Cultured meat
Cultured meat (also known by other names, see below) is a meat produced by "in vitro" cell cultures of animal cells. It is a form of cellular agriculture, with such agricultural methods being explored in the context of increased consumer demand for protein. Cultured meat is produced using tissue engineering techniques traditionally used in regenerative medicines. The concept of cultured meat was introduced to wider audiences by Jason Matheny in the early 2000s after he co-authored a paper on cultured meat production and created New Harvest, the world's first nonprofit organization dedicated to "in-vitro" meat  research. Cultured meat may have the potential to address substantial global problems of the environmental impact of meat production, animal welfare, food security and human health. Specifically, it can be thought of in the context of the mitigation of climate change. In 2013, professor Mark Post at Maastricht University pioneered a proof-of-concept for cultured meat by creating the first hamburger patty grown directly from cells. Since then, other cultured meat prototypes have gained media attention: SuperMeat opened a farm-to-fork restaurant called "The Chicken" in Tel Aviv to test consumer reaction to its "Chicken" burger, while the  world's first commercial sale of cell-cultured meat occurred in December 2020 at the Singapore restaurant "1880", where cultured meat manufactured by the US firm Eat Just was sold. While most efforts in the space focus on common meats such as pork, beef, and chicken which comprise the bulk of consumption in developed countries, some new companies such as Orbillion Bio have focused on high end or unusual meats including Elk, Lamb, Bison, and the prized Wagyu strain of beef. Avant Meats has brought cultured grouper fish to market as other companies have started to pursue cultivating additional fish  species and other seafood. The production process is constantly evolving, driven by multiple companies and research institutions. The applications of cultured meat have led to ethical, health, environmental, cultural, and economic discussions. In terms of market strength, data published by the non-governmental organization Good Food Institute found that in 2021 cultivated meat companies attracted $140 million in Europe alone. Nomenclature. Besides "cultured meat", the terms "healthy meat", "slaughter-free meat", "in vitro" "meat", "vat-grown meat", "lab-grown meat", "cell-based meat", "clean meat", "cultivated meat" and "synthetic meat" have been used to describe the product. "Artificial meat  is occasionally used, although that specific term has multiple definitions. Between 2016 and 2019, clean meat" gained traction. The Good Food Institute (GFI) coined the term in 2016, and in late 2018, the institute published research claiming that use of "clean" better reflected the production process and benefits. By 2018 it had surpassed "cultured" and ""in vitro"" in media mentions and Google searches. Some industry stakeholders felt that the term unnecessarily tarnished conventional meat producers, continuing to prefer "cell-based meat" as a neutral alternative. In September 2019, GFI announced new research which found that the term "cultivated meat" is  sufficiently descriptive and differentiating, possesses a high degree of neutrality, and ranks highly for consumer appeal. A September 2021 poll by GFI, indicated that the majority of industry CEOs have a preference for "cultivated meat", with 75 percent of 44 companies preferring it. History. Initial research. The theoretical possibility of growing meat in an industrial setting has long captured the public imagination. In a 1931 essay published by various periodicals and later included in his work "Thoughts and Adventures", British statesman Winston Churchill wrote: "We shall escape the absurdity of growing a whole chicken to eat the breast or wing , by growing these parts separately under a suitable medium." In the 1950s, Dutch researcher Willem van Eelen independently came up with the idea for cultured meat. As a prisoner of war during the Second World War, Van Eelen suffered from starvation, leading him to be passionate about food production and food security as an adult. He later attended the University of Amsterdam. At one point he attended a lecture discussing the prospects of preserved meat. Coupled with the discovery of cell lines earlier in the century, this enriched the idea of cultivated meat. "In vitro" cultivation of muscle fibers was  first performed successfully in 1971 when pathology professor Russel Ross cultured guinea-pig aorta. In 1991, Jon F. Vein of the United States filed for, and ultimately secured, patent for the production of tissue-engineered meat for human consumption, wherein muscle and fat would be grown in an integrated fashion to create food products. In 2001, University of Amsterdam dermatologist Wiete Westerhof, researcher and businessperson Willem van Eelen, and businessperson Willem van Kooten announced that they had filed for a worldwide patent on a process to produce cultured meat. In the process, a matrix of collagen is seeded with muscle  cells, which are then bathed in a nutritious solution and induced to divide. That same year, NASA began conducting experiments on cultured meat, with the intent of allowing far-traveling astronauts to grow meat without sacrificing storage. In partnership with Morris Benjaminson of Turro College, they were able to cultivate pieces of goldfish and later, turkey. In 2003, Oron Catts and Ionat Zurr of the Tissue Culture and Art Project and Harvard Medical School exhibited in Nantes a "steak" a few centimeters wide, grown from frog stem cells, which was cooked and eaten. The goal of the exhibition was to  start a conversation surrounding the ethics of cultured meat — "was it ever alive?", "was it ever killed?", "is it in any way disrespectful to an animal to throw it away?" In the early 2000s, American public health student Jason Matheny traveled to India and visited a chicken factory farm. From a public health perspective, he was appalled by the implications of this system for human consumers. Upon returning to the United States, Matheny teamed up with three scientists involved in NASA's effort to culture meat. The four commenced research on cultured meat. They published their findings in 2005, the  first peer-reviewed literature on the subject. In 2004, Matheny founded New Harvest, which aims to encourage development in the field by funding public research. In 2008, PETA offered a $1 million prize to the first company to bring cultured chicken meat to consumers by 2012. The contestant was required to complete two tasks before receiving the prize: "Produce a cultured chicken meat product that was indistinguishable from real chicken," and "Produce the product in large enough quantities to be competitively sold in at least 10 states." The contest was later extended until 4 March 2014. Since the challenge  was first announced in 2008, researchers around the world have made significant progress, although nothing has reached the mass market. The deadline eventually expired without a winner; however, the publicity around the topic brought cultured meat further into the eyes of scientists and animal welfare groups. In 2008, the Dutch government invested $4 million into experiments regarding cultured meat. The In Vitro Meat Consortium, a group formed by international researchers, held the first international conference on the matter, hosted by the Food Research Institute of Norway in April 2008. "Time" magazine declared cultured meat production to be one of  the 50 breakthrough ideas of 2009. In November 