Cultured Meat Production Research Articles

Effect of gelatin concentration and freezing temperature on porous scaffolds for cultured meat

Cultured meat is an emerging green alternative for livestock and poultry meat, and obtained from cell cultured in laboratories or factories. Three-dimensional (3D) scaffolds are widely used in cultured meat production by simulating extracellular matrix to support cell culture in vitro. Currently, the scaffolds used for the fabrication of cultured meat lack the research on porous structures, which limit cell growth and differentiation and simulation of distinct textural characteristics of traditional meat. Therefore, this study investigated the efficacy of food-grade scaffolds prepared with different gelatin concentrations and freezing temperatures to support cell attachment and growth. The result showed 6% gelatin scaffolds prepared at −80 °C or liquid nitrogen demonstrated suitable porous structures and promoted the proliferation and differentiation of muscle stem cells (MSCs) to improve the expression of genes and proteins (myogenin and myosin) related to myogenic progression. The scaffolds also created 3D-engineered cultured meat tissues that closely mimic the mechanical properties of traditional meat. This study provides the theoretical baseline for developing porous scaffolds for efficient cultured meat production in the meat substitute industry.

Global transcriptome profiles provide insights into muscle cell development and differentiation on microstructured marine biopolymer scaffolds for cultured meat production

Biomaterial scaffolds play a pivotal role in the advancement of cultured meat technology, facilitating essential processes like cell attachment, growth, specialization, and alignment. Currently, there exists limited knowledge concerning the creation of consumable scaffolds tailored for cultured meat applications. This investigation aimed to produce edible scaffolds featuring both smooth and patterned surfaces, utilizing biomaterials such as salmon gelatin, alginate, agarose and glycerol, pertinent to cultured meat and adhering to food safety protocols. The primary objective of this research was to uncover variations in transcriptomes profiles between flat and microstructured edible scaffolds fabricated from marine-derived biopolymers, leveraging high-throughput sequencing techniques. Expression analysis revealed noteworthy disparities in transcriptome profiles when comparing the flat and microstructured scaffold configurations against a control condition. Employing gene functional enrichment analysis for the microstructured versus flat scaffold conditions yielded substantial enrichment ratios, highlighting pertinent gene modules linked to the development of skeletal muscle. Notable functional aspects included filament sliding, muscle contraction, and the organization of sarcomeres. By shedding light on these intricate processes, this study offers insights into the fundamental mechanisms underpinning the generation of muscle-specific cultured meat.

Laminin as a Key Extracellular Matrix for Proliferation, Differentiation, and Maturation of Porcine Muscle Stem Cell Cultivation.

Extracellular matrix (ECM) proteins play a crucial role in culturing muscle stem cells (MuSCs). However, there is a lack of extensive research on how each of these proteins influences proliferation and differentiation of MuSCs from livestock animals. Therefore, we investigated the effects of various ECM coatings-collagen, fibronectin, gelatin, and laminin-on the proliferation, differentiation, and maturation of porcine MuSCs. Porcine MuSCs, isolated from 14-day-old Berkshire piglets, were cultured on ECM-coated plates, undergoing three days of proliferation followed by three days of differentiation. MuSCs on laminin showed higher proliferation rate than others (p<0.05). There was no significant difference in the mRNA expression levels of PAX7, MYF5, and MYOD among MuSCs on laminin, collagen, and fibronectin (p>0.05). During the differentiation period, MuSCs cultured on laminin exhibited a significantly higher differentiation rate, resulting in thicker myotubes compared to those on other ECMs (p<0.05). Also, MuSCs on laminin showed higher expression of mRNA related with maturated muscle fiber such as MYH1 and MYH4 corresponding to muscle fiber type IIx and muscle fiber type IIb, respectively, compared with MuSCs on other ECM coatings (p<0.05). In summary, our comparison of ECMs revealed that laminin significantly enhances MuSC proliferation and differentiation, outperforming other ECMs. Specifically, muscle fibers cultured on laminin exhibited a more mature phenotype. These findings underscore laminin's potential to advance in vitro muscle research and cultured meat production, highlighting its role in supporting rapid cell proliferation, higher differentiation rates, and the development of mature muscle fibers.

Industrial Research and Development on the Production Process and Quality of Cultured Meat Hold Significant Value: A Review.

