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Perspectives on scaling production of adipose tissue for food applications
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.
... Tissue constructs must nevertheless provide the flexibility to mature and sufficient cohesion to create a tissue-like structure (Figure 5). Several viable strategies for cultured fat production have been described in recent years and are reviewed extensively elsewhere (96,97). ...
... Regardless of the chosen strategy, the primary tissue engineering challenge remains ensuring proper gas and nutrient exchange, which limits the size of 3D constructs unless vascularization (or other channeling to the construct interior) can be achieved. Both solutions are possible on a laboratory scale but have cost, robustness, and scalability limitations that make them unsuitable for cultured meat applications without further development (96,97). ...
Cultured meat is an emerging biotechnology that aims to produce meat from animal cell culture, rather than from the raising and slaughtering of livestock, on environmental and animal welfare grounds. The detailed understanding and accurate manipulation of cell biology are critical to the design of cultured meat bioprocesses. Recent years have seen significant interest in this field, with numerous scientific and commercial breakthroughs. Nevertheless, these technologies remain at a nascent stage, and myriad challenges remain, spanning the entire bioprocess. From a cell biological perspective, these include the identification of suitable starting cell types, tuning of proliferation and differentiation conditions, and optimization of cell–biomaterial interactions to create nutritious, enticing foods. Here, we discuss the key advances and outstanding challenges in cultured meat, with a particular focus on cell biology, and argue that solving the remaining bottlenecks in a cost-effective, scalable fashion will require coordinated, concerted scientific efforts. Success will also require solutions to nonscientific challenges, including regulatory approval, consumer acceptance, and market feasibility. However, if these can be overcome, cultured meat technologies can revolutionize our approach to food.
Expected final online publication date for the Annual Review of Animal Biosciences, Volume 12 is February 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... While laboratory techniques (2D culture in tissue culture flasks) were used to produce the adipocytes for aggregation in this study, the concept of generating adipose tissues for food via the aggregation of individual fat cells should be applicable to cells generated in larger-scale bioreactor systems (Figure 7). For example, adherent preadipocytes could be proliferated and differentiated en masse in various fixed bed bioreactors, after which they are harvested (e.g., by spraying fluid onto the cells to detach them) (Leinonen et al., 2020;Pörtner and Platas Barradas, 2007;Hanley et al., 2014;Leung et al., 2022;Yuen et al., 2022). The largescale generation of adipocytes might also be achieved in suspension bioreactors using microcarrier, spheroid/aggregate approaches, or through cell adaptation to single cell suspension (Yuen et al., 2022;Frye and Patrick, 2006;Lohr et al., 2010;Nahmias and Cohen, 2020;Bellani et al., 2020). ...
... For example, adherent preadipocytes could be proliferated and differentiated en masse in various fixed bed bioreactors, after which they are harvested (e.g., by spraying fluid onto the cells to detach them) (Leinonen et al., 2020;Pörtner and Platas Barradas, 2007;Hanley et al., 2014;Leung et al., 2022;Yuen et al., 2022). The largescale generation of adipocytes might also be achieved in suspension bioreactors using microcarrier, spheroid/aggregate approaches, or through cell adaptation to single cell suspension (Yuen et al., 2022;Frye and Patrick, 2006;Lohr et al., 2010;Nahmias and Cohen, 2020;Bellani et al., 2020). Once produced, adipocytes can then be formed into structured cultured fat tissues by mixing with binder or crosslinking ingredients. ...
We present a method of producing bulk cell-cultured fat tissue for food applications. Mass transport limitations (nutrients, oxygen, waste diffusion) of macroscale 3D tissue culture are circumvented by initially culturing murine or porcine adipocytes in 2D, after which bulk fat tissue is produced by mechanically harvesting and aggregating the lipid-filled adipocytes into 3D constructs using alginate or transglutaminase binders. The 3D fat tissues were visually similar to fat tissue harvested from animals, with matching textures based on uniaxial compression tests. The mechanical properties of cultured fat tissues were based on binder choice and concentration, and changes in the fatty acid compositions of cellular triacylglyceride and phospholipids were observed after lipid supplementation (soybean oil) during in vitro culture. This approach of aggregating individual adipocytes into a bulk 3D tissue provides a scalable and versatile strategy to produce cultured fat tissue for food-related applications, thereby addressing a key obstacle in cultivated meat production.
... So far, the research on cultured meat has mainly focused on cultured muscle tissue [9,10]. However, fat tissue has an important influence on the sensory and texture properties of meat [11]. ...
... Large-scale expansion of ADSCs is one of the requisites for cultured fat [10]. In order to increase the scale expansion of ADSCs, it is necessary to improve the efficiency of the number of cells cultured in each unit of medium [34]. ...
Cultured meat is an innovative meat-production technology that does not rely on animal husbandry. As a new food component, cultured fat is of great significance to cultured meat. In this study, we isolated adipose-derived stem cells (ADSCs) and identified the purity by immunofluorescence staining of ADSC-specific surface marker proteins CD44 and CD29 and showed that most of the cells were positive for CD29 and CD44. In addition, we detected the expression of FABP4 and Plin1 to confirm that ADSCs differentiated into mature adipocytes at 10 days post-induction. Subsequently, the culture conditions of ADSCs on microcarriers (MCs) were optimized and showed that cell density of living cells reached their highest after 5 days when continuously stirring at 50 rpm. Finally, the expression of FABP4 and PPARγ was detected to confirm the adipogenic differentiation ability of ADSCs on 2D and 3D culture systems and showed that ADSCs maintained their adipogenic differentiation ability after expansion on MCs. In conclusion, this research demonstrated that reliance on MCs to expand ADSCs was a promising approach for production of cultured fat.
... Adipocytes have terminally differentiated and have lost their ability to divide. Hence, several primary adipogenic cells are used as seed cells in cultured meat research 7 . However, their adipogenic efficiency and proliferation potency decrease with the increasing number of passages, limiting their utility for cell-cultured fat research and large-scale production. ...
Adding adipose cells to cell-cultured meat can provide a distinctive aroma and juicy texture similar to real meat. However, a significant challenge still exists in obtaining seed cells that can be propagated for long periods, maintain their adipogenic potential, and reduce production costs. In this study, we present a cell strain derived from immortalized porcine preadipocytes that can be subculture for over 40 passages without losing differentiation capacity. This cell strain can be differentiated within 3D bioscaffolds to generate cell-cultured fat using fewer chemicals and less serum. Additionally, it can be expanded and differentiated on microcarriers with upscaled culture to reduce costs and labor. Moreover, it can co-differentiate with muscle precursor cells, producing a pattern similar to real meat. Therefore, our cell strain provides an exceptional model for studying and producing cell-cultured fat.