2009, scientists from the Netherlands announced they had managed to grow meat using cells from a live pig. "The Netherlands has always been a leader in food systems innovation and there is a real sense of pride in cultivated meat as a Dutch invention," Robert E. Jones of the Maastricht-based cellular agriculture company Mosa Meat has publicly remarked, looking back at the concept's history. First public trial. The first cultured beef burger patty was created by Mark Post at Maastricht University in 2013. It was made from over 20,000 thin  strands of muscle tissue, cost over $300,000 and needed 2 years to produce. The burger was tested on live television in London on 5 August 2013. It was cooked by chef Richard McGeown of Couch's Great House Restaurant, Polperro, Cornwall, and tasted by critics Hanni Rützler, a food researcher from the Future Food Studio, and Josh Schonwald. Rützler stated, "There is really a bite to it, there is quite some flavour with the browning. I know there is no fat in it so I didn't really know how juicy it would be, but there is quite some  intense taste; it's close to meat, it's not that juicy, but the consistency is perfect. This is meat to me... It's really something to bite on and I think the look is quite similar." Rützler added that even in a blind trial she would have taken the product for meat rather than a soya copy. Industry development. Between 2011 and 2017, many cultured meat startups were launched. Memphis Meats (now Upside Foods), a Silicon Valley startup founded by a cardiologist, launched a video in February 2016, showcasing its cultured beef meatball. In March 2017, it showcased chicken  tenders and duck a l'orange, the first cultured poultry shown to the public. Memphis Meats was later the subject of the 2020 "Meat the Future" documentary. An Israeli company, SuperMeat, ran a viral crowdfunding campaign in 2016, for its work on cultured chicken. Finless Foods, a San Francisco-based company working on cultured fish, was founded in June 2016. In March 2017 it commenced laboratory operations. Director Mike Selden said in July 2017 to expect cultured fish products on the market by the end of 2019. In March 2018, Eat Just (in 2011 founded as Hampton Creek in San Francisco , later known as Just, Inc.) claimed to be able to offer a consumer product from cultured meat by the end of 2018. According to CEO Josh Tetrick the technology was already there. JUST had about 130 employees and a research department of 55 scientists, where cultured meat from poultry, pork and beef was researched. JUST has received investments from Chinese billionaire Li Ka-shing, Yahoo! co-founder Jerry Yang and according to Tetrick also by Heineken International and others. Dutch startup Meatable, consisting of Krijn de Nood, Daan Luining, Ruud Out, Roger Pederson, Mark Kotter and Gordana Apic among others , reported in September 2018 that it had succeeded in growing meat using pluripotent stem cells from animal umbilical cords. Although such cells are reportedly difficult to work with, Meatable claimed to be able to direct them to behave to become muscle or fat cells as needed. The major advantage is that this technique bypasses fetal bovine serum, meaning that no animal has to be killed to produce meat. That month, an estimated 30 cultured meat startups operated across the world. Integriculture is a Japan-based company working on their CulNet system. Competitors included England based Multus Media and Canadian Future  Fields. In August 2019, five American startups announced the formation of the Alliance for Meat, Poultry & Seafood Innovation (AMPS Innovation), a coalition seeking to work with regulators to create a pathway to market for cultured meat and seafood. The founding members include Eat Just, Memphis Meats, Finless Foods, BlueNalu, and Fork & Goode. Similarly in December 2021, a group of 13 European and Israeli companies (Aleph Farms, Bluu Biosciences, Cubiq Foods, Future Meat, Gourmey, Higher Steaks, Ivy Farm, Meatable, Mirai Foods, Mosa Meat, Peace of Meat, SuperMeat, and Vital Meat) established Cellular Agriculture Europe, a Belgium-based association that sought to  'find common ground and speak with a shared voice for the good of the industry, consumers, and regulators'. In 2019, the Foieture project was launched in Belgium with the goal of developing cultured foie gras (the name is a portmanteau of 'foie' and 'future') by a consortium of 3 companies (cultured-meat startup Peace of Meat, small meat-seasoning company Solina, and small pâté-producing company Nauta) and 3 non-profit institutes (university KU Leuven, food industry innovation centre Flanders Food, and Bio Base Europe Pilot Plant). Peace of Meat stated in December 2019 that it intended to complete its  proof of concept in 2020, to produce its first prototype in 2022, and to go to market in 2023. That month, the Foieture project received a research grant of almost 3.6 million euros from the Innovation and Enterprise Agency of the Flemish Government. In May 2020, Peace of Meat's Austrian-born cofounder and scientific researcher Eva Sommer stated that the startup was then able to produce 20 grams of cultured fat at a cost of about 300 euros (€15,000/kg); the goal was to reduce the price to 6 euros per kilogram by 2030. Piece of Meat built  two laboratories in the Port of Antwerp. In late 2020, MeaTech acquired Peace of Meat for 15 million euros, and announced in May 2021 that it would build a new large-scale pilot plant in Antwerp by 2022. In 2019, Aleph Farms collaborated with 3D Bioprinting Solutions to culture meat on the International Space Station. This was done by extruding meat cells onto a scaffold using a 3D printer. In January 2020, "Quartz" found around 30 cultured meat startups, and that Memphis Meats, Just Inc. and Future Meat Technologies were the most advanced because they were building pilot plants. According  to "New Scientist" in May 2020, 60 start-ups were developing cultured meat. Some of these were technology suppliers. Growth media reportedly still cost "hundreds of dollars per litre, but for clean meat production to scale this needs to drop to around $1 a litre." In June 2020, Chinese government officials called for a national strategy to compete in cultured meat. In November 2020, Indian start-up Clear Meat claimed it had managed to cultivate chicken mince at the cost of only 800–850 Indian rupees (US$10.77–11.44), while a slaughtered processed chicken cost about 1000 rupees. Market entry. In  the European Union, novel foods such as cultured meat products have to go through a testing period of about 18 months during which a company must prove to the European Food Safety Authority (EFSA) that their product is safe. In November 2020, SuperMeat opened a 'test restaurant' in Ness Ziona, Israel right next to its pilot plant; journalists, experts and a small number of consumers could book an appointment to taste the novel food there, while looking through a glass window into the production facility on the other side. The restaurant was not yet fully open to the public, because  as of June 2021 SuperMeat still needed to wait for regulatory approval to start mass production for public consumption, and because the Covid-19 pandemic restricted restaurant operations. On 2 December 2020, the Singapore Food Agency approved the "chicken bites" produced by Eat Just for commercial sale. It marked the first time that a cultured meat product passed the safety review (which took 2 years) of a food regulator, and was widely regarded as a milestone for the industry. The chicken bits were scheduled for introduction in Singaporean restaurants. Restaurant "1880" became the first to