With the current interest in cultured meat, mammalian cell-based meat has mostly been unstructured. There is thus still a high demand for artificial steak-like meat. We demonstrate in vitro construction of engineered steak-like tissue assembled of three types of bovine cell fibers (muscle, fat, and vessel). Because actual meat is an aligned assembly of the fibers connected to the tendon for the actions of contraction and relaxation, tendon-gel integrated bioprinting was developed to construct tendon-like gels. In this study, a total of 72 fibers comprising 42 muscles, 28 adipose tissues, and 2 blood capillaries were constructed by tendon-gel integrated bioprinting and manually assembled to fabricate steak-like meat with a diameter of 5 mm and a length of 10 mm inspired by a meat cut. The developed tendon-gel integrated bioprinting here could be a promising technology for the fabrication of the desired types of steak-like cultured meats. Mammalian cell-based cultured meat has mostly been unstructured, leaving a demand for artificial steak-like meat. Here the authors present an assembled steak-like tissue of bovine skeletal muscle, adipose tissue, and blood capillary tissue fabricated by tendon-gel integrated printing technology.

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Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting

Cultured meat production requires the robust differentiation of satellite cells into mature muscle fibres without the use of animal-derived components. Current protocols induce myogenic differentiation in vitro through serum starvation, that is, an abrupt reduction in serum concentration. Here we used RNA sequencing to investigate the transcriptomic remodelling of bovine satellite cells during myogenic differentiation induced by serum starvation. We characterized canonical myogenic gene expression, and identified surface receptors upregulated during the early phase of differentiation, including IGF1R, TFRC and LPAR1. Supplementation of ligands to these receptors enabled the formulation of a chemically defined media that induced differentiation in the absence of serum starvation and/or transgene expression. Serum-free myogenic differentiation was of similar extent to that induced by serum starvation, as evaluated by transcriptome analysis, protein expression and the presence of a functional contractile apparatus. Moreover, the serum-free differentiation media supported the fabrication of three-dimensional bioartificial muscle constructs, demonstrating its suitability for cultured beef production. Bovine satellite cells undergoing myogenic differentiation in a chemically defined, serum-free medium are comparable to those undergoing serum starvation—demonstrating the suitability of this formulation for cultured meat production. 

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A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation

Cultivating meat from stem cells rather than by raising animals is a promising solution to concerns about the negative externalities of meat production. For cultivated meat to fully mimic conventional meat's organoleptic and nutritional properties, innovations in scaffolding technology are required. Many scaffolding technologies are already developed for use in biomedical tissue engineering. However, cultivated meat production comes with a unique set of constraints related to the scale and cost of production as well as the necessary attributes of the final product, such as texture and food safety. This review discusses the properties of vertebrate skeletal muscle that will need to be replicated in a successful product and the current state of scaffolding innovation within the cultivated meat industry, highlighting promising scaffold materials and techniques that can be applied to cultivated meat development. Recommendations are provided for future research into scaffolds capable of supporting the growth of high-quality meat while minimizing production costs. Although the development of appropriate scaffolds for cultivated meat is challenging, it is also tractable and provides novel opportunities to customize meat properties.

Scaffolding Biomaterials for 3D Cultivated Meat: Prospects and Challenges.

The functional capabilities of skeletal muscle are strongly correlated with its well-arranged microstructure, consisting of parallelly aligned myotubes. In case of extensive muscle loss, the endogenous regenerative capacity is hindered by scar tissue formation, which compromises the native muscle structure, ultimately leading to severe functional impairment. To address such an issue, skeletal muscle tissue engineering (SMTE) attempts to fabricate in vitro bioartificial muscle tissue constructs to assist and accelerate the regeneration process. Due to its dynamic nature, SMTE strategies must employ suitable biomaterials (combined with muscle progenitors) and proper 3D architectures. In light of this, 3D fiber-based strategies are gaining increasing interest for the generation of hydrogel microfibers as advanced skeletal muscle constructs. Indeed, hydrogels possess exceptional biomimetic properties, while the fiber-shaped morphology allows for the creation of geometrical cues to guarantee proper myoblast alignment. In this review, we summarize commonly used hydrogels in SMTE and their main properties, and we discuss the first efforts to engineer hydrogels to guide myoblast anisotropic orientation. Then, we focus on presenting the main hydrogel fiber-based techniques for SMTE, including molding, electrospinning, 3D bioprinting, extrusion, and microfluidic spinning. Furthermore, we describe the effect of external stimulation (i.e., mechanical and electrical) on such constructs and the application of hydrogel fiber-based methods on recapitulating complex skeletal muscle tissue interfaces. Finally, we discuss the future developments in the application of hydrogel microfibers for SMTE.