... Various sources for adipose tissue could be applied in cell culturing such as dedifferentiated fat (DFAT) cells harvested from mature adipocytes, adipose-derived stem cells, preadipocytes, mesenchymal stem cells, fibroadipogenic progenitors, bovine expanded potential stem cells and engineered cell lines (Fish et al., 2020;Yuen Jr et al., 2022). DFAT cells are multipotent and could maintain proliferation and adipogenicity for longer durations, even after 50 passages . ...
In-vitro meat production has entered into the phase of pilot-commercial scale production from the conceptual-laboratory phase. The main challenge for in-vitro meat production on a commercial scale is the very high cost of its production, mainly due to the cost of cell culture media, growth regulators, and the requirement of highly skilled manpower. The development of serum-free and animal-free culture media with plant, microbial, and fungi-derived compounds through recombinant technology and media recycling is crucial for scaling up in-vitro meat production and reducing the price of the end products. The proper design of bioreactors specific to in-vitro meat production, their automation, utilization of natural and edible scaffolds, and microcarriers made up of edible materials are the present focus of researchers. The co-culturing by proliferating various cells such as adipocytes, chondrocytes, fibroblasts, and endothelial cells are applied for imparting textural and organoleptic attributes to developed products similar to conventional meat. The industrial process to produce in-vitro meat needs a clear synergy between the biological, chemical, technical, and industrial fields because at the moment the main research focus is on the development and improvement of cell lines available to set up cell culture and culture media, bioreactors, cell lines, scaffolding, and biofabrication. The research on in vitro meat is limited by the fact that from the industry the protocols are not properly divulgated.
... When successful in large animal and eventually clinical studies, it will be an extra challenge to apply VATE on a large scale and not merely as a proof-of-concept. While upscaling of VATE for STR is reserved for the far future, it is interesting to see how concepts of scaling and industrialization of ATE take shape in other fields such as the food industry [372]. ...
Soft tissue defects are a common clinical challenge mostly caused by trauma, congenital anomalies and oncological surgery. Current soft tissue reconstruction (STR) options include synthetic materials (fillers and implants) and autologous adipose tissue transplantation through flap surgery and/or lipotransfer. Both reconstructive options hold important disadvantages to which vascularized adipose tissue engineering (VATE) strategies could offer solutions. In this review, we first summarized pivotal characteristics of functional adipose tissue (FAT) such as the structure, function, cell types, development and extracellular matrix (ECM). Next, we discussed relevant cell sources and how they are applied in different state-of-the-art VATE techniques. Herein, biomaterial scaffolds and hydrogels, ECMs, spheroids, organoids, cell sheets, 3D bioprinting and microfluidics are overviewed. Also, we included extracellular vesicles and emphasized their potential role in VATE. Lastly, current challenges and future perspectives in VATE are pointed out to help to pave the road towards clinical applications.
... Lipids (fat molecules) and adipocytes (fat cells) are two components of food, having many diff erences in terms of biological and food properties. Stem Cells (iPSCs) are another promising cell type that could be applicable for scalable production of adipocytes in the wide range of animal species [61,62]. While iPSCs feature exceptional growth properties, they are formed by including reprogramming genes in somatic cells [63]. ...
Cultivated meat (clean meat) is an emerging yet fast growing research field and industry with a great potential to overcome the limitations of traditional cattle meat production. Cultivated meat leverages the technologies of cell biology and tissue engineering, culturing multiple types of cells and assembling them into a tissue structure construct mimicking the muscle tissues of livestock animals. A sustainable cell source is the first and the utmost important component of cultivated meat technology. In this mini review, cell sources for the main cell types in cultivated meat (muscle cells and fat cells) are described. Stem cells with self-renewal and differentiation potential are the most prominent candidates. Progenitor stem cells from muscle tissues, mesenchymal stem cells isolated from many other tissues and induced Pluripotent Stem Cells (iPSCs) created from terminally differentiated cells have been used as cell sources for cultivated meat. To become a sustainable cell source, which can generate high quantity (extensive in vitro expansion) and high quality (stemness) cells for the making of cultivated meat, these cells still face the challenges and limitation intrinsically associated with in vitro culturing. The efforts and strategies to circumvent such limitations are also discussed.
... Significant emphasis has been placed in the field of study on improving the cellular biology component of these systems. Recently, new media that don't rely on animal products have been developed 9 , efficient design of experiments has been implemented to increase the speed at which these media formulations are optimized 10 , and cell lines themselves have been modified to increase protein or lipid production, improving nutrition and eventual taste of these cell systems 11 . On the side of texture, some have proposed using natural materials as scaffolds for cells that provide similar textures such as jackfruit 12 . ...
To meet the requirements for full production in cellular agriculture, a multi-disciplinary approach is required. New questions and understandings between different concepts, such as texture and cooking, need to be identified and how they correlate with cellular behavior and taste. This work focuses on understanding and developing photolithographic systems for scalable cellular agriculture. There were two specific research directions: 1) a traditional photolithography printing system was improved upon for mass manufacturing purposes and 2) mechanical analysis and degradation of key potential photolithographic biomaterials were tested for a food product. A novel digital rotational ultraviolet manufacturing (DRUM) process was developed, and initial photolithographic printing tests were carried out. GelMA hydrogels were tested in uniaxial compression and compared to a range of meat samples at 20°C using constant displacement speed control. The results showed effective mimicry under the defined environment to that of raw scallops, but not of higher order constructs. A soy-based resin was also tested in degradation 37°C for 0 to 28 days in phosphate buffered solution and ethanol solutions. Degradation was successfully mapped via thermomechanical means in dynamic mechanical analysis. Stability of the material was verified upon addition of EBC-1 enzyme. Initial values for the mechanical behavior of pure soya scaffolds at 100% density were too stiff and unable to match desired findings in literature. The achieved results showed promise as a proof of concept for this methodology and emphasizes the importance of standardization of results, ultimately leading to the conclusion that the requirements for these products will likely require complex modular, multi-step, multi-material systems at scale. Advisor: Michael P. Sealy
Cultured Meat (CM) is a growing field in cellular agriculture, driven by the environmental impact of conventional meat production, which contributes to climate change and occupies ≈70% of arable land. As demand for meat alternatives rises, research in this area expands. CM production relies on tissue engineering techniques, where a limited number of animal cells are cultured in vitro and processed to create meat‐like tissue comprising muscle and adipose components. Currently, CM is primarily produced on a small scale in pilot facilities. Producing a large cell mass based on suitable cell sources and bioreactors remains challenging. Advanced manufacturing methods and innovative materials are required to subsequently process this cell mass into CM products on a large scale. Consequently, CM is closely linked with biofabrication, a suite of technologies for precisely arranging cellular aggregates and cell‐material composites to construct specific structures, often using robotics. This review provides insights into contemporary biomedical biofabrication technologies, focusing on significant advancements in muscle and adipose tissue biofabrication for CM production. Novel materials for biofabricating CM are also discussed, emphasizing their edibility and incorporation of healthful components. Finally, initial studies on biofabricated CM are examined, addressing current limitations and future challenges for large‐scale production.
