Possibilities for an in vitro meat production system

Abstract

Meat produced in vitro has been proposed as a humane, safe and environmentally beneficial alternative to slaughtered animal flesh as a source of nutritional muscle tissue. The basic methodology of an in vitro meat production system (IMPS) involves culturing muscle tissue in a liquid medium on a large scale. Each component of the system offers an array of options which are described taking into account recent advances in relevant research. A major advantage of an IMPS is that the conditions are controlled and manipulatable. Limitations discussed include meeting nutritional requirements and large scale operation. The direction of further research and prospects regarding the future of in vitro meat production will be speculated.Industrial relevanceThe development of an alternative meat production system is driven by the growing demand for meat and the shrinking resources available to produce it by current methods. 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. In vitro produced meat products resembling the processed and comminuted meat products of today will be sooner to develop than those resembling traditional cuts of meat. While widening the scope of the meat industry in practices and products, the IMPS will reduce the need for agricultural resources to produce meat.

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Possibilities for an in vitro meat production system
I. Datar, M. Betti ⁎
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
abstractarticle info
Article history:
Received 28 June 2009
Accepted 11 October 2009
Editor Proof Receive Date 26 October 2009
Keywords:
In vitro meat
Myocyte culturing
Meat substitutes
Meat produced in vitro has been proposed as a humane, safe and environmentally beneficial alternative to
slaughtered animal flesh as a source of nutritional muscle tissue. The basic methodology of an in vitro meat
production system (IMPS) involves culturing muscle tissue in a liquid medium on a large scale. Each
component of the system offers an array of options which are described taking into account recent advances
in relevant research. A major advantage of an IMPS is that the conditions are controlled and manipulatable.
Limitations discussed include meeting nutritional requirements and large scale operation. The direction of
further research and prospects regarding the future of in vitro meat production will be speculated.
Industrial relevance: The development of an alternative meat production system is driven by the growing
demand for meat and the shrinking resources available to produce it by current methods. 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. In vitro produced meat
products resembling the processed and comminuted meat products of today will be sooner to develop than
those resembling traditional cuts of meat. While widening the scope of the meat industry in practices and
products, the IMPS will reduce the need for agricultural resources to produce meat.
© 2009 Elsevier Ltd. All rights reserved.
Contents
1. Introduction ............................................................... 14
2. Basic methodology ............................................................ 14
3. Cells................................................................... 15
3.1. Native muscle tissue formation................................................... 15
3.2. Possible cell types ......................................................... 15
3.2.1. Proposed cell types .................................................... 15
3.2.2. Adult stem cells ..................................................... 15
3.2.3. Dedifferentiated cells ................................................... 16
3.3. Replicative ability ......................................................... 16
4. Scaffold ................................................................. 16
4.1. Shape............................................................... 16
4.2. Texture and microstructure .................................................... 16
4.3. Composition ........................................................... 17
4.4. Scaffold removal. ......................................................... 17
5. Culture conditions ............................................................ 17
5.1. Growth media........................................................... 17
5.2. Regulatory factors ......................................................... 18
5.3. Contraction ............................................................ 18
6. Bioreactor ................................................................ 18
6.1. Medium perfusion ......................................................... 18
6.2. Oxygen carriers .......................................................... 18
7. Control and manipulation ........................................................ 18
Innovative Food Science and Emerging Technologies 11 (2010) 13–22
⁎Corresponding author. Tel.: +1 780 248 1598.
E-mail address: Mirko.Betti@ales.ualberta.ca (M. Betti).
1466-8564/$ –see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ifset.2009.10.007
Contents lists available at ScienceDirect
Innovative Food Science and Emerging Technologies
journal homepage: www.elsevier.com/locate/ifset

8. Limitations to the methodology ......................................................19
8.1. Nutritional value ..........................................................19
8.2. Metabolism ............................................................19
8.3. Large-scale operation........................................................19
9. Conclusion and future prospects ......................................................20
Acknowledgements.............................................................. 20
References ..................................................................20
1. Introduction
In light of the sizable negative effects of livestock production,
establishment of an in vitro meat production system (IMPS) is
becoming increasingly justifiable. Current meat production methods
are a major source of pollution and a significant consumer of fossil
fuels, land and water resources. 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 deforesta-
tion to create grazing land (Steinfeld et al., 2006). The production of
beef requires 15 500 m
3
/ton of water, while chicken requires
3918 m
3
/ton (Hoekstra & Chapagain, 2007) and 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 kg, 4 kg and
7 kg of grain, respectively (Rosegrant, Leach, & Gerpacio, 1999).
