Stem cells are unspecialized cells possessing self-renewal capacity, and the discovery of stem cells paved the way for in vitro cell production and the concept of cultured meat. These cells have the potential to divide through mitosis to renew to other cell types throughout the life span of the multicellular organism. Subsequently, stem cell divides to create a cell population that can either stay as stem cells or separate lineage with more characterized capabilities such as blood, muscle, and neuronal cells. Usually, there are two types of stem cells, i) embryonic stem cells and ii) undifferentiated/substantial/grown-up stem cells. The embryonic stem cell is determined from the embryos, while undifferentiated cells reside in a tissue or organ at the side of other divided cells. The basic three properties of the stem are i) capable of dividing and renewing, ii) unspecialized, and iii) differentiate to other cell types. The cell typically passes through various stages during differentiation and specializes at each step. The signals that trigger the differentiation process inside and outside stem cells are still to be deciphered. The epigenetic regulation of genes typically controls the internal signal, while external signals such as physical contact with neighboring cells, growth factors or chemicals (specific to various receptors of stem cell) secreted by other cells, and the microenvironment are the main driving force for stem cell differentiation.

Pluripotent stem cells are difficult to handle and culture for CBM research as they require more time and resources to proliferate and differentiate into mature cell types compared to primary adult stem cells. Nevertheless, a pluripotent stem cell has the inherent capability to increase in large numbers and become immortal. Pluripotent stem cells derived from non-muscle sources can be isolated from diverse domesticated animals and expanded as a myogenic cell source for CBM. Recently, chemically and genetically modified porcine pluripotent stem cells have been transformed into myogenic cells possessing the ability to differentiate into embryonic muscle fibers (Genovese N. J. et al., 2017). Pluripotent muscle stem cells are attractive possible source cells; any CBM produced from these pluripotent cells must be appropriately screened for safety before consumption.

On the other hand, primary adult stem cells offer the benefit of being simply achieved from a biopsy of any animal species, such as sheep, buffalo, or cow, to acquire a cell population for any meat product; however, their proliferative potential is limited (Ding, 2019). Depending on the stem cell types, these cells can be stimulated to differentiate into muscle or fat cells. MSCs are considered reliable cells for skeletal muscle recovery in vivo, and their self-renewal capability maintains the population of stem cells and the generation of enormous numbers of myogenic cells. These myogenic cells proliferate, divide, fuse, and help produce new myogenic fibers (Brack and Rando, 2012; Yin et al., 2013). Mark post presented the meat hamburger prototype that amplifies the myoblast progeny of MSCs (Post, 2014). Multipotent progenitor cells deriving from porcine skeletal muscle exhibit higher doubling capacity than MSCs, presenting them as better source cells for CBM production. However, these cells require additional growth factors and are not able to differentiate into skeletal muscle fibers as proficiently as MSCs can perform (Wilschut et al., 2008).

In 1961, Mauro first identified the bona fide satellite cells in frogs (Mauro, 1961). On appearance, satellite cells differ from muscle fiber in exhibiting chromatin-dense nuclei with minute cytoplasmic value. Satellite cells derive from the dermomyotome’s cell population and are between the basal membrane and sarcolemma (Gros et al., 2005). Satellite cells add new nuclei in the growing muscle fibers by fusing with adjacent fibers during peri- and postnatal development (Relaix et al., 2005; Biressi et al., 2007). Afterward, satellite cells may acquire a quiescent stage. Upon muscle injury, they are activated for further muscle growth and development (Biressi et al., 2007; Fu et al., 2015; Almada and Wagers, 2016). A preplating procedure usually isolates bovine satellite cells (Li et al., 2011; Will et al., 2015). The purity of isolated satellite cells through the preplating method without further purification steps can lead to 31% of cells (depending upon the fusion index) and 95% by DESMIN staining (Coles et al., 2015; Will et al., 2015). The purification of satellite cells may be improved by FACS/MACS and in combination with positive selection (CD29 and CD56) and negative selection (CD31 and CD45) (Figure 2). However, the degree of high positivity may be deduced by their PAX7 positivity (Ding et al., 2018).

The mainstay material for producing CBM is myoblasts (satellite cells), which are challenging to grow in vitro. However, myoblasts can readily differentiate into myotubes (immature muscle cells) and myofibrils under specific environments. To expedite the replication of skeletal muscle satellite cells in a lab, cells are attached to an immobile substratum, for example, scaffold or microbeads, which may be coated with protein (collagen, chitosan, and laminin) to imitate the natural tissue. The scaffolds are usually biodegradable, edible, and re-usable during the culture method (Stephens et al., 2018).