Chapter objectives:
• Illustrate the main steps involved in the use of 3D bioprinting for cell-based food products.
• Discuss the main technological challenges in 3D bioprinting for cultivated meat.
• Review the most relevant research in the tissue engineering field for bioprinting muscle and fat tissue.
• Recap the current state and interest in the growing industry of cultivated meat using 3D printing technology
The high population growth rate, massive animal food consumption, fast economic progress, and limited food resources could lead to a food crisis in the future. There is a huge requirement for dietary proteins including cultured meat is being progressed to fulfill the need for meat-derived proteins in the diet. However, production of cultured meat requires monitoring numerous bioprocess parameters. This review presents a comprehensive overview of various widely adopted techniques (optical, spectroscopic, electrochemical, capacitive, FETs, resistive, microscopy, and ultrasound) for monitoring physical, chemical, and biological parameters that can improve the bioprocess control in cultured meat. The methods, operating principle, merits/demerits, and the main open challenges are reviewed with the aim to support the readers in advancing knowledge on novel sensing systems for cultured meat applications.
Cultured meat is rapidly developing as an emerging meat production technology. Adipose tissue plays an essential role in the flavor of meat products. In this study, cultured fat was produced by cultured adipose-derived stem cells (ADSCs) based on collagen in vitro, with a 3D model. The research showed that ADSCs could attach to collagen hydrogels and differentiate into mature adipocytes. Texture analysis demonstrated that the springiness, cohesiveness, and resilience of cultured fat were consistent with porcine subcutaneous fat. Moreover, 28 volatile organic compounds (VOCs) were detected by headspace gas chromatography-ion mobility spectrometry. The relative contents of 17 VOCs in cultured fat were significantly higher than porcine subcutaneous fat and empty collagen hydrogels, and the relative contents of 5 VOCs in cultured fat were not significantly different from porcine subcutaneous fat. These findings assert the promising application of cultured fat in cultured meat production.
Cultured meat is an efficient, safe and sustainable meat production technology. Adipose-derived stem cell (ADSC) is a promising cell type for cultured meat. In vitro, obtaining numerous of ADSCs is a pivotal step for cultured meat. In this research, we demonstrated that the proliferation and adipogenic differentiation of ADSCs significantly decreased during serial passage. Then, senescence β-galactosidase (SA-β-gal) staining showed that the positive rate of P9 ADSCs was 7.74-fold than P3 ADSCs. Subsequently, RNA sequencing (RNA-seq) was performed for P3 and P9 ADSCs and found that PI3K-AKT pathway was up-regulated, but cell cycle and DNA repair pathway were down-regulated in P9 ADSCs. Then, N-Acetylcysteine (NAC) was added during long-term expansion and showed that NAC enhanced the ADSCs proliferation and maintained adipogenic differentiation. Finally, RNA-seq was performed for P9 ADSCs cultured with or without NAC and showed that NAC restored the cell cycle and DNA repair pathway in P9 ADSCs. These results highlighted that NAC was an excellent supplement for large-scale expansion of porcine ADSCs for cultured meat.
Cell culture media design is perhaps the most significant hurdle currently facing the commercialization of cultivated meat as an alternative source of dietary protein. Since media optimization for a specific culture system requires a significant amount of effort and investment, a major question remaining is whether media formulations can be easily shared across multiple production schemes for cells of different species and lineages. Here, we perform spent medium analysis to compare the specific nutrient utilization of primary embryonic chicken muscle precursor cells and fibroblasts to the murine C2C12 myoblast cell line. We demonstrate that these related cell types have significantly different nutrient utilization patterns collectively and on a per-cell basis, and that many components of conventional media do not appear to be depleted by the cells. Namely, glucose was not consumed as rapidly nor as completely by the chicken muscle precursors compared to other cells overall, and there were significant differences in specific consumption rates for several other key nutrients over the first day of culture. Ultimately, our results indicate that no one medium is likely ideal and cost effective to culture multiple cell types and that novel methods to streamline media optimization efforts will be important for the industry to develop. npj Science of Food (2022) 6:46 ; https://doi.
Cellular agriculture refers to a broad set of emerging technologies that draw upon research in genomics and synthetic biology to produce biological compounds. Much of the interest in cellular agriculture stems from its potential as a way of producing high-quality proteins and other nutrients with reduced environmental impact. Cellular agriculture techniques are rapidly nearing commercial scales of production, in part due to the application of knowledge and techniques produced through genomics research related to gene expression, editing, and genome-scale data analytics. However, much remains unknown and there is little rigorous evidence to test these assertions. This chapter applies the UN Sustainable Development Goals as a lens through which to examine protein production using cellular agriculture, to understand how it may contribute to the development of more sustainable and resilient food system. We examine two emerging approaches to cellular agriculture—cultured meat and fermentation-derived dairy—and explore both the complexity and knowledge gaps that need to be filled to ensure these tools are deployed to help create a more sustainable future for all. This chapter concludes by proposing an agenda for future research and policy development.
Cultured meat is an emerging technology that is friendly for the environment and animal welfare. As a novel food ingredient, cultured fat is essential for the flavor and nutrition of cultured meat. In this study, we purified adipose progenitor cell (APC) from freshly isolated porcine stromal vessel fraction (SVF) by fluorescence-activated cell sorting (FACS) and identified the transcriptome characteristics of APC by RNA sequencing (RNA-seq). The results showed that APC had characteristics of high-efficiency proliferation and adipogenic differentiation and was distinct from SVF cell in transcriptome profiles. Subsequently, APC was used to prepare cultured fat by 3D bioprinting and to evaluate the differences in fatty acid composition between cultured fat and porcine subcutaneous adipose tissue (pSAT). The results indicated that the fatty acid composition and content of cultured fat had a certain similarity with pSAT; specifically, the content of key monounsaturated fatty acid (MUFA) that create pork flavor in cultured fat, such as C18:1(n-12), C18:1(n-9) and C19:1(n-9)T, were close to that of pSAT. Therefore, this research indicated that APC is a promising candidate cell type for the production of cultured fat.
Cellular agriculture provides a potentially sustainable way of producing cultivated meat as an alternative protein source. In addition to muscle and connective tissue, fat is an important component of animal meat that contributes to taste, texture, tenderness, and nutritional profiles. However, while the biology of fat cells (adipocytes) is well studied, there is a lack of investigation on how adipocytes from agricultural species are isolated, produced, and incorporated as food constituents. Recently we compiled all protocols related to generation and analysis of adipose progenitors from bovine, porcine, chicken, other livestock and seafood species. In this review we summarize recent developments and present key scientific questions and challenges that need to be addressed in order to advance the biomanufacture of ‘alternative fat’.