Satisfying the demand for meat in the future will be a challenge if we
intend on maximizing the use of agricultural resources and reducing
greenhouse gas production, as Fiala (2008) calculates the amount of
total meat consumed worldwide in 2030 to be 72% higher than that
consumed in 2000 following current consumption patterns.
It is suspected that myocyte culturing would have a reduced water,
energy and land requirement because a) solely muscle tissue is
cultivated, bypassing the development of by-products and non-
skeletal muscle tissues; b) for the same mass of meat, tissue
cultivation is anticipated to be faster than growth to a slaughter-
ready age and c) in vitro meat production systems are capable of
increasing in volume vertically, making deforestation to create
pasture unnecessary. The controlled conditions would theoretically
eliminate product losses from infected and diseased animals. As a
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.
To provide meat globally, modern industrial meat production has
become a complex landscape of trade where feed production, animal
husbandry, processing and consumption may all take place in
different countries (Burke, Oleson, McCullough, & Gaskell, 2009).
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.
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 B
12
, and highly
bioavailable iron (Bender, 1992). The health benefits of meat are
countered by its association with cancer and cardiovascular disease
(Demeyer, Honikel, & De Smet, 2008), though these are the result of
overconsumption and high saturated fat content, not the muscle
tissue.
While it is possible that a cultured meat product could consist of a
variety of animal cell types, meat is being defined here as primarily
skeletal muscle tissue. 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 livestockmethods 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. The employment of aseptic technique throughout the culturing
process ensures that the meat product is free from contamination.
Controlled conditions also offer the capacity for manipulation to create
meat products with different nutritional, textural and taste profiles. This
can be accomplished by co-culturing with different cell types, medium
supplementation or genetic engineering.
Considering the benefits of an IMPS, is not surprising that a
number of parties have proposed (and patented) the methodology for
actualizing this idea (Vein, 2004; Van Eelen, van Kooten, & Westerhof,
1999; Edelman, McFarland, Mironov, & Matheny, 2005). This paper
introduces the techniques so far proposed. As of yet none of these
processes, though detailed, have been tested. This is partially because
livestock animal cell lines have not been well-established in vitro
(Talbot & Blomberg, 2008) and because growing muscle cells ex vivo
on a large scale it is certainly a vast and unexplored undertaking. The
technical demands of large scale production are unseen in the world
of medical research, where most efforts in growing tissue ex vivo have
been directed. The nutritional composition of ex vivo engineered
muscle tissue has not yet been paid much attention. As a result,
establishment of an IMPS is faced with many unique challenges so far
unexplored in the field of tissue engineering.
Breakthroughs in relevant research since the publishing of these
patents and proposals have widely altered the scope of designing an
IMPS. Because the proposed systems at present approximate to a
schematic with modifiable elements, the discrete elements will be
attended to individually, taking into account recent relevant scientific
developments. This will be followed by a discussion of some
drawbacks and limitations of the methodology, concluded with a
conjecture of the future of in vitro meat production, and suggestions
for further research.
2. Basic methodology
The methods so far proposed have a common set of elements. Each
proposal employs the growth of myoblasts or myosatellite cells on a
scaffold in a suspension culture medium within a bioreactor. Neither
of these cell types need to be stimulated to muscle cell lineages but
both have limited regenerative capacity. Van Eelen et al. (1999)
proposed growth of myocytes on a collagen meshwork, while
Edelman et al. (2005) suggest collagen beads as the scaffold. The
scaffold-based method can only produce a thin myocyte layer of 100–
200 µm thick on the scaffold in static culture due to diffusional
limitations (Carrier et al., 1999). As a result, the products of these
methods lack the structure of native muscle tissue, and therefore
could only be put towards processed meat products (Edelman et al.,
2005). To create a three-dimensional meat product, Van Eelen et al.
(1999) suggest layering several confluent sheets of myocyte culture.
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An alternative method of creating a three-dimensional productis the
expansion in volume of an explant of muscle tissue of animal origin.
Benjaminson, Gilchriest, and Lorenz (2002) were able to expand the
surface area of an explant of fish by growth in a medium containing
crude cell mixture; the resulting product was prepared and well-rated
by a food panel. This method also faces diffusional limitations and is
unlikely to translate well into a large scale operation.
3. Cells
It is worth mentioning that non-skeletal muscle cell types may have
relevance as a cultured meat product, but in vitro meat is being defined
here as a culture of primarily skeletal muscle tissue therefore cell types
destined to create this desired tissue type are discussed below.