serve cultured meat to customers on  Saturday 19 December 2020. In March 2022, cultured meat producers had reached the level of attempting to gain regulatory approval from European Union supranational institutions coming just before mass goods could be sold to consumers. Companies. Note: dates in italics refer to projected dates of achievement in the future; they may shift. Aside from these companies, non-profit organisations such as New Harvest, the Good Food Institute, ProVeg International and the Cellular Agriculture Society advocate for, fund and research cultured meat. Impossible Foods markets the Impossible Burger, which contains heme that the company claims gives the burger its bloody look  and taste. Their soy leghemoglobin is produced by taking the soybean gene that encodes the heme protein and transferring it to yeast. Pilot plants. Note: data in italics refer to unfinished projects or projected capacities in the future; they may shift. Process. Cell lines. Cellular agriculture requires cell lines, generally stem cells. Stem cells are undifferentiated cells which have the potential to become many or all of the required kinds of specialized cell types. Totipotent stem cells have the capacity to differentiate into all the different cell types found within the body. Pluripotent stem cells can mature into all cell  types save those in the placenta, and multipotent stem cells can differentiate into several specialized cells within one lineage. Unipotent stem cells can differentiate into one specific cell fate. While pluripotent stem cells would be an ideal source, the most prominent example of this subcategory is embryonic stem cells which — due to ethical issues — are controversial for use in research. As a result, scientists have developed induced pluripotent stem cells (iPSCs) — essentially multipotent blood and skin cells that have been regressed to a pluripotent state enabling them to differentiate into a greater range of cells. The alternative is using multipotent  adult stem cells that give rise to muscle cell lineages or unipotent progenitors which differentiate into muscle cells. Favourable characteristics of stem cells include immortality, proliferative ability, unreliance on adherence, serum independence and easy differentiation into tissue. However, the natural presence of such characteristics are likely to differ across cell species and origin. As such, "in vitro" cultivation must be adjusted to fill the exact needs of a specific cell line.  With regards to immortality, cells have a limit on the number of times they can divide that is dictated by their telomere cap — supplementary nucleotide bases added to the  end of their chromosomes. With each division, the telomere cap progressively shortens until nothing remains, in which case the cell ceases to divide. By inducing pluripotency, the telomere cap can be lengthened such that the cell divides indefinitely. Cell lines can be collected from a primary source, i.e., through a biopsy on an animal under local anesthesia. They could also be established from secondary sources such as cryopreserved cultures (cultures frozen after previous research). Growth medium. Once cell lines are established, they are immersed in a culture media to induce them to proliferate. Culture media are typically formulated from basal  media that provide cells with necessary carbohydrates, fats, proteins and salts. Once a cell consumes a sufficient amount, it divides and the population increases exponentially. Culture media can be supplemented with additives — for instance sera — that supply additional growth factors. Growth factors can be secreted proteins or steroids that are crucial in regulating cellular processes. Once differentiation begins, muscle fibres begin to contract and generate lactic acid. Cells' ability to absorb nutrients and proliferate in part depends on the pH of their environment. As lactic acid accumulates within the media, the environment will become progressively more acidic and falls below  the optimal pH. As a result, culture media must be frequently refreshed. This helps refresh the concentration of nutrients from the basal media. Scaffold. In the case of structured meat products — products that are characterized by their overall configuration as well as cell type — cells must be seeded to scaffolds. Scaffolds are essentially molds meant to reflect and encourage the cells to organize into a larger structure. When cells develop "in vivo", they are influenced by their interactions with the extracellular matrix (ECM). The ECM is the 3-dimensional mesh of glycoproteins, collagen and enzymes responsible for transmitting mechanical and  biochemical cues to the cell. Scaffolds need to simulate the characteristics of the ECM. Key properties: Porosity. Pores are minute openings on the surface of the scaffold. They can be created on the surface of the biomaterial in order to release cellular components that could interfere with tissue development. They also help diffuse gas and nutrients to the innermost layers of adherent cells which prevents developing a "necrotic center" (created when cells that are not in direct contact with the culture medium die due to a lack of nutrients). Vascularization. Vascular tissue found in plants contains the organs responsible for  internally transporting fluids. It forms natural topographies that provide a low cost way to promote cell alignment by replicating the natural physiological state of myoblasts. It may also help with gas and nutrient exchange. Biochemical properties. A scaffold's biochemical properties should be similar to those of the ECM. It must facilitate cell adhesion through textural qualities or chemical bonding. Additionally, it must produce the chemical cues that encourage cell differentiation. Alternatively, the material should be able to blend with other substances which have these functional qualities. Crystallinity. The degree of a material's crystallinity determines qualities such as rigidity . High crystallinity can be attributed to hydrogen bonding which in turn increases thermal stability, tensile strength (important for maintaining the scaffold's shape), water retention (important for hydrating the cells) and Young's modulus. Degradation. Certain materials degrade into compounds that are beneficial to cells, although this degradation can also be irrelevant or detrimental. Degradation allows easy removal of the scaffold from the finished product leaving only animal tissue — thereby increasing its resemblance to "in vivo" meat. This degradation can be induced by exposure to certain enzymes which do not impact the muscle tissue. Edibility. If scaffolds are unable to  be removed from the animal tissue, they must be edible to ensure consumer safety. It would be beneficial if they were to be made out of nutritious ingredients. Since 2010, academic research groups and companies have emerged in order to identify raw materials that have the characteristics of suitable scaffolds. Cellulose. Cellulose is the most abundant polymer in nature and provides the exoskeletons of plant leaves. Due to its abundance, it can be obtained at a relatively low cost. It is also versatile and biocompatible. Through a process called "decellularization", it is coated in a surfactant that creates pores. These  pores release the plant's cellular components, and it becomes decellularized plant tissue. This material has been extensively studied by the Pelling and Gaudette Groups at University of Ottawa and Worcester Polytechnic Institute, respectively. Through cross-linking (forming covalent bonds between individual polymer chains to hold them together) the plant tissue's