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Perspectives on scaling production of adipose tissue for food applications

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Abstract

With rising global demand for food proteins and significant environmental impact associated with conventional animal agriculture, it is important to develop sustainable alternatives to supplement existing meat production. Since fat is an important contributor to meat flavor, recapitulating this component in meat alternatives such as plant based and cell cultured meats is important. Here, we discuss the topic of cell cultured or tissue engineered fat, growing adipocytes in vitro that could imbue meat alternatives with the complex flavor and aromas of animal meat. We outline potential paths for the large scale production of in vitro cultured fat, including adipogenic precursors during cell proliferation, methods to adipogenically differentiate cells at scale, as well as strategies for converting differentiated adipocytes into 3D cultured fat tissues. We showcase the maturation of knowledge and technology behind cell sourcing and scaled proliferation, while also highlighting that adipogenic differentiation and 3D adipose tissue formation at scale need further research. We also provide some potential solutions for achieving adipose cell differentiation and tissue formation at scale based on contemporary research and the state of the field.

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Here, we established dedifferentiated fat (DFAT) cells from mature bovine adipocytes and then examined the effects of volatile fatty acids on the differentiation of these DFAT cells into adipocytes in vitro. When mature adipocytes were isolated from bovine adipose tissue and cultured using the ceiling culture method, they were dedifferentiated into fibroblast‐like cells without lipid droplets. These fibroblast‐like cells, termed bovine DFAT (b‐DFAT) cells, actively proliferated. After adipogenic induction, increased expression of adipocyte‐specific genes occurred in b‐DFAT cells and they redifferentiated into adipocytes with an accumulation of lipid droplets in their cytoplasm. The effects of volatile fatty acids on adipocyte differentiation in b‐DFAT cells were also examined. Specifically, acetate, butyrate, and propionate added to adipogenic induction medium significantly enhanced the adipogenesis of b‐DFAT cells compared with that observed in control cells; the addition of 10−3 mol of acetate enhanced adipogenesis of b‐DFAT cells to the greatest extent. These results suggest that b‐DFAT cells derived from bovine mature adipocytes are appropriate for the study of bovine adipocyte differentiation and that the optimum concentration treatment of acetate, a major energy source for ruminants, promotes adipogenesis of b‐DFAT cells in vitro.
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Muscle derived stem cells (MDSCs) and myoblast play an important role in myotube regeneration when muscle tissue is injured. However, these cells can be induced to differentiate into adipocytes once exposed to PPARγ activator like EPA and DHA that are highly suggested during pregnancy. The objective of this study aims at determining the identity of trans-differentiated cells by exploring the effect of EPA and DHA on C2C12 undergoing differentiation into brown and white adipocytes. DHA but not EPA committed C2C12 cells reprograming into white like adipocyte phenotype. Also, DHA promoted the expression of lipolysis regulating genes but had no effect on genes regulating β-oxidation referring to its implication in lipid re-esterification. Furthermore, DHA impaired C2C12 cells differentiation into brown adipocytes through reducing the thermogenic capacity and mitochondrial biogenesis of derived cells independent of UCP1. Accordingly, DHA treated groups showed an increased accumulation of lipid droplets and suppressed mitochondrial maximal respiration and spare respiratory capacity. EPA, on the other hand, reduced myogenesis regulating genes, but no significant differences were observed in the expression of adipogenesis key genes. Likewise, EPA suppressed the expression of WAT signature genes indicating that EPA and DHA have an independent role on white adipogensis. Unlike DHA treatment, EPA supplementation had no effect on the differential of C2C12 cells into brown adipocytes. In conclusion, DHA is a potent adipogenic and lipogenic factor that can change the metabolic profile of muscle cells by increasing myocellular fat.
