The penicillin concentration for tissue decontamination ranges from 50 to 100 IU/mL, streptomycin ranges from 100 to 500 µg/mL, and amphotericin B from 0.25 to 2.5 µg/mL31. These antibiotics were placed into Leibovitz’s 15 medium, phosphate-buffered saline solution , or artificial seawater for washing the tissue samples. Other antibiotics such as ampicillin, gentamycin, and kanamycin have also been used. It is important to note that decontamination might vary depending on the source of the tissues, as some tissues have a higher initial microbial load. For instance, digestive glands, gills, and the mantle are prone to more contamination than the heart or adductor muscle because these organs are primarily involved in filtration. In addition, some marine microbes and parasites carry a symbiotic relationship with the animal, leading to more contamination and making it difficult to find optimal decontamination conditions. Aside from serum-free media needs, environmental factors such as oxygen, salt, pH, osmolarity, and temperature must be optimized. Fish cells are generally adapted to low oxygen environments with hypoxia-response genes. Some fish cells only grow in 5% carbon dioxide, while others utilize anoxic or standard oxygen tension. A comprehensive study on muscle lactate dehydrogenase in warm-water fish and mammalian cells reported significant differences in metabolic activity dependent on pH. Generally, seafood cells grow at lower temperatures than mammalian cells, blueberry packaging making them good candidates for producing cell-cultivated seafood with lower energy inputs. There are different fully defined basal media available for seafood cell culture including Eagle’s Medium, Modified Eagle’s Medium , Medium 199 and Leibowitz’s 15 .
While there have been significant advances in the development of serum-free culture media for mammalian cell lines, there has been limited progress for fish cells. Serum-free media has been achieved for a few fish cell lines in the past, however, these formulations were not well-defined or were proprietary within companies, resulting in significant challenges in broadening their utility for cell-cultivated seafood. Serum-free media containing lactalbumin hydrolysate, trypticase-soy broth, bacto-peptone, dextrose, yeast isolate, polyvinylpyrrolidone, and non-essential amino acids were studied with different fish cell lines , and cell growth and morphology of the cells was similar to those that were grown in serum-containing media. Bioprocessing was utilized to convert different feedstocks including whole oysters , whole mussels , whole lugworms , black soldier flies and crickets to protein hydrolysates for growing fish cells. These hydrolysates were cytotoxic for Zebrafish cells at high concentrations regardless of serum concentration, while, at lower concentrations , all of the hydrolysates supported cell growth. Black soldier fly hydrolysates could replace serum and provided a cost-effective source of peptides. The use of modeling tools also has the potential to foster more rapid identification of key media and related conditions for seafood cell growth and differentiation. For example, through the use of Design of Experiments and/or AI, the development of a serum-free medium can be pursued. Protein hydrolysates from marine byproducts could also provide inexpensive and high quality proteins and amino acids to develop serum-free media.There is limited knowledge on the in vitro differentiation and maturation of fish, crustacean and mollusk cells into fat or muscle tissues.
To screen for myogenesis in mackerel cells as an example, a variety of methods were utilized [e.g., serum starvation, reduced serum, reduced serum plus additives, reduced serum with insulin, 1-oleoyl lysophosphatidic acid and transferrin, reduced serum medium with insulin-like growth factor 1, reduced serum medium plus additives with IGF-1; medium with extracellular signal-regulated kinase inhibitor. Myogenic potential was assessed via RTqPCR using primers based on genome sequences from southern bluefin tuna , myogenin, along with immunohistochemistry. Differentiation via paired-box protein 7 and myosin heavy chain immunostaining was observed in a continuous muscle cell line developed from Atlantic mackerel. The cell line also exhibited an adipocyte-like phenotype, which was confirmed via Oil Red O staining and quantification of neutral lipids. MEF2A, Mrf-4, MyoD and Myf-5 expression was reported in a muscle cell line developed from a freshwater fish during differentiation of muscle cell culture. However, more detailed studies need to be carried out to facilitate selection of the right cell type for cultivated aquatic food development. Images of mackerel cells are provided in Fig. 3.While suspension culture-based approaches may be sufficient for unstructured seafood products like surimi, tissue-like products that replicate some of the complexity of muscle tissue, including texture/mechanics and mouthfeel after cooking and oral mastication, will require more sophisticated methods to impart structure to the final product. A variety of approaches are utilized that mainly rely on scaffolds to facilitate the transport of oxygen, nutrients, and waste products as tissues mature . Approaches to scaffolding and tissue engineering for cell-cultivated meat have been reviewed elsewhere.