Cultured meat has been gaining popularity as a solution to the increasing problem of food insecurity. Although research on cultured meat started later compared to other alternative meats, the industry is growing rapidly every year, with developed products evaluated as being most similar to conventional meat. Studies on cultured meat production techniques, such as culturing new animal cells and developing medium sera and scaffolds, are being conducted intensively and diversely. However, active in-depth research on the quality characteristics of cultured meat, including studies on the sensory and storage properties that directly influence consumer preferences, is still lacking. Additionally, studies on the combination or ratio of fat cells to muscle cells and on the improvement of microbiota, protein degradation, and fatty acid degradation remain to be conducted. By actively investigating these research topics, we aim to verify the quality and safety of cultured meats, ultimately improving the consumer preference for cultured meat products.

Crusting-fabricated three-dimensional soy-based scaffolds for cultured meat production: A preliminary study

Crusting has been developed as a non-chemical and non-machine intensive scaffold fabrication method. This method is based on the self-assembling ability of soy biomolecules, allowing the fabrication of a three-dimensional network for cell growth. Preliminary characterization revealed differences in pore size, water absorption, and degradation between pure soy-based scaffold (Y2R) and with added glycerol (Y2G). The Fourier-transform infrared spectrum absorbance peaks of functional groups related to proteins, carbohydrates, and lipids hinted the integration of soy biomolecules potentially via the Maillard reaction, as supported by the visible browning of the scaffold surface. Microscopic images revealed aligned myotubes in both scaffolds, with Y2G myotubes having greater proximity after 72 h of proliferation. Both spontaneous and electro-stimulated contractions were recorded as early as 72 h in proliferation medium. Crusting-fabricated soy-based scaffolds can further be explored for its application in cultured meat production.

Cultivation of Bovine Mesenchymal Stem Cells on Plant-Based Scaffolds in a Macrofluidic Single-Use Bioreactor for Cultured Meat.

Reducing production costs, known as scaling, is a significant obstacle in the advancement of cultivated meat. The cultivation process hinges on several key components, e.g., cells, media, scaffolds, and bioreactors. This study demonstrates an innovative approach, departing from traditional stainless steel or glass bioreactors, by integrating food-grade plant-based scaffolds and thermoplastic film bioreactors. While thermoplastic films are commonly used for constructing fluidic systems, conventional welding methods are cost-prohibitive and lack rapid prototyping capabilities, thus inflating research and development expenses. The developed laser welding technique facilitates contamination-free and leakproof sealing of polyethylene films, enabling the efficient fabrication of macrofluidic systems with various designs and dimensions. By incorporating food-grade plant-based scaffolds, such as rice seeded with bovine mesenchymal stem cells, into these bioreactors, this study demonstrates sterile cell proliferation on scaffolds within macrofluidic systems. This approach not only reduces bioreactor prototyping and construction costs but also addresses the need for scalable solutions in both research and industrial settings. Integrating single-use bioreactors with minimal shear forces and incorporating macro carriers such as puffed rice may further enhance biomass production in a scaled-out model. The use of food-grade plant-based scaffolds aligns with sustainable practices in tissue engineering and cultured-meat production, emphasizing its suitability for diverse applications.

Production of Plant-Based, Film-Type Scaffolds Using Alginate and Corn Starch for the Culture of Bovine Myoblasts.

Natural scaffolds have been the cornerstone of tissue engineering for decades, providing ideal environments for cell growth within extracellular matrices. Previous studies have favored animal-derived materials, including collagen, gelatin, and laminin, owing to their superior effects in promoting cell attachment, proliferation, and differentiation compared to non-animal scaffolds, and used immortalized cell lines. However, for cultured meat production, non-animal-derived scaffolds with edible cells are preferred. Our study represents the first research to describe plant-derived, film-type scaffolds to overcome limitations associated with previously reported thick, gel-type scaffolds completely devoid of animal-derived materials. This approach has been employed to address the difficulties of fostering bovine muscle cell survival, migration, and differentiation in three-dimensional co-cultures. Primary bovine myoblasts from Bos Taurus Coreanae were harvested and seeded on alginate (Algi) or corn-derived alginate (AlgiC) scaffolds. Scaffold functionalities, including biocompatibility and the promotion of cell proliferation and differentiation, were evaluated using cell viability assays, immunofluorescence staining, and reverse transcription-quantitative polymerase chain reaction. Our results reveal a statistically significant 71.7% decrease in production time using film-type scaffolds relative to that for gel-type scaffolds, which can be maintained for up to 7 days. Film-type scaffolds enhanced initial cell attachment owing to their flatness and thinness relative to gel-type scaffolds. Algi and AlgiC film-type scaffolds both demonstrated low cytotoxicity over seven days of cell culture. Our findings indicated that PAX7 expression increased 16.5-fold in alginate scaffolds and 22.8-fold in AlgiC from day 1 to day 3. Moreover, at the differentiation stage on day 7, MHC expression was elevated 41.8-fold (Algi) and 32.7-fold (AlgiC), providing initial confirmation of the differentiation potential of bovine muscle cells. These findings suggest that both Algi and AlgiC film scaffolds are advantageous for cultured meat production.

