Edible, animal-derived meat serves as an excellent source of protein and a myriad of vitamins and minerals that are essential for the human diet. With rapid economic and population growth, the demand for meat and other sources of nutrition has increased exponentially worldwide; however, meat production has purported environmental impacts including increased pollution and greenhouse gas emissions, land degradation/water consumption, biodiversity loss, and negative effects on animal welfare.^1,2,3 Questions also remain concerning the sustainability of meat production⁴ and potential adverse effects on human health.⁵

Cultivated meat (i.e., cell-cultured meat) has been promoted as an alternative to the livestock industry to address many of the above dilemmas and touted as a more sustainable, safer, and healthier means of food production.⁶ However, regulatory hurdles must be overcome for cultivated meat to reach the commercial market, as cultivated meat has not yet been concluded safe for consumption in the U.S. The unique safety challenges of cell-cultivated meat include:

• The safety of the components utilized (the raw ingredients and the sources of cells, scaffolds, and bioreactors)
• The introduction of adventitious agents
• The presence of drug/chemical residues in the final food product.

Although implementation of protein alternatives has intensified in recent years, including fermentation and plant-based methods, the pursuit of alternatives to conventional meat has been a source of inspiration for decades. Cultivated meat has a competitive advantage, as it comes from the same source as conventional meat, and the technology to produce these products has grown exponentially in the past decade. In 2013, Dutch pharmacologist Mark Post created the first lab-grown hamburger,⁷ which consisted of over 20,000 thin strands of muscle tissue that took two years to make and cost $330,000.⁸ The first cultivated meat product approved by a regulatory body was Eat Just's cultured chicken by the Singapore Food Agency, with the sale of the Eat Just "chicken" nugget at the restaurant 1880 becoming the world's first commercial sale of cell-cultured meat.⁹ As of 2023, over 70 different companies around the world are working on commercial cultivated meat products.¹⁰

Regulation of Cell-Cultivated Meat

In November 2022, the U.S. Food and Drug Administration (FDA) ruled that cultured chicken cells, produced by the method described in CCC 000002, were safe for human consumption and gave the greenlight to a San Francisco-based startup company, Upside Foods.¹¹ Most recently, in March 2023, FDA also ruled that cultured chicken cells, made by Good Meat Inc. and produced by the method described in CCC 000001, were safe for human consumption.¹² For cultivated meat companies in the U.S., this sets a promising precedent for their prospective cultivated meat products. Moreover, the cost of cultivated meat production has decreased substantially when compared to the original six-figure budget for one cultivated meat burger back in 2013. The estimated cost as of late 2019 is $9.80 for an 8-ounce cultivated meat burger.¹³ Although not quite at the level of being competitive with conventional beef [$3.44 for a beef hamburger as of February 2023, according to the U.S. Department of Agriculture's (USDA's) Economic Research Service], the drastic reduction in price in approximately a decade indicates that cultivated meat products will likely become progressively more affordable to the consumer.

One of the biggest questions regarding the cultivated meat sector is how it will be regulated. Depending on what the food is and where it comes from, there are two regulatory bodies that monitor its safety: FDA and USDA. FDA is tasked with the safety of most conventional foods, including seafood [except Siluriformes (catfish)], in-shell eggs, and ingredients that are added to human food and animal feed; whereas USDA regulates the production of meat, poultry, and egg products. In March 2019, under a Memorandum of Understanding (MOU), FDA and USDA's Food Safety and Inspection Service (FSIS) agreed to jointly oversee food products incorporating cells isolated from livestock.¹⁴ Under this MOU, FDA is responsible for overseeing cell collecting, cell culturing, and premarket consultation on production processes. FSIS leads in the oversight of cell harvesting, packaging, and labeling. FDA and FSIS share oversight of harvesting of cultured cellular material.

Under sections 201(s) and 409 of the U.S. Federal Food, Drug, and Cosmetic Act (FD&CA), any substance that is intentionally added to food is a food additive and is subject to premarket review and approval by FDA through the food additive petition process. However, there is another pathway for regulatory compliance in which a substance is generally recognized, among qualified experts, as having been adequately shown to be safe under the conditions of its intended use. This path to regulatory compliance is known as Generally Recognized as Safe (GRAS). GRAS can be achieved by either establishing an ingredient as having a documented history of use before 1958 or through scientific procedures that establish the safety of the ingredient as determined by qualified experts. The basis for both a food additive petition and a conclusion of GRAS status is the safety profile of the ingredient when used as intended. To establish the safety in use, the following must be addressed: the proposed use, a detailed manufacturing process, compositional and specification analyses, and safety tests, which include in vivo and in vitro toxicity testing, genotoxicity testing, and allergenicity testing.