3.1. Native muscle tissue formation
During embryological development, committed muscle tissue
formation as seen in Fig. 1a) begins with mononucleated myoblasts
of limited proliferation capacity (Benjaminson et al., 2002). Myoblasts
fuse into a multinucleated myotube, which matures into a non-
proliferative myofiber (Campion, 1984). Postnatally, increases in
number of myofibers and number of nuclei per myofiber are kept
minimal, except in instances requiring repair or regeneration. In these
cases, myosatellite cells are responsible for generating new myofibers
or contributing additional myonuclei to existing ones (Fig. 1b; Le
Grand & Rudnicki, 2007). Located between the basal lamina and
sarcolemma of an associated myofiber, mononucleated myosatellite
cells are normally in a quiescent, non-dividing state (Hill, Wernig, &
Goldspink, 2003). When activated in vivo by weight-bearing stress or
injury, myosatellite cells asymmetrically divide into self-renewing
myoblasts and committed myofibers (Benjaminson et al., 2002;Le
Grand & Rudnicki, 2007).
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, Temm-Grove,
Sheehan, & Rice, 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).
3.2. Possible cell types
3.2.1. Proposed cell types
At the time when Van Eelen et al. (1999) and Edelman et al. (2005)
made their IMPS proposals, the two viable cell options were
embryonic stem (ES) cells or myosatellite cells. In theory, after the
ES 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 ES 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, Stem-Straeter, Beier, Bannasch, &
Stark, 2003). In addition to this, there are so far no “proven”bovine,
porcine, caprine nor ovine ES cell lines that have been established to
the degree of a biological reagent like that of human, monkey or
mouse ES cells (Talbot & Blomberg, 2008). Efforts invested into
establishing ungulate stem-cell lines over the past two decades have
been generally unsuccessful with difficulties arising in the recogni-
tion, isolation and differentiation of these cells (Keefer, Pant,
Blomberg, & Talbot, 2007).
Although myosatellite cells have the disadvantage of being a rare
muscle tissue cell type with limited regenerative potential, Bach et al.
(2003) indicate that they are the preferred source of primary myoblasts
because they recapitulate myogenesis more closely than immortal
myogenic cell lines. Myosatellite cells have been isolated and charac-
terized from the skeletal muscle tissue of cattle (Dodson, Martin,
Brannon, Mathison, & McFarland, 1987), chicken (Yablonka-Reuveni,
Quinn, & Nameroff, 1987), fish (Powell, Dodson, & Cloud, 1989), lambs
(Dodson, McFarland, Martin, & Brannon, 1986), pigs (Blanton, Grand,
McFarland, Robinson, & Bidwell, 1999; Wilschut, Jaksani, Van Den
Dolder, Haagsman, & Roelen, 2008), and turkeys (McFarland, Doumit, &
Minshall, 1988). Each animal species has its own benefits and
limitations as a cell source, and myosatellite cells isolated from different
muscles have different capabilities to proliferate, differentiate, or be
regulated by growth modifiers (Burton, Vierck, Krabbenhoft, Byrne, &
Dodson, 2000). Wilschut et al. (2008) have shown that 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.
Recent advances in tissue engineering and cell biology offer some
alternate cell options which may have practical applications with
multilineage potential allowing for co-culture development and with
suitability for large-scale operations.
3.2.2. Adult stem cells
Myosatellite cells are one example of an adult stem-cell type with
multilineage potential (Asakura, Komaki, & Rudnicki, 2001). Adult
Fig. 1. a) Prenatal myogenesis: Stem cells give rise to proliferative muscle precursor cells,
(myoblasts), which lose proliferative capability upon fusinginto multinucleate myotubes.
Myotubesundergo morphological changesto become mature non-proliferativemyofibers.
b) Postnatal/posthatchmyogenesis is for repair andregeneration of existingmuscle tissue.
Myofiber-associated myosatellite cells respond to weight-bearing stress or injury by
asymmetrically dividing into a self-renewing daughter cell and a nonproliferative
myofiber-committed cell.Committed cells can fusewith other committedcells to produce
new myofibers or add nuclei to e xisting myofibers.
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stem cells have been isolated from several different adult tissues
(Wagers & Weissman, 2004), but another cell type relevant to in vitro
meat production are adipose tissue-derived adult stem cells (ADSCs),
a rare population of multipotent cells found in adipose tissue (Gimble,
Katz, & Bunnell, 2007). Kim et al. (2006) have noted that these highly
expandable cells can be obtained relatively non-invasively from
subcutaneous fat and subsequently transdifferentiated to myogenic,
osteogenic, chondrogenic or adipogenic cell lineages.
