Meat Production from Stem Cells: Myth Or Reality

International Journal of Food Nutrition and Safety, 2013, 3(1): 29-54 International Journal of Food Nutrition and Safety ISSN: 2165-896X Journal homepage: www.ModernScientificPress.com/Journals/IJFNS.aspx Florida, USA Review Meat Production from Stem Cells: Myth or Reality

Heena Jalal 1, *, Mir Salahuddin 1, Humaira Gazalli 1

Division of Livestock Products Technology, Faculty of Veterinary Science and Animal Husbandry, SKUAST-K, Ganderbal-190006 (J & K), India

* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +91-8054478516.

Article history: Received 3 February 2013, Received in revised form 18 February 2013, Accepted 19 February 2013, Published 20 February 2013.

Abstract: The use of livestock for the production of food has always been an essential part of man’s existence on earth. To suit the meat demands of a modern society, animals are intensively kept and production is optimized disregarding the well being of animals. This results in herding of animals in confined spaces in unfavourable conditions. The adaptability of animals is not high enough to cope with this unnatural condition, and high stress levels are observed resulting in disease, abnormal behaviour and death. Another problem associated with mass animal production is the environmental problem caused by the enormous amounts of excrement the animals produce and which the environment subsequently has to deal with. Meat consumption around the world is growing at an incredible rate. The only way out is to increase the world's meat supply and help fight global hunger. The novel ways to increase food production will be needed. One way to get out of this predicament is to exploit the potential of modern biotechnology and process technology to produce meat from normal muscle progenitor cells in bioreactors at an industrial scale. he production of meat (muscle tissue) from cultured stem cells is a potential and promising alternative. Cultured meat could have financial, health, environmental and animal welfare advantages over traditional meat.

Keywords: meat; production; stem cells; biotechnology; muscle tissue; food; disease.

1. Introduction

The use of livestock for the production of food has always been an essential part of man’s existence on earth and its impact has until recent years been primarily positive, both economically and socially. However, current production methods are rather demanding. It has been estimated that the global population will increase from 6 billion people in 2000 to 9 billion people in the year 2050. It is anticipated that in the same period annual global meat production will rise from 228 to 465 million tonnes due to rising incomes, urbanization and growing populations (FAO, 2006). This means more and more people will begin eating meat. The latter is at the expense of food of plant origin such as cereals. These changes in consumption, together with sizeable population growth and urbanization, have led and will continue to lead to large increases in the total demand for animal products in many developing countries. In order for the population to be fed sufficiently more and more land is required for food produce. The natural sources are insufficient to fulfil the demand. Globally, 30% of the land surface is used for livestock production with 33% of arable land being used for growing livestock feed crops and 26% being used for grazing (Steinfeld et al., 2006). About 70% of the fresh water use and 20% of the energy consumption of mankind is directly or indirectly used for food production, of which a considerable proportion is used for the production of meat. Also the large amount of land currently required for animal production or the production of feed for the animals which cannot be used for alternative purposes such as growth of other crop, housing, recreation, wild nature and forests. To suit the meat demands of a modern society, animals are intensively kept and production is optimized disregarding the well being of animals being used, an efficient and cheap production system is required. This results in herding of animals in confined spaces in unfavourable conditions. The adaptability of animals is not high enough to cope with this unnatural condition, and high stress levels are observed resulting in disease, abnormal behaviour and death (Crok, 2003). Some animal races like pigs and chickens domesticated for meat production have such a high growth rate for muscle tissue that the cardiovascular system is incapable of providing satisfactory amounts of oxygen to the animal body, leading to health problems and in chickens even to preliminary death (Crok, 2003). This raises serious ethical questions. Another problem is that of animal disease epidemics and more serious threat is posed by the chicken flu, as this can lead to possible new influenza epidemics or even pandemics, which can kill millions of people (Webster, 2002). It is increasing the number of diseases and the consequences thereof for both animals and humans. Large scale slaughtering is currently required to fulfil the current food requirements and as a consequence of large scale disease outbreaks. We can take for example the recent large scale occurrence of porcine pest virus and mad cows disease. These diseases also result in loss of the meat for human consumption thus completely denying the purpose for which the animals

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 31 were being bred in the first place. Another problem associated with mass animal production is the environmental problem caused by the enormous amounts of excrement the animals produce and which the environment subsequently has to deal with. Greenhouse emissions and energy requirements also pose potential difficulties. The livestock sector contributes 18% of the anthropogenic greenhouse gas emissions and 37% of the anthropogenic methane emissions to the atmosphere worldwide (Steinfeld et al., 2006). The 18% of green house gas emission is currently produced by livestock which is more than the total emission of the transportation sector (FAO, 2006). The water use for livestock and accompanying feed crop production also has a dramatic effect on the environment such as a decrease in the fresh water supply, erosion and subsequent habitat and biodiversity loss (Asner et al., 2004; Savadogo et al., 2007). Thus, cost of current production process to the climate, the environment, the deforestation and animal welfare is too high to provide for this increasing demand in any sustainable way. On the other hand, humans are taxonomically omnivorous and meat provides several essential nutrients unavailable in plant sources. Meat is specifically valuable as a source of omega-3 fatty acids, vitamin B12, and highly bio-available iron (Bender, 1992). Under consumption of animal-derived products is closely associated with malnutrition in poorer areas of the world where access to plant derived food is also very limited. The average American currently consumes approximately 124 kg of meat each year. By contrast, the average worldwide consumption is 31 kg a year, with Bangladesh the lowest at 3 kg per person (FAO, 2006). This situation though is changing; meat consumption around the world is growing at an incredible rate. The only way out is to increase the world's meat supply and help fight global hunger. Traditional methods of assembly-line meat production require not only ever- increasing inputs of corn, soy, and other grains, but also enormous amounts of energy. Livestock production imposes a vast burden on global resources and it is one that the expanding world population can no longer sustain. It will not be possible to produce all that meat in an environmental and animal friendly way. With the world population forecast to hit 9 billion people by 2050 novel ways to increase food production will be needed. It is a tremendous political and economic challenge to change this grim scenario into a more sustainable one if we continue to base our meat consumption solely on production of animals. One way to get out of this predicament is to exploit the potential of modern biotechnology and process technology to produce meat from normal muscle progenitor cells in bioreactors at an industrial scale. If this production strategy were to replace a substantial part of the current meat production regime, this may allow development of a downsized animal production industry which can acquire a competitive edge in the upper-level meat market by documenting that it is ecologically sound and meets basic animal welfare requirements. Implementation of an in-vitro meat production system (IMPS) to complement existing meat production practices creates the opportunity for meat products of different characteristics to be put onto the market. While widening the scope of Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 32 the meat industry in practices and products, the IMPS will reduce the need for agricultural resources to produce meat. Widespread adoption of the technology also holds the potential to increase the world's meat supply, which could help fight global hunger. Thus, the production of meat (muscle tissue) from cultured stem cells is a potential and promising alternative.