mechanical properties can be changed so that it more closely resembles muscle tissue. This can also be done by blending plant tissue with other materials. On the other hand, decellularized plant tissue typically lacks mammalian biochemical cues, so it needs to be coated with compensatory functional proteins. C2C12  growth was not shown to change significantly between the bare scaffold and the same scaffold with a coating of collagen or gelatin proteins, however seeding efficiency (rate at which cells attach to the scaffold) improved. An advantage of decellularized plant tissue is the natural topography afforded by the leaf vasculature. This helps replicate the natural physiological state of the myoblasts which promotes cell alignment. The other ways of doing this are usually quite a bit more expensive including 3d printing, soft lithography and photolithography. Vascularization can also help overcome the 100–200 nm diffusion limit of culture medium into cells that  usually produce necrotic centres in muscle conglomerates. Another way to do this is by having a porous scaffold which supports angiogenesis (the development of new blood vessels). While this has been shown to work for Apple Hypanthium, not all plants are nearly as porous. The alternative to plant cellulose is bacterial cellulose which is typically more pure than plant cellulose as it is free from contaminants such as lignin and hemicellulose. Bacterial cellulose has more hydrogen bonding between its polymer strands and so it has greater crystallinity. It also has smaller microfibrils that allow it to retain more moisture and  have smaller pores. The substance can be produced using waste carbohydrates (which may allow it to be produced less expensively) and it adds juiciness and chewiness to emulsified meat (which would mean that even if it can't be taken out of the final product, it will contribute to the texture profile). Chitin. Chitin is nature's second most abundant polymer. It is found in the exoskeletons of crustaceans and fungi. As cellular agriculture is attempting to end reliance on animals, chitin derived from fungi is of greater interest. It has mostly been studied by Pelling Group. Chitosan is derived  from chitin in a process known as alkaline deacetylation (substituting out certain amino acid groups). The degree of this process determines the physical and chemical properties of the chitosan. Chitosan has antibacterial properties; in particular, it has bactericidal effects on planktonic bacteria and biofilms and a bacteria static effects on gram negative bacteria such as "E. coli". This is important as it neutralizes potentially harmful compounds without using antibiotics, which many consumers avoid. Chitosan's resemblance to glycosaminoglycans and internal interactions between glycoproteins and proteoglycans make it highly biocompatible. It can easily blend with other polymers in order to select  for more bioactive factors. One potential disadvantage of chitosan is that it degrades in the presence of lysozymes (naturally occurring enzymes). But, this can be resisted using deacetylation. This is not entirely negative, as the byproducts produced through degradation have anti-inflammatory and anti-bacterial properties. It is important to match the level that cells rely on the matrix for structure with degradation. Collagen. Collagen is a family of proteins that makes up the primary structure of human connective tissue. It is typically derived from bovine, porcine and murine sources. Cellular agriculture overcomes this dependency through the use of transgenic  organisms that are capable of producing the amino acid repeats that make up the collagen. Collagen naturally exists as collagen type I. It has been produced as porous hydrogels, composites and substrates with topographical cues and biochemical properties. Synthetic kinds of collagen have been produced through recombinant protein production — collagen type II and III, tropoelastin and fibronectin. One challenge with these proteins is that they can not be modified post translation. However, an alternative fibrillar protein has been isolated in microbes that lack collagen's biochemical cues, but has its kind of gene customizability. One focus of recombinant collagen production  is yield optimization — how it can be produced most effectively. Plants, in particular, tobacco look like the best option, however, bacteria and yeast are also viable alternatives. Textured soy protein is a soy flour product often used in plant-based meat that supports the growth of bovine cells. Its spongy texture enables efficient cell seeding and its porosity encourages oxygen transfer. Additionally, it degrades during cell differentiation into compounds that are beneficial to certain cells. Mycellium. Mycelium are the roots of mushrooms. Altast Foods Co. is using solid state fermentation to grow mushroom tissue on mycelium scaffolds. They harvest this  tissue and use it to create bacon analogs. Nanomaterials. Nanomaterials exhibit unique properties at the nanoscale. London-based Biomimetic Solutions is leveraging nanomaterials in order to create scaffolds. Cass Materials in Perth, Australia is using a dietary fibre called Nata de Coco (derived from coconuts) to create nanocellulose sponges for their BNC scaffold. Nata de Coco is biocompatible, has high porosity, facilitates cell adhesion and is biodegradable. Spinning. Immersion Jet Spinning is a method of creating scaffolds by spinning polymers into fibres, It was developed by the Parker Group at Harvard. Their platform uses centrifugal force to extrude a polymer  solution through an opening in a rotating reservoir. During extrusion, the solution forms a jet that elongates and aligns as it crosses the air gap. The jet is directed into a vortex-controlled precipitation bath that chemically cross links or precipitates polymer nanofibers. Adjusting air gap, rotation and the solution changes the diameter of the resulting fibres. This method can spin scaffolds out of PPTA, nylon, DNA and nanofiber sheets. A nanofibrous scaffold made from alginate and gelatin was able to support the growth of C2C12 cells. Rabbit and bovine aortic smooth muscle myoblasts were able to adhere to the  gelatin fibres. They formed aggregates on shorter fibres, and aligned tissue on the longer ones. Matrix Meats is using electrospinning — a process that uses electric force to turn charged polymers into fibres for scaffolds. Their scaffolds allowed meat marbling, are compatible with multiple cell lines, and are scalable. Additive manufacturing. Another proposed way of structuring muscle tissue is additive manufacturing. Such a technique was perfected for industrial applications in manufacturing objects made out of plastic, nylon, metal, glass and other synthetic materials. The most common variation of the process involves incrementally depositing a filament in layers onto a bed until  the object is completed. This method will most likely lend itself best to the application of cultured meat as opposed to other types such as binder jetting, material jetting or stereolithography that require a specific kind of resin or powder. A filament of muscle cells can be printed into a structure meant to resemble a finished meat product which can then be further processed for cell maturation. This technique has been demonstrated in a collaboration between 3D bioprinting solutions and Aleph Farms that used additive manufacturing to structure turkey cells on the International Space Station. 3D bioprinting has been used  to produce steak-like cultured meat, composed of three types of bovine cell fibers and with a structure of assembled of cell fibers that is similar to original meat.