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Background In the European Union proteins for food are largely animal-based consisting of meat and dairy products. Almost all soy but also a larger part of pulses and cereals consumed in the European Union are used for animal nutrition. While livestock is an important source of proteins, it also creates substantial environmental impacts. The food and feed system is closely linked to the planetary and health boundaries and a transformation to healthy diets will require substantial dietary shifts towards healthy foods, such as nuts, fruits, vegetables, and legumes. Results Extrudated vegetable meat alternatives consisting of protein combined with amaranth or buckwheat flour and a vegetable milk alternative made from lentil proteins were shown to have the potential to generate significantly less environmental impacts than their animal-based counterparts in most of the environmental indicators examined, taking into account both functional units (mass and protein content). The underlying field-to-fork LCA models include several variants for both plant and animal foods. The optimised plant-based foods show a clear potential for improvement in the environmental footprints. Conclusions Development of higher processed and therefore higher performing products is crucial for appealing to potential user groups beyond dedicated vegetarians and vegans and ultimately achieving market expansion. The Protein2Food project showed that prototypes made from Europe-grown legumes and pseudo-cereals are a valuable source for high-quality protein foods and despite being substantially processed they could help reduce the environmental impact of food consumption. This article is protected by copyright. All rights reserved.
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Intramuscular fatty deposits, which are seen in muscular dystrophies and with aging, negatively affect muscle function. The cells of origin of adipocytes constituting these fatty deposits are mesenchymal stromal cells, fibroadipogenic progenitors (FAPs). We uncover a molecular fate switch, involving miR-206 and the transcription factor Runx1, that controls FAP differentiation to adipocytes. Mice deficient in miR-206 exhibit increased adipogenesis following muscle injury. Adipogenic differentiation of FAPs is abrogated by miR-206 mimics. Using a labeled microRNA (miRNA) pull-down and sequencing (LAMP-seq), we identified Runx1 as a miR-206 target, with miR-206 repressing Runx1 translation. In the absence of miR-206 in FAPs, Runx1 occupancy near transcriptional start sites of adipogenic genes and expression of these genes increase. We demonstrate that miR-206 mimicry in vivo limits intramuscular fatty infiltration. Our results provide insight into the underlying molecular mechanisms of FAP fate determination and formation of harmful fatty deposits in skeletal muscle.
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https://authors.elsevier.com/c/1d0OB3QCo9bLFA Extruded meat substitutes, due to their high protein content, meat-like texture and meat processing compatibility, are very popular as the main ingredient of plant-based burger patties. The extrusion of plant-based proteins can be performed by two technologies: high moisture extrudates (HME) and low moisture texturized vegetable proteins (TVP). The largest difference between the technologies relates to the moisture content prevailing inside the extrusion barrel. The extrusion processes also vary in their throughput, and yields. Life cycle assessment (LCA) was performed to compare the environmental performance of the two extrusion technologies applied to two plant-based raw materials (soymeal and pumpkin seed flour). Additionally, the study compared plant-based burger patties to meat burger patties (beef, pork and chicken). The impact of plant-based burger patties was at least ten-fold lower than meat burger patties. TVP-production exhibited a higher environmental impact compared to HME (20-40% higher depending on the raw material). The best performing plant burger patties were HME-soy-patties, in contrast with the worst-performing plant TVP-soy patties. TVP-pumpkin seed patties presented lower impacts compared to TVP-soy ones.
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Introduction Obesity, defined as a condition of excessive fat accumulation in adipose tissue, is a global epidemic implicated in a myriad of processes deleterious to human health. It has become one of the leading impediments to public health globally. The study of obesity necessitates adipocyte models, which commonly employ a medium enriched with adipogenic hormones and fetal bovine serum (FBS) to culture terminal adipocytes. In the current study, we developed a novel protocol for serum-free differentiation of 3T3-L1 and ST2 pre-adipocytes using media enriched with free fatty acids (FFA) and bovine serum albumin (BSA). Differentiation was characterized by measuring FFA uptake and changes in expression of adipogenic genes. The novel protocol was also compared against the existing serum-inclusive method. Methods The National Institutes of Health (NIH)-3T3-L1 and ST2 pre-adipocyte cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% calf serum and 1% penicillin-streptomycin and Roswell Park Memorial Institute Medium (RPMI) with 10% FBS and 1% penicillin-streptomycin mixture, respectively, at 37℃, 5% CO2 in a humidified atmosphere. Differentiation was induced using a mixture of 0.25 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 10 µg/mL insulin, or 1% insulin-transferrin-selenium (ITS). Cells were cultured in serum-free media containing DMEM with BSA (2.5%) and lipid mixture 1 (LM1 1%) as well as serum-inclusive media enriched with 10% FBS. Total RNA was extracted, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using delta-delta Ct method, also known as the 2-∆∆Ct method. Ribosomal protein, large, P0 (RPLP0) was used as a house-keeping gene for quantitation of relative expressions. Results We observed an increase in fatty acid accumulation relative to controls using Oil Red O neutral lipid staining and spectrophotometry. This result was consistent with the effects of the serum-inclusive method. Differentiation was further confirmed by increased gene expression of adipogenic transcription factors - peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα); adipogenic genes - fatty acid-binding protein 4 (FABP4/aP2) and fatty acid translocase (FAT/CD36); and the lipogenic gene - perilipin by using quantitative RT-PCR. Conclusion Our data suggest that serum-free differentiation can significantly enhance the free fatty acid accumulation as well as adipogenic gene expression in both NIH-3T3-L1 and ST2 pre-adipocyte cells. Given the shortcomings of FBS, this method may provide advantages to the serum-inclusive protocols described previously.