Food Safety Aspects of Cultivated Meat

Cultivated meat has one advantage over conventional meat, as it does not have the susceptibility of foodborne pathogenic bacteria (i.e., Salmonella, Listeria, Campylobacter, and Escherichia coli) originating from an animal's digestive tract and feces;^15,16 however, the use of cell culture has its own safety considerations.

Genetic Alterations

One of the first things that must be considered is the type of cells utilized and their source(s). Primary cells can be acquired by isolation from the tissue of an animal, which can subsequently be used to produce a cell bank suitable for cultivated meat. Embryonic stem cells (pluripotent), adult stem cells (i.e., mesenchymal stem cells), or induced pluripotent stem cells (IPSCs, cells that are induced to dedifferentiate into a more "stem-like" state) can also be utilized, which can be directed into the desired cell types for the meat product. Often in cell culture, cells become immortalized, which includes overexpression of genes that encode the proteins like telomerase,¹⁷ the introduction of viral genes that deregulate the cell cycle,¹⁸ or by using a hybridoma technology that creates a hybrid with a naturally immortalized cell line.¹⁹ Cells immortalized by either of these methods can be specifically modified to accelerate the development of suitable cell lines, enhance the nutritional and flavor profile, or repress undesired characteristics by implementing genetic engineering.

The perpetual expansion of cell lines can lead to genetic alterations, either through genetic drift or as an inadvertent consequence of manipulation, which can lead to the permanent introduction, removal, or rearrangement of deoxyribonucleic acid (DNA). This can have unintentional consequences, such as cells becoming malignant (i.e., cancerous), through the upregulation of known oncogenes (i.e., c-Myc and Ras) or downregulation of tumor suppressor genes (i.e., p53, retinoblastoma protein, or phosphatase and tensin homolog).^20,21,22

Adventitious Agents

Another major safety consideration with continuous cell expansion is the introduction of adventitious agents (i.e., introduced unintentionally in the media). These include bacteria, fungi, and viruses that come from cell culture media, persistently or latently infected cells, or the environment.²³ A common source of adventitious agents in cell-cultured media is mycoplasma, which are small bacteria that infect 5–35 percent of current cell lines and are pathogenic.²⁴ Another source of adventitious agents from continuous cell expansion is from inadvertent production of bacterial or viral toxins. Often, genetic manipulation is accomplished by utilizing viral vectors such as those from adenoviruses or retroviruses, which can induce or integrate ectopic genes. Viral vectors are often overexpressed in a bacterial expression system and subsequently isolated to be used in these viral expression systems. These sources can introduce virulent proteins that can induce a toxigenic or immunogenic response when consumed.

FSIS has identified controls to mitigate against hazards related to producing conventional meat, which are comparable to certain hazards for producing meat from cell culture (i.e., microbiological contamination), as detailed in 9 CFR § 417.2 (a). The potential induction of random mutations that can produce nonfunctional or deleterious proteins (i.e., prions) is another significant consideration; however, this effect can be mitigated by using fresh vials of cells from cell banks.

Safety of Raw Components

Another question is whether the components used for production of cultivated meat are approved as food grade. The raw ingredients that are used for cell culture media must also be concluded as safe for use in food, as any component of cell culture media has the propensity of becoming a component of the final food product. Section 402 of the FD&CA considers a food adulterated if it contains any substance that is deemed unsafe within the meaning of section 409 of the FDC&A. This means that not only the cultivated meat, but all the ingredients utilized in the production of the final product, must be concluded as safe for their intended use. Cell culture medium is required for cells as it provides nutrients needed for cells to proliferate, which can also be tailored to meet the needs of human health in the final cultivated meat product. In general, cell culture media contains glucose, pyruvate, amino acids, minerals, vitamins, inorganic salts, and other nutrients that are required for most cells to grow and are commonly found in the human diet;²⁵ however, they may or may not be food grade.²⁶ There may also be unique components that are not found in food including fetal bovine serum (FBS), recombinant growth factors, antibiotics and other drugs, or other small molecules often utilized in cell culture media. The residual levels of these components, including any other additional additives, processing aids, and contaminants, must be considered and analyzed.

