Non Technical Summary
Issue: In the coming decades, cell-cultured meat is expected to play a significant role in the development of sustainable climate-friendly food systems to feed the growing population. Analysis of beef production finds that switching from conventional to cultured beef production could result in a 95% reduction in land use and 92% reduction in carbon footprint. However, to realize these impacts new innovations in cell-culture infrastructure are needed to address in the cost inefficiencies and scalability issues that currently hinder commercial progress in this market. Further, technical challenges remain with producing cultured meat that mimics the structured and thick characteristics of authentic meat products.Approach: CytoNest will address these gaps by developing a new cell culture system that enables the cost-effective generation of cultured meat products that mimic the texture and structure of authentic whole-cut meats. The proposed scaffolding technology offers customizable parameters, allowing the cultivation of meat with varying properties. The system is able to support the weeks-long cell culture process at high volume, making the technology well-positioned for commercial-scale cultured meat production. In this project, CytoNest will experiment with different edible scaffold materials (material that meat is grown on) in an effort to improve the sensory properties of cultivated meat while still maintaining scalability. The company will also investigate different orientations and parameters of the scaffold and the effect on meat structure development, for example, the amount of muscle/fat generated. Lastly, CytoNest will demonstrate scalability and proof of concept with the cultivation of immortalized bovine satellite cells (Beef).Ultimate Goals: Successful completion of these objectives will prepare CytoNest for future work focused on implementing the proposed technology at scale and demonstrating proof of concept with whole cut meat cultivation. Long-term, this technology will be extended for the development of cultured meat products, delivering a platform of scaffolds with meat-specific (e.g., beef, pork, poultry, salmon) architectures to support the growth of tissue structures mimicking those of the represented meat products. Beyond the cell-cultured meat market, we also anticipate applications for our 3D scaffolds in cellular therapeutics.
Animal Health Component
Research Effort Categories
Goals / Objectives
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 cm^3) 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 cm^3) 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.
FDA-approved edible materials listed in the GRAS list (Generally Recognized as Safe) will be selected to create formulations for rapid nanofiber fabrication using our proprietary and patented mechanical touch spun (TS) machine and further 3D scaffolding. Materials in GRAS database will be screened for feasibility to achieve nanofibers and their hydrodynamic stability in cell culture media. Different biopolymers such as polysaccharides (e.g., cellulose, starch acetate, sodium alginate, pectin, mannans, chitosan, agar, etc.) and proteins (e.g., zein, soy-based protein, casein, etc.) will be screened for compatibility with the immortalized bovine satellite and C2C12 cell lines. Strictly, aqueous solvent mixtures will be utilized to prepare the biopolymer formulations to be suitable for the future commercial-scale manufacturing.Fabricated nanofibers in the range of 500 nm - 2000 nm and casted thin films (100 - 500 nm) will be evaluated with respect to water-resistance and swelling behavior using optical observation and in situ atomic force microscopy (AFM). Fibers and films with a swelling ratio less than 10% of its original dimensions is considered a stable fiber for cell culture.Different 3DFS parameters such as fiber-fiber spacing, layer-layer spacing, fiber diameter, micro-perfusion channel gap will be researched thoroughly using the bovine and murine cell lines to establish the cell-fiber interactions and high-density cell proliferation towards thick tissue formation. We will evaluate materials in the GRAS list for potential approved food additives such as zein (Tm 96 oC), starch acetate (Tm 196 oC) etc. to use as a spacer material.C2C12s and will be used for 3D cell culture studies on nanofiber scaffolds consisting multiple layers embedded in a housing insert to fit with multi-well plates.Cell-fiber interactions such as cell adhesion, proliferation, cell morphology/phenotype, differentiation of immortalized bovine satellite cell line (iBSC) and C2C12s will be studied in detail. Scaffold parameters such as geometry, fiber-fiber distance, vasculature memetic micro-perfusion channels will be tuned to improve the connective tissue formation.Cell adhesion and subsequent proliferation will be measured using the metabolic assay PrestoBlue™, as well as through visualization with live-dead imaging kits. Cell densities during proliferation will be inferred through analysis of PrestoBlue™ signal over time, and the maximum cell density will be determined by a plateau of PrestoBlue™ signal and confirmed with high density (calcein AM staining covers over 80% of the observable fiber surface area) observed with live-dead imaging.As part of objective 2, Scaffold mass vs the tissue mass will be analyzed to establish the mechanics of the meat.After 7 and 14 days of serum starvation for muscle differentiation, scaffolds with/without cells and before/after cooking (in sous-vide) will be analyzed for mechanical properties (e.g., hardness, springiness, and cohesiveness) using the Warner-Bratzler shear force test and the double compression test.At minimum, 8 different architectures will be tested varying scaffold parameters such as fiber-fiber gap, 3D layer-layer gap, and vasculature mimetic microchannels. Upon cell proliferation, we will record the Warner-Bratzler shear force values.To evaluate sensory properties and flavor attributes, a sensory evaluation panel will be assembled to perform smell tests on cooked samples, with a negative control of scaffold without cells and a positive control of cooked animal meat. We will also perform lipidomics on lipids extracted from the tissue, to elucidate the fatty acids composition, with specific flavor emphasis on fatty acid profiles of phospholipids and triglycerides.Successful scaffold parameters based on above cell culture results will be used to fabricate large scaffolds with volume up to 300 cm^3Identify 3D scaffold geometry and engineered channel distribution promoting healthy culture growth, with scaffolds at ≥80% porosity; fiber/cell weight ratio analysis in the range of 0.1 - 0.6; successful development of large scaffold (15cm×10cm×2cm) with determined optimal properties.As part of the objective 3, a perfusion based dynamic cell culture system will be assembled to test the technology scalability.scaffold parameters will be optimized for compatibility and stability in physiological conditions, porosity and vasculature, biomechanical and biochemical cues, and geometry.Perfusion stress induced cell differentiation study will be conducted and cytoskeletal profiles will be evaluated for thick muscle formation.Cytoskeletal protein profiles will be evaluated for potential for thick muscle formation. Immunostaining and western blots will be used for the evaluation. For immunostaining, 3DFS will be removed from the perfusion system and the cells fixed using paraformaldehyde. Immunocytochemistry will then be performed, as described in the proposal Objective 1, Task 3. For western blot, cells from the 3D culture will be removed with trypsin and mechanical dissociation. Cells will then be lysed using RIPA lysis buffer with protease inhibitor. After SDS-PAGE and blot transfer, the proteins will be analyzed with antibodies for myosin heavy chain, desmin, myogenin, troponin-T, and beta-actin as a loading control. Semi-quantitative protein expression will be evaluated using analysis to quantify the ratios of the muscle protein bands to the loading control.