The greatest concern and matter of debate regarding adult stem cells
is their proneness to malignant transformation in long-term culture
(Lazennec & Jorgensen, 2008). Rubio et al. (2005) have found that
adipose tissue-derived adult stem cells immortalize at high frequency
and undergo spontaneous transformation in long-term (4–5months)
culturing, while evidence of adult stem cells remaining untransformed
have also been reported (Bernardo et al., 2007). In an in vitro meat
production system, re-harvesting of adultstem cells to minimizethe risk
of spontaneous transformation may be necessary. With this in mind,
harvesting ADSCs from subcutaneous fat is far less invasive than
collection of myosatellite cells from muscle tissue and samples can be
taken from certain organisms without causing substantial harm.
3.2.3. Dedifferentiated cells
Dedifferentiation is the reversion of a terminally differentiated cell
into a multipotent cell type. It has been reported by Matsumoto et al.
(2007) that mature adipocytes can be dedifferentiated in vitro into a
multipotent preadipocyte cell line termed dedifferentiated fat (DFAT)
cells. Following this, DFAT cells are capable of being transdifferen-
tiated into skeletal myocytes (Kazama, Fujie, Endo, & Kano, 2008). By
floating a piece of glass on top of a suspension of mature adipocytes,
buoyant mononucleated adipocytes attach to the upper surface and
either a) release their fat droplet and assume the fibroblast-like shape
of a DFAT cell or b) asymmetrically divide to produce one fibroblast-
shaped DFAT daughter cell (Matsumoto et al., 2007). Termed the
“ceiling culture”method, the process certainly seems achievable on an
industrial scale. Because mature adipocytes are the most common cell
type in adipose tissue, tissue samples will have high cell yields
compared to other cell options.
Cell dedifferentiation appears to be an attractive alternative to the
use of stem cells but Rizzino (2007) has put forth the argument that
many of the claims of transdifferentiation, dedifferention and multi-
potency of once terminally differentiated cells may be due to
abnormal processes resulting in cellular look-alikes.
3.3. Replicative ability
An IMPS requires many cell divisions to mass culture muscle
tissue, but most cells have a finite number of divisions in culture
before natural cell death; this number is termed the Hayflick limit.
Edelman et al. (2005) put forth the three possible means for
overcoming this limitation in an IMPS: a) regularly replenishing the
culture, b) using an immortal cell line or c) immortalizing a cell line.
Most cell types will require the first method though embryonic stem
cells fall into the second category. Use of the third strategy is
controversial because immortalization of cell lines requires genetic
manipulation. It is also important to draw parallels between the
genetic modifications that would facilitate large-scale production and
the mutations exhibited by cancerous cells.
Increasing the regenerative potential of cells without immortaliz-
ing them is one additional possibility. The Hayflick limit is determined
by telomere length. Telomeres are the guanine-rich repeats found at
the ends of linear chromosomes. Due to the linearity of the
chromosome and the mechanism by which replication occurs,
telomeres are shortened with every round of DNA replication (and
cell division). Telomere length therefore corresponds to the number
of divisions a cell type is capable of. This explains why a neonatal
source is best for harvesting myosatellite cells; very early in
development the cells still have long telomeres. Telomerase is a
ribozyme capable of lengthening telomeres, naturally found in
immortal cell lines. Many different cell types have been immortalized
with ectopic telomerase expression and have showed no signs of the
growth deregulation associated with cancer cells (Harley, 2002). Un-
fortunately, this helpful genetic alteration may be subject to consumer
rejection.
4. Scaffold
Scaffolding mechanisms differ in shape, composition and char-
acteristics to optimize muscle cell and tissue morphology. Myoblasts
are anchorage-dependent cells, capable of spontaneous contraction.
An ideal scaffold would have a large surface area for growth and
attachment, be flexible to allow for contraction, maximize medium
diffusion and be easily dissociated from the meat culture. A scaffold
that closely mimics the in vivo situation is best; myotubes
differentiate optimally on scaffold with a tissue-like stiffness (Engler
et al., 2004). The best materials would be natural and edible, though
inedible scaffold materials cannot be disregarded. Development of
new biomaterials offer additional characteristics that may be
beneficial for in vitro muscle tissue growth, such as fulfilling the
requirement of contraction for proliferation and differentiation (De
Deyne, 2000).
4.1. Shape
As mentioned before, Edelman et al. (2005) proposed beads made
of edible collagen as a substrate while 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. Conformation choice is
based primarily on maximizing surface area, which increases diffusion
and the amount of anchorage-dependent tissue that can be grown.
4.2. Texture and microstructure
Texturized surfaces can attend to specific requirements of muscle
cells, one of which is myofiber alignment. To mimic native muscle
architecture, Lam, Sim, Zhu, and Takayama (2006) cultured myoblasts
on a substrate with a wavy micropatterned surface and found that the
wave pattern aligned differentiated muscle cells while promoting
myoblast fusion to produce aligned myotubes. Myofiber organization is
important for the functional characteristics of muscle and the textural
characteristics of meat. Micropatterned surfaces could allow muscle
tissue cultured by the scaffold-based technique to assume a two-
dimensional structure more similar to that of meat of native origin.
