Animal-free Meat Biofabrication
Nutrition-related diseases, food borne illnesses, resource use and pollution and use of farm animals are some serious consequences associated with conventional meat production system and consumers have expressed growing concern over them. Biofabrication, production of complex living and non-living biological products, is a potential solution to reduce these ill effects of current meat production system. The industrial potential of biofabrication technology is far beyond the traditional medically oriented tissue engineering and organ printing and, in the long term, biofabrication can contribute to the development of novel biotechnologies that can dramatically transform traditional animal-based agriculture by inventing animal-free food, leather and fur products. In this study we review the possibility of producing in vitro meat using tissue-engineering techniques that may offer health and environmental advantages by reducing environmental pollution and land use associated with current meat production systems. Besides, reducing the animal suffering significantly, it will also ensure sustainable production of designer, chemically safe and disease free meat as the conditions in an in vitro meat production system are controlled and manipulatable. The techniques required to produce in vitro meat are not beyond imagination and the basic methodology of an in vitro meat production system (IMPS) involves culturing muscle tissue in a liquid medium on a large scale but the production of highly-structured, unprocessed meat faces considerably greater technical challenges and a great deal of research is still needed to establish a sustainable in vitro meat culturing system on an industrial scale. In the long term, tissue-engineered meat is the inescapable future of humanity. However, in the short term the extremely high prohibitive cost of the biofabrication of tissue-engineered meat is the main potential obstacle, although large-scale production and market penetration are usually associated with a dramatic price reduction.
July 09, 2010; Accepted: August 27, 2010;
Published: November 24, 2010
Conventional meat production system is a major source of pollution and a significant
consumer of fossil fuels, land and water resources. 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. World meat
production at present is contributing between 15 and 24% of total current greenhouse
gas emissions; a great proportion of this percentage is due to deforestation
to create grazing land (Steinfeld et al., 2006).
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 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). In addition, there is the problem of antibiotics being
used as growth promoters for animals kept in intensive farming. This use probably
contributes to the emergence of multi-drug-resistant strains of pathogenic bacteria
(Sanders, 1999). 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). Nutrition related diseases,
such as cardiovascular disease and diabetes, associated with the over-consumption
of animal fats are now responsible for a third of global mortality (WHO,
2001). Food-borne illnesses have become increasingly problematic, with a
six fold increase in gastro-enteritis and food poisoning in industrialized countries
in the last 20 years (Nicholson et al., 2000)
and the most common causes of food borne diseases in EU, USA and Canada are
contaminated meats and animal products (Barnard et al.,
1995; Mead et al., 1999; Nataro
and Kaper, 1998; European Food Safety Authority, 2006;
Fisher et al., 2006). It is anticipated that
by the year 2050 global population will increase from 6 billion (in 2000) to
9 billion people which will be accompanied by a rise in annual greenhouse gas
emissions from 11.2 to 19.7 gigatonne of carbondioxide, carbon equivalent and
in the same period annual global meat production will rise from 228 (in 2000)
to 465 million tonnes (Steinfeld et al., 2006).
With a growing population and great proportion of which facing starvation, it
no longer makes sense to contribute staple crops toward inefficient meat production,
where 1 kg poultry, pork and beef requires 2, 4 and 7 kg of grain, respectively
(Rosegrant et al., 1999). Thus, establishment
of an in vitro meat production system (IMPS) is becoming increasingly
justifiable in light of the sizable negative effects of current meat production
The idea of in vitro meat for human consumption is not new but was predicted
long back by Winston Churchill in the 1920s (Churchill, 1932).
In 1912, Alexis Carrel managed to keep a piece of chick heart muscle alive and
beating in a Petri dish. It was much later in the early 1950s when Willem van
Eelen of Netherlands 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 Eelens theoretical idea was patented. Some efforts have
already been put into culturing artificial meat. SymbioticA harvested muscle
biopsies from frogs and kept these tissues alive and growing in culture dishes
(Catts and Zurr, 2002). Other research initiatives have
also achieved keeping muscle tissue alive in a fungal medium, anticipating on
the infection risk associated with serum-based media. In 2002, a study involving
the use of muscle tissue from the common goldfish (Carassius auratus)
cultured in Petri dishes was published in which the possibilities of culturing
animal muscle protein for long term space flights or habituation of space stations
were explored (Benjaminson et al., 2002).
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
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 microcarrier 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 (Fig.