<ref name="10.1038/s41467-021-25236-9"></ref> Bioreactors. Scaffolds are placed inside bioreactors so that cell growth and specialization can occur. Bioreactors are large machines similar to brewery tanks which expose the cells to a large variety of environmental factors that are necessary to promote either proliferation or differentiation. The temperature of the bioreactor must replicate "in vivo" conditions. In the case of mammalian cells, this requires heating to 37 °C  (99 °F). Alternatively, insect cells can be grown at room temperature. Most bioreactors are maintained at 5% carbon dioxide. Cells can either be cultivated in continuous or fed-batch systems. The former entails inoculating and harvesting cells in a constant process so that there are always cells in the bioreactor. Fed-batch systems mean inoculating the cells, culturing them and harvesting them in a single period. Stirred tank bioreactors are the most widely used configuration. An impeller increases the flow, thereby homogenizing the culture media and a diffuser facilitates the exchange of oxygen into the media. This system is  generally used for suspended cultures but can be used for cells that require attachment to another surface if microcarriers are included. Fixed bed bioreactors are commonly used for adherent cultures. They feature strips of fibres that are packed together to form a bed to which cells can attach. Aerated culture media is circulated through the bed. In airlift bioreactors, the culture media is aerated into a gaseous form using air bubbles which are then scattered and dispersed amongst the cells. Perfusion bioreactors are common configurations for continuous cultivation. They continuously drain media saturated with lactic acid that is void of  nutrients and fill it with replenished media. Fermentation. The elements outlined above apply to the cultivation of animal muscle tissue. However, cellular agriculture includes "acellular agriculture", which involves the production of animal products synthesized of non-living material. Such products include milk, honey, eggs, cheese, and gelatin which are made of various proteins rather than cells. In such cases, these proteins must be fermented much like in recombinant protein production, alcohol brewing and the generation of many plant-based products like tofu, tempeh and sauerkraut. Proteins are coded for by specific genes, the genes coding for the protein of interest  are synthesized into a plasmid — a closed loop of double helical genetic information. This plasmid, called recombinant DNA, is then inserted into a bacterial specimen. For this to happen, the bacteria needs to be competent (i.e. able to accept foreign, extracellular DNA) and able to horizontally transfer genes (i.e. integrate the foreign genes into its own DNA). Horizontal gene transfer is significantly more challenging in eukaryotic organisms than prokaryotic organisms because the former have both a cell membrane and a nuclear membrane which the plasmid needs to penetrate whereas prokaryotic organisms only have a cell membrane. For this reason, prokaryotic  bacteria are often favoured. In order to make such a bacteria temporarily competent, it can be exposed to a salt such as calcium chloride, which neutralizes the negative charges on the cell membrane's phosphate heads as well as the negative charges on the plasmid to prevent the two from repelling. The bacteria can incubate in warm water, opening large pores on the cell surface through which the plasmid can enter. Next, the bacteria is fermented in sugar, which encourages it to grow and duplicate. In the process it expresses its DNA as well as the transferred plasmid resulting in  protein. Finally, the solution is purified to separate out the residual protein. This can be done by introducing an antibody raised against the protein of interest that will kill bacteria cells that do not contain the protein. Through centrifugation, the solution can be spun around an axis with sufficient force to separate solids from liquids. Alternatively it could be soaked in a buffered ionic solution that employs osmosis to leach the water from bacteria and kill them. Challenges. Growth factors. The culture media is an essential component of "in vitro" cultivation. It is responsible for providing the macromolecules, nutrients and  growth factors necessary for cell proliferation. Sourcing growth factors is one of the most challenging tasks of cellular agriculture. Traditionally, it involves the use of fetal bovine serum (FBS) which is a blood product extracted from fetal cows. Besides the argument that its production is unethical, it is also vitiates the independence of the use of animals. It is also the most costly constituent of cultured meat, priced at around $1000 per litre. Furthermore, chemical composition varies greatly depending on the animal, so it cannot be uniformly quantified chemically. FBS is employed because it conveniently mimics the process  of muscle development "in vivo". The growth factors needed for tissue development are predominantly provided through an animal's bloodstream, and no other known fluid can single-handedly deliver all these components. The current alternative is to generate each growth factor individually using recombinant protein production. In this process, the genes coding for the specific factor are integrated into bacteria which are then fermented. However, due to the added complexity of this process, it is particularly expensive. The ideal medium would be chemically quantifiable and accessible to ensure simplicity in production, cheap and not dependent on animals. It will most  likely be derived from plants and while this may reduce the possibility of transmitting infectious agents, it may induce allergic reactions in some consumers. Such culture sera may also require modifications specific to the cell line to which it is applied. Companies currently invested in developing effective plant based culture include Future Fields, Multus Media and Biftek. The Good Food Institute (GFI) put out a report in 2019 in support of the concept that cell-based "meat" could be produced at the same cost as ground beef and in 2021 they commissioned a report from CE Delft on the Techno -Economic Analysis of cultivated meat. Surface area. A common challenge to bioreactors and scaffolds is developing system configurations that enable all cells to gain exposure to culture media while simultaneously optimizing spatial requirements. In the cell proliferation phase, prior to the introduction of the scaffold, many cell types need to be attached to a surface to support growth. As such, cells must be grown in confluent monolayers only one cell thick which necessitates a lot of surface area. This poses practical challenges on large scales. As such, systems may incorporate microcarriers — small spherical beads of glass or other compatible material  that are suspended in the culture medium. Cells adhere to these microcarriers as they would to the sides of the bioreactor, which increases the amount of surface area. In the cell differentiation phase, the cells may be seeded to a scaffold and so do not require the use of microcarriers. However, in these instances, the density of the cells on the scaffold means that not all cells have an interface with culture media, leading to cell death and necrotic centers within the meat. When muscle is cultivated "in vivo", this issue is circumvented as the extracellular matrix delivers nutrients into  the muscle through blood vessels. As such, many emerging scaffolds aim to replicate such networks. Similarly, scaffolds must simulate many of the other characteristics