2. In-vitro Meat Production

In-vitro meat production refers to the practice of artificially growing animal tissue in laboratory. In-vitro meat production using stem cells appears as an appealing alternative for general meat production system through livestock. The idea was introduced to improve nutrition during space travel, but it has been further considered by the scientific community and supported by animal welfare groups. Studies have been undertaken and these concluded it is feasible to produce in-vitro meat, but the necessary techniques and prospective impacts need to be much further developed before it is produced in enough quantity to be tested. In-vitro meat (also known as cultured meat) is bioengineered meat made in the laboratory. It involves culturing myogenic cells in an environment that emulates the in vivo environment so that the cells proliferate, fuse, organize in three dimensions, and differentiate into skeletal muscle. It is real meat, but it is created from stem cells or myoblasts (precursors to muscle cells) in a Petri dish instead of from an actual animal. In-vitro meat can be made without any animal cruelty. It’s even PETA-approved. In 2008, the organization offered a $1 million prize to the first company that brings lab-grown chicken meat to consumers. In-vitro meat also has minimal ecological consequences. Research suggests that in-vitro meat production uses 90 percent less land and water than industrial practices, and produces 80 percent fewer greenhouse gas emissions. Sure, in-vitro meat does not address the underlying problem of our eating habits “we eat too much meat” but it would potentially improve some of the symptoms of our obsession with meat: global warming, pollution, excessive water use, land degradation, antibiotic-resistance, and disease. Cultured meat could have financial, health, environmental and animal welfare advantages over traditional meat. The idea: To produce animal meat, simply without using an animal. Starting cells are taken painlessly from live animals, they are put into a culture media where they start to proliferate and grow, independently from the animal. Theoretically, this process would be efficient enough to supply the global demand for meat. All this would happen without any genetic manipulation, i.e. without the need to interfere with the cells genetic sequences. Producing cultured meat for processed meat products, such as sausages, burgers and nuggets should be relatively simple, whereas cultured meat which should be more highly structured, such as for an in-vitro steak is considerably more of a challenge. A steak is made of muscle tissue which is threaded through with extremely long, fine capillaries which transport blood and nutrients directly to the cells. It is much more difficult to reproduce such a complex structure than it is

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 33 to put together the small balls of cells which grow to larger balls of cells which in turn become in-vitro chicken nuggets. In-vitro tissue engineering is not the same as genetic engineering, a common misconception. In tissue engineering natural cells from natural animals are used. Rather than changing Mother Nature, effort is made to imitate it. In-vitro meat differs from synthetic and artificial meat, which taste and have the texture of meat but do not consist of meat. In-vitro meat has been variously referred to as: test-tube meat, cultured meat, manufactured meat, vitro meat, laboratory meat.

3. Potential Advantages of Cultured Meat

Relative to conventional meat, cultured meat could offer a number of benefits: An in-vitro meat production system involves culturing muscle-like tissue in a liquid medium, therefore bypassing animal husbandry and slaughter. The controlled conditions of the IMPS are impossible to achieve by traditional livestock methods and therefore allow for a safer, healthier product. Myocyte culturing prevents the spread of animal-borne disease which may or may not affect meat products. Moreover, by reducing the amount of close quarter human-animal interaction, the incidence of epidemic zoonoses developing will decline. Due to strict quality control rules, such as Good Manufacturing Practice, that are impossible to introduce in modern animal farms, slaughterhouses, or meat packing plants, the chance of meat contamination and incidence of food borne disease could be significantly reduced. The controlled conditions would theoretically eliminate product losses from infected and diseased animals. In addition, the risks of exposure to pesticides, arsenic, dioxins, and hormones associated with conventional meat could be significantly reduced. Controlled conditions also offer the capacity for manipulation to create meat products with different nutritional, textural and taste profiles. With cultured meat, the ratio of saturated to poly-unsaturated fatty acids could be better controlled. This can be accomplished by co-culturing with different cell types, medium supplementation or genetic engineering. In-vitro system reduces animal use in the meat production system as theoretically a single farm animal may be used to produce the world’s meat supply. Furthermore, less livestock could lead to a decrease in intense land usage and green house gas emissions (Stamp et al., 2008; Williams et al., 2006). In theory, cells from captive, rare or endangered animals (or even cells from samples of extinct animals) could be used to produce exotic meats in cultures and thus a sustainable alternative to global trade of meats from rare and endangered animals will help in increasing wild populations of many species. As the biological structures in addition to muscle tissue are not required to produce meat in an in-vitro system, it reduces the amount of nutrients and energy needed for their growth and maintenance. In-vitro system significantly lowers time to grow the meat and takes several weeks instead of months (for chickens) or years (for pigs and cows) before the meat can be harvested and thus, the amount of feed and labour required per kg of in-vitro cultured meat is much lower. As a

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 34 method with little to no waste products or by-products and a minimized land and resource requirement, myocyte culturing could possibly alleviate the environmental burden exhibited by today's meat harvesting techniques. The long-term space missions, such as a settlement on the Moon or a flight to Mars, will likely involve some food production in-situ within a settlement or spacecraft, to reduce liftoff weight and its associated costs. There are other situations also in which it is costly to re-supply people with food and in which it is more economical to produce food in-situ. These include scientific stations in Polar Regions, troop encampments in isolated theatres of war and bunkers designed for long-term survival of personnel following a nuclear or biological attack. As laboratory produced meat does not come from a living animal, it therefore significantly minimizes the religious taboos like Jhatka, Jewish and Halal etc. (Pathak et al., 2008). It will not be possible to produce all the meat in an environmental and animal friendly way for the growing population and thus, there is a rather conventional meat market for in- vitro meat. The proteins produced using plants and fungi are animal friendly, sustainable and have been used to make a variety of good chief products but they lack a good texture and taste and such products are no solution for the craving for meat. Cultured meat will be safer than conventional meat and due to the non-sustainability of traditional meat production there is a huge market for this.