Electrospinning is the process of using electrical charge to extract
very fine fibers from liquids. Riboldi, Sampaolesi, Neuenschwander,
Cossu, and Mantero (2005) have suggested electrospun microfibrous
meshwork membranes as a scaffold for skeletal myocytes, as the
membranes offer high surface area to volume ratio and some elastic
properties. Electrospinning creates very smooth fibers, which may not
translate well into a good adhesive surface; Riboldi et al. (2005) have
shown thatcoating electrospun polymer fiberswith extracellularmatrix
proteins, such as collagen or fibronectin, promotes attachment by
ligand–receptor binding interactions. Electrospinning shows promise
for scaffold formation because the process is simple, controllable,
reproducible and capable of producing polymerswith special properties
by co-spinning (Riboldi et al., 2005). Microfibre organization can also
affect myofiber morphology. Electrofibers can be spun with nanometer
to micrometer width; this corresponds to the wave periodicity (6 µm
was found to be optimal by Lam et al., 2006) found to be beneficial for
myocyte alignment on a micropatterned surface. Knowing the effects of
16 I. Datar, M. Betti / Innovative Food Science and Emerging Technologies 11 (2010) 13–22

micropatterning, introducing electrospun fibers aligned at the correct
periodicity could theoretically align myofibers.
Beyond facilitating attachment and alignment, a scaffold capable of
increasing nutrient diffusion and medium circulation would prove
invaluable to the process. Noting that tissue thickness cannot exceed
100–200 µm without experiencing cell death, Borenstein et al. (2002)
microfabricated a synthetic vascular 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) created artificial networks of
channels in sheets of biodegradable, biocompatible polymer, then
seeded the network with endothelial cells. Following dissolution of
the polymer mold, successful proliferation could theoretically leave
behind a network of endothelial tissue 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. Unfortunately, at this moment creation
of these artificial vascular networks does not translate well into mass
production due to the microfabrication processes required.
4.3. Composition
Several different polymers could suffice as the scaffold material for
an in vitro meat production system. Examples of edible, naturally
derived polymers are collagen, cellulose, alginate or chitosan. These
polymers would be safe to leave in the meat product and could add a
textural quality. Edelman et al. (2005) have suggested that porous
beads made of these polymers capable of undergoing surface area
changes under different pH and temperature conditions could fulfill
the contraction requirement of myoblast cells.
By contrast, inedible polymers confer some interesting qualities
that can aid muscle tissue formation. The aforementioned micro-
patterned surfaces which can aid in myofiber alignment are inedible,
as are the thermoresponsive coatings described below. Jun, Jeong, and
Shin (2009) have found that growing myoblasts on electrically
conductive fibers induces their differentiation, forming more myo-
tubes of greater length without the addition of electrical stimulation.
Use of inedible scaffolding systems necessitates simple and non-
destructive techniques for removal of the culture from the scaffold.
4.4. Scaffold removal
A technical challenge of growing by the scaffold-based technique is
removal of the scaffolding system. Removal of confluent cultured cell
sheets is conventionally done enzymatically or mechanically, but these
two methods damage the cells and the extracellular matrix they may be
producing (Canavan, Cheng, Graham, Ratner, & Castner, 2005). Da Silva,
Mano, and Reis (2007) note that thermoresponsive coatings which
change from hydrophobic to hydrophilic at lowered temperatures can
release cultured cells and extracellular matrix as an intact sheet upon
cooling. Termed “thermal liftoff,”this method results in undamaged
sheets that maintain the ability to adhere if transferred onto another
substrate (da Silva et al., 2007). This opens the possibility of stacking
sheets to create a three-dimensional product.
Lam, Huang, Birla, and Takayama (2009) have presented a method
for detaching culture as a confluent sheet from a non-adhesive
micropatterned surface using the biodegradation of selective attach-
ment protein laminin.
It should be noted that culturing on a scaffold may not result in a
two-dimensional confluent “sheet”of culture. After scaffold removal,
the contractile forces exerted by the cytoskeleton of the myocyte are
no longer balanced by adhesion to a surface, causing 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. Lam et al. (2009) found
that use of a fibrin hydrogel was 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 as suggested by Van Eelen
et al. (1999), to create a three-dimensional product.
5. Culture conditions
Perhaps the most difficult task in designing an in vitro meat
production system is determining the best culture medium formula-
tion. The medium should support and promote growth while being
made of affordable, edible components available in large quantities.