Generation of Chicken Contractile Skeletal Muscle Structure Using Decellularized Plant Scaffolds.

Cultured meat is a meat analogue produced by in vitro cell culture, which can replace the conventional animal production system. Tissue engineering using myogenic cells and biomaterials is a core technology for cultured meat production. In this study, we provide an efficient and economical method to produce skeletal muscle tissue-like structures by culturing chicken myoblasts in a fetal bovine serum (FBS)-free medium and plant-derived scaffolds. An FBS-free medium supplemented with 10% horse serum (HS) and 5% chick embryo extract (CEE) was suitable for the proliferation and differentiation of chicken myoblasts. Decellularized celery scaffolds (Decelery), manufactured using 1% sodium dodecyl sulfate (SDS), were nontoxic to cells and supported myoblast proliferation and differentiation. Decelery could support the 3D culture of chicken myoblasts, which could adhere and coagulate to the surface of the Decelery and form MYH1E+ and F-actin+ myotubes. After 2 weeks of culture on Decelery, fully grown myoblasts completely covered the surface of the scaffolds and formed fiber-like myotube structures. They further differentiated to form spontaneously contracting myofiber-like myotubes on the scaffold surface, indicating that the Decelery scaffold system could support the formation of a functional mature myofiber structure. In addition, as the spontaneously contracting myofibers did not detach from the surface of the Decelery, the Decelery system is a suitable biomaterial for the long-term culture and maintenance of the myofiber structures.

Low-cost protein extracts and hydrolysates from plant-based agro-industrial waste: Inputs of interest for cultured meat

The main challenge in large-scale cultured meat production is replacing fetal bovine serum (FBS), an expensive, animal-derived component in the cell culture medium. This study explored the use of plant-based agro-industrial waste as an economical and nutritionally beneficial alternative to FBS. Soybean and peanut bran were processed to obtain extracts and protein hydrolysates rich in proteins, low molecular weight peptides, and amino acids. The results confirmed the presence of essential nutritional factors conducive to the growth and viability of animal cells in vitro. Protein extracts formed mainly by globulins and albumins resemble the composition of FBS. The hydrolysates contained a protein content similar to FBS and were particularly abundant in leucine and proline. The molecular weight distribution of the peptides indicated a high degree of hydrolysis. Cost analysis revealed that utilizing plant waste for protein inputs could be a favorable approach to reducing cultured meat production costs.

Revolutionizing meat processing: a nexus of technological advancements, sustainability, and cultured meat evolution

This thorough analysis traverses the ever-changing terrain of meat processing, revealing a story intertwined with technological innovations, environmentally friendly methods, and the revolutionary rise of cultured meat production. The amalgamation of sustainable polymers, sophisticated composite coatings, and potent antioxidant agents strikingly demonstrates the sector's dedication to novelty and ecological accountability. Diagrammatic depictions outline tactical approaches to lowering carbon emissions, highlighting the circular economy in terms of material recycling and the creative recycling of agricultural and food waste into environmentally acceptable packaging. Modern meat processing techniques, automation, and smart technology are all explored, emphasising waste minimization, energy efficiency, and sustainable practices.In terms of the future, the assessment offers a peek at how biotechnological developments and uses of nanotechnology will combine to transform how meat is produced. The integration of precision biotechnology, ethical concerns, and sustainability ushers a new era of responsible and creative food production, positioning the meat processing sector as a pathfinder in addressing consumer needs