of the extracellular matrix, most notably porosity, crystallinity, degradation, biocompatibility and functionality. Few materials that emulate all these characteristics have been identified, leading to the possibility of blending different materials with complementary properties. Research support. Cellular agriculture research does not have a significant basis of academic interest or funding streams. Consequently, the majority of research has been undertaken and funded by independent institutions. However, this is incrementally changing as not for profits drive support and interest . Notably, New Harvest has a fellowship program to support graduate students and groups at various academic institutions. However, a growing number of governments are funding research in cellular agriculture. In August 2020, the Grant Management Services of the European Commission awarded a €2.5 million grant to ORF Genetics. In August 2020, the Japanese Ministry of Economy, Trade and Industry granted Integriculture $2.2 million through their New Energy and Industrial Technology Development Organization. The European Union’s Horizon 2020 R&D funding framework awarded a €2.7 million grant to a consortium led by BioTech Foods. In 2021, the Spanish  government granted €3.7 million for Biotech Foods to investigate the potential health benefits of cellular agriculture. The National Science Foundation awarded a $3.55 million grant to a team of researchers at UC Davis for open-access cultured meat research. Non profits also drive support and interest in the field. Notably, New Harvest has a fellowship program to support the research of specific graduate students and groups at various academic institutions and the Good Food Institute funds open-access research through its Research Grant Program. Consumer acceptance. Consumer acceptance of the product is critical. A study looking at acceptance  of cultured meat in China, India, and the USA "found high levels of acceptance of clean meat in the three most populous countries worldwide." Several potential factors of consumer acceptance of cultured meat have been identified. Healthiness, safety, nutritional characteristics, sustainability, taste, and lower price, are all contributing factors. One study found that the use of highly technical language to explain cultured meat led to significantly more negative public attitude towards the concept. Transparently communicating the science is important, but "oversharing" the wrong aspects of the product could draw unfavourable attention to safety concerns. Thus one of the challenges in  how cultivated meat is marketed is striking the balance between transparency of the science behind it, but communicating it in a way that it does not evoke resistance. One study suggested that describing cultured meat in a way that emphasizes the final product rather than the production method was an effective way to improve acceptance. The role of nomenclature is also crucial. Although the 'lab-grown meat' portrayal of cultivated meat is favoured by media sensationalism, it has been opposed by industry leaders as it seeds an innately unnatural image of cultivated meat in consumer's perceptions. The use of  standardized descriptions would improve future research about consumer acceptance of cultured meat. Current studies have often reported drastically different rates of acceptance, despite similar survey populations. Lou Cooperhouse, CEO of BlueNalu, shared on the Red to Green Podcast that "cell-based" and "cell-cultured" were suitable terms to differentiate it from conventional meat whilst being clear about the process by which it was made. Global market acceptance has not been assessed. Studies are attempting to determine the current levels of consumer acceptance and identify methods to improve this value. Clear answers are not available, although one recent study reported that  consumers were willing to pay a premium for cultured meat. Low percentages of older adult populations have been reported to show acceptance for cultured meat. Green eating behavior, educational status, and food business, were cited as most important factors for this population. There is also a lack of studies relating the methods of producing cultured meat with its taste for the consuming public. Regulations. Singapore became the first country in the world to approve cultured meat for sale in 2020. The Singapore Food Agency has published guidance on its requirements for the safety assessment of novel foods, including specific requirements  on the information to be submitted for approval of cultivated meat products. Regulatory matters must also be sorted out. Prior to being available for sale, the European Union, Australia, New Zealand, the UK and Canada require approved novel food applications. Additionally, the European Union requires that cultured animal products and production must prove safety, by an approved company application, as of 1 January 2018. Within the United States, the FDA (Food and Drug Administration) and the USDA (United States Department of Agriculture) have agreed to jointly regulate cultured meat. Under the agreement, the FDA oversees cell collection, cell banks, and  cell growth and differentiation, while the USDA oversees the production and labeling of human food products derived from the cells. Differences from conventional meat. Health. Large-scale production of cultured meat may or may not require artificial growth hormones to be added to the culture for meat production. As cultured meat is grown in a sterile environment, there is no need for antibiotics. Today, the widespread use of antibiotics in conventional agriculture is the main driver of antibiotic resistance in humans. According to the World Health Organization, antimicrobial resistance represents “an increasingly serious threat to global public health that requires  action across all government sectors and society" – predicting up to 10 million deaths annually by 2050. Cultured meat could provide an effective solution to help mitigate this major risk to human health. Researchers have suggested that omega-3 fatty acids could be added to cultured meat as a health bonus. In a similar way, the omega-3 fatty acid content of conventional meat can be increased by altering what the animals are fed. Research is currently underway in Spain to develop cultivated meat with healthier fats, which could reduce cholesterol and the risk of colon cancer typically associated with red meat consumption . An issue of "Time" magazine suggested that the cell-cultured process may also decrease exposure of the meat to bacteria and disease. Due to the strictly controlled and predictable environment, cultured meat production has been compared to vertical farming. Some of its proponents have predicted that it will have similar benefits in terms of reducing exposure to dangerous chemicals like pesticides and fungicides, severe injuries, and wildlife. There is also a lack of research on the comparison on the health effects of production cultured meat with the industrial meat or the biologic organic meat ways of production. Artificiality. Although cultured  meat consists of animal muscle cells, fat and support cells, as well as blood vessels, that are the same as in traditional meat, some consumers may find the high-tech production process unacceptable. Cultured meat has been described as fake or "Frankenmeat". On the other hand, cultured meat can be produced without the artificial hormones, antibiotics, steroids, medicine, and GMOs commonly used in factory farmed meat and seafood, though not used on organic biologic production. If a cultured meat product is different in