Current Research, Industrialization Status, and Future Perspective of Cultured Meat

MSCs are muscle-derived adult stem cells that are responsible for the regenerative capacity of muscle following damage to myofibers. MSCs are characterized by rapid proliferation in a highly active state early in life, while the proportion entering a quiescent state increases with age (Mesires and Doumit, 2002). Myofibrils are composed of structures surrounded by an inner sarcolemma and an outer basement membrane, and the basal lamina, which is close to the myofibrils, has been identified as an extracellular matrix (ECM) that is in direct contact with MSCs and is involved in the maintenance of physiological functions and the development of skeletal muscle (Holmberg and Durbeej, 2013; Zhang et al., 2021). The basal lamina is composed mainly of type IV collagen, which plays a role in maintaining MSCs in a quiescent state by sequestering various growth factors and signaling molecules involved in their activation and proliferation (Kann et al., 2021; Sanes, 2003). Furthermore, quiescent MSCs located in the niche between the basal lamina and myofibrils have a fusiform morphology with little cytoplasm and organelles and have been shown to express MSC-specific genes, such as paired box protein 3 (Pax3) and Pax7, and myoblast determination protein 1 (MyoD) at the beginning of quiescence or proliferation entry (Fu et al., 2015; Kuang et al., 2006; Zhang et al., 2010).

Understanding the regeneration process of MSCs is necessary for cultured meat production, and the genes and signal transduction pathways that regulate proliferation and differentiation that have been widely reported to date are shown in Fig. 2. Pax3 is considered one of the important genes responsible for MSC survival during embryogenesis. It is also purported to be involved in the formation and underlying development of early muscles by affecting the expression of MyoD and myogenic factor 5 (Myf5) to regulate the development of limb muscles (MyoD) and peri-spinal and intercostal muscles (Myf5) in early embryos (Kablar et al., 1997). Pax7 is an essential gene for MSC maintenance, and individuals with Pax7 knockout show a decreased rate of muscle regeneration in muscle injury treatments and difficulty in generating MSCs (Kuang et al., 2006). In addition, Pax7 has been found to act as an antagonist of MyoD, resulting in an increased number of Pax7-positive cells in the muscles of individuals with MyoD knockout (Kuang et al., 2006; Olguin and Olwin, 2004; Seale et al., 2000).

Fig. 2. Gene regulation and signaling pathways in myogenesis.

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Activation of MSCs is an early step in myogenesis. When a muscle is damaged, the disruption of the basal plate and reorganization of the environment leads to interactions between signaling molecules that were previously sequestered by the basal plate and MSCs, leading to their activation (Li et al., 2018). Muscle formation is mainly regulated by myogenic regulatory factors (MRFs) expressed in activated MSCs. Some representative MRFs are MyoD, Myf5, myogenin, and muscle-specific regulatory factor 4 (MRF4, also known as Myf6; Kim et al., 2023a). Activated MSCs divide to produce satellite cell-derived myoblasts that continue to divide and proliferate before committing to differentiation and fusing to form myotubes, which then mature into myofibers. When satellite cells are activated, they initiate differential expression of MRFs depending on the asymmetry of cell orientation after division (Kuang et al., 2007). Accordingly, it has been shown that if the orientation of the cells formed after somatic cell division is on the myofibrillar side, they upregulate origin regulatory factors, such as MyoD and Myf5, whereas cells on the basal plate side do not express Myf5 and retain stemness (Kuang et al., 2007; Troy et al., 2012).

MyoD and Myf5 are genes that activate myogenin and MRF4 and participate in the late stages of muscle formation by influencing the fusion of myoblasts and the initiation of their final differentiation, leading to cell maturation and ultimately the formation of multinucleated myotubes (Cornelison et al., 2000; Hawke and Garry, 2001; Punch et al., 2009; Smith et al., 1993). MyoD has somewhat overlapping roles with myogenin, but when myogenin is deleted, MyoD is unable to take over its role, and individuals with myogenin deletion have been shown to die at birth due to impaired skeletal muscle formation (Adhikari et al., 2021; Nabeshima et al., 1993). It was also found that in C2C12 cultures with myogenin deletion, myomaker and myomixer, two genes that regulate the fusion of skeletal muscle, were significantly downregulated, leading to the inhibition of differentiation (Adhikari et al., 2021). MRF4 is an origin regulator that is predominantly expressed in fully differentiated muscle fibers and plays a role in maintaining the MSC pool. It has been reported that deletion of MRF4 can significantly reduce the number of Pax7-positive MSCs in postnatal individuals (Lazure et al., 2020).

