Alternatives for large-scale production of cultured beef: A review

Abstract

Cultured beef is a method where stem cells from skeletal muscle of cows are cultured in vitro to gain edible muscle tissue. For large-scale production of cultured beef, the culture technique needs to become more efficient than today's 2-dimensional (2D) standard technique that was used to make the first cultured hamburger. Options for efficient large-scale production of stem cells are to culture cells on microcarriers, either in suspension or in a packed bed bioreactor, or to culture aggregated cells in suspension. We discuss the pros and cons of these systems as well as the possibilities to use the systems for tissue culture. Either of the production systems needs to be optimized to achieve an efficient production of cultured beef. It is anticipated that the optimization of large-scale cell culture as performed for other stem cells can be translated into successful protocols for bovine satellite cells resulting in resource and cost efficient cultured beef.

Full text

Journal of Integrative Agriculture 2015, 14(2): 208–216
REVIEW
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Alternatives for large-scale production of cultured beef: A review
Matilda S M Moritz1, 2, Sanne E L Verbruggen1, Mark J Post1
1 Department of Physiology, Maastricht University, Maastricht 6229 ER, The Netherlands
2 Department of Physics, Chemistry and Biology, Linköping University, Linköping SE-581 83, Sweden
Abstract
Cultured beef is a method where stem cells from skeletal muscle of cows are cultured in vitro to gain edible muscle tissue.
For large-scale production of cultured beef, the culture technique needs to become more efcient than today’s 2-dimensional
(2D) standard technique that was used to make the rst cultured hamburger. Options for efcient large-scale production of
stem cells are to culture cells on microcarriers, either in suspension or in a packed bed bioreactor, or to culture aggregated
cells in suspension. We discuss the pros and cons of these systems as well as the possibilities to use the systems for
tissue culture. Either of the production systems needs to be optimized to achieve an efcient production of cultured beef.
It is anticipated that the optimization of large-scale cell culture as performed for other stem cells can be translated into
successful protocols for bovine satellite cells resulting in resource and cost efcient cultured beef.
Keywords: cultured beef, microcarriers, aggregated cells, packed bed bioreactor, cell culture
1. Introduction
In August 2013 we provided proof of principle that con-
sumption meat can be cultured using fairly standard tissue
engineering technology. Cells are typically grown in asks
with a at bottom and nourished with uid containing es-
sential nutrients, the so-called medium. Tissue engineer-
ing technology for cultured beef in particular relies on the
self-organizing capacity of skeletal muscle stem cells (i.e.,
satellite cells) when provided with a conducive hydrogel and
polar anchor points. The small-scale production of less than
5×1010 cells has been separated in a proliferation phase and
a differentiation phase and each of these phases has specic
requirements to be fullled for commercial application. The
separation into these two phases is dictated by different
medium requirements and by different anchoring of cells and
muscle bers and resultant mechanical conditioning. During
the proliferation phase, the cells grow in sheets (2-dimen-
sional, 2D) anchored to a surface, for instance the layers of
commercially available cell-factories. Satellite cells prolif-
erate well in a medium containing exceptionally high serum
concentrations of 30%. The differentiation phase starts with
reducing serum to 2% and placing the differentiating cells,
a.k.a myotubes, in a hydrogel that allows self-organization
into muscle bers (myooids, bioarticial muscle) between
two xed, rigid anchor points to which the cells can attach.
Protein synthesis, an important goal of meat engineering,
is stimulated by tension between the anchor points of the
bio-articial muscle (Vandenburgh et al. 1999).
The three major conditions for cultured beef to become
successful in replacing current livestock produced beef
are, 1) better efciency, 2) sustainability and 3) mimicry. In
Received 3 April, 2014 Accepted 4 July, 2014
Matilda S M Moritz, E-mail: matilda.moritz@maastrichtuniversity.
nl, matmo608@student.liu.se; Correspondence Mark J Post,
Tel: +31-43-3881200, Mobile: +31-6-46705558, Fax: +31-43-
3884166, E-mail: m.post@maastrichtuniversity.nl
© 2015, CAAS. All rights reserved. Published by Elsevier Ltd.
doi: 10.1016/S2095-3119(14)60889-3