Currently there are two detailed proposals based on emerging field of tissue
engineering (Boland et al., 2003; Zandonella,
2003) for using cell culture for producing in vitro meat. Both these
proposals are similar in nature and neither of the two has been tested. One
of the two proposals to create an in vitro meat production system has
been written by Vladimir Mironov for the NASA (Wolfson,
2002) while the other proposal has been written by Willem van Eelen who
also holds a worldwide patent for this system (Van Eelen
et al., 1999). However, Catts and Zurr (2002)
appear to have been the first to have actually produced meat by this method.
Both of these systems work by growing myoblasts in suspension in a culture medium.
Mironov proposal uses a bioreactor in which cells are grown together with collagen
spheres to provide a substrate onto which the myoblasts can attach and differentiate
whereas van Eelens proposal uses a collagen meshwork and the culture medium
is refreshed from time to time or percolated through the meshwork. Once differentiated
into myofibers, the mixture of collagen and muscle cells can be harvested and
used 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) meats 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.
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, explant surface area grew a surprising 79% in a weeks 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; Sample, 2002; Hukill,
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 (Wolfson, 2002). 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).
||Scaffold-based cultured meat production
|| Possible in vitro meat production scheme
Organ printing: The various problems associated with the current tissue engineering techniques are that they cannot provide consistency, vascularization, fat marbling or other elements of workable and suitably-tasting meat that are not simply versions of ground soft meat. A potential solution to such problems comes from research on producing organs for transplantation procedures known as organ printing.
Organ printing uses the principles of ordinary printing technology-the technology
used by inkjet printers to produce documents. Researchers use solutions containing
single cells or balls of cells and spray these cell mixtures onto gels that
act as printing paper. The paper can actually be removed through a simple heating
technique or could potentially be automatically degradable. What happens is
essentially that live cells are sprayed in layers to create any shape or structure
desired. After spraying these three dimensional structures, the cells fuse into
larger structures, such as rings and tubes or sheets. As a result, researchers
argue that the feasibility of producing entire organs through printing has been
proved. The organs would have not only the basic cellular structure of the organ
but would also include, built layer-by-layer, appropriate vascularization providing
a blood supply to the entire product. For applications focused on producing
meat, fat marbling could be added as well, providing taste and structure. Essentially,
sheets and tubes of appropriate cellular components could create any sort of
organ or tissue for transplantation or for consumption (Mironov
et al., 2003; Aldhous, 2010; Hopkins
and Dacey, 2008).
Biophotonics: Biophotonics is a new field that relies on the effects
of lasers to move particles of matter into certain organizational structures,
such as three-dimensional chessboard, or hexagonal arrays. In general, biophotonics
refers to the process of using light to bind together particles of matter and
the mechanisms of this field are still poorly understood. A surprising property
of interacting light, this phenomenon produces so called optical matter
in which the crystalline form of materials (such as polystyrene beads) can be
held together by nets of infrared light that will fall apart when the light
is removed. This is a phenomenon a step-up from optical tweezers that have been
used for years to rotate or otherwise move tiny particles in laboratories. This
has a binding effect among a group of particles that can lead them not only
to be moved one by one to specific locations but that can coax them to form
structures. Although, primarily sparking interest in medical technologies such
as separating cells, or delivering medicine or other microencapsulized substances
to individual cells, there is an intriguing possibility that such a technology
could be used for the production of tissues, including meat (Hopkins
and Dacey, 2008). Arrays of red blood cells and hamster ovaries have already
been created using this technology (Mullins, 2006).
Given the success of creating two-dimensional arrays, there is the possibility
of producing tissue formations that use only light to hold the cells together,
thus eliminating the need for scaffoldings (Hopkins and
Nanotechnology: The ability of optical tweezers to rotate or move tiny
particles has intrigued nanotechnologists, who have inventive plans for what
to do with the molecular scale sized robots they would like to create (but so
far, having few tools with which to make them). Nanotechnology (the production
and alteration of materials at the level of the atom and molecule) holds out
enormous possibilities and the holy grail of nanotechnology is some version
of an assembler, a robot the size of a molecule that would allow moving matter
at the atomic and molecular level. The obvious power of such a technology given
that everything is made of the same basic atoms but simply arranged in different
ways is that we would be able to construct virtually any substance we wanted
from scratch by putting together exactly the molecules we wanted. Interestingly,
one of the first examples given of the speculative technology of nanotechnology
was that of synthesized meat. Thus, technologies ranging from the actual to
the speculative promise a variety of ways to create real meat without killing
animals. Though still commercially infeasible at the moment or in some cases
technologically infeasible for several years to come, the point here is not
to be distracted by the fact that we cannot yet make use of these technologies
but rather to decide whether we should support the development of these technologies
(Hopkins and Dacey, 2008).