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Peroxisome proliferator‐activated receptor gamma (PPARγ) is a master regulator of adipogenesis and lipogenesis. To understand its roles in fiber formation and fat deposition in skeletal muscle, we successfully generated muscle‐specific overexpression of PPARγ in two pig models by random insertion and CRISPR/Cas9 transgenic cloning procedures. The content of intramuscular fat was significantly increased in PPARγ pigs while had no changes on lean meat ratio. PPARγ could promote adipocyte differentiation by activating adipocyte differentiating regulators such as FABP4 and CCAAT/enhancer‐binding protein (C/EBP), along with enhanced expression of LPL, FABP4, and PLIN1 to proceed fat deposition. Proteomics analyses demonstrated that oxidative metabolism of fatty acids and respiratory chain were activated in PPARγ pigs, thus, gathered more Ca2+ in PPARγ pigs. Raising of Ca2+ could result in increased phosphorylation of CAMKII and p38 MAPK in PPARγ pigs, which can stimulate MEF2 and PGC1α to affect fiber type and oxidative capacity. These results support that skeletal muscle‐specific overexpression of PPARγ can promote oxidative fiber formation and intramuscular fat deposition in pigs.
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An accurate in vitro model of human adipose tissue could assist in the study of adipocyte function and allow for better tools for screening new therapeutic compounds. Cell culture models on two-dimensional surfaces fall short of mimicking the three-dimensional in vivo adipose environment, while three-dimensional culture models are often unable to support long-term cell culture due, in part, to insufficient mass transport. Microfluidic systems have been explored for adipose tissue models. However, current systems have primarily focused on 2D cultured adipocytes. In this work, a 3D human adipose microtissue was engineered within a microfluidic system. Human adipose-derived stem cells (ADSCs) were used as the cell source for generating differentiated adipocytes. The ADSCs differentiated within the microfluidic system formed a dense lipid-loaded mass with the expression of adipose tissue genetic markers. Engineered adipose tissue showed a decreased adiponectin secretion and increased free fatty acid secretion with increasing shear stress. Adipogenesis markers were downregulated with increasing shear stress. Overall, this microfluidic system enables the on-chip differentiation and development of a functional 3D human adipose microtissue supported by the interstitial flow. This system could potentially serve as a platform for in vitro drug testing for adipose tissue-related diseases.
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Innovation in cultivated meat development has been rapidly accelerating in recent years because it holds the potential to help attenuate issues facing production of dietary protein for a growing world population. There are technical obstacles still hindering large‐scale commercialization of cultivated meat, of which many are related to the media that are used to culture the muscle, fat, and connective tissue cells. While animal cell culture media has been used and refined for roughly a century, it has not been specifically designed with the requirements of cultivated meat in mind. Perhaps the most common industrial use of animal cell culture is currently the production of therapeutic monoclonal antibodies, which sell for orders of magnitude more than meat. Successful production of cultivated meat requires media that is food grade with minimal cost, can regulate large‐scale cell proliferation and differentiation, has acceptable sensory qualities, and is animal ingredient‐free. Much insight into strategies for achieving media formulations with these qualities can be obtained from knowledge of conventional culture media applications and from the metabolic pathways involved in myogenesis and protein synthesis. In addition, application of principles used to optimize media for large‐scale microbial fermentation processes producing lower value commodity chemicals and food ingredients can also be instructive. As such, the present review shall provide an overview of the current understanding of cell culture media as it relates to cultivated meat.