4. Short History

The idea of culturing animal parts in-vitro for human consumption is not new. The idea of culturing muscle tissue in a lab ex-vivo already originates from the early nineteen hundreds. In 1912, Alexis Carrel managed to keep a piece of chick heart muscle alive and beating in a Petri dish. This experiment demonstrated that it was possible to keep muscle tissue alive outside the body, provided that it was nourished with suitable nutrients. Among other great thinkers, Winston Churchill predicted that it would be possible to grow chicken breasts and wings more efficiently without having to keep an actual chicken (Churchill, 1932). Although he predicted that it could be achieved within 50 years, his concept was not far off from reality today. In the Netherlands, it was Willem van Eelen in the early 1950s that independently had the idea of using tissue culture for the generation of meat products. Since at that time the concept of stem cells and the in-vitro culture of cells still had to emerge, it took until 1999 before van Eelen’s theoretical idea was patented. Van Eelen, Van Kooten and Westerhof hold a Dutch patent for this general approach to producing cultured meat. The Netherlands is the first country where work on cultured meat is being approached in a systematic way. However, Catts and Zurr appear to have been the first to have actually produced meat by this method. As part of a NASA- funded project, Morris A. Benjaminson (a biology professor at the Touro College School of Health Sciences and president of Zymotech Enterprises in Bay Shore, N.Y.) and his team were the first

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 35 researchers to have applied tissue engineering techniques to meat production. They placed skeletal muscle explants from goldfish (Carassius auratus) in a diverse culture media for 7 days and observed an increase in surface area between 5.2 and 13.8%. When explants were placed in a culture containing dissociated Carassius skeletal muscle cells (closely mimetic an in vivo structure) explants surface area increased by 79%. Prof. Benjaminson is not the only one working in the field. Dutch scientist, Dr. Henk Haagsman, Professor of Meat Sciences at the University of Utrecht and his research team, supported by million-dollar grants from the Dutch government, initiated a project in April 2002 and have succeeded in manufacturing, from stem cells of pigs, a kind of ground pork that could be used in burgers, sausages and pizza toppings etc within the next couple of years. Meat industry is also supporting this work. His assignment now is to determine whether the process will work on an industrial scale. Along with the University of Utrecht, the universities of Eindhoven and Amsterdam are working to cultivate muscles out of the stem cells. Cell biologists at Utrecht are searching for suitable stem cells and methods to let them grow into as much muscle as possible. Microbiologists at Amsterdam design the environment for growth. And tissue engineers at Eindhoven design bioreactors in which small muscle tissue grows quickly. Scientists are working on a variety of cell culture procedures. The cutting edge of in-vitro meat engineering is the attempt to get cells to grow as if they were inside a living animal. In biomedical research growing mammalian cells requires a nutritious medium that includes fetal bovine serum which is not only of animal origin but also very costly. Dutch researchers, mindful of the animal-friendly image cultured meat must maintain, have perfected a completely vegetarian growth medium, consisting of modified fungi, water, and glucose, making stem cells the only source of animal tissue in the process.

5. Requirements

In-vitro meat is an animal flesh product that has never been part of a living animal. The process works by taking precursor cells, typically embryonic stem cells or satellite cells from an animal via biopsy (or a piece of flesh from a slaughtered animal). Isolation requires mincing the muscles, followed by enzymatic treatment and subsequent separation of the cells by differential centrifugation. Putting them in a three-dimensional growth medium- a sort of scaffolding made of proteins. Bathed in a nutritional mix of glucose, amino acids and minerals, the stem cells multiply and differentiate into muscle cells, which eventually form muscle fibers. Those fibers are then harvested for a minced-meat product. For the growth of real muscle, however, the cells should grow "on the spot," which requires a perfusion system akin to a blood supply to deliver nutrients and oxygen close to the growing cells, as well as to remove the waste products. In addition, other cell types, such as adipocytes, need to be grown, and chemical messengers should provide clues to the growing tissue about the structure. Lastly,

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 36 muscle tissue needs to be physically stretched or exercised to properly develop. Thus, there are four basic premises on which cultured meat technology rests: (1) Starter Cells; (2) Growth Medium/ Culture Media; (3) Scaffold; (4) Bioreactor.

5.1. Starter Cells

Skeletal muscle is made up of several cell types. The proliferation, differentiation and fusion of the embryonic myoblasts form skeletal muscle tissue. In post natal stage this is done through the adult stem cells / satellite cells /myosatellite cells. Embryonic Stem (ES) cells can be obtained from the early developing stages of an embryo. Within a blastocyst there is a small group of about 30 cells called the inner cell mass, which will give rise to the hundreds of highly specialized cells needed to make up an adult organism. Embryonic stem cells are obtained from this inner cell mass. Embryonic stem cells can and do differentiate into all the specialized cells in the adult body. They could be induced to provide an unlimited source of specific and clinically important adult cells such as bone, muscle, liver or blood cells. There is an option of using embryonic stem cells for in-vitro meat production system. These cells have high potential to differentiate and proliferate. Culturing embryonic stem cells would be ideal for this purpose since these cells have an almost infinite self-renewal capacity and theoretically it is being said that one such cell line would be sufficient to literally feed the world. In theory, after the embryonic stem cell line is established, its unlimited regenerative potential eliminates the need to harvest more cells from embryos. But the harvests from these cells do not justify the effort required to force them to differentiate; the slow accumulation of genetic mutations over time may determine a maximum proliferation period for a useful long-term ES culture (Amit et al., 2000). While, embryonic stem cells are an attractive option for their unlimited proliferative capacity, these cells must be specifically stimulated to differentiate into myoblasts and may inaccurately recapitulate myogenesis (Bach et al., 2003). Although, embryonic stem cells have been cultured for many generations but so far it has not been possible to culture cell lines with unlimited self-renewal potential from pre- implantation embryos of farm animal species. Until now, true embryonic stem cell lines have only been generated from mouse, rhesus monkey, human and rat embryos (Talbot and Blomberg, 2008), but the social resistance to cultured meat obtained from mouse; rat or rhesus monkey will be considerable and will not result in a marketable product. The culture conditions required to keep mouse and human embryonic cells undifferentiated are different from the conditions that will be required for embryonic cells of farm animal species and fundamental research on the early development of embryos of these species can provide clues. Since it has been difficult, to derive Embryonic Stem (ES) cells from pre- implantation embryos of agricultural species. Alternatively, we can use adult stem cells from farm animal species and myosatellite cells are

