The creation of lab-grown fish starts with the preparation of the cells. Cells are the smallest building blocks of all living organisms. An ensemble of cells with similar structure and function makes up a tissue (Farley et al., 2012). In fish, cells are isolated from the edible part, which is the skeletal muscle tissue, which strongly affects the quality of the fish flesh. This protein source is composed of highly dense and closely packed muscle fibers. The longitudinal muscle fibers are organized into myofibers, which are produced through the fusion of the primary cells known as myosatellite cells (Figure 2) (Frontera and Ochala, 2015). Myosatellite cells are stem cells capable of developing into skeletal muscle tissue. Therefore, cells should be isolated from the muscle tissue of the target fish type. The isolation and culture conditions of the muscle cells vary depending on the fish type. There are various studies on the isolation of primary muscle cells (Table 1).

Cells alone do not have the ability to develop into functional tissue. Accordingly, a supportive platform has been introduced to guide the cells to mature into a whole tissue. In the field of TE, this platform is called a scaffold (Chan and Leong, 2008). Researchers developing lab grown fish have acquired TE techniques, including the fabrication of scaffolds. The isolated cells are grown on scaffolds to develop 3D-shaped tissues. The characteristics of the scaffold may affect the structure and texture quality of the final product. Therefore, the material and design of the scaffolds should be carefully considered. Figure 3 shows the type of scaffolds used to develop lab-grown meat or fish. Scaffolds are designed in various ways, including fibrous, sponge, mesh, and cell-laden types.

Since cells are essential in lab-grown fish, the cell culture medium is one of the most important factors. The early types of culture medium were composed of a balanced solution with pH, osmolarity, and salt concentration similar to that of bodily fluids (Xu et al., 2020; Yao and Asayama, 2017). Gradually, the culture medium was further developed by adding essential components, including glucose, amino acids, inorganic salts, vitamins, and buffers (Eagle, 1955). This is called the basal medium. However, additional supplements are required for long-term cell culture. The most common constituent is fetal bovine serum (FBS), which is derived from unborn calves at the slaughterhouse (Gstraunthaler, 2003). FBS is obtained from the fetuses of pregnant cows during the slaughter process. Blood is extracted using a vacuum pump, gravity, or massage by inserting a needle into the heart of the fetus, without anesthesia. This process, carried out within 5–35 min after slaughter, results in the fetus's brain function remaining intact due to their higher resistance to anoxia compared to adults. Consequently, it is highly likely that the fetuses survive and experience pain during the blood collection process (Jochems et al., 2002). This action raised ethical concerns related to animal welfare.

Collecting blood from a living fetus during the final third of its development violates EU Directive 2010/63/EU regulations. However, if the fetus is considered already dead prior to blood collection, its death by asphyxiation without anesthesia or euthanasia raises significant animal welfare concerns (Weber and Wagner, 2021). Other problematic issues regarding FBS are the large variability in quality and composition, the possibility of contamination, and the containment of toxic factors (van der Valk, 2022). The composition of FBS is not precisely defined and varies between batches. Furthermore, it is susceptible to contamination by viruses, mycoplasma, and prions. These factors present significant scientific challenges concerning the reproducibility of experiments and the safety of products utilizing FBS (Van der Valk et al., 2004).

Therefore, a replacement is necessary for the development of edible cell-cultured fish. Another reason for the need for an alternative to FBS is to reduce the production cost, which directly affects the price of the final product. The production cost of cultured meat is heavily influenced by the culture medium, which constitutes ~55 to 95% of the total cost. Containing 10–20% FBS, priced at approximately $1,000 per liter, this medium poses a major challenge to the commercial scalability of cultured meat production. Professor Mark Post has estimated that around 50 liters of FBS would be needed to produce a single cultured meat patty weighing 140 grams (Hubalek et al., 2022). As much as 30% of the lab-grown fish production cost can be attributed to the cost of the cell culture media (Garrison et al., 2022). So far, no corporation has formulated a fish cell culture medium with an FBS alternative.

To address these challenges, researchers are exploring alternatives such as plant-based proteins and recombinant proteins. Protein-rich sources, including soybeans, soy, and wheat, present a safe, environmentally sustainable, and nutritionally valuable alternative. Plant-derived peptides serve as an abundant source of amino acids, which are critical for protein synthesis and cellular function. These peptides contribute to enhancing cell growth, performance, differentiation, and sustainability in serum-free media (Amirvaresi and Ovissipour, 2024). However, the sensory characteristics (appearance, aroma, and flavor) are less preferred by consumers, necessitating additional processes to improve these attributes (Rubio et al., 2020).