Alternative proteins, such as cultivated meat, have recently attracted significant attention as novel and sustainable food. Fat tissue/cell is an important component of meat that makes organoleptic and nutritional contributions. Although adipocyte biology is relatively well investigated, there is limited focus on the specific techniques and strategies to produce cultivated fat from agricultural animals. In the assumed standard workflow, stem/progenitor cell lines are derived from tissues of animals, cultured for expansion, and differentiated into mature adipocytes. Here, we compile information from literature related to cell isolation, growth, differentiation, and analysis from bovine, porcine, chicken, other livestock, and seafood species. A diverse range of tissue sources, cell isolation methods, cell types, growth media, differentiation cocktails, and analytical methods for measuring adipogenic levels were used across species. Based on our analysis, we identify opportunities and challenges in advancing new technology era toward producing “alternative fat” that is suitable for human consumption.
Cultured meat is an emergent technology that cultivates cells in three-dimensional scaffolds to generate tissue for consumption. Fat makes an important contribution to the flavor and texture of traditional meat, but there are few reports on cultured fat. Here, we demonstrated the construction of cultured fat by inoculating porcine adipose-derived mesenchymal stem cell (ADSC) on peanut wire-drawing protein (PWP) scaffolds. First, we demonstrated that basic fibroblast growth factor (bFGF) promoted cell proliferation and maintained adipogenic differentiation ability. Then, we generated cultured fat and found that cultured fat decreased the texture of PWP scaffolds. Moreover, 43 volatile compounds were detected by headspace gas chromatography-ion mobility spectrometry (GC-IMS), of which 17 volatile compounds showed no significant differences between cultured fat and porcine subcutaneous adipose tissue (pSAT), which indicated that cultured fat and pSAT had certain similarities. Collectively, this research has great promise for improving the quality of cultured meat.
Animal cells are used in the manufacturing of complex biotherapeutic products since the 1980s. From its initial uses in biological research to its current importance in the biopharmaceutical industry, many types of culture media were developed: from serum-based media to serum-free to protein-free chemically defined media. The cultivation of animal cells economically has become the ultimate goal in the field of biomanufacturing. Serum serves as a source of amino acids, lipids, proteins and most importantly growth factors and hormones, which are essential for many cell types. However, the use of serum is unfavorable due to its high price tag, increased lot-to-lot variations and potential risk of microbial contamination. Efforts are progressively being made to replace serum with recombinant proteins such as growth factors, cytokines and hormones, as well as supplementation with lipids, vitamins, trace elements and hydrolysates. While hydrolysates are more complex, they provide a diverse source of nutrients to animal cells, with potential beneficial effects beyond the nutritional value. In this review, we discuss the use of hydrolysates in animal cell culture and briefly cover the composition of hydrolysates, mode of action and potential contaminants with some perspectives on its potential role in animal cell culture media formulations in the future.
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.
Self-amplifying RNA (saRNA) is a next-generation vaccine platform, but like all nucleic acids, requires a delivery vehicle to promote cellular uptake and protect the saRNA from degradation. To date, delivery platforms for saRNA have included lipid nanoparticles (LNP), polyplexes and cationic nanoemulsions; of these LNP are the most clinically advanced with the recent FDA approval of COVID-19 based-modified mRNA vaccines. While the effect of RNA on vaccine immunogenicity is well studied, the role of biomaterials in saRNA vaccine effectiveness is under investigated. Here, we tested saRNA formulated with either pABOL, a bioreducible polymer, or LNP, and characterized the protein expression and vaccine immunogenicity of both platforms. We observed that pABOL-formulated saRNA resulted in a higher magnitude of protein expression, but that the LNP formulations were overall more immunogenic. Furthermore, we observed that both the helper phospholipid and route of administration (intramuscular versus intranasal) of LNP impacted the vaccine immunogenicity of two model antigens (influenza hemagglutinin and SARS-CoV-2 spike protein). We observed that LNP administered intramuscularly, but not pABOL or LNP administered intranasally, resulted in increased acute interleukin-6 expression after vaccination. Overall, these results indicate that delivery systems and routes of administration may fulfill different delivery niches within the field of saRNA genetic medicines.
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 impact 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 life cycle assessment models include several variants for both plant and animal foods. The optimized 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 European‐grown legumes and pseudocereals are a valuable source for high‐quality protein foods, and despite being substantially processed they could help reduce the environmental impact of food consumption. © 2021 Society of Chemical Industry.
Abstract A new generation of plant-based meat alternatives—formulated to mimic the taste and nutritional composition of red meat—have attracted considerable consumer interest, research attention, and media coverage. This has raised questions of whether plant-based meat alternatives represent proper nutritional replacements to animal meat. The goal of our study was to use untargeted metabolomics to provide an in-depth comparison of the metabolite profiles a popular plant-based meat alternative (n = 18) and grass-fed ground beef (n = 18) matched for serving size (113 g) and fat content (14 g). Despite apparent similarities based on Nutrition Facts panels, our metabolomics analysis found that metabolite abundances between the plant-based meat alternative and grass-fed ground beef differed by 90% (171 out of 190 profiled metabolites; false discovery rate adjusted p
Bovine embryonic stem cells (bESCs) extend the lifespan of the transient pluripotent bovine inner cell mass in vitro. After years of research, derivation of stable bESCs was only recently reported. Although successful, bESC culture relies on complex culture conditions that require a custom-made base medium and mouse embryonic fibroblasts (MEF) feeders, limiting the widespread use of bESCs. We report here simplified bESC culture conditions based on replacing custom base medium with a commercially available alternative and eliminating the need for MEF feeders by using a chemically-defined substrate. bESC lines were cultured and derived using a base medium consisting of N2B27 supplements and 1% BSA (NBFR-bESCs). Newly derived bESC lines were easy to establish, simple to propagate and stable after long-term culture. These cells expressed pluripotency markers and actively proliferated for more than 35 passages while maintaining normal karyotype and the ability to differentiate into derivatives of all three germ lineages in embryoid bodies and teratomas. In addition, NBFR-bESCs grew for multiple passages in a feeder-free culture system based on vitronectin and Activin A medium supplementation while maintaining pluripotency. Simplified conditions will facilitate the use of bESCs for gene editing applications and pluripotency and lineage commitment studies.
Substitution of beef with alternative proteins is one practical trend taken by industry and consumers to reduce the negative impact of convenience products on the environment. Numerous products based on plant, insect and fungi proteins compete to replace beef burgers in an environmentally friendly and healthy way. At the same time, there is a lack of studies which assess different options from environmental impact perspective but also with consideration of production scales, recipes, nutritional values, and sensory properties. Therefore, the current study aimed to perform a holistic assessment (Life Cycle Assessment, sensory properties, and nutritional profile) of beef burgers in comparison to selected, alternative burgers, available on the market in Germany. The results indicated that alternative burgers based on plant, insect and mycoprotein biomass would be more environmentally friendly than beef burgers, but only a couple of them (insect-based and soy-based) have satisfying sensory or nutritional properties. Pea-based and mycoprotein-based burgers were perceived as average burgers from an environmental and sensorial perspective. The study demonstrated a sustainability differentiation system of meat analogs in convenience products using the multicriteria framework with inclusion of nutrient scoring, sensorial testing and Life Cycle Assessment.