Nutrient-rich medium is essential for growing satellite cells to provide a unique proliferation and differentiation phase, comprising antibiotics, antimicrobial agents, antifungal agents, and other chemicals to prevent contamination. Culture media is typically optimized with varying amounts of fetal bovine serum (5%–10%) to optimize the growth and differentiation of satellite cells in vitro. Some laboratories designed serum-free media or chemically-defined media for culturing the satellite cells; however, these media components are expensive and possess proprietary issues (Edelman et al., 2005).

Muscle cell culture implicates significant challenges on an industrial scale in a large bioreactor. Stephens et al. reported that roughly eight trillion muscle cells are required to produce 1 kg of protein from a traditional bioreactor possessing a capacity of 5000 L (Stephens et al., 2018). Cultured muscle cells may reach a thickness of around 200 μm. In the thick muscle layer, oxygen and essential nutrients may not be able to penetrate the inner layer of cells, and due to an insufficient supply of oxygen and nutrients, cells may begin to die (Jones, 2010). Muscle strips are harvested and processed at this stage, and several supplementary chemical compounds are added to augment nutritional value, color, texture, and flavor. The production of a particular cut of meat, like chops, steaks, or roasts, needs additional technology to give the correct shape and structure to the muscle cells.

The paraxial mesodermal progenitor cells during fetal development give rise to muscle tissue. Upon sequential development process, paraxial mesoderm differentiates into myoblast, and the ensuing process is regulated by numerous growth factors (Chal and Pourquié, 2017). Through cell-to-cell fusion, myoblast generates muscle tissue, and part of them inhabits underneath the basal lamina of myofiber, which converts into quiescent satellite cells during the postnatal period. During muscle injury, the quiescent muscle cells are activated to differentiate into myoblast, leading to muscle regeneration. The quiescent satellite cells are characterized by the expression of Pax7, while Myf5 and MyoD are absent (Kuang et al., 2007). Upregulation of Myf5 and MyoD and downregulation of Pax7 occur by myogenic satellite muscle cells and make them proliferating myoblast during muscle injury. Myf5 plays a significant role in myoblast proliferation, while MyoD has a principal function in differentiation (Asakura et al., 2007; Gayraud-Morel et al., 2007). These intrinsic factors can be exploited as muscle stem cell markers to explore the cellular states of the cell. Muscle stem cells possess surface and cytoskeletal proteins, for example, vascular cell adhesion molecule, neural cell adhesion molecule (also known as CD56), integrin α7, β1 (CD29), CD34, desmin, and SM/C-2.6 (Wang et al., 2014). The synchronization of intrinsic and extrinsic factors plays a significant role in the fate of muscle stem cells. Hence, to maintain the functioning of muscle stem cells in the lab, the physiological conditions of muscle stem cells should be provided by mimicking the in vivo stem cell niche in the form of extracellular matrix (ECM) and paracrine factors.

Skeletal muscle tissue comprises muscle fibers, connective tissues, and stem cell populations. Muscle stem cells usually reside on muscle fibers, and their isolation is a mainstay in upscaling in cellular agriculture, as described in the previous sections. Commonly used proteases for the purification of muscle stem cells are from muscle biopsy following physical dissociation, and meat mincing are trypsin, collagenase, pronase, and dispase. Numerous digestive enzymes with multiple combinations can be applied to digest muscle tissues. Collagenase and dispase have been extensively used as these enzymes specifically target ECM-containing collagen and fibronectin (Stenn et al., 1989).

Myogenesis is a highly ordered and complex process of MSCs. The process is regulated by the co-expression of paired box transcription factors (Pax3/Pax7) and myogenic regulatory factors (such as Myf5, Mrf4, MyoD, and myogenin) (Zammit and Beauchamp, 2001; Relaix et al., 2005; Baig et al., 2019). Myogenesis is illustrated by various factors such as cell cycle arrest, increased nuclear sizes, cell alignment, myogenic activation, multiple cell fusion, and peripheral localization (Chargé and Rudnicki, 2004). However, skeletal muscle regeneration depends on interactions between MSCs and their microenvironment composed of basal lamina and sarcolemma (Kuang et al., 2008).