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Vaccinology is shifting toward synthetic RNA platforms which allow for rapid, scalable, and cell-free manufacturing of prophylactic and therapeutic vaccines. The simple development pipeline is based on in vitro transcription of antigen-encoding sequences or immunotherapies as synthetic RNA transcripts, which are then formulated for delivery. This approach may enable a quicker response to emerging disease outbreaks, as is evident from the swift pursuit of RNA vaccine candidates for the global SARS-CoV-2 pandemic. Both conventional and self-amplifying RNAs have shown protective immunization in preclinical studies against multiple infectious diseases including influenza, RSV, Rabies, Ebola, and HIV-1. Self-amplifying RNAs have shown enhanced antigen expression at lower doses compared to conventional mRNA, suggesting this technology may improve immunization. This review will explore how self-amplifying RNAs are emerging as important vaccine candidates for infectious diseases, the advantages of synthetic manufacturing approaches, and their potential for preventing and treating chronic infections.
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Context. The texture of beef is highly important for the eating experience, and there is a continued interest in understanding the biochemical basis for the variation in texture between cattle and their meat cuts in order to improve and minimise variation in tenderness due to production and processing factors. Aims. The present study aimed to investigate the impact of characteristics of meat on Warner-Bratzler shear-force (WBSF) as an indicator of texture of beef as affected by breed type, age/feeding phase, and muscle. Methods. Seventy-five steers of three breed types (Angus, Hereford and Wagyu · Angus) were slaughtered after weaning 6 months old (n = 15), after backgrounding 17 months old (n = 30) and after feedlotting 25 months old (n = 30). At slaughter three muscles (M. supraspinatus, M. semitendinosus and M. longissimus lumborum) were sampled from each steer, and pH, intramuscular fat and collagen content, sarcomere length, and proteolysis (desmin degradation) were measured and used to explain the variation in WBSF after 7 and 14 days of aging. Key results. Meat from Hereford and Angus steers had higher WBSF after 7 days of aging compared with Wagyu · Angus steers, but after 14 days of aging there was only a difference between Hereford and Wagyu · Angus in the M. supraspinatus and M. semitendinosus. The WBSF of the young weaned steers and steers slaughtered after backgrounding were dependent on the degree of proteolysis in the muscles, whereas for steers slaughtered after feedlotting the content of collagen was more important for the WBSF. The amount of intramuscular fat had a significant impact on the differences in WBSF within the specific muscle studied. In contrast to the general dogma that WBSF increase with age, WBSF decreased in M. semitendinosus and M. longissimus lumborum from the weaned 6-month-old steers to the 25-month-old steers finished in feed-lot, whereas in M. supraspinatus the older feed-lot finished steers had a higher WBSF. Conclusion. The factors contributing to the Warner-Bratzler shear force of beef depends on the age/feeding phase of the animal and the muscle and less on the breed type. Implications. Optimisation of texture in beef through breeding and production should address different traits dependent on the age/feeding phase of the slaughter animal.
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Metabolic engineering of mammalian cells has to-date focused primarily on biopharmaceutical protein production or the manipulation of native metabolic processes towards therapeutic aims. However, significant potential exists for expanding these techniques to diverse applications by looking across the taxonomic tree to bioactive metabolites not synthesized in animals. Namely, cross-taxa metabolic engineering of mammalian cells could offer value in applications ranging food and nutrition to regenerative medicine or gene therapy. Towards the former, recent advances in meat production through cell culture suggest the potential to produce meat with fine cellular control, where tuning composition through cross-taxa metabolic engineering could enhance nutrition and food-functionality. Here we demonstrate this possibility by engineering primary bovine and immortalized murine muscle cells with prokaryotic enzymes to endogenously produce the antioxidant carotenoids phytoene, lycopene and β-carotene. These phytonutrients offer general nutritive value and protective effects against diseases associated with red and processed meat consumption, and so offer a promising proof-of-concept for nutritional engineering in cultured meat. We demonstrate the phenotypic integrity of engineered cells, the ability to tune carotenoid yields, and antioxidant functionality of these compounds in vitro towards both nutrition and food-quality objectives. Our results demonstrate the potential for tailoring the nutritional profile of cultured meats. They further lay a foundation for heterologous metabolic engineering of mammalian cells for applications outside of the clinical realm.
