The global population continues to expand quickly and is expected to reach nearly 10 billion people by 2050 [1]. Investment in innovations that allow for the expansion of the food sector, while minimizing the social compensation and ecological damages resulting from large-scale food production, will be necessary in order to meet future demands [2]. The current agro-food system has a considerable negative impact on the environment; livestock farming contributes to the occupation of large areas of land, in addition to high greenhouse gas emissions ^[3][4][3,4].

Given the challenges facing conventional meat production, traditional livestock farming is unlikely to meet the increased demand for meat required to sustain an increasing global population [5]. Cultured meat has advantages over other alternative proteins, such as plant-based proteins, algae, and insects. Because it is an animal-based protein, cultured meat can potentially replace animal products directly and thus, for example, overcome the concerns that some consumers may have about the taste and nutrition of plant-based proteins [6]. Therefore, cultured meat presents an exciting alternative dietary protein source due to its practicality, environmental and ecological benefits, and potential economic implications. An additional benefit of cultured meat is that the facilities necessary for its production do not require the extensive ecological conditions and availability of land suitable for grazing of traditional livestock farming. However, the production process necessary for large-scale commercial production will require technological advances, financial investment, regulatory guidance, and favorable market dynamics [2]. Moreover, the current method of meat production is challenged by ethical considerations concerning animal welfare and public health. Because cultured meat is almost exclusively produced in the laboratory, there is a much lower risk of acquiring zoonotic diseases than in conventional meat production practices, and animal welfare is preserved as no animal slaughtering is required for production [7].

Cultured meat production derives from tissue engineering practices used in regenerative medicine. This technology involves culturing precursor cells and their subsequent deposition in an organized and controlled manner so as to allow for the replication of the natural animal tissue under in vitro conditions, which can even be reflected in different cuts of meat. The process begins with a small biopsy from the source animal to obtain the required cells, followed by the proliferation of said cells in the appropriate culture media. These cells will then differentiate into muscle, fat, and connective tissue—products that will then be combined with different biomaterials to engender the 3D-shaped tissue ^[8][9][8,9]. Spinning techniques, cell stratification, and 3D bioprinting have been utilized to improve the construction of cultured tissue and its resultant characteristics, with the primary goal of increasing its similarity to in vivo animal tissue. These techniques can produce highly aligned fibers with a good distribution that resembles animal muscle tissue [10].

Among the 3D tissue production techniques mentioned above, 3D bioprinting has stood out as one of the primary methods for the biofabrication of cultured meat. This technique can improve the distribution of macromolecules and cells within the tissue, generating final products with improved organoleptic properties. This method allows for the precise deposition of cells, micronutrients, technological aids, and biomaterials in predefined locations and shapes and offers several advantages compared to other biofabrication methods ^[9][11][9,11]. The term “biofabrication” refers to products that are generated from the combination of biomaterials, living cells and their bioproducts, and other biomolecules, and that are structured through bioprinting or assembly [12].

Three-dimensional bioprinting is a technique very similar to conventional 3D printing. However, instead of traditional inks and materials, 3D bioprinting uses “bioink” which consists of hydrogels, a combination of biomaterials, cells, and other biomolecules of interest. According to the standard of the American Society for Testing and Materials (ASTM), 3D printing is mainly based on the following processes. In 3D bioprinting, the primary methods are extrusion-based, jetting-based, and vat photopolymerization-based bioprinting [13]. In extrusion-based bioprinting, the bioink is deposited with high precision, obtaining customized 3D structures with good structural integrity due to the continuous deposition of filaments. The entire process of bioprinting is carried out under the control of a computer [14]. Jetting-based bioprinting can produce ink droplets with controllable size and low volume, depositing the ink in specific locations with high precision and without contact. Employing this technique, it is possible to use a variety of biomaterials as well as the incorporation of living cells [15]. Finally, Vat polymerization-based bioprinting is an emerging technology in the biofabrication of scaffolds applied in tissue engineering, used for its high resolution compared to other bioprinting technologies [16].