Animal meat is composed of skeletal muscle tissues, so tissue engineering of skeletal muscle tissues has been exploited to produce CBM. Due to the non-proliferating capability of adult skeletal muscle cells, MSCs are utilized as a precursor for replication. MSCs exhibit high responsiveness and migratory abilities; MSCs are precarious for preserving skeletal muscle’s functional and structural integrities and are also accountable for muscle regeneration through a coordinated myogenic program (Lee et al., 2018). The discoveries of MSCs led to the production of cells in vitro and the development of CBM. Therefore, MSCs provide a viable source of cells for skeletal muscle recovery (in vivo). The ability of MSCs to self-renew and self-sustain the stem cell population and the production of an enormous number of myogenic cells, which again proliferate, multiply, and fuse to form new myofibers (Shaikh et al., 2021). MSCs are typically located between the basal lamina and sarcolemma and are active in regulating myofiber growth and development under the influence of myogenic regulatory factors (Ahmad et al., 2020; Shaikh et al., 2021). The first CBM production model was established on bovine MSC, and the principle is still applied in bioreactor-based cultured meat production (Verbruggen et al., 2018). In the following sub-section, we discussed the molecular parameters involved in the regulation of muscle and development.

Myokine is a cytokine comprising a molecular weight in the range of 5–20 kDa. During muscle contraction, skeletal muscle cell produce and release myokines. Table 1 briefly describes the function of myokines and cytokines implicated in myogenesis.

In vitro stem cells need a culture environment like growth media, cell substrates, antibiotics, antimycotic agents, and incubators. The culture environment provides optimum in vitro niche conditions to grow cells, mimicking in vivo (ECM, hormones, and cytokines). Recapitulations of media components are performed by exploiting synthetic chemicals and artificial devices. In the next section, we will discuss i) media, ii) cell substrates, iii) serum and their replacements, iv) antibiotics, and v) additional nutrients and supplements.

ECM is a multilayered environment that provides structural support, signals responses to injuries, helps cellular communication, and presents architectural preservation of skeletal muscle cells. During myogenesis, ECM interacts with, adheres to, safeguards muscle cell, assist in biochemical signaling, and offers structural support (Lee et al., 2018). Some of the ECM proteins assist in cell-matrix interactions and matrix assembly regulation (Gillies and Lieber, 2011). Besides its biological function, ECM comprises nutrients such as proteins (collagen) and glycosaminoglycans that impact the texture of tissue and overall meat quality (Table 2) (The Good Food Institute, 2022).

In myotubes’ developmental regulation during myogenic differentiation’s early stages, ECM is essential in regulating MSC’s phenotypic expression (Zhang et al., 2021). The basal lamina comprises a three-dimensional ECM network and is directly linked to MSC (Kuang et al., 2008). The majority of ECM comprises collagen fibers and proteoglycan matrix; however, ECM also contains elastin, fibronectin, and laminins (Thorsteinsdóttir et al., 2011). Three-dimensional scaffolds are critical to stabilize cells and impersonate the ECM during tissue formation. To augment the quality, taste, and tenderness of CBM, it may be advisable to co-culture preadipocytes with myoblast, owing to their effective increase in intramuscular fat content of cultures meat.

Collagen forms an intramuscular connective tissue network, and it is the most abundant fibrous protein in skeletal muscle (10% by weight) (Gillies and Lieber, 2011). Collagen provides elasticity, tensile strength, strengthens bones, regulates cell attachment, and role in differentiation (Ahmad et al., 2020). Collagens are necessary for the self-renewal of MSC and differentiation in vivo in mice. For example, the knockout of collagen VI impaired regenerating capacity of MSC following muscle injury (Urciuolo et al., 2013).

Collagens are found in numerous forms, and several of them have been revealed in skeletal muscles, such as fibrillar collagens I, III, V, IX, and XI. Collagen I and III account for more than 75% of total skeletal muscle collagen (McKee et al., 2019). Collagen and gelatins are widely applied in the pharmaceutical and food industries owing to their biodegradability, biocompatibility, and low antigenicity (Liu et al., 2015).