https://pubs.acs.org/doi/pdf/10.1021/acsbiomaterials.1c01145

Hydrogel-Based Fiber Biofabrication Techniques for Skeletal Muscle Tissue Engineering

Abstract Cultured meat is an emergent technology with the potential for significant environmental and animal welfare benefits. Accurate mimicry of traditional meat requires fat tissue; a key contributor to both the flavour and texture of meat. Here, we show that fibro-adipogenic progenitor cells (FAPs) are present in bovine muscle, and are transcriptionally and immunophenotypically distinct from satellite cells. These two cell types can be purified from a single muscle sample using a simple fluorescence-activated cell sorting (FACS) strategy. FAPs demonstrate high levels of adipogenic potential, as measured by gene expression changes and lipid accumulation, and can be proliferated for a large number of population doublings, demonstrating their suitability for a scalable cultured meat production process. Crucially, FAPs reach a mature level of adipogenic differentiation in three-dimensional, edible hydrogels. The resultant tissue accurately mimics traditional beef fat in terms of lipid profile and taste, and FAPs thus represent a promising candidate cell type for the production of cultured fat. 

/pdf/muscle-derived-fibro-adipogenic-progenitor-cells-for-3mi7ekk0.pdf

Muscle-derived fibro-adipogenic progenitor cells for production of cultured bovine adipose tissue

Owing to the increase in the global demand of meat, cultured meat technology is being developed to circumvent a shortage of meat in the future. However, methods for construction of millimetre-thick bovine muscle tissues with highly aligned myotubes have not yet been established. Here, we propose a culture method for constructing 3D-cultured bovine muscle tissue containing myotubes aligned along its long-axial direction, which contracted in response to electrical stimulation. First, we optimised the composition of biomaterials used in the construction and the electrical stimulation applied to the tissue during culture. Subsequently, we fabricated millimetre-thick bovine muscle tissues containing highly aligned myotubes by accumulating bovine myoblast-laden hydrogel modules. The microbial content of the bovine muscle tissue cultured for 14 days was below the detection limit, indicating that the muscle tissues were sterile, unlike commercial meat. Therefore, the proposed construction method for bovine muscle tissues will be useful for the production of clean cultured steak meat simulating real meat.

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Formation of contractile 3D bovine muscle tissue for construction of millimetre-thick cultured steak

Introduction This study aims to clarify how the unnaturalness of cellular agricultural products can be familiarized to society, using the case of the Japanese public's receptivity to cultured meats. Perceived unnaturalness is a key factor in the rejection of emerging technologies. While past studies have examined the explanatory factors involved in the public acceptance of cultured meats, the relationships among multiple factors have not been fully examined. Cultured meats and cellular agricultural products have been positively evaluated because they can contribute to future food sustainability, so the trade-off between perceived unnaturalness and sustainability is a significant issue for the public. Method This study uses a questionnaire survey with 2,000 Japanese respondents, which was conducted in 2019. Using a categorical data analysis approach, the strongest explanatory factors for receptivity were comprehensively searched among attitudes toward cultured meats, eating habits, demographics, and so on. Results and discussion The results indicated that perceived unnaturalness showed a strong explanatory power for the rejection of cultured meats, but awareness of world famine problems increased acceptance of cultured meat, if the degree of the respondents' concern for unnaturalness was moderate. The perceived animacy of non-human life forms is also associated with acceptance of cultured meat, which may reflect Japanese cultural values. These results suggest multiple pathways to overcoming the disgust of new food technologies in the social implementation process.

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Mai Furuhashi

Papers

Formation of contractile 3D bovine muscle tissue for construction of millimetre-thick cultured steak

How can the unnaturalness of cellular agricultural products be familiarized?: Modeling public attitudes toward cultured meats in Japan