Regulation of myogenic differentiation of myoblasts by groove structure for the design of cell culture meat scaffolds

As a meat substitute, cell cultured meat aims to simulate the composition of real meat to provide nutrient substance. The myotube formation efficiency of myoblasts in vitro is the key for simulating the structure and composition of muscle tissue. Therefore, this study is aimed to explore the most effective groove widths for myoblasts fusion and related molecular mechanism. The results of immunofluorescence showed that the grooves with a width range of 50 μm–1000 μm were beneficial to cell arrangement compared to flat structure. At the same time, the expression of myogenin (Myog) and myosin heavy chain 2 (Myh2) up-regulated. However, the cell fusion index of myoblasts was higher in grooves with a width of 100 μm, 200 μm and 300 μm. Furthermore, Cytochalasin D disrupted intracellular stress fibers and myotube formation. However, there were still many myotubes formed in the 200 μm groove with the addition of Cytochalasin D. The expression of fusion related genes was higher than the flat group. Moreover, incorporating groove structure into scaffolds of soybean protein isolate and pea protein isolate could enhance cell adhesion, activity and myogenic differentiation of myoblasts. This could provide ideas for scaffolds design of cultured meat products using groove structure.

Methods to Isolate Muscle Stem Cells for Cell-Based Cultured Meat Production: A Review.

Cultured meat production relies on various cell types, including muscle stem cells (MuSCs), embryonic stem cell lines, induced pluripotent cell lines, and naturally immortalized cell lines. MuSCs possess superior muscle differentiation capabilities compared to the other three cell lines, making them key for cultured meat development. Therefore, to produce cultured meat using MuSCs, they must first be effectively separated from muscles. At present, the methods used to isolate MuSCs from muscles include (1) the pre-plating method, using the ability of cells to adhere differently, which is a biological characteristic of MuSCs; (2) the density gradient centrifugation method, using the intrinsic density difference of cells, which is a physical characteristic of MuSCs; and (3) fluorescence- and magnetic-activated cell sorting methods, using the surface marker protein on the cell surface of MuSCs, which is a molecular characteristic of MuSCs. Further efficient and valuable methods for separating MuSCs are expected to be required as the cell-based cultured meat industry develops. Thus, we take a closer look at the four methods currently in use and discuss future development directions in this review.

Engineering Considerations on Large-Scale Cultured Meat Production.

In recent decades, cultured meat has received considerable interest as a sustainable alternative to traditional meat products, showing promise for addressing the inherent problems associated with conventional meat production. However, current limitations on the scalability of production and extremely high production costs have prevented their widespread adoption. Therefore, it is important to develop novel engineering strategies to overcome the current limitations in large-scale cultured meat production. Such engineering considerations have the potential for advancements in cultured meat production by providing innovative and effective solutions to the prevailing challenges. In this review, we discuss how engineering strategies have been utilized to advance cultured meat technology by categorizing the production processes of cultured meat into three distinct steps: (1) cell preparation; (2) cultured meat fabrication; and (3) cultured meat maturation. For each step, we provide a comprehensive discussion of the recent progress and its implications. In particular, we focused on the engineering considerations involved in each step of cultured meat production, with specific emphasis on large-scale production.

Metabolomic changes in culture media with varying passage numbers of pig muscle stem cell culture for cultured meat production

Selecting the primary cells in an optimal state for cultured meat production is a crucial challenge in commercializing cultured meat. We investigated the metabolomic changes in culture media according to passage numbers for indirectly assessing the state of primary cells. Pig skeletal muscle stem cells (PSCs) harvested from the biceps femoris muscles of 7-d-old crossbred pigs (Landrace × Yorkshire × Duroc, LYD) were used for cell characterization. Fresh media (FM) and spent media (SM) of PSCs during passages 1 to 3 in vitro culture were prepared for metabolomics analysis. SM was collected on the third day of proliferation for each passage of PSCs. Cell characterization analysis revealed that the proliferation rate was highest at passage 2; however, a significant loss of expression of myogenic marker genes was observed at passage 3. Based on metabolomic profiles of culture media, FM and SM groups (SM1, SM2, and SM3) were clearly separated by partial least squares-discriminant analysis. A total of seven differentially abundant metabolites (DAMs) were identified from FM and SM for each passage, based on the following criteria: P < 0.05, fold change > 1.5 or < 0.66, and a variable importance in projection score > 1.5. All seven DAMs and their interconnected metabolites might be primarily used as substrates for energy production and most of them were relatively abundant in SM3. Among the seven DAMs, the three potential biomarkers (γ-glutamyl-L-leucine, cytosine, and ketoleucine), which showed significant changes exclusively in SM3, each had an area under the curve value of 1. Therefore, monitoring the levels of these key metabolites in culture media could serve as a quality control measure for cultured meat production by enabling the indirect detection of suboptimal PSCs based on their proliferation ability.