CBM needs unique cell lines prepared from agriculturally effective systems to scale up on an industrial scale. One of the first daunting tasks in CBM is establishing its cell line, which can be used in each cycle to produce meat. Few firms (Kerafast, United States and ESCO ASTER, Singapore) manufacture cell lines of various species for commercial purposes. MACK1 (myoblast) adherent cell line derived from the mature muscle of Atlantic mackerel (Scomber scombrus) for useful cellular agriculture (Saad et al., 2022). ICAR-National Bureau of Fish Genetic Resources, India, has a repository of about a cell line of 50 fishes, especially Catla. Freshwater fish species of Bluegill fry cell line (adherent fibroblast), Rainbow trout, and embryonic cell line of Nile tilapia have already been established (NRFC, 2023). Researchers are manipulating the stem cell expression markers in a suitable culture medium to develop unique methods for myogenesis induction in the cell lines (Thorley et al., 2016). Presently, few labs are working on establishing particular cell lines for various organisms like bovine, seafood, and aquatic species (Better ways to start cultivating meat | Research (2020-2022) | GFI, 2021). One of the main obstacles in developing a cell line is the limited knowledge of surface genetic markers and the non-availability of species-specific antibodies to assist in identifying a suitable cell line. However, few firms have developed the cell lines of various species like chicken, fish and turkey, and are commercially available (Kerafast) (Kerafast, 2023). Due to limited growth of the primary existing cells (not growing beyond 30–40 passages) in the defined media, is the main reason for developing the cell lines. In November 2022, UPSIDE Foods reported the establishment of myoblast and fibroblast cell lines with demonstrated differentiation capacity in the suspension culture (Ding et al., 2018). Immortalization has been carried out through the introduction of a cis gene expressing chTERT. Based on their presented data, US FDA gave the green light to conduct further research to develop CBM.

Developing a cell-based fish cell line to meet the growing demand for alternative proteins has several advantages over mammalian and avian CBM approaches. Firstly, fish cells may undergo less senescence and have more doubling with regard to mammalian and avian species (excluding embryonic stem cells) (Klapper et al., 1998; Strecker et al., 2010; Graf et al., 2013; Kim et al., 2018). Secondly, fish cell lines are known for maintaining karyotypic stability with respect to mammalian and avian species (Barman et al., 2014; Fan et al., 2017). Thirdly, a fish cell may easily grow under atmospheric air and possess high intracellular buffering capacity (Bols and Lee, 1991; Rubio et al., 2019). In cell culture bioreactor avian and mammalian cells require carbon dioxide and bicarbonate to control the pH in addition to air, oxygen, and nitrogen (Warner et al., 2015). Managing only three gases (air, oxygen, and nitrogen) is an advantage of fish cells over mammalian cells as it simplifies scale-up challenges and mitigates the issue of CO₂ stripping (Sieblist et al., 2016).

Microcarriers are beads comprising various materials, porosities, and topographies that provide a surface for anchorage to the cells to hold (McKee and Chaudhry, 2017). Microcarriers offer a large surface-to-volume ratio and are perceived as critical for upscaling in CBM. Microcarriers suspended in a medium provide a 3D culture environment.

Since the inception of the microcarrier concept for the culture of adherent cells in 1967, numerous microcarriers have been established and commercialized (Van Wezel, 1967). Generally, microcarriers have been used for the expansion of cells fabricating molecules of interest, such as monoclonal antibodies, vaccines, and proteins (Phillips et al., 2008). In a recent development in the field of cell and gene therapy, emphasis has been given to developing microcarriers for the culture of human stem cells for therapeutic purposes (Cui et al., 2009; Chen et al., 2013; Gümüşderelioğlu et al., 2013; Li et al., 2016).

Microcarriers exploited for CBM production should fulfill the food regulation of the country while proposing optimal topography and surface chemical properties for target cell types. Preferably, microcarriers should be animal-free components to prevent the use of animal products throughout the production of CBM.

Microcarriers may also work as nutrient carriers, such as essential growth factors, amino acids, etc., to meet the satellite cell’s nutrient demand. This can help minimize the number of medium exchange steps and reduce the risk of contamination and cell loss. Successful loading of sol-gel-derived bioactive glass microcarriers in combination with basic FGF-2 and cytochrome c were sustainably released spanning several weeks (Perez et al., 2014). Sustained release and microencapsulation of bioactive molecules are currently given the utmost importance in the food industry (O’Neill et al., 2014; Shishir et al., 2018). The principle may also apply to microcarriers-based cell culture in meat production. Physical parameters like temperature and pH can be tuned to control in vitro release kinetics from loaded microcarriers (Zhou et al., 2018; Matsumoto et al., 2019).

Satellite cells are anchorage-dependent; thereby, these cells require microcarriers surface for the attachment. The attachment of cells is a critical parameter that affects the entire process of yield (Bock et al., 2009). Cell attachment encompasses the association between cell adhesion molecules and substrates on the surface of the microcarriers (Goldmann, 2012). The integrin protein regulates cell adherence (Derakhti et al., 2019). These are heterodimeric glycoproteins comprising a and b subunits, each with various isoforms depending on the expression of isoforms (Rowley et al., 1999). Integrins bind to a diverse class of proteins with different combinations like α₁β₁ has a specific affinity to collagen, α₅β₁ to fibronectin, and α_vβ₃ to vitronectin (Barczyk et al., 2010).