Recombinant proteins also offer potential solutions. The technology of genetically engineering microorganisms to produce growth factors enables the large-scale production of essential components for cell culture. While this approach is promising, there are potential risks associated with genetic engineering, and further research is required to enhance production efficiency.

An example of these advancements is the use of rapeseed protein isolate (RPI) derived from four types of plants as a replacement for expensive recombinant albumin. This method has shown promise in culturing and proliferating bovine satellite cells at a lower cost, demonstrating the feasibility of plant-based alternatives for serum-free media (Stout et al., 2023).

Skeletal muscle tissues are composed of specialized cells with the ability to shorten or contract, generating movement of the body. Unlike mammalian muscle structure, fish lack the tendon tissue that connects the muscle to the bone. Instead, fish skeletal muscle tissue is directly anchored to the skin and bone. Fish skeletal muscle can be divided into two different types: white muscle and red muscle (Figure 4). The color variation strongly depends on the amount of myoglobin present; red muscle has a high myoglobin content. Myoglobin is the main heme sarcoplasmic protein responsible for oxygen transport. The portion of red muscle in fish only accounts for < 10% of the total muscle mass. In most fish types, the largest red muscle mass is located near the lateral line below the skin. This muscle is recruited during slow to moderate continuous swimming speeds. In contrast to red muscle, white muscle is almost devoid of myoglobin, resulting in reduced oxygen-transport ability. Therefore, white muscle is used for fast, burst swimming, including predation and escape behavior, and fatigues rapidly. The fast muscle tissue corresponds to 70–90% of the entire muscle mass. This part of the fish becomes commercially important since it is the edible portion.

A fish filet is composed of W-shaped myomeres, which are muscle segments divided by myosepta, a connective tissue membrane consisting mainly of collagen fibers (Gemballa and Vogel, 2002; Van Leeuwen, 1999) (Figure 5). The myosepta dissolve easily upon cooking and turn into gelatin, which is why the “flakes” (muscle bundles in the myomere) tend to fall apart after cooking (Bricard et al., 2014) (Figure 6). Each myomere consists of muscle fibers (cells) organized in the form of separate bundles arranged in parallel. Compared to mammals, fish muscle fibers are relatively short. In fish, a single muscle fiber is approximately 20–100 mm in diameter and made up of myofibrils (1–2 mm in diameter). The unit of the myofibril is the sarcomere, which contains two sets of filaments, actin (thin filament) and myosin (thick filament). Not only the contractility of the muscle, but also the texture of fish strongly depends on the myofibrillar proteins, such as myosin and actin (Hatae et al., 1984, 1990; Rayment et al., 1993).

As mentioned above, fish is an essential part of a healthy diet, providing important nutrients, including protein and polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) (Li and Hu, 2009). Fish are the most diverse group among vertebrates, and the nutrient content can vary due to the species of fish, ecological factors (e.g., diet, environment, trophic level, metabolism), and sociocultural factors (e.g., cooking method, preparation method, parts consumed).

The consumer's decision to purchase meat depends on its quality, which can be sensed by factors such as appearance, taste, and freshness. In general, the meat quality is determined by the muscle fibers, intramuscular connective tissue, and intramuscular fat (Listrat et al., 2016). Because of the bonding of muscle fibers by the connective tissue, the softening of the fish meat is highly dependent on the extracellular matrix (ECM) components of the connective tissue, which is mainly collagen. Specifically, collagen type V in the ECM of the connective tissue is solubilized in postmortem fish muscle (Sato et al., 1997). Although the biochemical activity behind this postmortem tenderization is still unclear, Kubota et al. (2001) demonstrated that metalloproteinases are responsible for the postmortem tenderization of fish muscles. For these reasons, the storage method is of great importance.

The conventional method of manufacturing cell-based meat is based on the self-assembly of the cells. When cells are co-cultured in a suitable environment, such as in bioreactors, cells tend to organize and arrange themselves into complex structures without external manipulation. This approach has the potential to enhance the efficiency and scalability of cell-based meat production, making it more commercially viable. While self-assembling techniques show promise, ongoing research is needed to optimize the process and further improve the quality, taste, and nutritional composition of cell-based meat products.

3D bioprinting is an emerging field that holds great potential for various applications in medicine and TE. While significant progress has been made in the bioprinting of human tissues and organs, such as skin, cartilage, and bone, bioprinting of cell-cultured products is still in the early stages of development.