Nowadays, companies have been contributing to advancing studies related to the biofabrication of cultured meat using the 3D bioprinting technique. For example, 3D Bioprinting Solutions, in partnership with KFC (Kentucky Fried Chicken), plans to produce and market cultured nuggets. 3D Bio-Tissues Ltd (3DBT) has also been partnered with CPI (Independent Center for Technological Innovation) and the United Kingdom government’s High-Value Manufacturing Catapult to improve cell culture media for the cultured meat industry. Japan’s Nissin Food Holdings is partnering with the University of Tokyo to develop printed meat cubes [17]. In addition to cultured meat, a wide variety of food types have been successfully manufactured using 3D printing technology, including chocolate, cakes, and breads [18].

2. Status of Cultured Meat in in the World

Considering that China is the world’s largest consumer of conventional meat ^[19][42], a shortage of traditional meat could soon present a major sustainable development challenge for the country. As domestic meat production is increasing at a rate lower than the demand, China must keep exploring and adopting more effective alternatives ^[20][22]. China’s Ministry of Agriculture and Rural Affairs has included cultured meat and other “future foods” such as plant-based eggs as part of its food security project to overcome this challenge. This strategic initiative could accelerate the country’s regulatory timeline for cultured meat, drive more research and investment in the alternative protein industry, and encourage wider consumer acceptance ^[21][22][23][43,44,45]. India’s leadership in cultured meat may reflect a national policy aimed at further developing the manufacturing sector, namely, the “Make in India” policy. This policy includes new initiatives that stimulate foreign direct investment and protect intellectual property (Creative India), facilitating business and thus promoting the creation of technology-based industrial start-ups (Start-up India) ^[24][46]. Mumbai was the first city in the world to host a research center for laboratory-grown “clean meat”, namely, the Center for Excellence in Cellular Agriculture, based on the culture of animal cells extracted via a painless procedure. The priority of their studies is to promote technological advancement in this industry with the development and optimization of the most relevant cell lines ^[25][47]. Australia has become the latest country to join the emerging laboratory cultured meat industry, with two producers. One producer is Vow Food, a biotechnology start-up based in Sydney that has attracted about USD 20 million in venture capital. Vow Food intends to reproduce meats that are already produced and marketed on a large scale in Australia, with a focus on premium quality ^[26][48]. According to Glen Neal, general manager of risk management and intelligence at FSANZ (Food Standards Australia and New Zealand), culture meat could be on shelves in 2023 ^[27][49]. In Japan, companies are already working to bring cultured meat to local consumers. To ensure the successful integration of the new product into the population, Japan’s Ministry of Health has assembled a team of researchers to investigate its safety. The team will help to advise the authorities on any health risks associated with cultured meat and the regulations that will be needed for this sector ^[28][29][50,51]. Recently, the Israeli biotechnology company “Future Meat” (Jerusalem, Israel) opened its first factory which produces meat grown from chicken-, pork-, and lamb-derived cells ^[25][47]. This achievement marks a major step along the path of technological development for the cultivated meat market, serving as a catalyst to developing the product on an industrial scale. Another Israeli company, “MeaTech 3D Ltd.” (Ness Ziona, Israel), announced the printing of a cultured steak of 104 g made from fat and muscle cells using its own 3D bioprinting technology. It is believed to be one of the largest cultured steaks produced in recent years ^[30][52]. In December 2020, the Singapore Food Agency (SFA) (Singapore) was the first regulatory authority in the world to approve the commercialization of a cultured meat product, after it was considered safe for consumption ^[31][53]. According to a study by Singapore Management University (SMU) (Singapore), conducted with locals and USA consumers, the product was better accepted by Singaporeans than Americans. This greater acceptance by Singaporeans may be a reflection of a local cultural trait called “Kiasuism” which describes a desire to be pioneer when compared to other nations, expressed by a greater acceptance of new foods ^[32][54]. In 2012, the United Nations’ Food and Agriculture Organization (FAO) (Rome, Italy) estimated that global demand for meat would reach 455 million tons by 2050 (increasing 76% compared to 2005) ^[33][55]. This increase in demand, combined with sustainability policies, has been revolutionizing production processes, especially in food chain sectors, as can be seen in the emergence of cultured meat production ^[34][56]. Traditional meat production has some disadvantages when compared to this new technology, such as animal slaughtering, inadequate breeding environments, the transmission of some diseases, and the generation of greenhouse gases (GHG) such as methane (CH₄) and nitrous oxide (N₂O) ^[35][36][57,58].