Signals that regulate the stemness of MSCs are known to include p38α/β mitogen-activated protein kinase (MAPK) or Notch. First, inhibition of p38 has been reported to induce self-renewal of MSCs by blocking the MyoD expression pathway and maintaining Pax7 expression along with inhibition of cell cycle entry to sustain an undifferentiated and proliferative state (Ding et al., 2018; Li et al., 2023; Troy et al., 2012). Among Notch signaling components, Notch1 is activated upon muscle injury in vivo by binding to myofilament ligands to induce cell cycle exit, Notch2 is activated in MSCs to maintain the stemness of the MSC population by inhibiting differentiation, and Notch3 has been shown to inhibit the p38α/β MAPK pathway to suppress myocyte enhancer factor 2 (MEF2) expression associated with differentiation (Conboy and Rando, 2002; Gagan et al., 2012; Jo et al., 2022).

It has been reported that activated MSCs are proliferation-induced and differentiation-inhibited by the phosphoinositide 3-kinase (PI3K)/Akt pathway or the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway (Li et al., 2023; Mohammadabadi et al., 2021; Ohashi et al., 2015). Growth factors known to be involved in PI3K/Akt activation include fibroblast growth factor (FGF), insulin-like growth factor (IGF)-1/2, hepatocyte growth factor (HGF)/c-Met, epidermal growth factor (EGF), and interleukin-6/Janus kinase 2/signal transducer and activator of transcription 3 (IL-6/JAK2/STAT3). These factors have been shown to act as activators of mammalian target of rapamycin complex 1 (mTORC1), which can regulate the proliferation of muscle progenitor cells (Brandt et al., 2018; Holterman and Rudnicki, 2005; Lu et al., 2017; Messersmith et al., 2021; Ohashi et al., 2015; Ornitz and Itoh, 2015; Relaix et al., 2021; Rhoads et al., 2016; Wang et al., 2023a). Furthermore, it has been confirmed that EGF and FGF are involved in the ERK1/2 pathway, one of the MAPK family signaling pathways, which can activate myoblast proliferation and impair the initiation and maintenance of differentiation (Li et al., 2023; Mohammadabadi et al., 2021; Ohashi et al., 2015). Additionally, the Wnt pathway can activate both mTORC1/2, with mTORC1 regulating metabolism in response to environmental factors (growth factors, amino acids, energy, and stress) and mTORC2 involved in the maintenance of MSC populations through phosphatase family pathways (Oh and Jacinto, 2011; Rion et al., 2019; Wei et al., 2019).

The p38α/β MAPK pathway activates MEF2 and plays a major role in the differentiation of myoblasts. Myotube formation is inhibited when MEF2 is removed because of the involvement of MEF2 in the proliferation and differentiation of MSCs (Chen et al., 2017; Shao et al., 2022; Wang et al., 2018). Furthermore, when the mTOR pathway is inhibited by rapamycin in MSC cultures, the expression of myogenic genes (Pax7, Myf5, and MyoG) is inhibited, indicating that the mTOR pathway is essential for the proliferation and differentiation of MSCs (Zhang et al., 2015). In addition, previous studies on MSC differentiation have shown that Wnt1 and Wnt7a signaling, along with activation of the Wnt/β-catenin pathway, increases β-catenin to induce myogenic differentiation of mesenchymal stem cells and activate the myogenic regulators Myf5 and MyoD to influence skeletal muscle development (Eng et al., 2013; Zhu et al., 2022). Signals that inhibit differentiation include ERK, myostatin, and protein kinase A (PKA), with myostatin reported to inhibit muscle formation by co-inhibiting the Akt pathway and PKA reported to induce proteolytic cleavage to produce factors that inhibit MEF2 signaling (Backs et al., 2011; Mohammadabadi et al., 2021; Trendelenburg et al., 2009).

The nuclear factor of activated T-cells (NFAT) can activate signaling molecules that regulate the fusion of myoblasts and myotubes, such as MEF2 and IL-4, by the calcineurin and p38/MAPK pathways; however, PKA has been reported to prevent premature differentiation of myoblasts by rephosphorylating MEF2 and NFAT while inhibiting their differentiation (Horsley et al., 2003; Knight and Kothary, 2011; McKinsey et al., 2002; Stork and Schmitt, 2002; Wu et al., 2007; Yue et al., 2023). In addition, it has been shown that mTOR regulates the proliferation of MSCs but can also regulate myotube fusion by both kinase-dependent and -independent pathways (Park and Chen, 2005).