The differences in requirements for scaffolds for seafood vs. terrestrial meat can be divided into two broad categories: those related to the cell requirements and those related to the effects of the scaffold on the organoleptic properties of the final product. Because scaffolds play a crucial role in delivering cues to the cells as they proliferate, differentiate, and mature, scaffolds that are appropriate for use with cells from one taxonomic group may not be optimal for another. Therefore, optimization of scaffold stiffness, topography, or surface functionalization may require significant differences between terrestrial animals, fish, and aquatic invertebrates. Scaffolds can also impact the acceptability of the final product due to texture, taste and flavor. For example, the melting temperature of fish collagen differs from that of collagen from terrestrial animals, with important impacts on cooking fish muscle, thus, the thermal properties of scaffolds for cell-cultivated seafood will need to be carefully considered. In addition, the 3D geometry of muscles from terrestrial animals, fish, crustaceans, and mollusks are different and need design considerations with scaffolds for whole cut cell-cultivated products. One of the earliest investigations into cell-cultivated meat or seafood was a NASA-funded study that demonstrated the in vitro expansion of goldfish muscle explants co-cultured with brown bullhead fibroblasts. While research into cell-cultivated seafood over the subsequent two decades has lagged behind that of cell-cultivated terrestrial meat, several recent studies have demonstrated progress. While this is an early example of a scaffold-free cell cultivated seafood prototype, there is precedent for the use of scaffold-free techniques in both academic and commercial efforts at producing cell-cultivated terrestrial meat, but less so for seafood-related goals. Recent advances with scaffold-free alternatives were reported for livestock-derived adipocyte cell cultures in 2D, that could also be applied to seafood cell cultures; the 2D systems were consolidated into 3D tissues via post cell growth aggregation using food grade cross-linking enzymes like transglutaminase or a gelling agent. The use of 3D bio-printing to produce cell-cultivated meat products has been a focus due to the level of control over structure, and this strategy was also applied for the formation of cell-cultivated large yellow croaker prototypes by printing with a bioink consisting of gelatin, alginate, and primary croaker satellite cells into a tissue-like structure. Microcarriers as scaffolds in suspension are also utilized towards cell production goals and scalability in cell-cultivated seafood production, providing large surface/volume ratios. These can have a temporary role or become part of the final product when developed from edible sources . Cells grown in the 2D environment inside or on the surface of the MCs can provide a smooth transition from flasks and bioreactors to finalized 3D tissue outcomes. Different types of marine polymers could be used for cell-cultivated seafood including hydrogels from algal sources, chitosan extracted from marine exoskeletons, blueberry packaging box and gelatin from underutilized species such as jellyfish, fish skin and seafood byproducts, which can also provide specific colors and flavors. In addition, extracellular matrix proteins and lipids can be integrated into the process via scaffolds and can have a significant role in the sensory and textural properties of fish meat. A recent study illustrated that some of the established lipid structure approaches, such as oleogels, could be integrated with cells cultured on microcarriers to form 3D structures simulating meat products. These approaches of combining structured fats with fibrous tissue scaffolds could enable the development of muscle-like fish products, however, there have been few studies reported in the literature to date. Cellular aggregates as self-scaffolding outcomes can also be pursued as a robust option for increasing biomass.Scaling-up using bioreactors for the 3D cell production environment is a major bottleneck for the cell-cultivated meat industry.