3. Biofabrication of Cultured Meat

3.1. Animal Source

Despite its benefits, producing biofabrication cultured meat takes time and effort. The first challenge lies in choosing an appropriate animal for the cell biopsy. The selection of the donor animal should not be random, as numerous factors affect satellite cells (adult skeletal muscle stem cells), such as age, sex, and rearing conditions ^[37][59]. Advances in engineering production are required in order to improve cell culture efficiency and scalability and tissue engineering to select biomaterials and form 3D meat products that mimic their animal-derived equivalents. Furthermore, advances in food engineering and nutrition are still needed in order to make a final product palatable, sensorially acceptable, and nutritionally interesting ^[38][60].

3.2. Cells

The main challenge in tissue culture for meat production is the obtention of an ideal cell source since a large number of homogeneous initiator cells is essential to promoting cell proliferation and differentiation effectively ^[39][61]. The process of cell proliferation and expansion is directly related to the industrial scalability of cultured meat production ^[40][62]. Currently, some of the most studied cell lines used in cultured meat production are satellite cells ^[41][42][43][63,64,65], pluripotent stem cells ^[44][66], mesenchymal stem cells (MSCs) ^[45][67], and dedifferentiated fat cells (DFAT) ^[46][68]. Since all the cell lines used in cultured meat are adherent, it is necessary to have an appropriate matrix serving as a support (scaffold) in order that the cells can successfully grow and differentiate ^[47][69]. Hydrogels are widely used as a cellular matrix in bioinks, and they can be adapted to mimic or replace native tissue due to their high water content, mechanical properties, and 3D network structure ^[48][49][70,71]. Bioinks must have specific properties for tissue engineering, such as biodegradability, biocompatibility, printability, high mechanical integrity, chemical stability, non-immunogenicity, non-toxicity, and insolubility in the cell culture medium ^[50][72]. These bioinks are made by combining cells and structural-based biomaterials to biofabricate tissue-like constructs ^[51][52][73,74].