In conclusion, an understanding of the various gene expression and signaling processes within MSCs for cultured meat production is required, and further research is needed to control and regulate cell cycle arrest and activation, proliferation, differentiation, and even fusion.

MSCs can be obtained by biopsy of muscle tissue from living animals and by harvesting muscle tissue from animals immediately after slaughter. The most used processes for harvested muscle tissue are disinfection, removal of fat and connective tissue, fragmentation, digestive enzyme treatment, sequential filtration, centrifugation, pre-culture, and finally, cell recovery to obtain primary cells (Lee et al., 2021). The obtained primary cells are then subjected to immunofluorescence staining or polymerase chain reaction to determine the proportion of MSCs from the proportion of progenitor regulatory factors in the primary cells. Typically, Pax7 and MyoD are used to determine the purity of MSCs, and by comparing their expression levels, the activation of the MSCs used in the experiment can be determined (Ding et al., 2017; Kim et al., 2023a; Pasut et al., 2013). In addition, flow cytometry methods, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), can be used to obtain pure MSCs labeled with MSC-specific markers, which can then be proliferated to sufficient quantities for use in cultured meat experiments and production (Ding et al., 2018; Gromova et al., 2015; Kim et al., 2023a; Motohashi et al., 2014).

The culture of MSCs has been performed since before the 1990s, and the methods can be broadly divided into two types: culture of isolated single strands of muscle fibers and culture of cells isolated from enzymatically treated muscle tissue (Anderson and Pilipowicz, 2002; Bischoff, 1986; Doumit and Merkel, 1992; McFarland et al., 1988). Fetal bovine serum (FBS) is a key ingredient added to the basal medium for culture, but the exact nature of FBS is still poorly understood, and commercialization of cultured meat is currently limited by the need to replace FBS completely (Lee et al., 2022; Lee et al., 2023a). It is difficult to avoid the ethical issues associated with the production of FBS, as more than 2 million bovine fetuses derived from slaughtered mothers are used for FBS production each year (Lee et al., 2022). In addition to ethical concerns, the high price of FBS has led numerous research teams to investigate serum-free media as an alternative to FBS, and along with research to refine the active ingredients of FBS, results support that serum can be effectively replaced with proteins required for cell growth or a combination of various growth factors (Messmer et al., 2022; Schenzle et al., 2022; Skrivergaard et al., 2023; Stout et al., 2022; Stout et al., 2023). Furthermore, to meet halal standards, the use of blood in cultured meat production is also limited (Hamdan et al., 2018). However, challenges remain, such as the use of recombinant growth factors in the preparation of serum-free media or chemically composed media and the cost of expensive additives (Stout et al., 2022; Stout et al., 2023).

Once the medium in which the cells are to be cultured is prepared, the method of culturing the cells must be chosen according to each cell type. Cell culture techniques for cultured meat production can be broadly divided into adherent culture and floating culture, and among the cells, MSCs and fibroblasts have been studied, as well as adipocytes (Bodiou et al., 2020; Ge et al., 2023; Humbird, 2021; Lee et al., 2021). Approximately 10¹⁴ cells and 10,000 L of culture medium are required to produce 1 t of cultured meat, assuming a cell density of 10⁷ cells/mL in the bioreactor (Guan et al., 2021). However, the larger the bioreactor size, the higher the stirring intensity needed to maintain a homogeneous environment in the vessel, which can lead to shear stresses of a magnitude that can cause cell damage (Allan et al., 2019). In a modeling study of cultured meat production scenarios, it was emphasized that optimal cell selection to reduce the consumption rate of medium, completely replace or decrease the cost of growth factors, and increase the size of perfusable bioreactors are necessary for mass production environments (Risner et al., 2021).

As such, cultured meat is a tissue engineering technique under investigation based on the theory that the self-renewal ability of MSCs can be harnessed to produce dozens of times the amount of muscle tissue from a small piece of muscle. Cultured meat is one of the promising future technologies that can be used as an important source of meat for some countries because it is less sensitive to climatic conditions than conventional meat production, but there is a need to improve economic issues, such as cell acquisition, mass production and cost, and the amount of culture fluid and energy required for production compared to real meat. Additionally, research is being conducted worldwide to improve the qualitative limitations, such as flavor, texture and structure, meat color, and nutritional content, which are different from those of real meat.