Significance
Bovine embryonic stem cells and pluripotent stem cells hold the potential to substantially advance biotechnology and agriculture. We report the establishment of bovine expanded potential stem cells (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer (SCNT). EPSCs have broader developmental potential to generate embryonic and extraembryonic cell lineages. bEPSCs express high levels of pluripotency genes, propagate robustly in single cell passaging, are genetically stable, and permit efficient precise gene editing. They differentiate in vitro and in chimeras to both the embryonic and extraembryonic cell lineages. Importantly, genetically modified bEPSCs can be used as donors in SCNT or cloning.
Meat analogue products are considered to help consumers reducing their meat consumption. Their key success factor is their high similarity in sensory properties compared to meat. Even though the structure and texture characteristics of meat are well documented, dedicated methods used to analyse meat analogues are limited still. This review summarises texture and structure analysis methods of meat and meat analogues: mechanical testing; for example Texture Profile Analysis, spectroscopy; for example NMR and imaging techniques; for example hyperspectral imaging. Furthermore, the advantages and limitations of each texture and structure method are described. Finally, characterizations aspects specific to meat analogues are discussed. Promising methods for future research are described that have potential to get more insight into the fibers of meat analogues and the structure development during thermomechanical processing of meat analogues.
Industrial relevance
To be commercially successful for large groups of consumers, alternatives for meat should be highly similar to meat. That is why meat analogues should resemble existing meat in their texture. It is thus important to understand the texture properties with the help of relevant techniques, such as mechanical, spectroscopy and imaging techniques. In this manuscript, we describe promising texture methods for characterization of properties specific to meat analogues. The development of novel techniques to quantify meat analogue properties will stimulate the development of meat analogues that satisfy the values and wishes of consumers.
The development of soft tissue regeneration has recently gained importance due to safety concerns about artificial breast implants. Current autologous fat graft implantations can result in up to 90% of volume loss in long-term outcomes due to their limited revascularization. Adipose tissue has a highly vascularized structure which enables its proper homeostasis as well as its endocrine function. Mature adipocytes surrounded by a dense vascular network are the specific features required for efficient regeneration of the adipose tissue to perform host anastomosis after its implantation. Recently, bioprinting has been introduced as a promising solution to recreate in vitro this architecture in large-scale tissues. However, the in vitro induction of both the angiogenesis and adipogenesis differentiations from stem cells yields limited maturation states for these two pathways. To overcome these issues, we report a novel method for obtaining a fully vascularized adipose tissue reconstruction using supporting bath bioprinting. For the first time, directly isolated mature adipocytes encapsulated in a bioink containing physiological collagen microfibers (CMF) were bioprinted in a gellan gum supporting bath. These multilayered bioprinted tissues retained high viability even after 7 days of culture. Moreover, the functionality was also confirmed by the maintenance of fatty acid uptake from mature adipocytes. Therefore, this method of constructing fully functional adipose tissue regeneration holds promise for future clinical applications.
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.
Objectives
“Cultured food” is focused worldwide as “the third stage in meat production system” after hunting and livestock farming, and a sustainable food production system. In this study, we attempted to fabricate a three-dimensional (3-D) tissue by co-cultivation of animal cells with photosynthetic autotrophic microalgae so as to produce thicker and healthy cultured foods.
Results
Metabolism and damage of co-cultured tissues fabricated by microalgae, Chlorella vulgaris (C. vulgaris), and C2C12 cells were compared to monoculture tissues fabricated by C2C12 animal cells alone. Although the metabolism of monoculture tissue showed anaerobic respiration (ratio of lactate production to glucose consumption, LG ratio: 2.01 ± 0.15), that of the co-culture tissue partially changed to efficient aerobic respiration (LG ratio: 1.58 ± 0.14). In addition, the amount of ammonia in the culture media decreased markedly by co-cultivation. The release of lactate dehydrogenase from the thicker tissue was one-seventh in the co-cultivation, showing improved tissue damage. The co-cultivation with microalgae improved the culture condition of thicker tissues, resulting in the fabrication/maintenance of 200–400 µm-thickness tissues. The co-cultured tissue fabricated by microalgae and animal cells was not only rich in nutrients but also enabled thicker tissue fabrication without tissue damage as compared to tissue fabricated by animal cells alone.
Conclusions
This tissue fabrication system by co-culture of microalgae and animal cells will be a valuable tool for the production of thicker and healthy cultured food.
Current research of avian adipogenesis has been dependent on primary preadipocytes culture due to the lack of commercially available immortal preadipocyte cell lines in avian species. In addition to primary stromal vascular cells, primary chicken embryonic fibroblasts (CEF) were suggested as new in vitro models for adipogenesis study, because CEF can be differentiated into adipocytes by a combination of fatty acids and insulin (FI), or all-trans retinoic acid (atRA) alone in the media containing chicken serum (CS). However, there are decreases in differentiation of primary cells due to diverse population of cell types and low adipogenic potential of cells after passages. In the current study, adipogenic differentiation of DF-1 cells, immortal fibroblasts derived from an embryonic chicken, was tested with four different medium; 10% fetal bovine serum (FBS), 10% CS, 10% CS with FI, and 10% CS with FI and atRA. Lipid droplets stained with Oil-Red-O were not shown in DF-1 cells under 10% FBS, appeared with very small sizes under 10% CS, significantly increased under 10% CS with FI, and most significantly accumulated under 10% CS with FI and atRA. In addition, expressions of markers for adipogenesis (Znf423, C/ebpβ, Pparγ, and Fabp4), fatty acid uptake (CD36), triglyceride synthesis (Gpd1, Dgat2), and lipid droplet stabilization (Plin1) were significantly up-regulated by supplementation of 10% CS with FI and atRA. Morphological evidence for formation of lipid droplets and dramatic induction of adipogenic marker genes support the adipogenic potential of DF-1 cells, offering DF-1 cells as a new cell model to investigate various research involving avian adipogenesis.