Many growth factors present in media (i.e., insulin) are degraded in the stomach by proteases and cannot be absorbed into systemic circulation as fully active proteins. However, the presence of digestion-resistant proteins, which can lead to toxicity and allergenicity, is still possible.²⁷ Required specifications for final product residues (heavy metals, natural toxins, agricultural or veterinary chemicals, environmental contaminants) are established for conventional livestock or aquaculture products in many jurisdictions.^28,29 The metabolic fate of these substances when consumed at the levels present in the final product must also be determined. Moreover, under the Delaney clause, the cultivated meat cannot contain any substances that are known to be carcinogenic to animals. However, this applies only to items that are intentionally added to food. If contaminants (i.e., things unintentionally present) are present below the threshold of regulation (TOR) (21 CFR § 170.39), are already consumed in foods at a level equal to or above the level in the final product, or are not considered a safety concern at the level found within the food, then they may be exempt from regulation.

Scaffolds

Along with the cells being able to grow, they must also be able to form a viable tissue that emulates conventional meat in flavor, palatability, and texture. In a biological setting, cells exist within a complex matrix of proteins and other connective molecules known collectively as the extracellular matrix (ECM). Cells interact with and get cues from the ECM, which leads to distinct downstream signaling that tells a cell how to behave and interact/influence neighboring cells. To emulate the microenvironment in which the cultivate meat cells normally grow, scaffolds are utilized. Scaffolds may comprise natural materials such as polysaccharides (i.e., cellulose, chitosan, alginate), proteins (i.e., collagen, gelatin, elastin), synthetic materials (polyethylene glycol and polyacrylamide), or complex natural composites (i.e., mycelium, lignin, decellularized plant tissue).³⁰ Scaffolds may also contain recombinant proteins and/or small molecules present at low concentrations.³¹ These scaffolds must be safe to consume and only contain an acceptable level of residues and contaminants, especially if remaining in the final cultivated meat product, which is usually the case. However, in some instances, the scaffold may be removed from the final product and be considered a processing aid. In both instances, it should be evident that the material in the scaffold is safe to consume.

Microcarriers

Bioreactors are automated, closed systems that contain culture media and proper nutrients, which are maintained at optimal temperature and gas exchange to emulate the native biological conditions. Most cells in cultivated meat require anchorage-dependent growth (as demonstrated above by the necessity of a scaffold) to form the desired cell type and to prevent a type of programmed cell death known as anoikis. Although pluripotent stem cells can be grown in anchorage-independent conditions, they are typically dissociated into single cell suspensions to prevent differentiation or to prevent cell death (i.e., inhibition of Rho kinase).³² Another way to prevent anoikis is by utilizing microcarriers, which are small (100–400 µm diameter) bead-like structures that emulate the ECM and enable cells to attach.

Microcarriers are advantageous in bioreactor culture because they provide a large surface area to volume ratio, permitting high densities of cells relative to 2D culture and can be used in flexible (i.e., batch, fed-batch, perfusion) and controllable bioprocessing pipelines.³³ Expansion of cells using microcarriers occurs either by the addition of more microcarriers where cells undergo bead-to-bead transfer,³⁴ or via enzymatic dissociation and passaging of cells to larger vessels via a seed train process.³⁴ These bioreactors must be massive in size to meet the economic demand, which leads to shear stress that can tear cells apart. Moreover, these bioreactors can produce large amounts of metabolites, growth factors, and other substances at much higher levels than those that are present in an animal. Bioreactors may also induce genetic and epigenetic drift, which can influence protein expression, as well as lead to the induction retrotransposable elements such as endogenous retroviral elements (ERVs) or other species-specific viruses.³⁵ Although the likelihood of these events is very low for biotechnology, the lack of these still need to be confirmed by the use of assays such as polymerase chain reaction (PCR) or chromatin immunoprecipitation (ChIP) assays.³⁵

Conclusion

Major advances have been made in cell culture and biotechnology that have made the use of culturing animal cells into consumable meat feasible. However, major regulatory and safety hurdles still must be overcome to bring these products to market. Cultivated meat manufacturers must critically evaluate the safety of the cell types used, the ingredients utilized, and other small molecules that may become integrated into the final meat product. The path for cultivated meat being provided to the consumer is promising; however, the frontier of cultivated meat commercialization has many regulatory and safety obstacles that each company will need to address to make widespread cultivated meat consumption a reality.

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