Bovine MSC isolation techniques for cultured meat production reported in 2023 are shown in Table 3. The goal of the isolation process is to obtain the raw material for cultured meat. The isolation techniques used can be broadly categorized into 1) enzymatic reactions and centrifugation to obtain MSCs and pre-culture and 2) flow cytometry to increase the purity of the MSCs.

First, an enzymatic reaction is performed to obtain primary cells from muscle tissue. The cells are minced to increase the surface area, and connective tissue is removed to facilitate the reaction. Enzymes used for MSC isolation include collagenase, dispase, trypsin, and pronase in various concentrations. Centrifugation is a method that uses centrifugal force and density gradients to remove unwanted tissue and isolate desired cells. In the isolation process of MSCs, the centrifugal acceleration was 76–1,200×g, and the time was generally around 5–15 min.

The cells obtained by enzymatic reaction and centrifugation are primary cells. Cell pre-plating or purification techniques, such as FACS and MACS, are employed to increase the purity of MSCs. Pre-culture is a technique for isolating specific cells from a mixture of different cell types, effectively increasing the purity of MSCs by exploiting differences in the adhesion properties of primary intracellular fibroblasts and MSCs (Richler and Yaffe, 1970). The preincubation time used in the isolation of bovine MSCs reported in 2023 was 1–3 h. Fibroblasts begin to adhere 5 min after incubation and adhere to surfaces faster than MSCs, indicating that a relatively high purity of Pax7- or MyoD-positive cells can be obtained using the preincubation process (Table 3; Kim et al., 2022; Xu et al., 2018; Yoshioka et al., 2020). In other studies, preincubation conditions have been shown to vary from 5 min to 24 h after fibroblasts begin to adhere (Table 3). One of the effective methods for rat MSCs was preincubation for up to 10 min with shaking every 5 min (Yoshioka et al., 2020). For chicken MSCs, it was up to 2 h with shaking every 8 min after 2 h of rest, indicating that the preincubation conditions may also vary depending on species-specific cell characteristics (Kim et al., 2022).

Even without the pre-culture step, the purity of the MSCs can be increased by using cell sorting techniques, such as FACS and MACS. Some drawbacks of the flow cytometry-based isolation process are that it requires expensive equipment and reagents, trained professionals, and is cumbersome because of sorter-induced cellular stress, such as high-pressure jets, high voltage, and laser exposure during the isolation process, and cytotoxicity that can occur when using specific markers (Lopez and Hulspas, 2020). Although FACS has the advantage of being able to separate cells based on their size or three-dimensional features using fluorescent labeling, it has the disadvantage of expensive equipment and long analysis times. MACS uses magnetic particles to sort cells more than four times faster than FACS and is less expensive, but it is difficult to apply to cells that are susceptible to magnetism or cannot be labeled (Gerashchenko, 2011).

Both FACS and MACS label cells with clusters of differentiation (CD), which are specific markers of MSCs, and each uses a fluorescent agent for FACS and magnetic particles for MACS. Specific markers for MSCs used for cell labeling are species-specific, but some examples are integrin α7, vascular cell adhesion protein 1 (Vcam1), and differentiation clusters, such as CD29 (integrin β1), CD34 (hematopoietic stem cell marker), CD56 (neural cell adhesion molecule), and CD82 (4-transmembrane glycoprotein) (Castiglioni et al., 2014; Uezumi et al., 2016; Yoshioka et al., 2020). After sorting the MSCs, they can then be cultured to check the expression of Pax7 or MyoD to confirm the purity of the isolated MSCs, and the proliferation and differentiation capacity of the cells can be assessed.

In conclusion, the cell biological characteristics necessary for the isolation of MSCs from each animal species have not yet been fully identified, and comprehensive research is limited by the lack of standardization of separation methods, which is an obstacle to industrialization.

