Ph.D Thesis


Ph.D StudentBen-Arye Tom
SubjectBovine Skeletal Muscle Tissue Engineering
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisor PROF. Shulamit Levenberg


Abstract

Increasing public awareness towards foodborne illnesses, factory farming and the ecological footprint of the meat industry, has generated the need for animal-free meat alternatives. In the last decade, scientists have begun to leverage the tools accumulated in the fields of stem cells and tissue engineering towards the development of cultured meat, meat produced from cell cultures.

In tissue engineering, the physical and biochemical features of the native tissue are mimicked; cells and biomaterials are integrated under suitable culture conditions to form mature tissues. Cells are cultured inside scaffolds, 3D cell-compatible environments that facilitate tissue development. Multicellular seeding of cells with specific functions can recapitulate key components of the native tissue and facilitate cell-cell interactions. For cultured meat production, scaffolds should edible and resemble meat texture and composition. Tissues should be developed from farm animal cells, and include cells that produce extracellular matrix (ECM), a key component of the skeletal muscle tissues.

We hypothesized that textured soy protein (TSP), an edible, porous, protein-based meat substitute, can be used as a novel cultured meat scaffold. TSP pore size, porosity, pore interconnectivity and surface area were measured using confocal microscopy and micro-CT, showing that TSP pore size and pore interconnectivity are suitable for cell culture. High seeding efficiency (>80%), cell attachment, proliferation and spread into the scaffold thickness were observed on the TSP scaffolds. Cell seeding on a 1cm thick unmodified TSP sample showed that cell seeding can penetrate TSP thickness. Also, while the majority of TSP pores are suitable for cell culture, several large pores are interspersed throughout the TSP, which could positionally be used for media perfusion. Since ethanol-based scaffold sterilization is not suitable for scale-up and food production, it was replaced with gamma radiation.

Bovine skeletal muscle tissues were generated inside TSP scaffolds by multicellular seeding of bovine satellite cells (BSC), bovine aortic smooth muscle cells (BSMC) and bovine skeletal muscle microvascular endothelial cells (BEC). Expansion media (LLM1) and Differentiation media (LMM2) were optimized for 3D myogenesis, significantly improving myotube size, shape complexity and coverage on PLLA-PLGA scaffolds. LLM1 significantly improved BSC coverage on TSP scaffolds from 18% to 72%, while also improving cell morphology. The combination of LLM1 and LLM2 facilitated myotube formation on the TSP scaffold. Co-culture with BSMC and tri-culture with BSMC and BEC improved myogenin expression, collagen deposition and scaffold weight.

Proteome analysis provided an in-depth quantification of the contribution of co-culture with BSMC to the development of complex ECM development on PLLA-PLGA scaffolds. TGF-ß, Collagen and laminin clusters and a network of ECM related enzymes, chaperons and regulatory molecules were significantly elevated (>75%). The majority of enriched gene ontology (GO) annotations in the co-culture were ECM related.

To conclude, TSP was found to be a promising candidate for cultured meat production. LLM1 and LLM2 media compositions were optimized for enhanced 3D myogenesis. BSC co-culture with BSMC and Tri-culture with BSMC and BEC improved muscle tissue development. Proteome analysis provided strong evidence for BSMC role in complex ECM production.