Future efforts in culturing meat will have to address the limitations of current techniques through advances that make cultured cells, scaffolds, culture media and growth factors edible and affordable.
Cells: In vitro meat can be produced by culturing the cells from
farm animal species in large quantities starting from a relatively small number
and 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
however; 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.
However, different efforts invested into establishing ungulate stem-cell lines
over the past two decades have been generally unsuccessful with difficulties
arising in the recognition, isolation and differentiation of these cells (Keefer
et al., 2007). 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.
Alternatively, we can use adult stem cells from farm animal species and myosatellite
cells are one example of an adult stem-cell type with multilineage potential
(Askura et al., 2001). Adult stem cells have
been isolated from several different adult tissues (Wagers
and Weissman, 2004) but their in vitro proliferation capacity is
not unlimited and can proliferate in vitro for several months at most.
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. A rare population of multipotent
cells found in adipose tissue known as adipose tissue-derived adult stem cells
(ADSCs) is another relevant cell type for in vitro meat production (Gimble
et al., 2007) which can be obtained from subcutaneous fat and subsequently
transdifferentiated to myogenic, osteogenic, chondrogenic or adipogenic cell
lineages (Kim et al., 2006). However, adult stem
cells are prone to malignant transformation in long-term culture (Lazennec
and Jorgensen, 2008) that is the greatest matter of debate. It has been
observed that adipose tissue-derived adult stem cells immortalize at high frequency
and undergo spontaneous transformation in long-term (4-5 months) culturing (Rubio
et al., 2005), while evidence of adult stem cells remaining untransformed
have also been reported (Bernardo et al., 2007).
To minimize the risk of spontaneous transformation, re-harvesting of adult stem
cells may be necessary in an in vitro meat production system and as such
obtaining ADSCs from subcutaneous fat is far less invasive than collection of
myosatellite cells from muscle tissue.
Matsumoto et al. (2008) reported that mature
adipocytes can be dedifferentiated in vitro into a multipotent preadipocyte
cell line known as dedifferentiated fat (DFAT) cells, reversion of a terminally
differentiated cell into a multipotent cell type. These DFAT cells are capable
of being transdifferentiated into skeletal myocytes (Kazama
et al., 2008) and appear to be an attractive alternative to the use
of stem cells. This process known as ceiling culture method certainly seems
achievable on an industrial scale but Rizzino (2007)
has put forth the argument that many of the claims of transdifferentiation,
dedifferentiation and multipotency of once terminally differentiated cells may
be due to abnormal processes resulting in cellular look-alikes.
Fields: Proliferation and differentiation of myoblasts have been found
to be affected by the mechanical, electromagnetic, gravitational and fluid flow
fields (Kosnik et al., 2003, De
Deyne, 2000). Repetitive stretch and relaxation equal to 10% of length,
six times per hour increase differentiation into myotubes (Powell
et al., 2002). Myoblasts seeded with magnetic microparticles induced
differentiation by placing them in a magnetic field without adding special growth
factors or any conditioned medium (Yuge and Kataoka, 2000).
Electrical stimulation also contributes to differentiation, as well as sarcomere
formation within established myotubes (Kosnik et al.,
Scaffolds: As myoblasts are anchorage-dependent cells, a substratum
or scaffold must be provided for proliferation and differentiation to occur
(Stoker et al., 1968). Scaffolding mechanisms
differ in shape, composition and characteristics to optimize muscle cell and
tissue morphology. 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
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 nondestructive techniques for removal
of the culture from the scaffold.
Furthermore, there are greater technical challenges in developing a scaffold
for large and highly structured meats due to the absence of vascular system.
There is a need to build a branching network from an edible, elastic and porous
material, through which nutrients can be perfused and myoblasts and other cell
types can then attach to this network. Edelman et al.
(2005) acknowledge that a cast of an existing vascularization network, such
as that in native muscle tissue, can be used to create a collagen network mimicking
native vessel architecture. Taking this a step further, Borenstein
et al. (2002) has proposed an approach to create such a network by
creating a cast onto which a collagen solution or a biocompatible polymer is
spread and after solidification seeding the network with endothelial cells.