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 37 one example of an adult stem-cell type (Askura et al., 2001). Adult stem cells have been isolated from several different adult tissues (Wagers and Weissman, 2004). These cells also have the capacity to differentiate into skeletal muscle cells, although not very efficiently but for now, these are the most promising cell type for use in the production of cultured meat. Satellite cells are small mononuclear progenitor cells with virtually no cytoplasm found in mature muscle. They are found sandwiched between the basement membrane and sarcolemma (cell membrane) of individual muscle fibers. These cells represent the oldest known adult stem cell niche, and are involved in the normal growth of muscle, as well as regeneration following injury or disease. In undamaged muscle, the majority of satellite cells are quiescent; they neither differentiate nor undergo cell division. In response to mechanical strain, satellite cells become activated. Activated satellite cells initially proliferate as skeletal myoblasts before undergoing myogenic differentiation. Myosatellite cells are a very small proportion (1-5%) of the cell population of muscle tissue, and this percentage is dependent on muscle fiber composition and organism age (Allen et al., 1997). As an organism ages, the regenerative potential of its myosatellite cell population also decreases rapidly. As a result, the cells that offer optimal regenerative potential and myofiber morphology in-vitro (longer myofibers and greater myofiber density) must be harvested from neonates (Delo et al., 2008). According to Bach et al. (2003) myosatellite cells are the preferred source of primary myoblasts although, they have the disadvantage of being a rare muscle tissue cell type with limited regenerative potential because they recapitulate myogenesis more closely than immortal myogenic cell lines. Myosatellite cells isolated from different animal species have different benefits and limitations as a cell source and that isolated from different muscles have different capabilities to proliferate, differentiate, or be regulated by growth modifiers (Burton et al., 2000). Myosatellite cells have been isolated and characterized from the skeletal muscle tissue of cattle (Dodson et al., 1987), chicken (Yablonka-Reuveni et al., 1987), fish (Powell et al., 1989), lambs (Dodson et al., 1986), pigs (Blanton et al., 1999; Wilschut et al., 2008) and turkeys (McFarland et al., 1988). Porcine muscle progenitor cells have the potential for multilineage differentiation into adipogenic, osteogenic and chondrogenic lineages, which may play a role in the development of co-cultures (Wilschut et al., 2008). Advanced technology in tissue engineering and cell biology offer some alternate cell options having practical applications and multilineage potential allowing for co-culture development with suitability for large-scale operations. Adult stem cell plasticity and transdifferentiation: A number of experiments have suggested that certain adult stem cell types are pluripotent. This ability to differentiate into multiple cell types is called plasticity or transdifferentiation. The following list offers examples of adult stem cell plasticity that have been reported during the past few years. (a) Hematopoietic stem cells may differentiate into three major types of brain cells (neurons, oligodendrocytes, and astrocytes); skeletal muscle cells; cardiac muscle cells; and liver cells. (b) Bone marrow stromal cells may differentiate into cardiac Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 38 muscle cells and skeletal muscle cells. (c) Brain stem cells may differentiate into blood cells and skeletal muscle cells. In 2006, researchers made another breakthrough by identifying conditions that would allow some specialized adult cells to be reprogrammed genetically to assume a stem cell-like state. This new type of stem cell, called induced pluripotent stem cells (iPSCs).

5.2. Growth Medium/Culture Media

Perhaps the most difficult task in designing an in-vitro meat production system is determining the best culture medium formulation. The medium should support and promote growth while being made of affordable, edible components available in large quantities. To enjoy many of the potential advantages over conventional meat production, cultured meat would need to employ an affordable medium system. Such a medium must contain the necessary nutritional components and be presented in a form freely available to myoblasts and accompanying cells, as no digestive system is involved. Animal sera are from adult, newborn or fetal source, with fetal bovine serum being the standard supplement for cell culture media (Coecke et al., 2005). Because of its in vivo source, it can have a large number of constituents in highly variable composition and potentially introduce pathogenic agents (Shah, 1999). The harvest of fetal bovine serum also raises ethical concern. Commercially available serum replacements and serum-free culture media offer some more realistic options for culturing mammalian cells in-vitro. Serum-free media reduce operating costs and process variability while lessening the potential source of infectious agents (Froud, 1999). Improvements in the composition of commercially available cell culture media have enhanced our ability to successfully culture many types of animal cells. Serum-free media have been developed to support in-vitro myosatellite cell cultures from the turkey (McFarland et al., 1991), sheep (Dodson and Mathison, 1988) and pig (Doumit et al., 1993). Variations among different serum-free media outline the fact that satellite cells from different species have different requirements and respond differentially to certain additives (Dodson et al., 1996). Ultroser G is an example of a commercially available serum substitute specially designed to replace fetal bovine serum for growth of anchorage-dependent cells in-vitro. It has a consistent composition containing growth factors, binding proteins, adhesin factors, vitamins, hormones and mineral trace elements, all necessary for eukaryotic cell growth. It has one-fifth the protein content of serum (Pope et al., 1987), yet growth of mammalian skeletal myocytes with serum substitute Ultroser G showed more maturation over the same period of time, longer lasting viability and longer myotubes with more localized nuclei (Benders et al., 1991). While Ultroser G has many beneficial effects on the growth and maturation of muscle tissue in-vitro, its costliness may make this an unlikely candidate for scale up. In addition, while the Ultroser G and other commercially available

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 39 serum replacements may have advantageous effects on the growth of tissues, their exact formulations are protected by commercial copyright and an evaluation of their suitability on a large scale can only be determined by cost and effects. In most cases, serum-free media are supplemented with purified proteins of animal origin (Merten, 1999). Benjaminson et al. (2002), in their investigations with fish explants, found that maitake mushroom extracts were comparable to serum as a growth medium in promoting explants surface area expansion. A cheap, rich serum is necessary for an in-vitro meat production system; it is possible that amino acid-rich mushroom extracts could be applied here. And it has recently been shown that lipids such as sphingosine-1-phosphate can replace serum in supporting the growth and differentiation of embryonic tissue explants (W. Scott Argraves, Medical University of South Carolina, personal communication, 22 May 2004). The development of an appropriate serum- free media completely free of any animal-derived components appears ideal, but the potential for allergens from plant-derived proteins are a risk factor to be mindful of. In addition to supplying proper nutrition to growing muscle cells in culture, it is necessary to provide an appropriate array of growth factors. Growth factors are synthesized and released by muscle cells themselves and, in tissues, are also provided by other cell types locally (paracrine effects) and non-locally (endocrine effects). The liver is the primary source of circulating insulin-like growth factor-I. Typically, investigators initiate differentiation and fusion of myoblasts by lowering the levels of mitogenic growth factors. The proliferating cells then commence synthesis of insulin-like growth factor-II, which leads to differentiation and formation of multinucleated myotubes. So, the successful system must be capable of changing the growth factor composition of the media. Creating an optimal cocktail of hormones, and growth factors is a complex undertaking, one which requires an extensive amount of investigation. Extrinsic regulatory factor selection must be specific to the chosen cell type and species, as myosatellite cells for instance, of different species respond differentially to the same regulatory factors (Burton et al., 2000). It is also likely that the formulation may be required to change over the course of the culturing process. For instance, the proliferation period may require one certain combination of growth factors and hormones while the differentiation and maturation period may require a different set. A multitude of regulatory factors have been identified as being capable of inducing myosatellite cell proliferation (Cheng et al., 2006), and the regulation of meat animal-derived myosatellite cells by hormones, polypeptide growth factors and extracellular matrix proteins has also been investigated (Dodson et al., 1996; Doumit et al., 1993). Purified growth factors or hormones may be supplemented into the media from an external source such as transgenic bacterial, plant or animal species which produce recombinant proteins (Houdebine, 2009). Alternatively, a sort of synthetic paracrine signalling system can be arranged so that co- cultured cell types (a feeder layer) can secrete growth factors which can promote cell growth and proliferation in neighbouring cells. Co-cultured hepatocytes for instance could provide insulin-like Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 40 growth factors which stimulate myoblast proliferation and differentiation (Cen et al., 2008) as well as myosatellite cell proliferation in several meat-animal species in vitro (Dodson et al., 1996). Human growth hormone, routine produced from a transgenic bacterial source can be supplemented into the medium to stimulate production of insulin-like growth factors by hepatocytes. Alternatively, autocrine growth factor signalling can play a role, as certain muscle cell-secreted growth factors such as insulin- like growth factor II stimulate myocyte maturation (Wilson et al., 2003). Similarly, growth factor production can occur in genetically engineered muscle cells.

