Cultured meat, or cell-based meat, offers a sustainable alternative to traditional meat production by using stem cells to produce edible tissues in vitro. While initial research focused on skeletal muscle production, intramuscular fat ('marbling') is crucial for meat's flavor, texture, and palatability. Plant-based alternatives struggle to replicate the distinct lipid profile and melting temperature of animal fat, highlighting the need for methods to produce cultured fat. Current adipose tissue engineering methods face limitations in adipogenic maturity, reliance on animal-derived scaffolds, and scalability issues. Various cell types can undergo adipogenic differentiation, but in vivo intramuscular fat primarily originates from fibro-adipogenic progenitor cells (FAPs). FAPs, found in various species including bovine, are marked by PDGFRa expression and play a role in muscle regeneration and pathological fat deposition. This study investigates the potential of bovine muscle-derived FAPs for cultured fat production, focusing on their purification, expansion, differentiation, and the resulting fat tissue's characteristics.

Existing literature demonstrates the challenges in producing cultured fat suitable for meat alternatives. Studies have explored various cell types for adipogenic differentiation, including mesenchymal stem cells (MSCs) from bone and fat, de-differentiated fat (DFAT) cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Some research suggests satellite cells (SCs) may also contribute, but in vivo, FAPs are the primary source of intramuscular fat. Previous research has identified and characterized FAPs in various species, noting their expression of cell surface receptors like PDGFRa. However, their precise role in skeletal muscle biology and their potential for cultured meat applications remained under-explored. The existing methods for cultured fat production often involve animal-based scaffolds, limiting scalability and posing ethical concerns.

Bovine skeletal muscle samples were obtained from a registered abattoir. Muscle-derived cells were isolated using collagenase digestion, filtered, and treated with ACK erythrocyte lysis buffer. Fluorescence-activated cell sorting (FACS) was used to purify FAPs (CD29+, CD31/45/56-) and SCs (CD29+, CD56+) based on cell surface markers. RT-qPCR and RNA sequencing were employed to characterize the transcriptomic differences between FAPs and SCs. Myogenic and adipogenic differentiation potentials were compared in 2D culture using serum starvation and adipogenic differentiation medium (ADM), respectively. For 3D culture, FAPs were encapsulated in alginate hydrogels and cultured as microfibers in ADM. Lipid accumulation was assessed using BODIPY staining and quantified using image analysis. The proliferative capacity of FAPs was evaluated in 2D and in Cytodex 1 microcarrier-based spinner flask cultures. Sensory analysis (appearance, aroma, taste, texture) was performed by volunteers on cultured fat compared to traditional beef fat. Lipidomic analysis using HILIC LC-MS/MS compared the lipid profiles of cultured fat and traditional fat tissues. Statistical analyses included t-tests, one-way ANOVA, and two-way ANOVA with Bonferroni's correction.

FAPs were successfully purified from bovine muscle using a FACS-based strategy, yielding approximately 10⁵ cells per gram of tissue. Transcriptomic analysis revealed significant differences between FAPs and SCs, with FAPs showing upregulation of adipogenic markers (PDGFRA, PPARG, SCARA5) and downregulation of myogenic markers (PAX7, NCAM1). In 2D cultures, FAPs showed significantly higher adipogenic differentiation potential compared to SCs, with greater lipid droplet accumulation and upregulation of adipocyte marker genes. In 3D alginate hydrogels, FAPs formed mature adipocytes with unilocular lipid droplets, demonstrating significantly higher lipid accumulation upon ADM treatment compared to 2D cultures. FAPs exhibited robust proliferative capacity exceeding 30 population doublings in 2D culture and demonstrated efficient expansion in microcarrier-based spinner flasks. Sensory analysis indicated that cultured fat from FAPs had a creamy consistency and a discernible 'beefy' flavor. Lipidomic analysis showed that the triglyceride composition of cultured fat became increasingly similar to traditional subcutaneous fat during adipogenic differentiation, with a higher proportion of monounsaturated fatty acids (MUFAs) and lower levels of saturated and polyunsaturated fatty acids compared to traditional fat and muscle.

This study successfully demonstrated the potential of bovine muscle-derived FAPs as a source for cultured fat production. The ability to purify FAPs concurrently with SCs from a single muscle sample is advantageous for a commercially viable bioprocess. The transcriptomic and immunophenotypic characterization of FAPs aids in optimizing purification strategies. The use of edible alginate hydrogels addresses the limitations of animal-derived scaffolds in previous studies. The results show that cultured fat derived from FAPs closely mimics traditional beef fat in both its lipid profile and sensory characteristics, suggesting its potential as a novel food ingredient. The robust proliferative capacity of FAPs in both 2D and upscaled 3D cultures supports the feasibility of large-scale production.

This study establishes the feasibility of producing cultured fat tissue from bovine muscle-derived FAPs. FAPs offer advantages in terms of ease of acquisition, high adipogenic potential, and the ability to closely mimic traditional beef fat in lipid profile and taste. While challenges remain, such as maintaining differentiation potential at higher population doublings and optimizing fully food-compatible production protocols, this work represents a significant step towards making cultured meat a commercially viable and consumer-acceptable product. Future research should focus on optimizing culture conditions to extend the cellular lifespan and reduce donor-to-donor variability, investigating the pathways involved in the loss of stemness during prolonged passaging and exploring further modifications of fatty acid composition to potentially enhance nutritional value.

The study used a limited number of volunteers in the sensory analysis, requiring further blind taste testing with a larger group for more robust conclusions. The observed drop in adipogenic differentiation potential at later population doublings needs further investigation to improve the longevity of FAPs in culture. The study also noted some variability in lipid accumulation across different donor animals. While alginate hydrogels provide a suitable biocompatible scaffold, exploring other edible biomaterials might further enhance adipogenic differentiation and the resulting tissue’s structure. Finally, the study focused on subcutaneous fat for comparison; comparing with intramuscular fat would provide more direct insights.