Animal-free scaffold from brown algae provides a three-dimensional cell growth and differentiation environment for steak-like cultivated meat

Scaffolds for the production of cultivated meat, a promising sustainable meat alternative, should exhibit physical and chemical properties that enable three-dimensional animal cell culture, along with biological characteristics that support cell attachment, proliferation, and differentiation. Additionally, the scaffold should be crafted from edible materials and offer textural similarities to meat and have minimal influence on flavor and taste. Herein, an edible alginate-based alginate-cellulose hydrogel (ACe-gel) scaffold derived from the brown alga Undaria pinnatifida is developed. In terms of physical characteristics, the scaffold had porosity (119.5 ± 37.2 μm) and moisture-holding capacity (73.03 ± 3.82, 68.66 ± 9.54, and 84.17 ± 9.94 at 25 °C, 37 °C, and 60 °C, respectively) suitable for three-dimensional culture and differentiation of bovine muscle stem cells (bMuSCs). Accordingly, the scaffold was superior to a commercial alginate scaffold in terms of the attachment and proliferation of bMuSCs (5.5-fold over 72 h), and its performance was comparable with that of a lyophilized collagen scaffold (7.8-fold over 72 h, compared with the pure alginate). The bMuSCs cultured on the ACe-gel scaffold were capable of differentiating into muscle fibers, as verified by gene expression profile analysis. Furthermore, the scaffold exhibited minimal heavy metal contents and distinct seaweed odorants, while the stress-strain characteristics of the scaffold cultured with bMuSC (Young's modulus of raw ACe-gel: 285.19 ± 83.37 kPa, cooked ACe-gel meat: 880.60 ± 485.60 kPa) closely resembled that of meat (raw beef: 267.76 ± 156.42 kPa, cooked beef: 1331.94 ± 762.43 kPa). These findings highlight that the seaweed-derived and animal-free ACe-gel scaffold has strong potential for utilization as a food technology for cultured meat production in the future.

Highly efficient isolation and 3D printing of fibroblasts for cultured meat production

Fibroblasts are important components of animal tissues such as muscle and skin, as they are the major producers of various matrix proteins. Matrix proteins such as collagen play an important role in meat products by providing unique nutrition, texture, and flavor. Cultured meat is an innovative meat alternative produced by culturing animal cells, but currently, relatively few studies have been conducted using fibroblasts as seed cells for cultured meat manufacturing. In this work, we first developed an innovative digestion-friction method for isolating fibroblasts from porcine skin efficiently and cost-effectively. After optimizing the enzymatic digestion and physical friction conditions, 2.39 ± 0.28 × 105 fibroblasts were obtained from 1 cm2 of porcine skin tissue, which was about 9 times higher than the conventional tissue explant method. In addition, we identified an edible bio-ink composed of gelatin and chitosan that has good printing properties and supports fibroblast adhesion and growth. Furthermore, we fabricated fibroblast-based cultured meat by 3D printing with an initial cell density of 1.0 × 107 mL−1 and evaluated its texture and nutritional properties. This work provides valuable insights and references for introducing fibroblasts into the production of cultured meat that is more comparable to structured animal meat.

Effect of Crude Polysaccharides from Ecklonia cava Hydrolysate on Cell Proliferation and Differentiation of Hanwoo Muscle Stem Cells for Cultured Meat Production.