Antibiotic resistance profiles of Escherichia coli were investigated in an intensive pig production system in the uMgungundlovu District, South Africa, using the ‘farm-to-fork’ approach. Four hundred seventeen (417) samples were collected from pig and pig products at different points (farm, transport, and abattoir). E. coli was isolated and enumerated using the Colilert® 18/Quanti-Tray® 2000 system. Ten isolates from each Quanti-tray were selected randomly and putatively identified on eosin methylene blue agar. Real-time PCR targeting the uidA gene was used to confirm isolates to the genus level. The Kirby–Bauer disc diffusion method was used to determine the isolates’ antibiotic susceptibility profiles against 20 antibiotics. A total of 1044 confirmed E. coli isolates were obtained across the three critical points in the food chain. Resistance was observed to all the antibiotics tested with the highest and lowest rates obtained against tetracycline (88.5%) and meropenem (0.2%), respectively. Resistance was also observed to chloramphenicol (71.4%), ampicillin (71.1%), trimethoprim-sulfamethoxazole (61.3%), amoxicillin-clavulanate (43.8%), cephalexin (34.3%), azithromycin (23.9%), nalidixic acid (22.1%), cefoxitin (21.1%), ceftriaxone (18.9%), ciprofloxacin (17.3%), cefotaxime (16.9%), gentamicin (15.5%), cefepime (13.8%), ceftazidime (9.8%), amikacin (3.4%), piperacillin-tazobactam (1.2%), tigecycline (0.9%), and imipenem (0.3%). Multidrug resistance (MDR) was observed in 71.2% of the resistant isolates with an overall multiple antibiotic resistance (MAR) index of 0.25, indicating exposure to high antibiotic use environments at the farm level. A high percentage of resistance was observed to growth promoters and antibiotics approved for veterinary medicine in South Africa. Of concern was resistance to critically important antibiotics for animal and human use and the watch and reserve categories of antibiotics. This could have adverse animal and human health consequences from a food safety perspective, necessitating efficient antibiotic stewardship and guidelines to streamline antibiotic use in the food-animal production chain.
This review will explore the four major pillars required for design and development of an saRNA vaccine: Antigen design, vector design, non-viral delivery systems, and manufacturing (both saRNA and lipid nanoparticles (LNP)). We report on the major innovations, preclinical and clinical data reported in the last five years and will discuss future prospects.
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.
Background
Human induced pluripotent stem cells (hiPSCs) hold enormous promise in accelerating breakthroughs in understanding human development, drug screening, disease modeling, and cell and gene therapies. Their potential, however, has been bottlenecked in a mostly laboratory setting due to bioprocess challenges in the scale-up of large quantities of high-quality cells for clinical and manufacturing purposes. While several studies have investigated the production of hiPSCs in bioreactors, the use of conventional horizontal-impeller, paddle, and rocking-wave mixing mechanisms have demonstrated unfavorable hydrodynamic environments for hiPSC growth and quality maintenance. This study focused on using computational fluid dynamics (CFD) modeling to aid in characterizing and optimizing the use of vertical-wheel bioreactors for hiPSC production.
Methods
The vertical-wheel bioreactor was modeled with CFD simulation software Fluent at agitation rates between 20 and 100 rpm. These models produced fluid flow patterns that mapped out a hydrodynamic environment to guide in the development of hiPSC inoculation and in-vessel aggregate dissociation protocols. The effect of single-cell inoculation on aggregate formation and growth was tested at select CFD-modeled agitation rates and feeding regimes in the vertical-wheel bioreactor. An in-vessel dissociation protocol was developed through the testing of various proteolytic enzymes and agitation exposure times.
Results
CFD modeling demonstrated the unique flow pattern and homogeneous distribution of hydrodynamic forces produced in the vertical-wheel bioreactor, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. We developed a scalable, single-cell inoculation protocol for the culture of hiPSCs as aggregates in vertical-wheel bioreactors, achieving over 30-fold expansion in 6 days without sacrificing cell quality. We have also provided the first published protocol for in-vessel hiPSC aggregate dissociation, permitting the entire bioreactor volume to be harvested into single cells for serial passaging into larger scale reactors. Importantly, the cells harvested and re-inoculated into scaled-up vertical-wheel bioreactors not only maintained consistent growth kinetics, they maintained a normal karyotype and pluripotent characterization and function.
Conclusions
Taken together, these protocols provide a feasible solution for the culture of high-quality hiPSCs at a clinical and manufacturing scale by overcoming some of the major documented bioprocess bottlenecks.
Tissue engineering has attracted significant attention since the 1980s, and the applications of tissue engineering have been expanding. To produce a cell-dense tissue, cell sheet technology has been studied as a promising strategy. Fundamental techniques involving tissue engineering are mainly introduced in this review. First, the technologies to fabricate a cell sheet were reviewed. Although temperature-responsive polymer-based technique was a trigger to establish and spread cell sheet technology, other methodologies for cell sheet fabrication have also been reported. Second, the methods to improve the function of the cell sheet were investigated. Adding electrical and mechanical stimulation on muscle-type cells, building 3D structures, and co-culturing with other cell species can be possible strategies for imitating the physiological situation under in vitro conditions, resulting in improved functions. Finally, culture methods to promote vasculogenesis in the layered cell sheets were introduced with in vivo, ex vivo, and in vitro bioreactors. We believe the present review that shows and compares the fundamental technologies and recent advances for cell-sheet-based tissue engineering should promote further development of tissue engineering. The development of cell sheet technology should promote many bioengineering applications.
Interest in animal cell-based meat (ACBM) or laboratory-grown meat has been increasing; however, the economic viability of these potential products has not been thoroughly vetted. Recent studies suggest monoclonal antibody production technology can be adapted for the industrialization of ACBM production. This study provides a scenario-based assessment of the projected cost per kilogram of ACBM produced in the United States based on cellular metabolic requirements and process/chemical engineering conventions. A sensitivity analysis of the model identified the nine most influential cost factors for ACBM production out of 67 initial parameters. The results indicate that technological performance will need to approach technical limits for ACBM to achieve profitably as a commodity. However, the model also suggests that low-volume high-value specialty products could be viable based on current technology.
Demand for animal protein is rising globally and has been facilitated by the expansion of intensive farming. However, intensive animal production relies on the regular use of antimicrobials to maintain health and productivity on farms. The routine use of antimicrobials fuels the development of antimicrobial resistance, a growing threat for the health of humans and animals. Monitoring global trends in antimicrobial use is essential to track progress associated with antimicrobial stewardship efforts across regions. We collected antimicrobial sales data for chicken, cattle, and pig systems in 41 countries in 2017 and projected global antimicrobial consumption from 2017 to 2030. We used multivariate regression models and estimated global antimicrobial sales in 2017 at 93,309 tonnes (95% CI: 64,443, 149,886). Globally, sales are expected to rise by 11.5% in 2030 to 104,079 tonnes (95% CI: 69,062, 172,711). All continents are expected to increase their antimicrobial use. Our results show lower global antimicrobial sales in 2030 compared to previous estimates, owing to recent reports of decrease in antimicrobial use, in particular in China, the world’s largest consumer. Countries exporting a large proportion of their production are more likely to report their antimicrobial sales data than countries with small export markets.