Medium composition will be a substantial cost determinant if not
solely for the fact that large quantities will be required.
5.1. Growth media
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. 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).
Serum-free media have been developed to support in vitro
myosatellite cell cultures from the turkey (McFarland, Pesall, Norberg,
& Dvoracek, 1991), sheep (Dodson & Mathison, 1988)andpig(Doumit,
Cook, & Merkel, 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,
McFarland, Grant, Doumit, & Velleman, 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 (Duque et al., 2003).
It has one-fifth the protein content of serum (Pope, Harrison, Wilson,
Breen, & Cummins, 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, van Kuppevelt, Oosterhof, & Veerkamp,
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 serum replacements may have advanta-
geous 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 costand 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 mushroom extracts were comparable to serum
as a growth medium in promoting explant 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. The development of an appropriate serum-free media com-
pletely 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.
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5.2. Regulatory factors
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 specificto
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 combina-
tion 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 supple-
mented 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 growth factors
which stimulate myoblast proliferation and differentiation (Cen,
Zhang, Huang, Yang, & Xie, 2008) as well as myosatellite cell pro-
liferation in several meat-animal species in vitro (Dodson et al., 1996)
Human growth hormone, routine produced from a transgenic bacte-
rial source can be supplemented into the medium to stimulate pro-
duction 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, Hsieh, & Rotwein,
2003). Similarly, growth factor production can occur in genetically
engineered muscle cells.
5.3. Contraction
Regular contraction is a necessity for skeletal muscle. It promotes
differentiation and healthy myofiber morphology while preventing
atrophy. Muscle in vivo is innervated, allowing for regular, controlled
contraction. An in vitro system would necessarily culture denervated
muscle tissue, so contraction must be stimulated by alternate means.
Cha, Park, Noh, and Suh (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 fulfill the requirement of
providing contraction. This in mind, de Deyne (2000) noted that
external mechanical contraction is less effective than electrical
stimulation in promoting muscle development. Electrical stimulation,
feasible on a large scale, induces contraction internally as opposed to
passively and aids in differentiation and sarcomere formation. As
mentioned before, even growth on electrically conductive fibers
without application of electrical stimulation sufficed in reaping the
benefits of induced contraction (Jun et al., 2009).
6. Bioreactor
Achieving adequate perfusion of the cultured tissue is 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 hemoglobin-
provided oxygen supply.
6.1. Medium perfusion
The design of a bioreactor is inten ded to promote the growth of tissue
cultures which accurately resemble native tissue architecture while
providing an environment which allows for increased culture volumes.
The cylindrical wall of rotating wall vessel bioreactors rotates 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). This creates
a laminar flow of the medium 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, Wendt, & Herberer, 2004).
Direct perfusion bioreactors flow medium through a porous scaffold
with gas exchange taking place in an external fluid loop (Carrier et al.,
2002). This type of bioreactor appears more appropriate for scaffold-
based myocyte cultivation. They offer high mass transfer but also
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 vasculariz-
ing 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. This is
certainly a method that can increase the diffusional limitation of
tissue thickness beyond 100-200 µm in vitro.
6.2. Oxygen carriers
Cell viability and density positively correlate with the oxygen
gradient in statically grown tissue cultures (Radisic et al., 2008).
Oxygen carriers can be supplemented to the medium to maintain high
oxygen concentrations in solution, similar to that of blood. There are
two distinct varieties of oxygen carrier: those which are modified
versions of hemoglobin and those which are chemically inert,
artificially produced perfluorochemicals (PFCs) (Lowe, 2006a).
Though many chemically modified hemoglobins have been devel-
oped, their bovine or human source makes them an unlikely candidate
for scale up. Alternatively, human hemoglobin has been produced by
genetically modified plants (Dieryck et al., 1997) and microorganisms
(Zuckerman, Doyle, Gorczynski, & Rosenthal, 1998). Efforts to produce
heme proteins and blood substitute components using the microorgan-
isms Escherichia coli,Pichia pastis,andAspergillus niger are underway as
these organisms are already used to commercially produce human
pharmaceuticals and food additives (Lowe, 2006b).
Perfluorochemicals (PFCs) dissolve large volumes of oxygen and
therefore can perform the same function as hemoglobin. To be miscible
in aqueous conditions PFCs must be emulsified andemulsifications have
been available for use in vitro and in vivo (Lowe, 2006a). Shine et al.
(2005) note that medical PFCs are extremely potent greenhouse gases
on a per molecule basis and though they presently make a trivial
contribution to climate change, increasing their use requires careful
consideration. Development of an artificial blood is an active area of
research and many applicable options are likely to arise with time.