5.3. Co-culturing

Commonly, cell cultures are expanded and differentiated in monoculture, which creates a well- controlled environment without the interaction of different cell types. Once stem cells are differentiated into myoblasts, these cells are specialized to produce contractile proteins but produce only little extracellular matrix. The in-vivo situation is distinctly different from this. In skeletal muscle, nerve cells, cells forming blood vessels and fibroblasts that form the basal membrane are ubiquitously present. Therefore, other cells likely need to be introduced to engineer muscle. Extracellular matrix is mainly produced by fibroblasts residing in the muscle, which could be beneficial to add to the culture system (Brady et al., 2008). Accruing evidence suggests that the basal membrane is paramount in directing regeneration and controlled growth in many tissues, including skeletal muscle. It regulates the activity of locally active growth factors such as fibroblast growth factor (FGF), transforming growth factor beta (TFG-P) and hepatocyte growth factor (HGF). Since the presence of extracellular matrix (ECM) gives meat its texture and bite, co-culturing primary myotubes with fibroblasts may be a good way to improve the maturation and to produce a meat-like texture at the same time. However, co- cultures of fibroblasts and myoblasts involve the risk of fibroblasts overgrowing the myoblasts, due to the difference in growth rate. Co-culturing has been shown to be very effective in the in-vitro engineering of skeletal muscle when the highly proliferative fibroblasts are seeded as a low percentage of the total cell population. Next to fibroblasts, regular consumption meat also contains fat and a vasculature. Possibly, co-culture with fat cells should also be considered (Edelman et al., 2005). The problem of vascularisation is a general issue in tissue engineering. Tissue engineering is currently at the level in which we can only produce thin tissues because of passive diffusion limitations. To overcome the tissue thickness limit of 100-200 μm, a vasculature needs to be created (Jain et al., 2005). Proof of concept for endothelial networks within engineered tissues has been provided (Levenberg et al., 2005) but reproducible and routine incorporation of vascular networks in a co- culture system will pose a special challenge.

5.4. Contraction/Conditioning

In the body, regular activity of the skeletal muscle is a potent stimulus for increase of muscle mass. It promotes differentiation and healthy myofiber morphology while preventing atrophy. Muscle in vivo is innervated, allowing for regular, controlled contraction. Upon contraction, myotubes produce the IGF-1 splice variant mechano growth factor (MGF) which increases protein synthesis in the myotubes. Furthermore, internal forces that occur inside the myotubes during contraction are potent regulators of the cytoskeletal arrangement. An in-vitro system would necessarily culture denervated muscle tissue, so contraction must be stimulated by alternate means. The creation of native-like tissue architecture with the capacity of active force generation is crucial in the process towards tissue engineered muscle, and consequently also important for in vitro meat production. However, the advances made in culturing of engineered muscle constructs have not yet resulted in satisfactory products. An important hurdle that still has to be overcome is the inability of muscle cells to fully mature within these engineered muscle constructs. Although biochemical stimuli may be more important in the initial differentiation process, biophysical stimuli have proven to be crucial in the maturation towards functional tissue with native-like properties (Kosnik et al., 2003).

5.5. Biophysical Conditioning

Regarding the relatively poor development of sarcomeres in vitro (Engler et al., 2004), indicated by a lack or limited level of maturation specific cross striations, biochemical stimulation alone may not be sufficient in the maturation process towards fully functional engineered muscle constructs. It appears that in addition to biochemical stimuli, biophysical stimulation is required for full muscle maturation and function. In culture, it takes some time before myotubes have matured enough to contract in such a way that they exert significant intracellular forces. Stretch can be applied to the culture to yield a similar effect on the immature myotubes. This procedure has the added advantage that the myotubes will align in the direction of the applied stretch, which will make electrical stimulation more effective, and it does stimulate the production of ECM proteins by the fibroblasts in the culture. Cha et al. (2006) have found that administration of cyclic mechanical strain to a highly porous scaffold sheet promotes differentiation and alignment of smooth muscle cells. Edelman et al. (2005) and Van Eelen et al. (1999) proposed mechanical stretching of scaffolds and expandable scaffold beads to fulfil the requirement of providing contraction. Mechanical stimulation is an important biophysical stimulus in myogenesis (Vandenburgh and Karlisch, 1989). Mechanotransduction, the process through which cells react to mechanical stimuli, is a complex but increasingly understood mechanism (Burkholder, 2007; Hinz, 2006). Cells attach to the insoluble meshwork of extracellular matrix proteins mainly by means of the family of integrin receptors (Juliano and Haskill, 1993), transmitting the applied force to the cytoskeleton. The resulting series of events

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 42 shows parallels to growth factor receptor signalling pathways, which ultimately lead to changes in cell behaviour, such as proliferation and differentiation (Burkholder, 2007). Different mechanical stimulation regimes affect muscle growth and maturation. The application of static mechanical stretch to myoblasts in vitro resulted in a facilitated alignment and fusion of myotubes, and also resulted in hypertrophy of the myotubes (Vandenburgh and Karlisch, 1989). In addition, cyclic strain activates quiescent satellite cells (Tatsumi et al., 2001) and increases proliferation of myoblasts (Kook et al., 2008). These results indicate that mechanical stimulation protocols affect both proliferation and differentiation of muscle cells. The applied stimulation should be tuned very precisely to reach the desired effect. Percentage of applied stretch, frequency of the stimulus and timing in the differentiation process are all parameters that presumably influence the outcome of the given stimulus.