appearance, taste, smell, texture, or other factors, it may not be commercially competitive with conventionally produced meat . The lack of bone and cardiovascular system is a disadvantage for dishes where these parts make appreciable culinary contributions. However, the lack of bones and/or blood may make many traditional meat preparations, such as buffalo wings, more palatable to some people. Furthermore, blood and bones could potentially be cultured in the future. Environment. Animal production for food is a major causes of air/water pollution and carbon emissions. Significant questions have been raised about whether the traditional industry can meet the rapidly increasing demands for meat. Cultured meat may provide an environmentally conscious alternative to traditional meat production. The environmental  impacts of cultured meat are expected to be significantly lower than from animal husbandry. For every hectare that is used for vertical farming and/or cultured meat manufacturing, anywhere between 10 and 20 hectares of land may be returned to its natural state. Vertical farms (in addition to cultured meat facilities) could exploit methane digesters to generate a portion of its electrical needs. Methane digesters could be built on site to transform the organic waste generated at the facility into biogas which is generally composed of 65% methane. This biogas could be burned to generate electricity for the greenhouse or a  series of bioreactors. One study reported that cultured meat was "potentially ... much more efficient and environmentally-friendly". It generated only 4% of greenhouse gas emissions, reduced the energy needs of meat production by up to 45%, and required only 2% of the land that the global meat/livestock industry does. In Tuomisto's life cycle analysis claimed that producing 1000 kg of meat conventionally requires "26–33 GJ energy, 367–521 m³ water, 190–230 m² land, and emits 1900–2240 kg CO2-eq GHG emissions". On the other hand, producing the same quantity of meat "in vitro" has "7–45% lower energy use… 78–96 % lower GHG emissions, 99% lower land use, and 82–96% lower water use". The latest study by independent research firm CE Delft shows that — compared with conventional beef — cultured meat may cause up to 92% less greenhouse gas emissions if renewable energy is used in the production process, 93% less pollution, up to 95% less land use and 78% less water. Skeptic Margaret Mellon of the Union of Concerned Scientists speculates that the energy and fossil fuel requirements of large-scale cultured meat production may be more environmentally destructive than producing food off the land. However, S.L. Davis speculated that both  vertical farming in urban areas and the activity of cultured meat facilities may cause relatively little harm to the wildlife that live around the facilities. Dickson Despommier speculated that natural resources may be spared from depletion due to vertical farming and cultured meat. One study reported that conventional farming kills ten wild animals per hectare each year. Role of genetic modification. Techniques of genetic engineering, such as insertion, deletion, silencing, activation, or mutation of a gene, are not required to produce cultured meat. Cultured meat production allows the biological processes that normally occur within an animal to occur without the  animal. Since cultured meat is grown in a controlled, artificial environment, some have commented that cultured meat more closely resembles hydroponic vegetables, rather than genetically modified vegetables. More research is underway on cultured meat, and although cultured meat does not require genetic engineering, researchers may employ such techniques to improve quality and sustainability. Fortifying cultured meat with nutrients such as beneficial fatty acids is one improvement that can be facilitated through genetic modification. The same improvement can be made without genetic modification, by manipulating the conditions of the culture medium. Genetic modification may be able to enhance muscle cell proliferation . The introduction of myogenic regulatory factors, growth factors, or other gene products into muscle cells may increase production over that of conventional meat. To avoid the use of any animal products, the use of photosynthetic algae and cyanobacteria has been proposed to produce the main ingredients for the culture media, as opposed to fetal bovine or horse serum. Some researchers propose that the ability of algae and cyanobacteria to produce ingredients for culture media can be improved with certain technologies, most likely not excluding genetic engineering. Ethical. Australian bioethicist Julian Savulescu said, "Artificial meat stops cruelty to animals, is better  for the environment, could be safer and more efficient, and even healthier. We have a moral obligation to support this kind of research. It gets the ethical two thumbs up." Animal welfare groups are generally in favor of cultured meat, because the culture process does not include a nervous system and therefore does not involve pain or infringement of rights. Reactions of vegetarians to cultured meat vary. Some feel the cultured meat presented to the public in August 2013 was not vegetarian because fetal bovine serum was used in the growth medium. However, since then, cultured meat has been grown  with a medium that does not involve bovine serum. Philosopher Carlo Alvaro argues that the question of the morality of eating "in vitro" meat has been discussed only in terms of convenience. Alvaro proposes a virtue-oriented approach, suggesting that the determination to produce cultured meat stems from unvirtuous motives, i.e., "lack of temperance and misunderstanding of the role of food in human flourishing." Some have proposed independent inquiries into the standards, laws, and regulations for cultured meat. Just as with many other foods, cultured meat needs technically sophisticated production methods that may be difficult for some communities, meaning they  would lack self-sufficiency and be dependent on global food corporations. However, some projects are focusing on making cell ag accessible to all. The open source cellular agriculture initiative Shojinmeat Project has for instance a bottom-up approach, teaching members of the project to cultivate DIY cultured meat at home. Establishing a similar parallel with cultured meat, some environmental activists claim that adopting a vegetarian diet may be a way of focusing on personal actions and righteous gestures rather than systemic change. Environmentalist Dave Riley states that "being meatless and guiltless seems seductively simple while environmental destruction rages around us , and notes that Mollison insists that vegetarianism drives animals from the edible landscape so that their contribution to the food chain is lost". Religious considerations. Jewish rabbinical authorities disagree whether cultured meat is kosher, meaning acceptable under Jewish law and practice. One factor is the nature of the animal from which the cells are sourced, whether it is a kosher or non-kosher species and whether, if the cells were taken from a dead animal, slaughter in accordance with religious practice had taken place prior to the extraction of cells. Most authorities agree that if the original cells were taken  from a religiously slaughtered animal then the meat cultured from it will be kosher. Depending on the nature of the cells, it may be determined to be kosher even when taken from a live animal, and some have argued that it would be kosher even if coming from non-kosher animals such as pigs. Islamic dietary practices must also be considered. The