MSCs obtained during the isolation process will multiply in number in a properly conditioned growth medium. The proliferation process is directly related to the yield of cultured meat, and various studies have been conducted to improve the proliferation efficiency. First, the basal media commonly used for MSC culture are Ham’s F-10, Dulbecco’s modified Eagle’s medium (DMEM), and DMEM/F12, with bovine fetal serum added to the media at a concentration of 10%–20% (v/v) in most cases (Table 4). Basal media is a solution of basic nutritional components (e.g., amino acids, glucose, lipids, nucleic acid bases, inorganic salts, vitamins, buffers, pH indicators) formulated in a certain proportion according to the culture conditions of the desired cells. In the culture of MSCs, the basal medium and serum concentrations are known to be closely related to the cell proliferation rate and myotube formation (McFarland et al., 1988). In a broiler MSC culture experiment based on culture medium composition, DMEM was found to be more effective than McCoy’s 5A medium in terms of proliferation rate and MRF expression (Flees et al., 2022). In addition, the common view that a low glucose content is effective for chicken and bovine MSC proliferation when using DMEM as basal medium was confirmed (Flees et al., 2022; Zygmunt et al., 2023).

As adherent cells, MSCs require an ECM-based coating for proliferation and differentiation. Representative ECMs used for bovine MSC proliferation have been shown to be gelatin, collagen I, laminin, and Matrigel (Table 4). Integrin α7β1, which is present in the cell membrane of MSCs, binds to collagen and laminin, and laminin induces the proliferation and migration of satellite cells (Öcalan et al., 1988; Sanes, 2003). However, C2C12 cells cultured on plates coated with ECM proteins had a better proliferation rate compared to highly elastic coatings, such as collagen I/laminin/fibronectin hydrogels, which were not conducive to inducing proliferation of MSCs (Palade et al., 2019).

Growth factors are cell signaling proteins. For MSCs, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and HGF are commonly used in culture (Table 4). Typically, bFGF is added to the proliferation medium at a concentration of 5–10 ng/mL. Accordingly, in bovine MSC cultures, a bFGF content of 10 ng/mL in the medium led to a faster proliferation rate than when the bFGF content was 5 ng/mL (Zygmunt et al., 2023). In addition, the expression of various endothelial cell-derived growth factors (IGF-1, HGF, bFGF, and VEGF) can stimulate MSCs to proliferate and regenerate muscle (Yamamoto et al., 2020; Zygmunt et al., 2023).

However, research into cultured meat using FBS remains prevalent. Even when serum-free media is used, various culture ingredients, such as basal media, coating agents, and recombinant proteins (growth factors, hormones), are employed. To address this issue, the development of natural product-derived media that meet food regulatory requirements is underway. However, industrialization is inevitably delayed because companies and research teams cannot disclose it due to competitiveness.

MSC differentiation is commonly achieved by removing the proliferation medium and replacing it with the differentiation medium once the cells have reached a sufficient cell density in the proliferation medium (Ding et al., 2017). The differentiation process typically uses media containing 2% FBS or horse serum to induce serum starvation (Table 5). Serum starvation is often chosen to induce differentiation of MSCs (Pirkmajer and Chibalin, 2011). Induction of differentiation in studies published in 2023 was mainly performed at cell densities above 70% confluence, and the duration of differentiation varied by 1–10 d depending on the experimental conditions, but only one study was identified that varied the serum concentration within the culture period (Table 5).

When a low-serum environment is used to induce differentiation of MSCs, extensive changes occur at the transcript level, with upregulation of progenitor transcription factors and markers associated with differentiation identified during the differentiation process (Dmitriev et al., 2013; Messmer et al., 2022). Transient and mild levels of serum starvation (15% serum, v/v) induce autophagy, which can promote cell metabolism and differentiation, but 5% serum starvation induces excessive autophagy, leading to cell death (Wang et al., 2023b).

A hypoxic environment (1%–10% O₂) in MSC culture can create conditions that mimic oxygen saturation in mature skeletal muscle. Moreover, a hypoxic environment (2% O₂) upregulates the myogenic regulators Pax7, Myf5, and MyoD, and intermittent hypoxic exposure increases the expression of VEGF released from MSCs (Koning et al., 2011; Nagahisa and Miyata, 2018; Urbani et al., 2012). The hypoxia-induced factor-1 signaling pathway, which is expressed in response to hypoxic conditions, is thought to be involved in the regulation of myoblast proliferation and differentiation. Under a hypoxic environment (1% O₂), broiler MSC cultures exhibited a decrease in the level of MyoD-positive cells along with changes in the transcriptome profile (Jung et al., 2024; Li et al., 2007).

However, serum starvation tends to be the preferred method for induction of differentiation compared to hypoxic environments, and the signaling pathways involved and their effects on differentiation remain poorly understood. Furthermore, the regulations regarding cultured meat are extensive and do not clearly differentiate between cultured meat with or without differentiated tissue, leading to confusion within the industry.