3.3. Biomaterials

It is in this context that biomaterials are applied. Several supporting biomaterials have been used successfully in adipose tissue engineering and may be viable options for cultured meat production. The material used for cultured meat must be biocompatible and biodegradable, safe for consumption, and mimic the texture of traditional meat ^[53][75]. Amongst the different potential biomaterials, biopolymers stand out. This material class has been extensively studied in the biofabrication of cultured meat as they provide a temporary mechanical support structure for cell adhesion and proliferation, simulating the extracellular matrix (ECM) ^[54][76]. Biopolymers can be divided into two categories: the naturals and the synthetics. However, it is essential to note that biopolymers derived from animals should be avoided because one of the principal drivers for cultured meat production is animal welfare ^[55][56][77,78]. For this reason, biopolymers derived from algae and plants have been widely studied for their application in cultured meat production. The main biopolymers derived from algae used in this process are alginate ^{[57][58][59][60]}[79,80,81,82], agarose ^[55][61][77,83] and Konjac gum ^[62][84], and derived from plants are soy protein ^[63][64][65][85,86,87], glutenin ^[66][88], starch ^[67][68][89,90], and bacterial cellulose ^[69][91]. Alginate was the biomaterial with the highest co-occurrence in the network generated from the keywords search, linked with the term “bioink”. This polymer is a natural polysaccharide extracted from brown algae and has been used as a matrix in the 3D cell culture, providing support for cell growth and integration ^[70][92]. Alginate is also widely used in the food industry as an additive ^[71][93]. Due to its capacity to produce hydrogels via ionic crosslinking, alginate is one of the most promising and most applied natural biopolymers in tissue engineering ^[72][73][94,95] and, consequently, in the biofabrication of cultured meat. However, pure alginate lacks functional groups that stimulate cell adhesion and proliferation. It also has low mechanical properties ^[60][74][82,96]. Alternative studies have shown that combining alginate with other biomaterials—for example, collagen and gelatin—revealed promising results, mainly improving cell adhesion and proliferation. However, the use of animal-derived materials is contrary to the concept of cultured meat, where the main purpose is to avoid the slaughter of animals. Therefore, studies using biomaterials from other sources, such as plants, algae, and microorganisms, have been conducted ^[62][84]. Ianovic et al. ^[75][33] used the 3D printing technique to print scaffolds using modified alginate-based bioinks, enriched with isolated pea and soy protein. The bioinks enriched with pea and soy proteins showed better stability and rigidity than those produced with modified alginate, increasing muscle cell proliferation by up to 90%. These results may be of great interest in the field of cultured meat biofabrication.

3.4. 3D Bioprinting

Three-dimensional bioprinting is one of the most promising technologies with which to biofabricate scaffolds for tissue engineering, both for regenerative medicine and for the food sector, and especially with respect to the biofabrication of cultured meat ^[76][97]. The main 3D bioprinting techniques used for the biofabrication of scaffolds loaded with cells are extrusion, jetting, and Vat photopolymerization. The extrusion-based bioprinting technique can be pneumatically driven (air pressure) or mechanically driven (piston or screw). This technique is the most commonly used for various applications in tissue engineering due to its versatility and wide availability ^[77][98]. The main advantages of the extrusion-based method are its scalability and ability to print a wide range of high-viscosity materials and high cell concentrations ^[78][99]. However, high-viscosity fluids are the most suitable for this technique, providing adequate mechanical support for 3D scaffolds ^[79][100]. The jetting-based bioprinting technique is capable of printing biological materials with optimized speed, accuracy, and resolution ^[80][101]. Despite its advantages, this technique can only be used for low-viscosity materials, which leads to a tendency towards nozzle clogging ^[81][102]. In vat photopolymerization-based bioprinting, noted for its stereolithography technique, it is possible to obtain 3D scaffolds with high