Research on 3D bioprinting of cultured meat has been gaining attention in recent years as a potential solution to address sustainability and ethical concerns associated with traditional livestock farming. However, there have been limited studies on the 3D bioprinting of cell-based fish or meat. Jeong et al. (2022) used a digital light processing-based (DLP) printing technology to produce steak-type cultured meat. Bovine embryonic fibroblasts in the gelatin methacryloyl (GelMA) scaffold were differentiated into muscle cells and adipocytes to resemble real meat. The cultured meat maintained its shape after frying and cutting. In another study, Kang et al. (2021) endeavored to create meat-like tissue with a hierarchical structure resembling natural meat by assembling cell fibers. In this manufacturing process, tendon-gel integrated bioprinting technology was used to fabricate cultured steak composed of muscle, fat, and vascular tissue. This approach allowed for the precise placement of cell fibers to mimic the arrangement found in real meat. According to Ben-Arye et al. (2020), a 3D-printed scaffold composed of soy protein can support bovine satellite cells to become bovine muscle tissue for cell-based meat. Not only provided the 3D scaffold a supportive platform, but it also offered an environment for the cells to grow and organize into muscle tissue.

It is important to note that 3D bioprinting of cultured meat is still in its early stages, and there are many technical and regulatory challenges to overcome. The complexity of mimicking the intricate structure and texture of conventional meat poses significant hurdles. However, ongoing R&D efforts are expected to advance the field and pave the way for the commercial production of cultured meat in the future. Table 2 summarizes the advantages and limitations of conventional methods and 3D bioprinting in the production of cultured meat and fish.

In 2013, a Dutch company called Mosa Meat, led by Dr. Mark Post, showcased the world's first cell-cultured burger created using muscle cells extracted from a cow and grown in a laboratory, marking a significant milestone in the development of cultured meat. In December 2020, the Singapore Food Agency (SFA) became the first regulatory body to approve a cultured meat product, granting Eat Just permission to sell its cell-cultured chicken. This lab-grown chicken nugget, available at Huber's Butchery in Singapore for around $14, underwent a regulatory review process lasting over 2 years.

Since then, progress has continued worldwide. In June 2023, the United States Department of Agriculture (USDA) approved the sale of cell-cultured chicken produced by Upside Foods and Eat Just, representing a major step toward commercialization in the U.S. market. In January 2024, Israel approved a cell-cultured beef product developed by Aleph Farms, expanding global acceptance of cultured meat. Additionally, Vow, an Australian start-up, developed a cultured quail meat product that received approval from the Singapore Food Agency in April 2024, marking Singapore's second approval and the fourth globally for cultured meat.

Despite these advancements, cell-based fish, including cell-based seafood, has not yet received regulatory approval in any country. Research and development efforts continue, and regulatory approval for such products is expected as the technology and frameworks evolve.

According to consulting firm AT Kearney, cell-based meat could substitute 30% of the global meat market by 2030 and increase to 60% by 2040, while the conventional meat supply could drop more than 33%. The Good Food Institute (GFI), a non-profit organization focused on alternative proteins, estimates that the global cultivated meat market could reach $25 billion by 2030. It is important to note that these Figures and projections are subject to change as the market evolves, and various factors can influence the actual market size, including regulatory developments, consumer acceptance, production scalability, and R&D investment. Experts envision that this invisible market could become a reality if price parity and consumer acceptance can be overcome.

The price of a product strongly affects the willingness of customers to buy it and, in turn, consumers' faith in a business enterprise. The cost of cultured meat is expected to decrease over time as the industry progresses and technology advances. Several factors contribute to cost reduction in the production of cultured meat.

To reduce the unit production cost of cultured meat, it is essential to develop large-scale production technologies utilizing bioreactors or microcarriers. Additionally, automation of production processes is necessary to lower labor costs and maximize operational efficiency (Pajčin et al., 2022).

While regulatory milestones, such as approvals by the SFA, USDA, and FDA, mark significant progress, resistance remains. Shortly after the U.S. approvals in 2023, seven states introduced legislation banning cultured meat, citing ethical and economic concerns. Similarly, Italy and France have proposed bills prohibiting the production and sale of lab-grown meat. These contrasting regulatory stances highlight the complex socio-political and cultural challenges facing cultured meat.

Additionally, discussions are ongoing regarding the safety of cultured meat products and their production facilities, which could significantly impact the development and commercialization of the cultured meat industry. For cell-based seafood, current efforts focus on high-value species such as fish maw. Despite cost reductions, production costs remain significantly higher than those of traditional seafood products, limiting commercialization potential (Rubio et al., 2019; Stephens et al., 2018).