ARTICLES https://doi.org/10.1038/s43016-020-0046-5 1 Department of Biomedical Engineering, Technion – Israel Institute of Technology, Haifa, Israel. 2 Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa, Israel. 3 Aleph Farms Ltd, Ashdod, Israel. 4 These authors contributed equally: Tom Ben-Arye, Yulia Shandalov. ✉ e-mail: shulamit@bm.technion.ac.il C ell-based meat (CBM)—also termed cultured meat, culti- vated meat or clean meat—is an evolving technology with the aim of producing meat from cell cultures 1,2 . The development of this technology is motivated by sustainability issues related to traditional animal-based meat production methods, including the overuse of antibiotics, food and water security, food safety, environ- mental footprints and poor animal welfare conditions 3 . CBM tech- nology may also enable new fields of culinary meat innovation 3,4 . Tissue-engineering techniques, such as seeding cells inside an environment that recapitulates the native tissue conditions, are required to replicate muscle development in vitro. Vascularized skeletal muscle tissues can be generated inside three-dimensional (3D) scaffolds by co-culture of muscle cells, endothelial cells and supporting cells 5–9 . Skeletal muscle progenitors—the primary cell type required for this technique 10 —differentiate into muscle fibres under starvation conditions in the presence of insulin-like growth factor 1 (IGF-1) 11,12 . Their immediate native environment is com- posed of a honeycomb-like structure called the perimysium, which is filled with a soft gel called the endomysium 13 . The endomysium can be recapitulated using simple biological gels, such as fibrin and collagen, and extracellular matrix (ECM)-producing supporting cells, such as fibroblasts. During tissue development, the gels and supporting cells reorganize and a complex tissue-related ECM can be deposited 14 . While it is feasible for supporting cells to produce an amorphous endomysium-like ECM during tissue maturation in vitro, the perimysium honeycomb structure should be added externally as a porous, edible scaffold that also provides mechani- cal support to the developing tissue. Since the scaffold constitutes a main component of the final product, it should resemble the composition and properties of meat. Muscle tissues are abundant in aligned microvascular networks formed by endothelial cells 15,16 . While endothelial cells might not have a direct effect on meat qual- ity, they regulate skeletal muscle tissue behaviour via angiocrine signalling 17–19 . Textured soy protein (TSP) is a porous, food-grade, inexpensive byproduct of soybean oil processing that was invented in 1960 20 . It is frequently used as a raw material in meat substitutes, due to its texture and high protein content (>50%) 21 , which both improve its nutritional value and provide anchor points for cell adherence; therefore, it can be used as a scaffold. Its porosity is an important scaffolding feature that facilitates tissue development throughout the volume of the 3D scaffold 22 . TSP can be tailored to various sizes and shapes, which will be useful for scale-up processes (for exam- ple, adjustment to bioreactor geometry) in future CBM produc- tion. These properties render TSP a suitable candidate for processes aimed at engineering tissues for human consumption. Therefore, we aimed to create a bovine muscle tissue on an edible scaffold made of TSP. Results TSP scaffold characterization. TSP is a dry, porous, protein-based material (Fig. 1a) that expands and becomes softer when soaked in water (Supplementary Fig. 1). Two TSP scaffolds were tested, TSP-1 and TSP-2, containing 69% and 53% protein, respectively. Scanning electron microscopy (SEM) images showed that the fun- damental structure of the TSP-1 scaffold, on the dimensions of cell-surface interactions 23 , contained spheres of 36 ± 7 nm (Fig. 1b). Analysis of the TSP-1 pore size distribution (Fig. 1c) showed that the vast majority of pores were small (88% <50 µm), several were medium sized (11% in the range 50–400 µm) and a small percentage Textured soy protein scaffolds enable the generation of three-dimensional bovine skeletal muscle tissue for cell-based meat Tom Ben-Arye 1,2,4 , Yulia Shandalov  1,4 , Shahar Ben-Shaul 1 , Shira Landau 1 , Yedidya Zagury 1 , Iris Ianovici 1 , Neta Lavon 3 and Shulamit Levenberg  1,2,3 ✉ 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 pro- teins 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. NATURE FOOD | www.nature.com/natfood