5.6. Scaffolds

As myoblasts are anchorage-dependent cells, a substratum or scaffold must be provided for proliferation and differentiation to occur (Stoker et al., 1968). An ideal scaffold must have a large surface area for growth and attachment, be flexible to allow for contraction as myoblasts are capable of spontaneous contraction, maximize medium diffusion and be easily dissociated from the meat culture. A best scaffold is one that mimics the in-vivo situation as myotubes differentiate optimally on scaffold with a tissue-like stiffness (Engler et al., 2004) and its by-products must be edible and natural and may be derived from non-animal sources, though inedible scaffold materials cannot be disregarded. New biomaterials may be developed that offer additional characteristics, such as fulfilling the requirement of contraction for proliferation and differentiation (De Deyne, 2000). Thus, challenge is to develop a scaffold that can mechanically stretch attached cells to stimulate differentiation and a flexible substratum to prevent detachment of developing myotubes that will normally undergo spontaneous contraction. Edelman et al. (2005) proposed porous beads made of edible collagen as a substrate while as Van Eelen et al. (1999) proposed a collagen meshwork described as a collagen sponge of bovine origin. The tribeculate structure of the sponge allows for increased surface area and diffusion, but may impede harvesting of the tissue culture. Other possible scaffold forms include large elastic sheets or an array of long, thin filaments. Cytodex-3 micro-carrier beads have been used as scaffolds in rotary bioreactors but these beads have no stretching potential. One elegant approach to mechanically stretch myoblasts would be to use edible, stimuli-sensitive porous microspheres made from cellulose, alginate, chitosan, or collagen (Edelman et al., 2005) that undergo, at minimum, a 10% change in surface area following small changes in temperature or pH. Once myoblasts attach to the spheres, they could be stretched periodically provided such variation in the pH or temperature would not negatively affect cell proliferation, adhesion and growth. Jun et al. (2009) have found that growing myoblasts on electrically

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 43 conductive fibers induces their differentiation, forming more myotubes of greater length without the addition of electrical stimulation but use of such inedible scaffolding systems necessitates simple and non-destructive techniques for removal of the culture from the scaffold. Another important factor is the texture and microstructure of scaffolds as texturized surfaces can attend to specific requirements of muscle cells, one of which is myofiber alignment. This myofiber organization is an important determinant for the functional characteristics of muscle and the textural characteristics of meat. Lam et al. (2006) cultured myoblasts on a substrate with a wavy micro patterned surface to mimic native muscle architecture and found that the wave pattern aligned differentiated muscle cells while promoting myoblast fusion to produce aligned myotubes. While using scaffold-based techniques for meat culturing, micro patterned surfaces could allow muscle tissue to assume a two dimensional structure more similar to that of meat of native origin. Production of meat by the scaffold-based technique faces a technical challenge of removal of the scaffolding system. Confluent cultured cell sheets are conventionally removed enzymatically or mechanically, but these two methods damage the cells and the extracellular matrix they may be producing (Canavan et al., 2005). However, thermo responsive coatings which change from hydrophobic to hydrophilic at lowered temperatures can release cultured cells and extracellular matrix as an intact sheet upon cooling (Da Silva et al., 2007). This method known as thermal liftoff, results in undamaged sheets that maintain the ability to adhere if transferred onto another substrate (Da Silva et al., 2007) and opens the possibility of stacking sheets to create a three-dimensional product. To remove the culture as a sheet, a hydrophilic membrane or gel placed on the apical surface of the culture before detachment can provide physical support and use of a fibrin hydrogel is ideal for skeletal muscle tissue because cells can migrate, proliferate and produce their own extracellular matrix within it while degrading excess fibrin. These two-dimensional sheets could be stacked to create a three-dimensional product as suggested by Van Eelen et al. (1999).

5.7. Bioreactor

It is in the bioreactor that everything comes together; the cells, the culture medium and the scaffold. Nutrients and oxygen need to be delivered close to each growing cell, on the scale of millimetres. In animals this job is handled by blood vessels. A bioreactor should emulate this function in an efficient manner. To grow thick slabs of muscle, there is need to establish good blood supply within the growing tissue cells. Scientists have thought of ways of getting around the blood supply problem with one suggestion of using a bioreactor with a branching network of hundreds of tiny edible tubes that act like artificial capillaries to convey nutrients to the growing meat. Through fluctuations in temperature an environment is created which can be likened to a fitness centre with movement training

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 44 for the muscle cells. During the past few years a large variety of simple, often disposable, stirred tank type of reactors has been described, often for use at an industrial scale. Proper reactor design can add a lot to the rate of oxygen transfer. It is most ideal to grow cells in a chemostats. By continuous supply of fresh medium and removal of cells plus medium, a constant, time-independent, environment can be created which gives optimal opportunity to characterize metabolic processes. This system also allows continuous supply of cells. Nevertheless, chemostats are challenging devices for use of slow-growing mammalian cells. Generally, batch cultures and chemostats do not result in very high cell densities. This parameter can be further increased by running so-called fed-batch cultures, in which after an initial phase of exponential growth the limiting nutrient is added in a steady supply. Achieving adequate perfusion of the cultured tissue is a key to producing large culture quantities. To construct viable tissue greater than 100-200 μm in thickness it is necessary to have adequate oxygen perfusion during cell seeding and cultivation on the scaffold (Radisic et al., 2008). Adequate oxygen perfusion is mediated by: (a) Bioreactors which increase mass transport between culture medium and cells, along with; (b) The use of oxygen carriers to mimic haemoglobin, providing oxygen supply. Efforts to produce heme proteins and blood substitute components using the microorganisms Escherichia coli, Pichia pastis, and Aspergillus niger are underway as these organisms are already used to commercially produce human pharmaceuticals and food additives (Lowe, 2006). Development of an artificial blood is an active area of research and many applicable options are likely to arise with time. Much recent skeletal muscle tissue engineering research has employed NASA rotating bioreactors. Their chief advantages are that cells are in near-continuous suspension, fluid shear is minimal, and suspension is possible for tissue assemblies up to 1 centimetre. These bioreactors can sustain biomass concentrations up to 108 cells per mL. Research size rotating bioreactors (10 to 250 mL) have been scaled up to three litres and, theoretically, scale up to industrial sizes should not affect the physics of the system. Industrial scales are already available for low-shear particle-based bio film reactors, allowing biomass concentrations as high as 30 kg/m3.

