S., S.B.-S., N.L. and S.Levenberg conceived of and designed the experiments. T.B.-A., Y.S., S.B.-S., S.Landau, Y.Z., I.I. and N.L. performed the experiments. T.B.-A., Y.S., S.B.-S., S.Landau, N.L. and S.Levenberg analysed the data. T.B.-A., Y.S., S.Landau and S.Levenberg wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Shulamit Levenberg.

Ethics declarations

Competing interests

This research was sponsored by Aleph Farms. S.Levenberg is the chief scientific officer and N.L. is the vice president of research and development of Aleph Farms.

Additional information

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

Extended data

Extended Data Fig. 1 Micro-CT analysis of Porosity, connectivity and surface area per volume of TSP1, TSP2.

Surface area calculation assumes a scaffold density of 0.65 gr/ml. Scaffold density was assessed based on scaffold height and weight measurements, assuming the scaffold diameter is 6 mm.

Extended Data Fig. 2 Proteome analysis.

Extracellular matrix and myogenesis proteins upregulated in co-cultures versus BSC mono-cultures.

Extended Data Fig. 3 Enriched gene ontology (GO) terms of proteins elevated in co-cultures of Bovine satellite cells with bovine smooth muscle cells.

Including all DE proteins.

Extended Data Fig. 4 Number of differentially expressed proteins in each gene ontology (GO) cluster & average fold Change (FC).

In reference to figure 6. Averaging was performed on the log2 scale, to prevent bias towards elevated proteins.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7.

Reporting Summary

Supplementary Video 1

Micro-CT TSP-1 reconstruction.

Supplementary Video 2

Fibroblast (red) proliferation on TSP scaffold over 21 days.

Supplementary Table 1

A full factorial statistical analysis (2 × 23) of the effect of the proliferation media (LLM1 versus control), IGF-1 in the differentiation medium (+IGF-1 versus −IGF-1) and EGF in the differentiation medium (+EGF versus −EGF) on myotube coverage, average myotube area and myotube complexity.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ben-Arye, T., Shandalov, Y., Ben-Shaul, S. et al. Textured soy protein scaffolds enable the generation of three-dimensional bovine skeletal muscle tissue for cell-based meat. Nat Food 1, 210–220 (2020). https://doi.org/10.1038/s43016-020-0046-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s43016-020-0046-5

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research