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TISSUE ENGINEERING Volume 11, Number 5/6, 2005 © Mary Ann Liebert, Inc…
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TISSUE ENGINEERING
Volume 11, Number 5/6, 2005
© Mary Ann Liebert, Inc.
Commentary
In Vitro-Cultured Meat Production
P.D. EDELMAN, M.Sc.,1 D.C. MCFARLAND, Ph.D.,2 V.A. MIRONOV, Ph.D., M.D.,3
and J.G. MATHENY, M.P.H.4
INTRODUCTION work or microcarrier beads, and then perfused with a cul-
ture medium in a stationary or rotating bioreactor. By in-
A LTHOUGH MEAT has enjoyed sustained popularity as
a foodstuff, consumers have expressed growing
concern over some consequences of meat consumption
troducing a variety of environmental cues, these cells fuse
into myotubes, which can then differentiate into my-
ofibers.2 The resulting myofibers may then be harvested,
and production. These include nutrition-related diseases, cooked, and consumed as meat. van Eelen, van Kooten,
foodborne illnesses, resource use and pollution, and use and Westerhof hold a Dutch patent for this general ap-
of farm animals. Here we review the possibility of pro- proach to producing cultured meat.3 However, Catts and
ducing edible animal muscle (i.e., meat) in vitro, using Zurr appear to have been the first to have actually pro-
tissue-engineering techniques. Such "cultured meat" duced meat by this method.4
could enjoy some health and environmental advantages A scaffold-based technique may be appropriate for
over conventional meat, and the techniques required to producing processed (ground, boneless) meats, such as
produce it are not beyond imagination. To tissue engi- hamburger or sausage. But it is not suitable for produc-
neers this subject is of interest as cultured meat produc- ing highly structured meats, such as steaks. To produce
tion is an application of tissue-engineering principles these, one would need a more ambitious approach, cre-
whose technical challenges may be less formidable than ating structured muscle tissue as self-organizing con-
those facing many clinical applications. structs5 or proliferating existing muscle tissue in vitro.
The latter technique was employed by Benjaminson,
Gilchriest, and Lorenz, the first researchers to have ap-
CULTURED MEAT PRODUCTION plied tissue-engineering techniques to meat production.6
They placed skeletal muscle explants from goldfish
Most edible animal meat is made of skeletal muscle (Carassius auratus) in diverse culture media for 7 days
tissue. The idea that skeletal muscle tissue-engineering and observed an increase in surface area between 5.2 and
techniques could be applied to produce edible meat dates 13.8%. When the explants were placed in a culture con-
back at least 70 years,1 but has been seriously pursued taining dissociated Carassius skeletal muscle cells, ex-
by only three groups of researchers. Their efforts can be plant surface area increased by 79%.
divided roughly into scaffold-based and self-organizing Explants have the advantage of containing all the cells
techniques. that make up muscle in their corresponding proportions,
In scaffold-based techniques, embryonic myoblasts or thus closely mimicking an in vivo structure. However,
adult skeletal muscle satellite cells are proliferated, at- lack of blood circulation in these explants makes sub-
tached to a scaffold or carrier such as a collagen mesh- stantial growth impossible, as cells become necrotic if
1Wageningen, The Netherlands.
2Department of Animal and Range Sciences, South Dakota State University, Brookings, South Dakota.
3Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina.
4Department of Agricultural and Resource Economics, University of Maryland, College Park, Maryland.
659
660 EDELMAN ET AL.
separated for long periods by more than 0.5 mm from a stance, back-of-the-envelope calculations suggest that a
nutrient supply.5 single parent cell with a Hayflick limit of 75 could the-
Future efforts in culturing meat will have to address oretically satisfy the current annual global demand for
the limitations of current techniques through advances meat.) For other species, it may be necessary to prolif-
that make cultured cells, scaffolds, culture media, and erate a sufficient number of stem cells in culture before
growth factors edible and affordable. differentiation into myoblasts--or to use cells transfected
with the telomerase gene, that have higher Hayflick
limits.
