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Goals:In the proposed work, we aim to establish an edible scaffold and 3D cell culture protocol suitable for the bovine cell line to develop a high-density cell culture tissue, mimicking the beef product in the whole-cut format. A perfusion based dynamic cell culture system with a liter capacity will be assembled and tested for scalability of the cultured meat production using fiber-based scaffolds. The quality and mechanics of the obtained cultivated meat tissues will be assessed in comparison with animal harvested meat tissues.Objective 1: Investigate cell-fiber interactions.Cell specific edible material formulations will be researched to improve the cell adhesion and growth on fibers with immortalized bovine satellite cell lines (iBSCs). We will explore an array of edible materials for use in our nanofiber scaffolds specifically for an immortalized bovine satellite cell line. While our previous versions used cellulose as a base material, we seek materials to promote cell adhesion and growth at scale, as well as improve organoleptic properties of meat. For each formulation and the derived 3D scaffold, we will test iBSC proliferation and differentiation toward 3D tissue formation, with C2C12 murine myoblasts as a control cell line, given its extensive use and characterization in muscle tissue engineering. Muscle cell differentiation and analysis will be pursued, including cytoskeletal and muscle differentiation protein profiles.Objective 2: Determine the scaffold parameters for meat structure development.iBSCs and C2C12s will be studied with small (8 cm3) edible nanofiber scaffolds in multi-well plates for the time-dependent changes in the meat development process on fibers to support cell differentiation into muscle phenotype, along with an appropriate mass balance for food. 3D scaffold parameters such as fiber-fiber gap, 3D layer-layer gap, and vasculature mimetic microchannels will be varied to research the healthy tissue development over the prolonged cell culture of 2 - 10 weeks. Ultimately, we will use the findings to guide the fabrication of a large scaffold (>300 cm3) that can be used to study the mechanics of the meat generated in the process.Objective 3: A perfusion-based dynamic culture system to validate the scalability assumptions.A perfusion based dynamic cell culture system will be integrated with multi-well plates and a 1,000 mL vessel to optimize scaffold parameters for compatibility and stability in physiological conditions, porosity and vasculature memetic channels, biomechanical and biochemical cues, and geometry. 3D cell culture conditions for iBSCs will be optimized in collaboration with the Kaplan Lab at Tufts University. We will investigate the effect of serum on cell differentiation and growth as well as the potential for perfusion stress induced differentiation. C2C12s will be considered as a control, though the bioreactor design will ideally be developed for the iBSCs, as they are more relevant to cultured meat. Successful completion of these objective will position us for a Phase II research efforts where we plan to implement our technology at scale and demonstrate proof of concept with whole cut meat cultivation.

Yadavalli, N.; Kaplan, DA, .; Minko, SE, .
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