speed and high resolution ^[37][59]. This technique is based on the polymerization of light-sensitive polymers. UV radiation used in conventional stereolithography can be harmful to cells and can trigger mutations ^[82][103]. In common, these 3D bioprinting techniques have limitations according to the properties of the bioinks, such as their viscosity, directly impacting the bioprinting quality of the material ^[79][83][84][100,104,105]. Despite the numerous advantages of 3D bioprinting, the success of this technology depends directly on a few factors, including the biomaterial properties, the crosslinking process, print fidelity, stability, and cell viability, as well as the physical, mechanical, and rheological properties of bioinks ^[85][86][106,107]. One of the most challenging properties in the 3D bioprinting process is cell viability. In addition to the technical challenges, 3D bioprinting presents ethical issues that need to be addressed, such as intellectual property issues, safety regulations, and concerns about misuse of the technology ^[87][108]. In the extrusion-based bioprinting process, the bioink is extruded through a needle under pressure, which can cause shear stresses between the bioink and the wall of the printing nozzle. If these stresses exceed a specific limit, they can rupture cell membranes, causing reduced cell viability ^[88][109]. Chand et al. ^[89][110] used computational simulation to evaluate the maximum shear stress during the extrusion-based bioprinting process. They concluded that in addition to the shear stress that normally occurs in the printing process, the geometry of the nozzle also influences cell viability. In the jetting-based bioprinting process, there are few detailed studies on the impact of droplet velocity and volume on cell viability. Ng et al. ^[90][111] used a thermal inkjet bioprinting system to evaluate the influence of drop impact speed and drop volume on cell viability and proliferation. They observed that increasing the concentration of cells in the bioink resulted in slower droplet impact speed, providing greater cell viability and improved print quality by reducing droplet splatter. It was concluded that due to the limited use of biomaterials compatible with the method, in addition to its photopolymerization process, the control of impact velocity and droplet evaporation directly influences cell proliferation and viability [16]. The cross-linking process is another fundamental factor for 3D bioprinting and is directly related to the mechanical properties of 3D structures, as well as to the gelation kinetics, post-print structure fidelity, and viability of encapsulated cells ^[91][112]. The most common cross-linking mechanisms applied in the 3D bioprinting field of bioinks are ionic cross-linking, UV photopolymerization, and physical cross-linking ^[92][113]. Ionic cross-linking occurs between polymer chains and oppositely charged divalent or multivalent ions. A typical example of ionic crosslinking is the electrostatic interaction between negatively and positively charged alginate chains with Ca²⁺ ions ^[93][114]. Photocrosslinking has been widely used in 3D bioprinting as it provides spatial and temporal control over the gelation of bioinks ^[94][115]. Ultraviolet light sources are most commonly used in the photocrosslinking of bioprinted materials ^[95][116]. Usually, photoinitiators are used, and their choice is fundamental in the bioprinting of bioinks. The absorption peak of the photoinitiator is related to the wavelength used. The wavelength has a direct impact on cell viability bioprinted bioinks. Therefore, depending on the wavelength, harm can be done to a cell’s nuclear DNA, causing genomic and carcinogenic mutations ^[96][117]. To overcome the effects of UV light on cell viability, researchers have investigated the use of visible light in photocrosslinking. Normally, visible light does not impact the biological conditions of bioprinted scaffolds ^[97][118]. Finally, in physical crosslinking, no crosslinking agent is incorporated into the reaction. This is an advantage of physical crosslinking, as the use of crosslinking agents can affect the integrity of the substances ^[98][119], in addition to their potential cytotoxicity to living cells. The main parameters that can promote physical crosslinking are pH variation, temperature, thermodynamics, and physical–chemical interactions: for example, hydrophobic interactions, hydrogen bonding, and charge interaction ^[99][120].