Following dissolution of the polymer mold, successful proliferation could theoretically
leave behind a network of endothelial tissue, a branched network of micro-channels,
which can be stacked onto each other to form a three-dimensional network onto
which one could grow myocytes. A synthetic vascular system would then require
a circulation pumping system and a soluble oxygen carrier in the medium to be
fully functional. But at this moment creation of these artificial vascular networks
does not translate well into mass production due to the microfabrication processes
required. Alternatively, Benjaminson et al. (2002)
proposed an attempt to create a highly structured meat without a scaffold by
solving the vascularization problem through controlled angiogenesis of explants.
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 micropatterned 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, micropatterned surfaces could allow muscle tissue to assume
a two dimensional structure more similar to that of meat of native origin. Riboldi
et al. (2005) utilized electrospinning, a process that uses electrical
charge to extract very fine fibers from liquids, by using electrospun microfibrous
meshwork membranes as a scaffold for skeletal myocytes. These membranes offer
high surface area to volume ratio in addition to some elastic properties. Electrospinning
creates very smooth fibers, which may not translate well into a good adhesive
surface and coating electrospun polymer fibers with extracellular matrix proteins,
such as collagen or fibronectin, promotes attachment by ligand-receptor binding
interactions (Riboldi et al., 2005). Electrospinning
shows promise for scaffold formation because the process is simple, controllable,
reproducible and capable of producing polymers with special properties by co-spinning
(Riboldi et al., 2005).
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, thermoresponsive 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.
Lam et al. (2009) have presented a method for
detaching culture as a confluent sheet from a non-adhesive micropatterned surface
using the biodegradation of selective attachment protein laminin. However, culturing
on a scaffold may not result in a two-dimensional confluent sheet of culture.
The contractile forces exerted after scaffold removal by the cytoskeleton of
the myocyte are no longer balanced by adhesion to a surface that causes the
cell lawn to contract and aggregate, forming a detached multicellular spheroid
(Da Silva et al., 2007). 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
(Lam et al., 2009). These two-dimensional sheets
could be stacked to create a three-dimensional product as suggested by Van
Eelen et al. (1999).
Industrial bioreactors: Production of in vitro meat for processed
meat based products will require large-scale culturing in large bioreactors
as stem cells and skeletal muscle cells require a solid surface for culturing
and a large surface area is needed for the generation of sufficient number of
muscle cells. Cultured meat production is likely to require the development
of new bioreactors that maintain low shear and uniform perfusion at large volumes
(Pathak et al., 2008). The bioreactor designing
is intended to promote the growth of tissue cultures which accurately resemble
native tissue architecture and provides an environment which allows for increased
culture volumes. A laminar flow of the medium is created in rotating wall vessel
bioreactors by rotating the cylindrical wall at a speed that balances centrifugal
force, drag force and gravitational force, leaving the three-dimensional culture
submerged in the medium in a perpetual free fall state (Carrier
et al., 1999) which improves diffusion with high mass transfer rates
at minimal levels of shear stress, producing three dimensional tissues with
structures very similar to those in vivo (Martin
et al., 2004). Direct perfusion bioreactors appear more appropriate
for scaffold based myocyte cultivation and flow medium through a porous scaffold
with gas exchange taking place in an external fluid loop (Carrier
et al., 2002). Besides offering high mass transfer they also offer
significant shear stress, so determining an appropriate flow rate is essential
(Martin et al., 2004). Direct perfusion bioreactors
are also used for high-density, uniform myocyte cell seeding (Radisic
et al., 2003). Another method of increasing medium perfusion is by
vascularizing the tissue being grown. Levenberg et al.
(2005) had induced endothelial vessel networks in skeletal muscle tissue
constructs by using a co-culture of myoblasts, embryonic fibroblasts and endothelial
cells co-seeded onto a highly porous biodegradable scaffold. Research size rotating
bioreactors have been scaled up to three liters and, theoretically, scale up
to industrial sizes should not affect the physics of the system.