Adipose-derived stem cells (ADSCs) can maintain self-renewal and enhanced multidifferentiation potential through the release of a variety of paracrine factors and extracellular vesicles, allowing them to repair damaged organs and tissues. Consequently, considerable attention has increasingly been paid to their application in tissue engineering and organ regeneration. Here, we provide a comprehensive overview of the current status of ADSC preparation, including harvesting, isolation, and identification. The advances in preclinical and clinical evidence-based ADSC therapy for bone, cartilage, myocardium, liver, and nervous system regeneration as well as skin wound healing are also summarized. Notably, the perspectives, potential challenges, and future directions for ADSC-related researches are discussed. We hope that this review can provide comprehensive and standardized guidelines for the safe and effective application of ADSCs to achieve predictable and desired therapeutic effects.
Antimicrobial resistance leads to failure of clinical antimicrobial therapy, and has raised urgent global public health concern. Humans can acquire antimicrobial resistance from drugs through the food chain or the environment (contaminated water, air, soil, or manure). While antimicrobials have been regular supplements in animal feed that maintain health and improve productivity of livestock, their over-use in feeding forage has led to a rise in antibacterial resistance. This review summarizes the current use of antimicrobials in livestock, the harmful effects of antimicrobial resistance, and the comprehensive combat measures.
Great importance is being given to the impact our food supply chain and consumers' food habits are having on the environment, human health, and animal welfare. One of the latest developments aiming at positively changing the food ecosystem is represented by cultured meat. This form of cellular agriculture has the objective to generate slaughter-free meat products starting from the cultivation of few cells harvested from the animal tissue of interest. As a consequence, a large number of cells has to be generated at a reasonable cost. Just to give an idea of the scale, there were billions of cells just in a bite of the first cultured-meat burger. Thus, one of the major challenges faced by the scientists involved in this new ambitious and fascinating field, is how to efficiently scale-up cell manufacture. Considering the great potential presented by cultured meat, audiences from different backgrounds are very interested in this topic and eager to be informed of the challenges and possible solutions in this area. In light of this, we will provide an overview of the main existing bioprocessing technologies used to scale-up adherent cells at a small and large scale. Thus, giving a brief technical description of these bioprocesses, with the main associated advantages and disadvantages. Moreover, we will introduce an alternative solution we believe has the potential to revolutionize the way adherent cells are grown, helping cultured meat become a reality.
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.
The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.
Advanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.
Abstract The development of the induced pluripotent stem cells (iPSCs) technology has revolutionized the world on the establishment of pluripotent stem cells (PSCs) across a great variety of animal species. Generation of iPSCs from domesticated animals would provide unrestricted cell resources for the study of embryonic development and cell differentiation of these species, for screening and establishing desired traits for sustainable agricultural production, and as veterinary and preclinical therapeutic tools for animal and human diseases. Induced PSCs from domesticated animals thus harbor enormous scientific, economical, and societal values. Although much progress has been made toward the generation of PSCs from these species, major obstacles remain precluding the exclamation of the establishment of bona fide iPSCs. The most prominent of them remain the inability of these cells to silence exogenous reprogramming factors, the obvious reliance on exogenous factors for their self-renewal, and the restricted development potential in vivo. In this review, we summarize the history and current progress in domestic farm animal iPSC generation, with a focus on swine, ruminants (cattle, ovine, and caprine), horses, and avian species (quails and chickens). We also discuss the problems associated with the farm animal iPSCs and potential future directions toward the complete reprogramming of somatic cells from farm animals.
Background:
In China, although buffaloes are abundant, beef is mainly obtained from cattle, and this preference is mainly attributed to the low intramuscular fat (IMF) content of buffalo. Genetic factors are an important driver that affects IMF deposition.
Results:
To reveal the intrinsic factors responsible for the low IMF content of buffalo, mRNA expression patterns in muscle and adipose tissue between buffalo and cattle were characterized by RNA sequencing analysis. The IMF content in Nanyang cattle was higher than that in Xinyang buffalo. A total of 1566 mRNAs expressed in adipose tissue showed differential expression between the longissimus dorsi muscles of buffalo and cattle. Functional annotation suggested a difference in the glycolysis/gluconeogenesis pathway between the two species. The results of RT-qPCR analysis and gain-of-function experiments confirmed the positive association between the IMF content and phosphoenolpyruvate carboxykinase 1 (PCK1) expression in buffalo. In both mouse C2C12 cells and cultured bovine myocytes, the activity of the PCK1 promoter in buffalo is lower than that in cattle. However, in mouse 3T3-L1 adipocytes and cultured bovine adipocytes, the activity of PCK1 in buffalo promoter is higher than that in cattle.
Conclusions:
These results indicate the important role of PCK1 in buffalo IMF deposition and illustrate the differences between buffalo and cattle promoter activity that drive PCK1 expression. This research helps to establish a foundation for further studies investigating IMF deposition in buffalo.
Policy Issues in Genetically Modified Crops: A Global Perspective contains both theoretical and empirical evidence of a broad range of aspects of GM crop policies throughout the world. Emphasizing world agriculture production and ethics of GM crops, the book balances insights into the various discussions around the use of GM crops including soil health, effects on animals, environmental sustainability impact, and ethical issues. The book presents aspects of GM crop policies and prevailing controversies throughout the world, in 5 sections containing 23 chapters. Beginning with the discussion of the policies related to GM crops, the book dives deep into issues related to food insecurity, agricultural sustainability, food safety, and environmental risks. Section 5 also captures the recent advances in agricultural biotechnology encompassing research trends, the nano-biotech approach to plant genetic engineering, and other transformation techniques in crop development.
Perilipin 1 (PLIN1) is a protein encoded by the PLIN1 gene in eukaryotes. PLIN1 is a member of the PAT protein family, a family of proteins related to lipid droplet (LD) surface proteins. PLIN1 phosphorylation plays a vital role during fat metabolism of adipose tissue lipolysis and fat storage in adipocytes. However, to further explore the regulation of the PLIN1 gene on the proliferation, differentiation and lipid metabolism of bovine adipocytes. In this study, the mRNA expression of PLIN1, at day six, was the highest during bovine adipocyte differentiation. Moreover, PLIN1 can promote the proliferation and differentiation of preadipocytes in cattle. On the sixth day, after transfection with, and overexpression of, the PLIN1 gene in bovine preadipocytes via adenovirus, cell samples were collected, and transcriptome sequencing was performed. A total of 1923 differentially expressed genes were detected. Through GO and KEGG pathway analysis, the differentially expressed genes were established to be mainly enriched in the AMPK, Wnt, and PPAR signaling pathways related to fat proliferation and differentiation. In conclusion, at the transcriptional level, PLIN1 plays an important role in regulating fat proliferation and metabolism. Additionally, the sequencing results screened new differentially expressed genes related to fat metabolism, providing theoretical support for molecular breeding of Qinchuan beef cattle.