Biofabrication of Cultured Meat Using 3D Bioprinting

Three-dimensional bioprinting technology allows for the deposition of bioinks in a layer-by-layer manner, with precise control of the functional component deposition and spatial arrangement ^[100][121]. Three-dimensional bioprinting has several advantages in the biofabrication of cultured meat, such as the possibility of meeting specific needs in terms of calories, nutrients, shape, texture, and flavor of the product ^[101][122]. The texturing and flavoring of cultured meat comes from two essential steps in the production process: the first phase occurs before the 3D bioprinting process, and consists of cell proliferation, commonly performed in a bioreactor, aiming at the obtention of the maximum possible number of cells; the second phase takes place after 3D bioprinting, in which the bioprinted scaffolds already containing the cells are taken to the bioreactor for cell differentiation and tissue maturation ^[102][103][123,124].

3.5. Bioreactor

The bioreactor promotes a favorable environment for cell expansion and supports the diffusion of nutrients through the pores of the scaffolds ^[104][125]. It allows for large-scale cell culture with filtering and replacement medium process throughout the proliferation stage ^[105][126]. In recent years, bioreactors have been developed to monitor and mechanically stimulate cell growth. Cacopardo and Ahluwalia ^[106][127] redesigned a bioreactor to reproduce the mechanical changes of a liver microenvironment and, at the same time, to monitor the mechanical properties of cell-loaded gelatin hydrogels in vitro. The authors observed that increases in scaffold elasticity are related to an increase in cellular stress. According to the authors, this study can be implemented to model other processes, and thus evaluate the parameters that influence tissue stiffness, the complex system of cell division, ECM differentiation and synthesis, and density and crosslinking. Todros et al. ^[107][128] designed a bioreactor for the radial stimulation of porcine-derived diaphragmatic scaffolds with the aim of promoting cell alignment and the development of radially oriented muscle fibers. Regarding the biofabrication of cultured meat, cell and fiber alignment influences the quality of the meat product, mainly with respect to its texture. Therefore, bioreactors with monitoring and mechanical stimulation represent a positive development in the field of cultured meat, inspiring other researchers.

4. Technological Challenges

The three-dimensional bioprinting of food is one of the emerging technologies of Industry 4.0, enabling the production of on-demand, complex, and personalized food ^[108][129]. This technology has several advantages in terms of health, economy, and the environment, with the potential to revolutionize the manufacturing of alternative food sources. Through the use of this technology, an improvement in the functional and nutritional properties of printed foods is expected with the advent of Industry 4.0 innovations, thus increasing the chance of consumer acceptance ^[109][130]. While 3D bioprinting is a promising technology in the biofabrication of cultured meat, it is still at an early stage, with many challenges still to overcome, as it requires non-animal-derived scaffold compositions that are both printable and edible ^[75][33], in addition to challenges related to regulation, scalability, acceptance, and cost. Another much discussed point is the continuous supply of cells and/or tissues obtained from animals via biopsy and the methods involved ^[110][131]. In addition, the use of fetal bovine serum (FBS) in cell culture media is contrary to the basic principle of cultured meat. It is necessary to find reliable alternatives to FBS, ensuring sustainability and ethical development ^[37][59]. The prospect is that in the future, with viable biotechnological alternatives and regulation of the technology, the biofabrication of cultured meat via 3D bioprinting will be scalable, sustainable, and efficient, with the expectation that cultured meat will have texture and flavor characteristics reminiscent of, or even better than, natural meat, as well as being more economically viable.

5. Acceptance and Regulation

In addition to the technical challenges to be overcome in the biofabrication of cultured meat, such as with respect to the appearance, structure, texture, flavor, and nutritional composition of cultured meat ^[111][132], cultured meat production must consider several social issues, including consumer appeal and acceptance ^[112][113][133,134]. Acceptance of cultured meat has been the subject of studies in several countries ^{[114][115][116]}[135,136,137]. Pakseresht, Kaliji, and Canavari ^[117][138] performed a systematic review which aimed to identify, evaluate, and summarize the empirical results of published studies, providing a broad overview of recent empirical evidence on consumer acceptance of cultured meat. For the review, the authors considered the WoS, Science Direct, and Scopus databases throughout 2008–2020. After screening, 43 articles met the selection criteria defined by the authors. They concluded that the most important factors influencing consumer acceptance/rejection of cultured meat include public awareness, perceived naturalness, and food-related risk perception. Ethical and environmental concerns have made consumers willing to pay higher prices to purchase meat substitutes, but not necessarily cultured meats. In addition, food neophobias related to safety, health, and uncertainty also appear to be important barriers to wide acceptance of this technology. Another challenge facing the production of cultured meat that was evident is the regulation of this product. Regulatory approaches differ substantially between countries and continents ^[118][139]. The Good Food Institute (GFI) has encouraged the development of alternative food sources among research groups and companies from different countries ^[119][140]. The GFI surveyed countries that are using existing food regulations or developing new regulations to evaluate novel products such as cultured meat ^[120][141]. Details of this survey by country and region are presented in Table 1. There is still no definitive regulation for the production and marketing of cultured meat. Many regulatory issues still need to be addressed through legislation and policies, such as food fraud and mislabeling. In recent years, administrative and public bodies have promoted debates and meetings with the aim of structuring appropriate regulation ^[121][142].