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Most studies on the acquisition of advantageous traits in transgenic animals only focus on monogenic traits. In practical applications, transgenic animals need to possess multiple advantages. Therefore, multiple genes need to be edited simultaneously. CRISPR/Cas9 technology has been widely used in many research fields. However, few studies on endogenous gene mutation and simultaneous exogenous gene insertion performed via CRISPR/Cas9 technology are available. In this study, the CRISPR/Cas9 technology was used to achieve myostatin (MSTN) point mutation and simultaneous peroxisome proliferator‐activated receptor‐γ (PPARγ) site‐directed knockin in the bovine genome. The feasibility of this gene editing strategy was verified on a myoblast model. The same gene editing strategy was used to construct a mutant myoblast model with MSTN mutation and simultaneous PPARγ knockin. Quantitative reverse‐transcription polymerase chain reaction, immunofluorescence staining, and western blot analyses were used to detect the expression levels of MSTN and PPARγ in the mutant myoblast. Results showed that this strategy can inhibit the expression of MSTN and promote the expression of PPARγ. The cell counting kit‐8 cell proliferation analysis, 5‐ethynyl‐2′‐deoxyuridine cell proliferation analysis, myotube fusion index statistics, oil red O staining, and triglyceride content detection revealed that the proliferation, myogenic differentiation, and adipogenic transdifferentiation abilities of the mutant myoblasts were higher than those of the wild myoblasts. Finally, transgenic bovine embryos were obtained via somatic cell nuclear transfer. This study provides a breeding material and technical strategy to breed high‐quality bovine and a gene editing method to realize the mutation of endogenous genes and simultaneous insertion of exogenous genes in genomes.
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The ability to form tissue-like constructs that have high cell density, proper cell-cell contacts and cell-ECM interactions is critical for many applications including tissue models for drug discovery and tissue regeneration. Self-assembly methods have been used to create tissue like constructs with high cellular density and well-defined microstructure in the form of spheroids, organoids, or cell sheets. Cell sheets have a particularly interesting format for in vitro models as they possess 3D architecture and high cellular density but also are easy to visualize and conduct assays. Till now, the preparation of these sheets has involved culturing on specialized substrates that can be triggered by temperature or phase change (hydrophobic to hydrophilic) to release cells growing on them. Here a new technique is proposed that allows delamination of cells and secreted ECM and rapid self-assembly into a cell sheet on routine tissue culture plates using a simple pH trigger and without the need to use responsive surfaces or external electrical and magnetic stimuli. This technique can be used with cells capable of syncytialization and fusion such as skeletal muscle and placenta cells. Using C2C12 myoblast cells, we show that the pH trigger induces a rapid delamination of the cells as a continuous layer that self-assembles into a thick dense sheet. The delamination process has little effect on cell viability and maturation and preserves the ECM components that allow sheets to adhere to each other within a short incubation time enabling formation of thicker constructs when multiple sheets are stacked (double- and quadruple-layer constructs are formed here as supposed to single-layer constructs). We also show that another pH trigger (basic) can maintain the sheets in the planar configuration which makes them suitable for subsequent handling. This simple method can be used to form in vitro models for skeletal muscle and placental tissues.
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Background The environmental impact of meat consumption requires immediate action. Cultured meat—which is emerging through technologies to grow meat ex vivo—has exciting potential to offset the burden of livestock agriculture by providing an alternative method to sustainably produce meat without requiring individuals to become vegetarian. However, consumer uptake of cultured meat may be challenged by negative public perceptions. Scope and approach In this Review, we assert that the academic sector can play a vital role by understanding and communicating the science of cultured meat to the public. Here, we discuss how crosstalk between the science and technology of cultured meat and the behavioral sciences will be critical to overcome challenges in public perceptions, and ultimately to realize the environmental benefits of cultured meat. We identify research and outreach priorities for the academic sector as well as potential policy actions to achieve the maximum benefits of cultured meat for planetary health.