Adequate perfusion of the cultured tissue is key to produce large culture quantities
and it is necessary to have adequate oxygen perfusion during cell seeding and
cultivation on the scaffold as cell viability and density positively correlate
with the oxygen gradient in statically grown tissue cultures (Radisic
et al., 2008). Adequate oxygen perfusion is mediated by bioreactors
which increase mass transport between culture medium and cells and by the use
of oxygen carriers to mimic hemoglobin provided oxygen supply to maintain high
oxygen concentrations in solution, similar to that of blood. Oxygen carriers
are either modified versions of hemoglobin or artificially produced perfluorochemicals
(PFCs) that are chemically inert (Lowe, 2006). Many
chemically modified hemoglobins have been developed but their bovine or human
source makes them an unfit candidate and alternatively, human hemoglobin has
been produced by genetically modified plants (Dieryck et
al., 1997) and microorganisms (Zuckerman et al.,
Culture media and growth factors
Safe media for culturing of stem cells: In vitro meat would
need an affordable medium system to enjoy its potential advantages over conventional
meat production and that medium must contain the necessary nutritional components
available in free form to myoblasts and accompanying cells. Myoblast culturing
usually takes place in animal sera, a costly media that does not lend itself
well to consumer acceptance or large-scale use. 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 and
for the generation of an animal-free protein product, the addition of fetal
calf serum to the cells would not be an option and it is therefore essential
to develop a serum-free culture medium. 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 and 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 containing growth factors, binding
proteins, adhesin factors, vitamins, hormones, mineral trace elements and has
been designed specially to replace fetal bovine serum for growth of anchorage-dependent
cells in vitro (Duque et al., 2003). Benjaminson
et al. (2002) succeeded in using a serum-free medium made from maitake
mushroom extract that achieved higher rates of growth than fetal bovine serum
and recently it has been shown that lipids such as sphingosine-1-phosphate can
replace serum in supporting the growth and differentiation of embryonic tissue
explants. In most cases, serum-free media are supplemented with purified proteins
of animal origin (Merten, 1999).
Indeed such media have already been generated and are available from various companies for biomedical purposes; however, their price is incompatible with the generation of an affordable edible product. Therefore, a cell culture medium has to be developed that does not contain products of animal origin and enables culturing of cells at an affordable price.
For stem cell culturing it is important that these cells remain undifferentiated
and maintain their capacity to proliferate and for the production of cultured
meat a specific and efficient differentiation process initiated with specific
growth factors is needed. An appropriate array of growth factors is required
to growing muscle cells in culture in addition to proper nutrition and these
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 myosatellite cells of different species
respond differentially to the same regulatory factors (Burton
et al., 2000) and as such extrinsic regulatory factors must be specific
to the chosen cell type and species. Furthermore, formulation may be required
to change over the course of the culturing process from proliferation period
to the differentiation and maturation period, requiring different set of factors.
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 can secrete growth factors which
can promote cell growth and proliferation in neighbouring cells. Appropriate
co-culture systems like hepatocytes may be developed to provide growth factors
necessary for cultured muscle production that provide insulin-like 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). Typically, investigators initiate differentiation and fusion of myoblasts
by lowering the levels of mitogenic growth factors and the proliferating cells
then commence synthesis of insulin-like growth factor-II, which leads to differentiation
and formation of multinucleated myotubes (Florini et
al., 1991) and stimulate myocyte maturation (Wilson
et al., 2003). So, the successful system must be capable of changing
the growth factor composition of the media. Currently, the most efficient method
to let (mouse) stem cells differentiate into skeletal muscle cells is to culture
them in a medium that contains 2% horse serum instead of 10 or 20% fetal calf
serum but for the generation of cultured meat, however, it is essential that
the cells are cultured and differentiated without animal products, so a chemically
defined culture medium has to be developed that enables the differentiation
of stem cells to skeletal muscle cells.
Need of cultured meat: Meat composition and quality can be controlled
by manipulating the composition of the culture medium and co-culturing with
other cell types. The flavor, fatty acid composition, fat content and ratio
of saturated to poly-unsaturated fatty acids of the cultured meat can be better
controlled. Moreover, health aspects of the meat can be enhanced by adding factors
to the culture medium which might have an advantageous effect on the health,
like certain types of vitamins (Van Eelen et al.,
||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)
||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. In addition, the risks
of exposure to pesticides, arsenic, dioxins and hormones associated with
conventional meat could also be significantly reduced
||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 in many countries
||In vitro system reduces animal use in the meat production
system as theoretically a single farm animal may be used to produce the
worlds meat supply
||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 labor required per kg of in vitro cultured meat is much
||Bioreactors for in vitro meat production, unlike farm
animals, do not need extra space and can be stacked up in a fabric hall.