6. Methods so far Proposed

Meat is already cultured on small and early scales using a variety of basic procedures, including techniques that use scaffolds and those that rely on self-organization (Edelman et al., 2005). The different design approaches for an in-vitro meat production system, all of which are designed to overcome the diffusion barrier, range from those currently in use (scaffold/cell culture based and self organizing/tissue culture techniques) to the more speculative possibilities.

6.1. Scaffolding Techniques

In scaffold-based techniques, embryonic myoblasts or adult skeletal muscle satellite cells are proliferated, attached to a scaffold or carrier such as a collagen meshwork or micro carrier beads and then perfused with a culture medium in a stationary or rotating bioreactor. By introducing a variety of environmental cues, these cells fuse into myotubes, which can then differentiate into myofibers (Kosnik et al., 2003). The resulting myofibers may then be harvested, cooked and consumed as meat. Other forms of scaffolding could also be used, for example, growing muscle tissue on large sheets of edible or easily separable material. The muscle tissue could be processed after being rolled up to suitable thicknesses (Edelman et al., 2005). While, these kinds of techniques work for producing ground processed (boneless) meat with soft consistency, they do not lend themselves to highly structured meats like steaks. However, cells can also be grown in substrates that allow for the development of self-organizing constructs that produce more rigid structures.

6.2. Self-organizing Tissue Culture

To produce highly structured meats, one would need a more ambitious approach, creating structured muscle tissue as self-organizing constructs (Dennis and Kosnik, 2000) or proliferating existing muscle tissue in vitro, like Benjaminson et al. (2002) who cultured Gold fish (Carassius auratus) muscle explants. They took slices of goldfish tissue, minced and centrifuged them to form pellets, placed them in Petri dishes in a nutrient medium and grew them for 7 days. The explanted tissue grew nearly 14% when using fetal bovine serum as the nutrient medium and over 13% when using Maitake mushroom extract. When the explants were placed in a culture containing dissociated Carassius skeletal muscle cells, explants surface area grew a surprising 79% in a week’s time. After a week, the explants and their newly grown tissue, which looked like fresh fish filets, were cooked (marinated in olive oil and garlic and deep-fried) and presented to a panel for observation. The panel reported that the fish looked and smelled good enough to eat (Benjaminson et al., 2002; Britt, 2002; Hukill, 2006; Sample, 2002). Tissue culture techniques have the advantages that explants contain all the tissues which make up meat in the right proportions and closely mimics in-vivo situation. However, lack of blood circulation in these explants makes substantial growth impossible, as cells become necrotic if separated for long periods by more than 0.5 mm from a nutrient supply (Dennis and Kosnik, 2000). According to Vladimir Mironov entirely artificial muscle can be created with tissue engineering techniques by a branching network of edible porous polymer through which nutrients are perfused and myoblasts and other cell types can attach. Such a design using the artificial capillaries for the purpose of tissue-engineering has been proposed (Zandonella, 2003). Like the myooids, it is possible to co- culture the myoblasts with other cell types to create a more realistic muscle structure which can be organized in much the same way as real muscles (Dennis and Kosnik, 2000; Dennis et al., 2001;

Kosnik et al., 2001).

7. Consumer Acceptability

The consumption of meat by humans is as old as the human species, and perhaps even older, as is implied by the fact that most hominid primates consume significant amounts of meat (Nestle, 1999). However, meat was always obtained by killing an animal. The in vitro culturing of meat is a completely novel technique, and cultured meat can be regarded as a novel product. To be implemented as large scale commercial meat production system the in-vitro meat would have to be accepted by consumers. For that it is necessary that the taste and texture of in-vitro meat closely resemble the natural meat. The fact that meat would not exactly resemble natural meat is offset by the fact that there is large market for boneless and skin less meat and use of meat for processed meat products such as hamburgers and sausages. It would also appeal to increasing number of people concerned about food security and also to those who are against killing of animals for meat. The argument that such meat is not natural is countered by the response that many foods consumed today are also engineered. Some beverages and cheese are prime examples. It is also pointed out that the way meat is produced today is far removed from traditional methods. There is a ‘yuck’ factor involved with producing any novel food. Criticism over the minor ‘yuck’ factor associated with vat-grown animal protein is countered and compared to what goes on in the meat industry. Since competition to reduce the price of meat, eggs, and dairy products has driven the worldwide trend to replace small family farms with large warehouses, animals are typically confined in crowded cages or pens or in restrictive stalls. The clamorous animal rights group, People for the Ethical Treatment of Animals (PETA), calls it “the best thing since sliced bread” arguing that “anything that takes the cruelty out of meat-eating is good.” It is anticipated that consumers will almost certainly accept cultured meat regardless of metaphysical reservations. A stem-cell burger will have no E. coli, no Salmonella etc. It will have no fat or only fat put there as an additive. And cultured meat will taste the same as animal meat because the protein content (which imparts taste) will be the same, say the researchers. Everything said and done, production of in-vitro meat seems like an excellent idea, with the potential to ease the burden on the environment from meat production, reduce greenhouse gas emissions and improve human health. However there is an intuitive reluctance to reject ignaling d foods. It is this factor which could lead to non acceptance of in-vitro meat. Animals are still used. The stem cells originate from an animal sample, regardless of how many times it is allowed to regenerate in a laboratory. Furthermore, growth factors required for ignaling the cells to differentiate into muscle cells are collected from blood samples of the animals during the fetal stage. No animals are killed from any of these collections, but they would still be needed for production of specific cells, which some still consider

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 47 inhumane. Many fear there may still be underlying long-term health risks that trials conducted by the FDA and other organizations would miss. In addition, there is a concern that the quality of the meat will suffer and have an unnatural taste and texture. Some people disagree with a transition to in-vitro meat as a way to substitute animal meat because it would cause a flood in the market for farmers wishing to get rid of livestock in order to pursue agricultural production and ultimately result in a massacre of the current livestock. This would potentially cause even more inhumane deaths for the animals. Some believe that stem cell technology can be better used to gain a clearer understanding of how animals produce meat rather than attempting to replicate what animals do. It is said that every time humans mess around the ecological heritage there are always unintended side effects that come from it.

7.1. Suggestions to Make in-vitro Meat More Acceptable

(a) Raise customer awareness about the environmentally unsustainable nature of meat production today. (b) Underline the benefits to the environment and the economic benefits that would accrue from the usage of in-vitro meat. (c) Stress on the health advantages of having in-vitro meat. (d) Stress should be laid on the fact that use of in-vitro meat would reduce the atrocities being committed on animals, like overfeeding, confining in small spaces and use of antibiotics and hormones to accelerate growth, in the name of commercial production of meat. (e) Pertinent question of food security can also be used to sway public opinion in favour of in-vitro meat.