Recently, Norris et al. developed edible microcarriers possessing tunable mechanics as well as a surface topology for CBM (Norris et al., 2022). They are made-up microcarriers employing gelatin and food-grade crosslinking enzyme (transglutaminase). The inexpensive method does not require the application of any synthetic polymeric materials, instead needs small crosslinking agents or modified chemical groups. The scalable process to produce edible microcarriers by exploiting water-in-oil emulsions enables readily fabricating hydrogel microparticles with a spherical shape and smooth surface.

The crucial aspects of the scale-up are bioprocess modeling and optimization, for which spinner flasks act as small-scale systems, especially in defining mass and energy transfer models. Because of this, attempts were made to mimic the 3D environment in the spinner flasks for the animal cell culture by internal mixing (Verbruggen et al., 2018). There are different lab-scale reactors for use in tissue engineering. Biopharmaceuticals are stirred-tank, hollow fiber, roller bottles, rocking bed, fluidized-bed and fixed (packed) bed systems (Odeleye et al., 2020). Vijay Singh first described the rocking bed bioreactor or wave reactor intended for the cultivation of animal cells (Singh, 1999). Wave bioreactor contains a flexible polymeric bag with special ports for allowing us to introduce air, oxygen, or medium or to withdraw samples under sterile conditions. Fluidized bed reactors (FBRs) are rarely applied in tissue culture, but homogenous bed expansion behavior, related to good mass transfer characteristics, as well as lower shear stress, compared to stirred tank reactors and simpler scale-up procedures, makes them advantageous to others in the production of the cultured meat (Ellis et al., 2005). In hollow fiber bioreactors, hollow fibers act as a semi-permeable membrane by allowing the water and nutrients for cell growth while removing metabolic products and serve as a cell immobilization base by not allowing cells to pass through it (Baba and Sankai, 2017).

The large-scale production of cultured meat requires bioreactors. The ultimate bioreactor will have properties like mass transfer, oxygen level, shear stress, and medium flow at optimum levels to produce high output and support the cells with the scaffold. Bioreactors with specific functions are available. Studies have been carried out on bioreactors, like rotating wall vessel bioreactors, direct perfusion bioreactors, and microcarrier-based bioreactors for producing cultured meat. Porous scaffolds that allow media flow through it are used in direct perfusion reactors, and gas exchange will happen in a fluid loop located externally. These reactors maintain the scaffolds-based culture with high mass transfer and great shear stress (Carrier et al., 2002). For the differentiation phase, animal cell immobilization on an edible scaffold or MCs with perfusion mode operation method is a promising approach for the production of CM (Allan et al., 2019; Bellani et al., 2020; Letti et al., 2021). The limitations of these bioreactors are membrane fouling which leads to heterogeneous cell growth, mass transport limitations, and non-uniform nutrient and inhibitor gradients (Yang et al., 2006; Detzel et al., 2010).

In vivo-like conditions are maintained in rotating wall vessel bioreactors by regulating the rotating speed in a row, balances the centrifugal force, drag force, and gravitational force, and finally allowing the 3D culture to be submerged in the medium (van der Weele and Tramper, 2014). This type of reactor bears high mass transfer with reduced shear stress. A well-mixed environment that controls bioprocess conditions precisely, such as pH value, dissolved oxygen concentration, and concentration of nutrients in the cell culture broth, will be provided in Stirred tank reactors (Badenes et al., 2016; Bellani et al., 2020; Hanga et al., 2021). Bovine adipose-derived stem cells are used as precursor cells for both adipose and muscle cells in a single-use, disposable STR vessel (Mobius CellReady 3 L, MilliporeSigma, Burlington, MA, United States) to expand the cells in 100 mL-spinner flask to 3 L-STR resulted in a notable fold increase of 114.19 ± 1.07 for bovine adipose-derived stem cell number (Hanga et al., 2021).

Microcarrier-based reactors support the cells in 3D environments. Microcarriers are used in two ways one is suspension, and the other is packed bed reactors. In the case of packed bed reactors, the medium flow should be continuous and oxygenated before entering the reactor; the limitation of this type of reactor is it is only useful for up to 30 L of volume. In the case of suspension-based microcarriers, the cells should grow on them, and the characteristics of the cell will vary according to the seeding density. The formation of aggregates and shear stress due to agitation are the major problems with these reactors (Moritz et al., 2015).