7. Control and manipulation
An IMPS offers a level of control unattainable by traditional livestock
methods of producing meat. Myocyte culturing prevents the uncon-
trollable, unpredictable complications present in livestock production
includingthe spread of disease among animals and the development of
zoonoses. The diseases of concern in industrial agriculture which have
18 I. Datar, M. Betti / Innovative Food Science and Emerging Technologies 11 (2010) 13–22

become increasingly more difficult to contain with expanding interna-
tional trade—avian flu, swine flu, foot-and-mouth disease, and bovine
spongiform encephalopathy—will not threaten an IMPS. In addition, the
problems caused by pre-slaughter environment: pale, soft, exudative
(PSE) and dark, firm, dry meat (DFD), would not exist in the products of
myocyte culturing.
While the controlled conditions in vitro allow the likelihood of
unpredictable complications to be minimized, they can be manipu-
lated to intentionally create products with differing qualities. With the
advent of functional and enriched foods, consumers are more willing
to try products that have been altered to have particular nutritional
characteristics (Korhonen, 2002; Burdock, Carabin, & Griffiths, 2006).
By co-culture, medium formulation or genetic engineering, it is
theoretically possible to create products with different tastes, textures
and nutrient profiles.
Fat content is an example of a nutritional quality that can be
manipulated. The greatest criticism against meat is the high saturated
fat content which contributes to cardiovascular disease. In vitro,
saturated fat content is determined by the amount of adipocyte co-
culture present; without co-culture, pure myocyte culture would
produce a product rich in nutritionally beneficial fatty acids from
phospholipids. Furthermore, genetic manipulation of myocytes could
allow the fatty acid composition to be altered to enrich for particularly
beneficial fatty acids such as those which are omega-3 polyunsaturated.
While in theory nutrient content can be altered, one major
obstacle likely to postpone the development of an IMPS is ensuring
that the product has the full complement of nutrients available in
meat harvested in vivo.
8. Limitations to the methodology
The scientific development of an IMPS is most hindered by the facts
that a) a major tissue will be cultured in the absence of in vivo
homeostatic regulation and b) that the process needs to be carried out
on a large scale. The lack of homeostasis affects the nutritional value of
the meat product, as often other organ systems are involved in nutrient
absorption and distribution in the live organism. Muscle tissue is also a
highly metabolic tissue and the products of metabolism need to be
removed or recycled at a pace matching the provision of reactants.
Large-scale tissue culturing is a concept crucial to thedevelopment of an
IMPS but is as of yet unexplored in the field of tissue engineering.
8.1. Nutritional value
In addition to having high protein content with the full complement
of amino acids, meat is an exclusive sourceof several essentialnutrients.
It is necessary that an in vitro grown meat product meets if not exceeds
the nutritional value of traditional meat products to be competitive on
the market. Nutrients found in meat in vivo which are not synthesized
by muscle cells must be supplemented.
For instance, the essential vitamin B12 is synthesized exclusively
by certain species of gut-colonizing bacteria and is therefore found
solely in food products of animal origin. Supplementation of
crystalline vitamin B
12
produced commercially by biosynthetic
microbial fermentation would be necessary in an in vitro meat
product grown in an aseptic environment.
Iron in meat is present as the Fe(II) ion in the highly bioavailable
form of heme, the prosthetic group found in myoglobin. To provide
iron to growing myocytes in a bioavailable form, Fe(III) ions bound to
the plasma binding protein transferrin will have to be supplemented
to the culture medium. By transferrin-mediated iron transport, iron
can enter the myocyte mitochondria and be incorporated into heme
synthesis and subsequent myoglobin synthesis (Aisen, Enns, &
Wessling-Resnick, 2001). It must be noted that levels of transferrin
must be closely monitored, so as not to allow free ferric or ferrous ions
to be present in the medium, as they can readily catalyze the
production of damaging reactive oxygen species in aerobic environ-
ments (Papanikolaou & Pantopoulos, 2005). In addition, Graber and
Woodworth (1986) found that myoglobin levels are not significant in
culture until a stable population of myotubes has formed, a fact which
can help determine the optimal growth time necessary before harvest.
Vitamin B
12
and heme iron are two especially important and
exclusively meat-source nutrients of several. 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 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.
8.2. Metabolism
An IMPS lacks the organ systems that maintain homeostasis in an
organism and therefore metabolism needs to be carefully and strictly
monitored. Myocytes must be metabolizing aerobically to prevent
acidification of the culture medium with lactic acid. Oxygen must be
readily available to myocyte culture to prevent hypoxia and the
acidification of medium, two situations which are damaging to the
cells. As mentioned above, adequate oxygenation is dependent on the
ability of the bioreactor to enhance perfusion and the availability of
oxygen in the medium. Adequately providing the chemical and physical
requirements for metabolism while removing damaging waste products
can be a difficult task, with the possibility of unforeseen shortcomings.