209
Matilda S M Moritz et al. Journal of Integrative Agriculture 2015, 14(2): 208–216
order to reduce resources required for beef production, the
culture system needs to be more efcient than livestock
produced beef in converting vegetable proteins from feed
into edible animal proteins. This so-called bioconversion
rate is estimated to be a meager 15% for cattle (Egbert and
Borders 2006), which is the lowest for domesticated animals
providing staple meats. Sustainability is also an absolute
requirement. In the particular case of cultured beef, this
means that the production cannot involve animal products
such as fetal bovine serum or bovine collagen hydrogel,
because we will not be able to source them if cultured beef
becomes successful and global livestock volume is greatly
reduced. Mimicry, meaning that the eventual product needs
to be sufciently similar in taste, texture and appearance
to livestock beef that it can serve as a widely acceptable
alternative, is also a condition for success.
Cell and tissue culture in their current states are not
efcient processes in terms of energy, water and feedstock
expenditure as they have been primarily employed for sci-
entic and medical applications, and were considered less
dependent on cost and resources effectiveness than any
food application. Also, the scale of cell and tissue manu-
facturing for food would trump scientic and medical tissue
production by several orders of magnitude offering a new
perspective on current production, resource management
and pricing (Post and van der Weele 2014).
In this review, we focus on scale and efciency of cell
and tissue production for cultured beef applications. Con-
siderations on the other requirements, sustainability and
mimicry, are beyond the scope of this review. First, we will
discuss systems to scale up cell and tissue production and
second we will focus on the resource efciency of cell culture
in general and that of the large-scale systems for anchor
dependent cells in particular.
2. Large scale cell production systems
The goal of a large-scale cell production system is to gen-
erate a large amount of cells with the smallest possible
amount of resources (i.e., culture medium) and minimal
handling and preferably in a short time. For the very large-
scale cultivation of stem cells for food, suspension cultures
in bioreactors are required. To achieve high density cultures
in suspensions there are two alternatives: 1) cultivation on
microcarriers or 2) cultivation in aggregated form as cell
aggregates (Reuveny 1990; Steiner et al. 2010; Abbasal-
izadeh et al. 2012). Microcarriers are beads to which cells
can adhere and grow in apposition. Cell aggregates are
clumps of cells that grow in 3D and serve as anchors for
their neighbors, while the aggregates themselves remain in
suspension. Microcarriers or beads can also be static in a
bioreactor with uidized media in a system called a packed
bed bioreactor (PBR). A basic overview of the three large-
scale production systems can be seen in Fig. 1.
2.1. Microcarriers in suspension
Microcarriers are beads to which cells can attach and grow
by apposition, much the same way as if they are grown on
at surfaces. They are typically 100–200 µm in diameter
Fig. 1 Overview of the three possible large-scale systems for cultured beef.