Islamic Institute of Orange County, California said, "There does not appear to be any objection to eating this type of cultured meat." In addition, Abdul Qahir Qamar of the International Islamic Fiqh Academy said that cultured meat "will  not be considered meat from live animals, but will be cultured meat." As long as the cells are not from pigs, dogs, and other haram animals, the meat would be considered vegetative and "similar to yogurt and fermented pickles." Hinduism typically excludes the consumption of beef, such as steak and burgers. Chandra Kaushik, president of the Hindu Mahasabha, said about cultured beef that he would "not accept it being traded in a marketplace in any form or being used for a commercial purpose." Catholicism, which excludes eating meat in certain days along the year (Lent, Holy Week), has not pronounced  on whether cultivated meat is banned (as it happens with meat) or not (as with any other food as vegetables or fish). Economic. At the moment, cultured meat is significantly more costly than conventional meat. However,in a March 2015 interview, Post said that the marginal cost of his team's original €250,000 burger was now €8.00. He estimated that technological advancements would allow the product to be cost-competitive to traditionally sourced beef in approximately ten years. In 2018, Memphis Meats reduced the cost of production to $1,700 per pound. In 2019, Eat Just said it  cost about US$50 to produce one chicken nugget. The company’s cultured chicken nuggets, now available at Singapore restaurant 1880, cost around US$17. A study by independent research firm CE Delft found that production costs for cultivated meat could fall to $5.66/kg by 2030. Farmers. A scientific paper published in "Front. Sustain. Food Syst." addresses the social and economic opportunities and challenges of cultured and plant-based meat for rural producers. According to this research, cellular agriculture offers “opportunities such as growing crops as ingredients for feedstock for cultured meat; raising animals for genetic material for  cultured meat; producing cultured meat in bioreactors at the farm level; transitioning into new sectors; new market opportunities for blended and hybrid animal- and alt-meat products; and new value around regenerative or high-animal welfare farming.” However, some challenges are also identified, with possible “loss of livelihood or income for ranchers and livestock producers and for farmers growing crops for animal feed; barriers to transitioning into emerging alt-meat sectors; and the possibility of exclusion from those sectors.” Some farmers already see the potential of cellular agriculture. For instance, Illtud Dunsford comes from a long line of farmers in  Wales and established his cultured meat company Cellular Agriculture Ltd in 2016. Continuing development. Education. In 2015, Maastricht University hosted the first International Conference on Cultured Meat. New Harvest — a 501(c)(3) research institute — as well as The Good Food Institute host annual conferences to convene industry leaders, scientists, investors, and potential collaborators. The two organizations also fund public research and produce educational content. Organizations such as the Cellular Agriculture Society, Cellular Agriculture Canada, Cellular Agriculture France, Cellular Agriculture Australia and Cellular agriculture New Zealand were founded to advocate for cultured meat in their respective countries. Publications such as Cell Agri  and the Protein Report have also emerged in order to provide updates concerning the technology and business within the field. Research. Research continues on many fronts, including entomoculture, interactome maps of cardiac tissue, substrate design, scaffold design, nutritional profile, reaction kinetics, transport phenomena, mass transfer limitations and metabolic stoichiometric requirements. and bioprinting process. Accelerators and incubators. Multiple venture capital firms and accelerator/incubator programs focus on assisting cultured technology startups, or plant-based protein companies in general. The Big Idea Ventures (BIV) Venture Capital firm launched their New Protein Fund to invest in emerging cell and plant-based food companies  in New York and Singapore. They invested in MeliBio, Actual Veggies, Biftek.co, Orbillion Bio, Yoconut, Evo, WildFor and Novel Farms. Indie Bio is a biology oriented accelerator program that has invested in Memphis Meats, Geltor, New Age Meats and Finless Foods. In popular culture. Cultured meat has often featured in science fiction. The earliest mention may be in "Two Planets" (1897) by Kurd Lasswitz, where "synthetic meat" is one of the varieties of synthetic food introduced on Earth by Martians. Other notable books mentioning artificial meat include "Ashes, Ashes" (1943) by René Barjavel; "The Space Merchants" (1952) by Frederik Pohl  and C.M. Kornbluth; "The Restaurant at the End of the Universe" (1980) by Douglas Adams; "Le Transperceneige (Snowpiercer)" (1982) by Jacques Lob and Jean-Marc Rochette; "Neuromancer" (1984) by William Gibson; "Oryx and Crake" (2003) by Margaret Atwood; "Deadstock" (2007) by Jeffrey Thomas; "Accelerando" (2005) by Charles Stross; "Ware Tetralogy" by Rudy Rucker; "Divergent" (2011) by Veronica Roth; and the Vorkosigan Saga (1986-2018) by Lois McMaster Bujold. In film, artificial meat has featured prominently in Giulio Questi's 1968 drama "La morte ha fatto l'uovo" ("Death Laid an Egg") and Claude Zidi's 1976 comedy "L'aile ou la cuisse  (The Wing or the Thigh"). "Man-made" chickens also appear in David Lynch's 1977 surrealist horror, "Eraserhead". Most recently, it was also featured prominently as the central theme of the movie "Antiviral" (2012). The Starship "Enterprise" from the TV and movie franchise "Star Trek" apparently provides a synthetic meat, although crews from "The Next Generation" and later use replicators. In the ABC sitcom "Better Off Ted" (2009–2010), the episode "Heroes" features Phil (Jonathan Slavin) and Lem (Malcolm Barrett) trying to grow cowless beef. In the videogame "Project Eden", the player characters investigate a cultured meat company called Real Meat . In the movie "Galaxy Quest" during the dinner scene, Tim Allen's character refers to his steak tasting like "real Iowa beef". In "The Expanse" "vat-grown" meat is produced to feed the people who live on spaceships/space stations away from Earth, due to the exorbitant cost of importing real meat. Cultured meat was a subject on an episode of the Colbert Report on 17 March 2009. In February 2014, a biotech startup called BiteLabs ran a campaign to generate popular support for artisanal salami made with meat cultured from celebrity tissue samples. The campaign became popular on Twitter , where users tweeted at celebrities asking them to donate muscle cells to the project. Media reactions to BiteLabs variously identified the startup as a satire on startup culture, celebrity culture, or as a discussion prompt on bioethical concerns. While BiteLabs claimed to be inspired by the success of Sergey Brin's burger, the company is seen as an example of critical design rather than an actual business venture. In late 2016, cultured meat was involved in a case in the episode "How The Sausage Is Made" of CBS show "Elementary". Cultured meat was profiled in the 2020 Canadian documentary film  Meat the Future. In the 2020 video game "Cyberpunk 2077", multiple cultured meat products are for sale, due to the high cost of natural meat. This includes "EEZYBEEF", made from "in vitro" cultured muscle cells taken from cattle, and the flatworm culture based "Orgiatic" which comes in several flavors.