Thus, nutritional costs for in vitro cultured meat will be significantly
lower and the decrease in costs of resources, labor and land may be compensated
by the extra costs of a stricter hygiene regime, stricter control, computer
||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 lift-off 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 theaters of war and bunkers designed for long-term survival
of personnel following a nuclear or biological attack
||Need for other protein sources demands production of cultured
meat and because it is, unlike the other products, animal-derived and with
respect to composition most like meat, it may be the preferred alternative
A definite market is available for meat substitutes. Examples are legume-based
and mycoprotein-based meat substitutes. A small market comprising the vegetarians
that do not eat meat for ethical reasons is also available. However, it is still
unclear if animal suffering minimized through in vitro meat production
actually convinces all the vegetarians (Pathak et al.,
||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
||One of the most important reasons to produce in vitro
meat would be consumer demand. More and more people are interested in cultured
meat and it can be a very successful product
||The comparatively minimal land requirement of an in vitro
meat production system allows meat production and processing to take place
domestically in countries which would normally rely on imported meats. By
bringing the stages of the meat production process closer together spatially
and temporally, meat supply can be better determined by demand
||Other factors like potential impact on reducing cardiovascular
diseases, greenhouse gas emissions and liberation of land for nature (including
wild animals), prevention of animal suffering and food scarcity that can
be expected with an increasing world population
Objections to cultured meat: Hopkins and Dacey (2008)
defined the following objections to cultured meat:
Danger: Worry about the danger of consuming untested (or even tested) novel materials.
Cannibalism: The ability to culture human muscle tissue leading to victimless
cannibalism (Peterson, 2006; Mcilroy,
Reality of meat: Some people may feel that in vitro meat is not real meat and they will think of it as artificial meat or synthetic meat and not the real thing. As such, they depreciate the value of the meat in the same way they would look down on artificial flowers or synthetic diamonds.
Naturalness: The quality of being unnatural is the primary objection to cultured meat.
Yuck factor: Ever since Leon Kasss call for making human cloning
illegal largely as a result of the wisdom of repugnance or the less nobly known
yuck factor, people have paid more attention to the reaction of disgust in trying
to judge whether a new and especially biotechnological, process is morally permissible
and whether it should be legally permissible (Kass, 1997).
Technological fix is moral cowardice: One argument against this whole approach is about seeking a technological fix at all.
Wrong moral motivations: A related argument against promoting cultured meat is that this would be motivated by selfishness when we should be self-sacrificing and virtuous. That is, refusing to stop eating meat until a new technology relieves our discomfort is hardly morally laudable.
Delay moral change: Another worry might be that hoping for a technological fix will make people more at ease with meat-eating now, will make them think they do not need to change anything now because in the near future technology will solve their difficulties.
The lives of food animals are better than nothing: One objection that is already familiar from critiques of ethical vegetarianism is that animals lives will go better, paradoxically, in a world with something like the present meat industry, than in a world with universal or widespread vegetarianism.
Taint of the source: Another argument is that in vitro meat will
use original cells gathered from some animal in a morally suspect way and that
the use of such cells will morally taint all future generations of tissue (Hawthorne,
Animal integrity: Novel consideration brought up for the purposes of exonerating some peoples moral intuitions that animals should not be biotechnologically altered is that 1 of animal integrity. In regards to a suggestion that chickens might be genetically altered into insentient lumps of flesh, producing eggs and meat, some have argued that such alterations are intuitively wrong and this sense of wrong can be captured by the idea that the integrity of the animal has been violated.
Dominion versus reverence: A final, more sweeping version of the objection from respect and dignity, which applies to many different types of technological manipulations, goes like this. To revere a creature (or perhaps the world in general) we must accept what is given about it rather than transforming its nature.
In vitro meat production is a sustainable and safer system and could
offer a number of benefits. With cultured meat, the composition, flavour and
functional role of meat could be better controlled; the incidence of food borne
disease could be significantly reduced; and resources could be used more efficiently.
Cultured meat has the potential to make eating animals unnecessary, even while
satisfying all the nutritional and hedonic requirements of meat eaters (Hopkins
and Dacey, 2008). It also has the potential to greatly reduce animal suffering.
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. In vitro meat
production on an industrial scale is feasible only when a relatively cost-effective
process creating a product qualitatively competitive with existing meat products
is established and provided with governmental subsidization like that provided
to other agribusinesses.
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