CELLS
Skeletal muscle is a tissue consisting of several cell FIELDS
types. Skeletal muscle fibers are formed by the prolifer-
ation, differentiation, and fusion of embryonic myoblasts Mechanical, electromagnetic, gravitational, and fluid
and, in the postnatal animal, satellite cells, to form large flow fields have been found to affect the proliferation and
multinucleated syncytia.7 Attempts to force skeletal mus- differentiation of myoblasts.2,14 Powell and others found
cle fibers to proliferate are typically counterproductive, that repetitive stretch and relaxation equal to 10% of
as most myonuclei remain postmitotic.2 Embryonic stem length, six times per hour, increased differentiation into
cells have the drawback that despite the high prolifera- myotubes.15 Yuge and Kataoka seeded myoblasts with
tion and differentiation potential, considerable effort magnetic microparticles and induced differentiation by
must be applied to force them to differentiate and cell placing them in a magnetic field, without adding special
yields from harvests are low. Moreover, it is not clear growth factors or any conditioned medium.16 Electrical
whether embryonic stem cells forced to commit to a stimulation also contributes to differentiation, as well as
skeletal muscle lineage will have the proliferative char- sarcomere formation within established myotubes.2,14
acteristics of embryonic stem cells, or become indistin-
guishable from myoblasts. Thus the most practical cell
source for cultured meat is probably embryonic myo- SCAFFOLDS
blasts or postnatal/posthatch skeletal muscle cells called
satellite cells. Myoblasts are attachment dependent, meaning that a
Satellite cells with high proliferative potential have substratum or scaffold must be provided for proliferation
been isolated and characterized from the skeletal muscle and differentiation to occur.17 For cultured meat, a scaf-
of chickens, turkeys, pigs, lambs, and cattle.812 In each fold and its by-products must be edible and may be de-
case medium conditions have been established by these rived from nonanimal sources. A further challenge is to
investigators to support the proliferation and differentia- develop a scaffold that can mechanically stretch attached
tion of cells to form immature muscle fibers called cells to stimulate differentiation. A flexible substratum is
myotubes in culture. also necessary to prevent detachment of developing my-
The simplest cultured meat system would likely use a otubes that will normally undergo spontaneous contrac-
single myogenic cell line from one of these animals, or tion.
a coculture with fat cells. After culture and harvest, cells Cytodex-3 microcarrier beads have been used as scaf-
might then be prepared for consumption as a processed folds in rotary bioreactors. However, these beads have
meat. To replicate the taste and texture of unprocessed no stretching potential. One approach to mechanically
meats is a more ambitious goal, as vascular cells would stretch myoblasts would be to use edible, stimulus-sen-
be needed and fibroblasts for the production of connec- sitive porous microspheres made from cellulose, alginate,
tive tissue. Moreover, these would have to be properly chitosan, or collagen that undergo, at minimum, a 10%
organized in a three-dimensional structure. A proper change in surface area after small changes in tempera-
growth factor milieu would be essential to direct the con- ture or pH. Once myoblasts attach to the spheres, they
struction of a structured skeletal muscle tissue. could be stretched periodically. It is not clear how the
It is unclear how much cultured meat a single cell could variation in pH or temperature, or the differential me-
yield. Cells in culture are believed to undergo a fixed chanical stresses that bead curvature imposes on cells,
number of doublings, called the Hayflick limit. The would affect cell proliferation, adhesion, and growth.
Hayflick limits of farm animal muscle progenitor cells Theoretically, giant sheets of muscle tissue could be
have not been well established. It has been shown that cultured on thin membranes or arrays of narrowly spaced
satellite cells cloned from turkey breast muscle express fibers. The sheets could be mechanically conditioned by
telomerase.13 This finding suggests that some domestic minimal stretch to induce development of aligned
animal satellite cells may generate enough daughter cells myotubes. The membranes or fibers could be extracted
to produce huge quantities of cultured meat. (For in- from the meat (e.g., a thermoresponsive polymer could
IN VITRO-CULTURED MEAT PRODUCTION 661
be used, and the muscle biofilm separated from the sub- thesized and released by muscle cells themselves and, in
strate with a change of temperature). Or they could be tissues, are also provided by other cell types locally
made from edible material. The freed sheets could then (paracrine effects) and nonlocally (endocrine effects).
be rolled up to a substantial thickness and processed.* The liver is the primary source of circulating insulin-like
Developing a scaffold for unprocessed meats presents growth factor I. Appropriate coculture systems may be
greater technical challenges, because of the need for vas- developed such that liver cells (hepatocytes) provide
cularization. It may be possible to build a branching net- growth factors necessary for cultured muscle (meat) pro-
work from an edible, elastic, and porous material, through duction. Typically, investigators initiate differentiation
which nutrients are perfused. Myoblasts and other cell and fusion of myoblasts by lowering the levels of mito-
types can then attach to this network. Approaches to cre- genic growth factors. The proliferating cells then com-
ating such a network for the purpose of tissue engineer- mence synthesis of insulin-like growth factor II, which
ing have been proposed by creating a cast onto which a leads to differentiation and formation of multinucleated
collagen solution or a biocompatible polymer is spread. myotubes.19 So, the successful system must be capable
After solidification, the original material is dissolved, of changing the growth factor composition of the
leaving a branched network of microchannels behind, medium.