For instance, co-culturing hepatocytes to convert lactate back to the
glucose via the Cori cycle can be one method of preventing lactic
acidification of medium in the eventof anaerobic conditions. This can be
complicated, however, by the individual metabolic needs of hepato-
cytes, which may not coincide well with those of myocytes.
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.
8.3. Large-scale operation
The development of a large-scale IMPS facility with the capability
to mass produce meat at a rate comparable to traditional slaughter-
houses is greatly hindered by the lack of investigation into large-scale
culturing. Presently, “large”pieces of cultured tissue are measured on
a centimetre scale with most relevant tissue engineering efforts have
been put towards the medical application of in vivo tissue repair.
Vladimir Mironov (Medical University of South Carolina, personal
communication, 19 April 2009) has stated that an industrial scale
bioreactor would need to be at least three to five storeys to produce
industrially relevant amounts of cultured meat. The best source of
inspiration for mass production at this time would be to look towards
the pharmaceutical industry and microbial biotechnology where
living organisms in bioreactors “manufacture”purified chemicals.
Though the mass production of purified molecules is quite different
from the mass production of cultured tissue, some of the technology
and methodology may prove relevant.
While stem-cell lines are defined by the ability to be propagated in
culture indefinitely while maintaining broad plasticity, a great criticism
of stem cells in long-term culturing is spontaneous transformation.
19I. Datar, M. Betti / Innovative Food Science and Emerging Technologies 11 (2010) 13–22

9. Conclusion and future prospects
Developments in regenerative medicine, stem-cell research and
biomaterial engineering in the past decade have yielded highly
applicable, viable options which can aid in the scientific development
of an IMPS. One possible in vitro meat production scheme is shown in
Fig. 2. With the scaffold-technique being a practicable method for
scale up, the materials and techniques now available make the IMPS
seem more achievable, however, there remain several aspects of the
concept yet to be investigated.
Because nearly all research applicable to the development of an IMPS
is intended for biomedical application, many of the investigations into
multipotent and myogenic cell lineages have been done with human,
mouse or ratcell lines, with relatively few studies describing cell lines of
agriculturally relevant animals. Adult stem cells and dedifferentiated
cells are recently innovated cell types which require further exploration
into their safety and suitability for an in vitro meat production system.
The biological aspects of an IMPS need to be further investigated to
ensure that the resulting tissue closely mimics native muscle tissue
morphologically and nutritionally. In addition, relevant studies have so
far focused on small-scale applications, with pertinent biological
research focused towards tissue replacement in vivo. Undoubtedly, a
major hindrance in development of the science of an IMPS is based
around application on a massproduction scale, where little research has
been done. Many of the cell lines, scaffold materials and medium
components described are unreasonable for scale up economically, and
are often complicated by other issues such as environmental impact and
safety. It is absolutely necessary that anin vitro meatproduction system
be developed according to the rules and regulations of good cell culture
practice (GCCP) (Coecke et al., 2005) as well as current good
manufacturing practice (cGMP) as pertaining to food and drug
production.
The greatest stumbling block comes with the commercial
implementation of an IMPS, where cost-effectiveness and consumer
acceptance determine if cultured myocyte tissue will become a
significant meat alternative on the market. A preliminary economics
study reviewing the financial viability of in vitro grown meat
estimated the cost of manufacturing to be Euro 3500/ton, but note
that because such technology has not yet been developed, this
estimation could be largely inaccurate (eXmoor Pharma Concepts,
2008). In vitro meat production on an industrial scale is feasible only
when a relatively cost-effective process creating a product qualita-
tively competitive with existing meat products is established and
provided with governmental subsidization like that provided to other
agribusinesses.
Culturedmeat can certainlyhave an applicationin ground, processed
foods such as hamburgers or hotdogs as a main component or as an
additive. In this form, the textural shortcomings of the in vitro grown
productwill not compromise the finalprocessed productand one would
expect greater consumer acceptance. The second goal of cultured meat
is to create three-dimensional products resemblingtraditional cuts with
proper textural characteristics.
Acknowledgements
Thanks to Jason G. Matheny, director of New Harvest for his support
and Vladimir Mironov of the Medical University of South Carolina and
Bernard A.J. Roelen of the Utrecht University for helpful comments on
the manuscript. The Authors also express their gratitude to Prof. Carlo
Pelanda (University of Georgia) and to his book "Futurizzazione"
(Sperling & Kupfer, 2003) for inspiring this work.
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