Adipose tissue is contemplated as a dynamic organ that plays key roles in the human body. Adipogenesis is the process by which adipocytes develop from adipose-derived stem cells to form the adipose tissue. Adipose-derived stem cells' differentiation serves well beyond the simple goal of producing new adipocytes. Indeed, with the current immense biotechnological advances, the most critical role of adipose-derived stem cells remains their tremendous potential in the field of regenerative medicine. This review focuses on examining the physiological importance of adipogenesis, the current approaches that are employed to model this tightly controlled phenomenon, and the crucial role of adipogenesis in elucidating the pathophysiology and potential treatment modalities of human diseases. The future of adipogenesis is centered around its crucial role in regenerative and personalized medicine.
In vitro adipose tissue models can be used to provide insight into fundamental aspects of adipose physiology. These systems may serve as replacements for animal models, which are often poor predictors of obesity and metabolic diseases in humans. Adipose tissue consists of a rich vasculature that is essential to its function. However, the study of endothelial cell-adipocyte interactions has been challenging due to differences in culture conditions required for the survival and function of each cell type. To address this issue, we performed an extensive evaluation of the cell culture media composition to identify the conditions optimal for the co-culture of endothelial cells and adipocytes. The effects of individual media factors on cell survival, proliferation, and differentiation were systematically explored. Several media factors were determined to disrupt the co-culture system. Optimized culture conditions were identified and used to generate a vascularized human adipose microtissue. An interconnected vascular network was established within an adipose micro-tissue, and the networks were anastomosed with perfused channels to form a functional network. In conclusion, media conditions were identified that enabled endothelial cell-adipocyte co-culture and were used to support the formation of a vascularized adipose tissue within a microfluidic device.
The global cell culture market is experiencing significant growth due to the rapid advancement in antibody-based and cell-based therapies. Both rely on the capacity of different living factories, namely prokaryotic and eukaryotic cells, plants or animals for reliable and mass production. The ability to improve production yield is of important concern. Among many strategies pursued, optimizing the complex nutritional requirements for cell growth and protein production has been frequently performed via culture media component titration and serum replacement. The addition of specific ingredients into culture media to modulate host cells’ metabolism has also recently been explored. In this study, we examined the use of extracted bioactive components of the microalgae Chlorella vulgaris, termed chlorella growth factor (CGF), as a cell culture additive for serum replacement and protein expression induction. We first established a chemical fingerprint of CGF using ultraviolet-visible spectroscopy and liquid chromatography-mass spectrometry and evaluated its ability to enhance cell proliferation in mammalian host cells. CGF successfully promoted the growth of Chinese hamster ovary (CHO) and mesenchymal stem cells (MSC), in both 2D and 3D cell cultures under reduced serum conditions for up to 21 days. In addition, CGF preserved cell functions as evident by an increase in protein expression in CHO cells and the maintenance of stem cell phenotype in MSC. Taken together, our results suggest that CGF is a viable culture media additive and growth matrix component, with wide ranging applications in biotechnology and tissue engineering.
The topic of plant‐based meat alternatives (PBMAs) has been discussed for several decades, but it has only recently become one of the hottest topics in the food and research communities. With the purpose of investigating the current situation of scientific research on PBMA and determining future research opportunities, the driving forces for PBMA development, a brief history of its progression, key technologies required for production, and the resulting consumer attitudes are summarized. Environmental, human health, and animal welfare concerns are the main factors that have driven the development of PBMA. Although its history can trace back to ancient Asian civilizations, the first generation of PBMA originated in 1960s and a new generation of PBMA designed for carnivore was developed in recently years. Structuring methods such as extrusion and shear cell techniques have been widely studied, but improvements toward the overall appearance and flavor, biological and chemical safety control, as well as the selection of protein sources are also very important for PBMA production. The consumer acceptance of PBMA remains unsatisfactory but is continually improving. Based on those knowledge, future research opportunities include developing more effective strategies for consumer education, providing more scientific evidence for the health properties of PBMA, finding more suitable protein sources to improve the quality of the final products, improving the appearance and flavor, further examining and securing the chemical safety, exploring the structure formation mechanism during the extraction or shearing processes, and developing methods and standards for a quality evaluation of PBMA.
The objective was to examine the effects of Taiwanese green propolis (TGP) ethanol extract on adipocyte differentiation and lipolysis. TGP ethanol extract significantly enhanced the differentiation of murine mesenchymal stem cells into adipocytes, and this was accompanied by an increase in intracellular triglyceride content and adiponectin levels. TGP ethanol extract significantly increased the expression of adipogenic transcription factor-associated genes in murine mesenchymal stem cells after hormonal induction of adipogenesis. TGP ethanol extract was able to alleviate norepinephrine-induced lipolysis in differentiated adipocytes, which was accompanied by a decrease in lipolytic gene expression. Besides, TGP ethanol extract could reduce the tumor necrosis factor-α (TNF-α)-mediated inhibition of adiponectin gene expression and secretion during adipogenic differentiation of mesenchymal stem cells and in differentiated adipocytes. These results demonstrate that TGP ethanol extract enhances adipocyte differentiation and is capable of reversing the inhibitory effects of TNF-α on adipocyte differentiation and adiponectin expression in differentiated adipocytes.
Complex interaction between genetics, epigenetics, environment, and nutrition affect the physiological activities of adipose tissues and their dysfunctions, which lead to several metabolic diseases including obesity or type 2 diabetes. Here, adipogenesis appears to be a process characterized by an intricate network that involves many transcription factors and long noncoding RNAs (lncRNAs) that regulate gene expression. LncRNAs are being investigated to determine their contribution to adipose tissue development and function. LncRNAs possess multiple cellular functions, and they regulate chromatin remodeling, along with transcriptional and post-transcriptional events; in this way, they affect gene expression. New investigations have demonstrated the pivotal role of these molecules in modulating white and brown/beige adipogenic tissue development and activity. This review aims to provide an update on the role of lncRNAs in adipogenesis and adipose tissue function to promote identification of new drug targets for treating obesity and related metabolic diseases.
Overcoming pandemics, such as the current Covid‐19 outbreak, requires the manufacture of several billion doses of vaccines within months. This is an extremely challenging task given the constraints in small‐scale manufacturing for clinical trials, clinical testing timelines involving multiple phases and large‐scale drug substance and drug product manufacturing. To tackle these challenges, regulatory processes are fast‐tracked, and rapid‐response manufacturing platform technologies are used. Here, we evaluate the current progress, challenges ahead and potential solutions for providing vaccines for pandemic response at an unprecedented scale and rate. Emerging rapid‐response vaccine platform technologies, especially RNA platforms, offer a high productivity estimated at over 1 billion doses per year with a small manufacturing footprint and low capital cost facilities. The self‐amplifying RNA (saRNA) drug product cost is estimated at below 1 USD/dose. These manufacturing processes and facilities can be decentralized to facilitate production, distribution, but also raw material supply. The RNA platform technology can be complemented by an a priori Quality by Design analysis aided by computational modeling in order to assure product quality and further speed up the regulatory approval processes when these platforms are used for epidemic or pandemic response in the future.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.