Ecklonia cava, a brown seaweed native to the East Asian coast, is known for its unique composition, including polysaccharides, polyphenols, and phlorotannins. Fucoidan is a sulfated polysaccharide widely used as a functional ingredient in foods. This study obtained crude polysaccharides (ECC_CPS) from E. cava celluclast enzymatic hydrolysate using ethanol precipitation. ECC_CPS increased cell viability during the proliferation of Hanwoo muscle satellite cells (HMSCs). The effect of ECC_CPS on the expression of proliferation-related markers was confirmed as MYF5 and MYOD expression significantly increased, whereas PAX7 expression was maintained. The evaluation of cell migration activity has a major impact on cell proliferation and differentiation, and the cell migration index significantly increased with ECC_CPS treatment (p < 0.01). This was related to the HGF/MET pathway and FAK pathway. Treatment with ECC_CPS promoted differentiation at the cell differentiation stage, thereby increasing the expression of differentiation markers, such as MYH2, MYH7, and MYOG (p < 0.001 or p < 0.01). Therefore, our findings imply that crude polysaccharide obtained from E. cava can be an additive ingredient that enhances the proliferation and differentiation of muscle satellite cells used in the manufacture of cultured meat products.

Non-animal protein hydrolysates from agro-industrial wastes: A prospect of alternative inputs for cultured meat

In light of the growing demand for alternative protein sources, laboratory-grown meat has been proposed as a potential solution to the challenges posed by conventional meat production. Cultured meat does not require animal slaughter and uses sustainable production methods, contributing to animal welfare, human health, and environmental sustainability. However, some challenges still need to be addressed in cultured meat production, such as the use of fetal bovine serum for medium supplementation. This ingredient has limited availability, increases production costs, and raises ethical concerns. This review explores the potential of non-animal protein hydrolysates derived from agro-industrial wastes as substitutes for critical components of fetal bovine serum in cultured meat production. Despite the lack of standardization of hydrolysate composition, the potential benefits of this alternative protein source may outweigh its disadvantages. Future research holds promise for increasing the accessibility of cultured meat.

κ-Carrageenan/konjac glucomannan composite hydrogel-based 3D porcine cultured meat production

Alternative technology for cultured meat production holds the potential to alleviate ethical, environmental, and public health concerns stemming from conventional husbandry. As a key food component, fat plays a crucial role in the sensory characteristics and nutritional value of meat products. However, current adipose tissue engineering protocols have so far proven unsuitable for edible cultured fat production. In this study, hybrid hydrogels based on Κ-Carrageenan (Car) and konjac glucomannan (KGM) were designed as a culture scaffold for adipocytes. The edible hydrogel with excellent biocompatibility, food safety, and low cost was demonstrated to support cell proliferation and allow the formation of tissue-like cell spheres. Furthermore, both porcine subcutaneous pre-adipocytes (PreAs) and fibro-adipogenic progenitors (FAPs) acquired mature adipocyte characteristics and higher lipid accumulation in Car-KGM (CK) hydrogel, suggesting its superior ability to support in vitro adipogenesis than conventional monolayer culture and alginate (Alg)-based 3D culture. Taken together, the excellent nature of CK hydrogel makes it a versatile platform for improving the molecular and cellular phenotypes of a variety of adipocyte models. The established spheroid model opens possibilities for broad applications in cultivated fat and represents a viable strategy for the manufacture of ‘snowflake pork’.

Quality Control of Stem Cell-Based Cultured Meat According to Specific Differentiation Abilities.

The demand for stem cell-based cultured meat as an alternative protein source is increasing in response to global food scarcity. However, the definition of quality controls, including appropriate growth factors and cell characteristics, remains incomplete. Cluster of differentiation (CD) 29 is ubiquitously expressed in bovine muscle tissue and is a marker of progenitor cells in cultured meat. However, CD29+ cells are naturally heterogeneous, and this quality control issue must be resolved. In this study, the aim was to identify the subpopulation of the CD29+ cell population with potential utility in cultured meat production. The CD29+ cell population exhibited heterogeneity, discernible through the CD44 and CD344 markers. CD29+CD44-CD344- cells displayed the ability for long-term culture, demonstrating high adipogenic potential and substantial lipid droplet accumulation, even within 3D cultures. Conversely, CD29+CD44+ cells exhibited rapid proliferation but were not viable for prolonged culture. Using cells suitable for adipocyte and muscle differentiation, we successfully designed meat buds, especially those rich in fat. Collectively, the identification and comprehension of distinct cell populations within bovine tissues contribute to quality control predictions in meat production. They also aid in establishing a stable and reliable cultured meat production technique.