which can be stacked onto each other to form a three-di-
mensional network.18 However, this approach does not
lend itself to mass production. BIOREACTORS
Alternatively, one could attempt to create a highly
structured meat without a scaffold. Benjaminson, Gil- The importance of bioreactor design to tissue engi-
chriest, and Lorenz proposed solving the vascularization neering has been discussed elsewhere.20,21 Cultured meat
problem through controlled angiogenesis of explants.6 production is likely to require the development of new
bioreactors that maintain low shear and uniform perfu-
sion at large volumes. Much skeletal muscle tissue engi-
CULTURE MEDIA AND neering research has employed NASA rotating bio-
GROWTH FACTORS reactors. Their chief advantages are that cells are in
near-continuous suspension, fluid shear is minimal, and
To enjoy many of the potential advantages over con- suspension is possible for tissue assemblies up to 1 cm.
ventional meat production, cultured meat would need to These bioreactors can sustain biomass concentrations up
employ an affordable medium system. Such a medium to 108 cells/mL. Research-size rotating bioreactors (10 to
must contain the necessary nutritional components and 250 mL) have been scaled up to 3 L and, theoretically,
be presented in a form freely available to myoblasts and scale-up to industrial sizes should not affect the physics
accompanying cells, as no digestive system is involved. of the system. Industrial scales are already available for
Improvements in the composition of commercially avail- low-shear particle-based biofilm reactors, allowing bio-
able cell culture media have enhanced our ability to suc- mass concentrations as high as 30 kg/m3.22
cessfully culture many types of animal cells.
McFarland and others developed a serum-free medium
that supported the proliferation of turkey satellite cells in CONCLUSIONS
culture.8 Kosnik, Dennis, and Vandenburgh refer to
serum-free media developed by Allen et al., Dollenmeier Relative to conventional meat, cultured meat could of-
et al., and Ham et al.2 Benjaminson and others succeeded fer a number of benefits. With cultured meat, the ratio of
in using a serum-free medium made from maitake mush- saturated to polyunsaturated fatty acids could be better
room extract that achieved higher rates of growth than controlled; the incidence of foodborne disease could be
fetal bovine serum.6 And it has been shown that lipids significantly reduced; and resources could be used more
such as sphingosine 1-phosphate can replace serum in efficiently, as biological structures required for locomo-
supporting the growth and differentiation of embryonic tion and reproduction would not have to be grown or sup-
tissue explants (W.S. Argraves, Medical University of ported. Whether or not cultured meat is economically
South Carolina, Charleston, SC; personal communica- practical is a different question. A number of tissue en-
tion, May 22, 2004). gineers have speculated on its prospects.23
In addition to supplying proper nutrition to growing Cultured meat, at least of the scaffold-based variety,
muscle cells in culture, it is necessary to provide an ap- appears technically feasible. However, significant chal-
propriate array of growth factors. Growth factors are syn- lenges remain before it could be produced economically.
Most of these challenges are common to skeletal muscle
tissue engineering, generally. The environmental cues
* We owe this suggestion to an anonymous reviewer. needed to promote myofiber development are not well
662 EDELMAN ET AL.
understood. And it is not clear which, among them, are tion of satellite cells from bovine muscles. J. Tissue Cul-
essential to cultured meat production. Still, it is safe to ture Methods 10, 233, 1986.
assume that the level of functionality needed for most 12. Dodson, M.V., Martin, E.L., Brannon, M.A., Mathison,
clinical applications of muscle tissue engineering exceeds B.A., and McFarland, D.C. Optimization of bovine satel-
lite cell-derived myotube formation in vitro. Tissue Cell
that needed to produce cultured meat with nutritional and
19, 159, 1987.
aesthetic properties sufficiently similar to those of con-
13. Mozdziak, P.E., McFarland, D.C., and Schultz, E. Telo-
ventional meat. Thus, cultured meat should present fewer meric profiles and telomerase activity in turkey satellite cell
technical challenges than functional engineered muscle. clones with different growth rates. Biochim. Biophys. Acta
Future research is likely to be most fruitful if focused 1492, 362, 2000.
on developing scaffold-based techniques appropriate for 14. De Deyne, P.G. Formation of sarcomeres in developing
processed meat products, and affordable, nonserum myotubes: Role of mechanical stretch and contractile acti-
media needed to support them. vation. Am. J. Physiol. Cell Physiol. 279, C1801, 2000.
15. Powell, C.A., Smiley, B.L., Mills, J., and Vandenburgh,
H.H. Mechanical stimulation improves tissue-engineered
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