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

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.

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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.

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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

Affiliations

Authors

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.

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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.

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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

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