Single-cell analysis of bovine muscle-derived cell types for cultured meat production.

Cultured meat technologies leverage the proliferation and differentiation of animal-derived stem cells ex vivo to produce edible tissues for human consumption in a sustainable fashion. However, skeletal muscle is a dynamic and highly complex tissue, involving the interplay of numerous mono- and multinucleated cells, including muscle fibers, satellite cells (SCs) and fibro-adipogenic progenitors (FAPs), and recreation of the tissue in vitro thus requires the characterization and manipulation of a broad range of cell types. Here, we use a single-cell RNA sequencing approach to characterize cellular heterogeneity within bovine muscle and muscle-derived cell cultures over time. Using this data, we identify numerous distinct cell types, and develop robust protocols for the easy purification and proliferation of several of these populations. We note overgrowth of undesirable cell types within heterogeneous proliferative cultures as a barrier to efficient cultured meat production, and use transcriptomics to identify conditions that favor the growth of SCs in the context of serum-free medium. Combining RNA velocities computed in silico with time-resolved flow cytometric analysis, we characterize dynamic subpopulations and transitions between active, quiescent, and committed states of SCs, and demonstrate methods for modulation of these states during long-term proliferative cultures. This work provides an important reference for advancing our knowledge of bovine skeletal muscle biology, and its application in the development of cultured meat technologies.

A Robust Aqueous Core-Shell-Shell Coconut-like Nanostructure for Stimuli-Responsive Delivery of Hydrophilic Cargo.

Conventional delivery systems for hydrophilic material still face critical challenges toward practical applications, including poor retention abilities, lack of stimulus responsiveness, and low bioavailability. Here, we propose a robust encapsulation strategy for hydrophilic cargo to produce a wide class of aqueous core-shell-shell coconut-like nanostructures featuring excellent stability and multifunctionality. The numerous active groups (-SH, -NH2, and -COOH) of the protein-polysaccharide wall material enable the formation of shell-cross-linked nanocapsules enclosing a liquid water droplet during acoustic cavitation. A subsequent pH switch can trigger the generation of an additional shell through the direct deposition of non-cross-linked protein back onto the cross-linked surface. Using anthocyanin as a model hydrophilic bioactive, these nanocapsules show high encapsulation efficiency, loading content, tolerance to environmental stresses, biocompatibility, and high cellular uptake. Moreover, the composite double shells driven by both covalent bonding and electrostatics provide the nanocapsules with pH/redox dual stimuli-responsive behavior. Our approach is also feasible for any shell material that can be cross-linked via ultrasonication, offering the potential to encapsulate diverse hydrophilic functional components, including bioactive molecules, nanocomplexes, and water-dispersible inorganic nanomaterials. Further development of this strategy should hold promise for designing versatile nanoengineered core-shell-shell nanoplatforms for various applications, such as the oral absorption of hydrophilic drugs/nutraceuticals and the smart delivery of therapeutics.