7.2. Economics of Production

The cost of conventional meat production fluctuates due to feed prices, disease and veterinary needs, weather, and the lags in production response due to changing demands. As a result, profit margins for conventional meat production are highly volatile, although producers and consumers are shielded to some extent by public subsidies. The costs of cultured meat are unknown. A major threat for the implementation of the technology comprises the production costs. Obviously the price of cultured meat should not differ too much from the price of traditional meat. If cultivated with nutrient solutions that are currently used for biomedical applications, the cost of producing one pound of in- vitro meat runs anywhere from $1,000 to $10,000. It is believed that in-vitro meat can compete with conventional meat by using nutrients from plant or fungal sources, which could bring the cost down to about $1 per pound. According to a recent economic analysis, conducted to determine whether the production of in-vitro meat is likely to be economically viable when compared with the factory gate prices, say, for chicken, it will be possible to produce in-vitro meat in large quantities for less than Euro 3,300-3,500 ($5,200-5,500) a ton, which the analysis claims is cost competitive with European

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 48 beef prices. On this basis it makes sense to continue to invest in developing the process technology for the efficient large scale production of muscle tissue from stem cells and developing cheaper and larger volume sources of growth medium that do not contain animal products. Advances in the technical aspects of cultured meat production will greatly improve efficiency and lower costs significantly. Theoretically, cultured meat could afford higher resource and labour efficiencies, which could translate into lower costs, if cultured meat were produced at scale with an affordable medium. Secondly, it has been predicted that the price of regular meat will rise significantly, as the meat demand will only increase with the growing world population while there is simply not enough feed for the animals. It is therefore expected that the price of regular meat will only increase, whereas it is predicted that the price of in vitro meat will decrease as the market becomes bigger. In any case, it is unlikely that cultured meat will soon compete with conventional meat in ordinary markets. However, there are technologies now found in virtually every household computer, the internet, freeze-dried foods that originally cost too much for mass acceptance. They found their first applications in space and defence projects. Only after reductions in cost by several orders of magnitude were they mass-produced. This could be true of cultured meat, as well.

8. Challenges

Cultured meat technology is still in its infancy. Cultured meat production is not a commercial reality yet, but researchers believe it could be possible and economically feasible. However, there are a number of technical obstacles that have to be overcome before cultured meat can be a compelling substitute for conventional meat. Growing mammalian cells in sufficient volumes to produce a meat- like product for a price which is competitive with meat poses many challenges. The problem boils down to producing a cell-culture medium in large enough quantities at a low enough price. Growing mammalian cells in suspension in 20 k litre bioreactors is currently possible but expensive and increasing the volume will present challenges. Growing cells in a 3D matrix is still in the research stage. The biology around the choice of cell, the optimum conditions for growth and the method to differentiate into the final product. Need for sterile engineering over a considerable scale. Mammalian cells grow slowly and contaminants will out compete them. Plant must be cleanable and sterilizable, clean in place (CIP) and sterilize in place (SIP) systems will be needed, standard operating procedures (SOPs) will be required and raw materials will need to be pharmaceutical grade. Sophisticated instrumentation will be required to measure and control conditions inside the reactors. A great challenge in producing a competitive in-vitro grown meat product is ensuring that all necessary nutrients are present. Dietary minerals and vitamins not synthesized by myocytes will often

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 49 require binding proteins in medium and effective transport mechanisms for entry into the cells. Knowledge of the complex metabolism of each crucial vitamin and mineral is necessary to develop a nutritionally valuable meat product. While determining the proper nutrient profile will be a major hurdle to overcome, it comes with the knowledge of how to manipulate the culturing system to make nutritionally tailored products. Adequately providing the physical and chemical requirements for metabolism while removing damaging waste products, can be a difficult task with the possibility of unforeseen shortcomings. Also important to consider is that muscle tissue and meat are biochemically (and therefore qualitatively) dissimilar. The metabolic reactions that proceed post-slaughter: anaerobic glycolysis, lactic acid accumulation, protein denaturation and enzymatic proteolysis, are responsible for producing the textural quality, taste and appearance of meat. One uncertainty concerning metabolism is whether these typical post-slaughter reactions will occur in cultured meat after harvest in like manner to properly convert cultured muscle tissue to meat as it is traditionally defined. Furthermore the source of secondary ingredients, i.e. those substances which are not directly involved in cell growth, should be considered. These include collagen and polymers (for protein spheres, scaffolds). Collagen sources derived from animals directly would defeat the purpose of the IMPS. Recombinant collagen sources are available, but these suffer from a bad consumer acceptance.

9. Spin-off

It is anticipated that research on cultured meat will have a significant spin-off, primarily for the biomedical industry. Four areas of spin-off can be discerned: (1) Generation of stem cells from pigs (and other farm animals); (2) Production of tissue culture media that do not contain animal products; (3) Increased knowledge on aspects of tissue engineering; (4) Specific know-how on (industrial scale) bioreactors.

10. Conclusions and Future Prospects

Man’s impact on the planet Earth has become alarmingly clear in the past decades. Numerous reports have been produced with frightening scenarios of the future and indeed without a change of policies there is a distinct possibility that our planet will become inhabitable. Energy consumption and the livestock sector in particular have been identified as the leading drivers of climate change, deforestation, pollution and reduction of biodiversity. Simultaneously, the livestock sector is extremely important for the agricultural economy and obviously for the human diet. Different solutions can, and should be, developed to reduce the environmental impact of this sector, one of those being the (partial) replacement of traditional meat with an edible product made from animal proteins produced by cultured cells. The possibility of making an edible meat product from cultured cells without the use of

Copyright © 2013 by Modern Scientific Press Company, Florida, USA Int. J. Food Nutr. Saf. 2013, 3(1): 29-54 50 animals may provide a change in agriculture and society at large. The infrastructure that should accompany the implementation of such a technology is still lacking and has to be developed. Cultured meat has the potential to make eating animals unnecessary, even while satisfying all the nutritional and hedonic requirements of meat eaters. Since, crucial knowledge is still lacking on the biology and technology, it may be concluded that commercial production of cultured meat is as yet not possible and the focus must be on filling these gaps in knowledge. Proponents say it will ultimately be a more efficient way to make animal meat. There are arguments in favour and against and may be some religious and ethical concerns which need to be addressed. But from a scientific perspective, the sky is the limit when it comes to meat production in the laboratory.

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