Most approaches being pursued are based on variations with stirred tank bioreactors derived from pharmaceutical industry designs, with a focus on cost reductions via simplified designs or those requiring lower energy impacts. These systems apply to cultivated meat and seafood alike. Other approaches generally being pursued in the field include hollow fiber-based bioreactors. In all cases, the costs of scaling are related to media, microcarriers, clean rooms, bioreactor hardware and labor. Innovative approaches will be required to reduce the cost of scaling up. For example, there are many unutilized nutrients and growth factors, which could be recovered and returned to the bioreactor after removing cell metabolites. This could be achieved using different approaches such as growing plants on the spent media to generate additional biomass for use in the production process, utilizing microbial communities for metabolic support to reduce inhibitor byproducts, along with more traditional selective membranes to isolate, recover and re-use key growth factors. Reductions in ammonia can be pursued using microorganisms and chemicals, which can help sustain cultures with reduced media changes or specific nutrient feeding. Glutamine substitutes including α-ketoglutarate , glutamate and pyruvate had a positive impact on cell proliferation and differentiation by reducing the rate of ammonia production. For instance, proliferation media containing αKG improved primary bovine fibro-adipogenic progenitor cell proliferation, while significantly reducing ammonia production rate due to the antioxidative and ammonia scavenging properties of αKG.In the US, both the Food and Drug Administration and the United States Department of Agriculture have established a joint agreement to address cell-cultivated meat and seafood safety and regulations. The FDA oversees cell collection, cell banks, cell growth and differentiation for all the seafood organisms, while the USDA/Food Safety and Inspection Service evaluates the products after harvest onwards for catfish. Codex Alimentarius also recently initiated programs on developing Hazard Analysis Critical Control Point and Good Manufacturing Practices for cell-cultivated meat and seafood. Complementary to regulatory organizations, the Food and Agriculture Organization , and the World Health Organization developed the first comprehensive food safety document that covers cell-cultivated seafood. This document outlines the food safety risks including zoonotic risks from cell lines and the production environment, biological contamination risks from initial cell sources to production, and risks from unwanted residues and novel inputs during production and processing of cell-cultivated meat products. These risk factors are combined with a food safety plan to address the challenges and regulatory requirements of both the FDA and the USDA along each step of cell-cultivated seafood production . Critical Control Points are biological, chemical, allergen and physical issues that need to be used for developing preventive controls. In the cell culture environment, bacteria can rapidly outgrow the animal cells, with additional hazards from other organisms including viruses, prions, fungi, protozoa and parasites. Escherichia coli, Listeria monocytogenes, Salmonella spp., Aeromonas hydrophyla, Vibrio spp., and Mycoplasma spp. are some of the most common bacterial contaminants in foods. Chemicals may be added intentionally or unintentionally to the production process and can pose food safety risks. These chemicals include antibiotics, drugs, sanitizers, cryoprotective agents, leachable chemicals , surfactants, and anti-foaming agents. There are approved chemicals listed by FDA that can be used for cell culture, but for new production processes these potential contaminants will need to be tracked. Physical hazards include objects, debris, plastics, and microplastics. A major issue with seafood is the allergens, with different types of proteins and allergens in fish and shellfish. For example, the major allergens in fish are parvalbumins, while in shellfish, tropomyosin, arginine kinase, and myosin light chain are the main allergens. Cellular aquaculture has the potential to reduce allergenicity in seafood by selectively growing specific cell types to avoid allergenic components. This can also be achieved through genetic modifications, such as using RNA Interference techniques to knock out causative genes. Additionally, the incorporation of food-grade additives like creatine or ethylenediamine tetra-acetic acid into the cell culture media may offer a route to address allergen-related issues by modulating the expression of parvalbumin, thereby reducing allergenicity.Cell-cultivated seafood industries need to comply with preventive controls rules established by the Food Safety Modernization Act .