210 Matilda S M Moritz et al. Journal of Integrative Agriculture 2015, 14(2): 208–216
and made of polystyrene so that they oat in the medium.
To ensure mixture of nutrients and gases, the medium is
agitated by an impeller, gas ow or rotation of the bioreactor.
There are several factors that need to be considered for
retaining the proliferation phase of satellite cells in cell sus-
pension. One important aspect is inter-dependency of cells
through their proximity. Cells in culture depend on growth
factors that come from the medium but also from the cells
themselves. The growth factors and cytokines produced
during metabolic activity trigger neighboring cells resulting
in increased growth (Greene and Allen 1991; Tatsumi et al.
1998; Troy et al. 2012). For microcarrier cell culture this
means that a low initial seeding concentration (cells per
bead) can cause a lower growth rate compared to high
seeding concentration but there is also a maximum density
of cells when they reach conuency on the beads. The
cells also seem to form aggregates, which on microcarriers
can build big clusters (Molnar et al. 1997). To overcome
clusters, new beads can be added which make the cells
transfer and colonize the new beads as well, a phenomenon
known as bead-to-bead transfer (Wang and Ouyang 1999a;
Dürrschmid et al. 2003). To add new beads for bead-to-bead
transfer of cells also offers a convenient scalable production
of cells on microcarriers. Mesenchymal and pluripotent stem
cells cultured on microcarriers, earlier reviewed by Sart et al.
(2013), can be produced this way in large-scale.
Satellite cells grow, proliferate and differentiate on micro-
carriers (Molnar et al. 1997). By just changing the medium
in the bioreactor differentiation was initialized, however with
less myotube formation, a hallmark of the initial stage of
differentiation into muscle cells. Molnar et al. (1997) con-
cluded therefore that satellite cells show slower expansion
in bioreactor cultures compared to layered cell factories.
Optimization is therefore still necessary.
A mouse cell line of muscle cells have also been tested
to grow, proliferate and differentiate on microcarriers (Torgan
et al. 2000). In this study, cells were cultured either in a
microgravity bioreactor or in a teon bag. In both systems,
myotubes developed showing that the differentiation phase
can occur on microcarriers in much the same way it occurs
on at surfaces. Results differed in the two culture conditions
with less myotube formation in the microgravity bioreactor,
probably as a result of different hydrodynamics that inu-
enced the cells. In this study, it was also observed aggre-
gate formation of the cells on microcarriers. Prevention of
aggregation might be necessary for myotube formation. As
aggregation is never observed in at surface cell factories,
it is reasonable to assume that increasing the diameter of
the microcarriers will prevent aggregate formation.
The observation that differentiation on beads occurs
however suggests that proliferation and differentiation phase
do not have to be separated, which would save additional
handling of the cell culture. One of the important parameters
in microcarrier-based cell culture is shear stress imparted
on the cells by medium agitation.
The formation of muscle skeletal tissue on microcarriers
may be possible since the coating of the microcarrier reas-
sembles the hydrogel that is needed for self-organization.
If the microcarriers are spherical or cylindrical, bioarticial
muscle (BAMs) might also be formed around the microcar-
riers through self-anchoring, in much the same way as we
cultured BAMs for the hamburger. It has previously been re-
ported that myoblasts cultured on microcarriers cannot only
differentiate into myotubes, but they also mature to muscles
in a static condition (Bardouille et al. 2001). It remains to be
determined if these muscle bers are sufciently oriented
and anchored to develop in full-edged muscle bers like in
separate dedicated differentiation bioreactors. In addition,
muscle bers need to be harvested from the microcarriers,
which imposes another level of complexity on the design
of the beads. Harvest from microcarriers could be done
by changing temperature (Tamura et al. 2012), or through
electronically induced shape change of the microcarriers
(Persson et al. 2011). Alternatively, the beads themselves
are edible and become an integrated and perhaps partly
degraded part of the skeletal muscle tissue, obviating the
need for harvesting the muscle by sequestering them from
the microcarriers.
Thus, it is anticipated that for optimal cell production,
microcarriers have to be tailored for bovine satellite cells by
coating, surface modication, size and perhaps composition
(Sart et al. 2013).
2.2. Cell aggregates
Successful suspension cultures with aggregated stem cells
have been developed (Cormier et al. 2006). Many studies of
stem cells in suspension are on human embryonic stem cells
(hESC) or human induced pluripotent stem cells (hiPSC)
reprogrammed to allow suspension culture by treatment with
cytokines that delay differentiation or with a Rho-associated
protein kinase-inhibitor (ROCKi) to delay apoptosis (Amit
et al. 2010; Larijani et al. 2011; Fluri et al. 2012).
For aggregated cells in suspension, cell density is of
utmost importance, as well as medium composition and
parameters of mixing through agitation (Abbasalizadeh
et al. 2012; Chen et al. 2012). Metabolic activities of the
cells in aggregates depend on the initial cell density with
high initial cell densities being preferable to assure colo-
nization of all cells (Abbasalizadeh et al. 2012). Growth
media formulation is also an important aspect, which can
change properties of cell cultures. The ROCKi is commonly
used to inhibit apoptosis and increase proliferation in cell
aggregates suspended in medium (Watanabe et al. 2007).

211
Matilda S M Moritz et al. Journal of Integrative Agriculture 2015, 14(2): 208–216
ROCKi treatment is not ideal for cells in food applications
since protein expression is diminished even after withdrawal
of the treatment (Krawetz et al. 2011).
Size of the aggregates is an important determinant of
successful culture. Ideally, aggregates are not too large
with a fairly homogeneous size distribution. By changing
the agitation of the medium, size can be contained and
shear stress on the cells decreased (Zweigerdt et al. 2011;
Abbasalizadeh et al. 2012). Another method to prevent the
occurrence of large aggregates is to split the cell aggre-
gates at regular intervals by passaging methods, although
inevitable cell loss with each passage might off-set the
benets from reducing aggregate size (Singh et al. 2010;
Amit et al. 2011; Chen et al. 2012). Passaging refers to the
dissociating cells from the surface and redistributing them
at lower densities in fresh medium to boost the next phase
of growth. Higher cell densities have been described for
aggregate systems than for microcarrier systems (Table 1).
A combination of aggregated cells on microcarriers
have also been tested and seem better than single cells
on microcarriers (Phillips et al. 2008), although reported
cell expansion was still lower than with aggregates or
non-aggregate microcarriers alone (Amit et al. 2011; Park
et al. 2014). It appears however, that aggregates on mi-
crocarriers could be an option for expansion to large-scale
because the aggregated state protected the cells from stress
and decreased the lag phase during bead-to-bead transfer
(Boudreault et al. 2001).
A synthetic, biodegradable scaffold that serves as sup-
port for the cells is required for differentiation and myober
formation. After having proliferated, the cells have to be
transferred to a second bioreactor system for differentiation
and tissue generation. One option might be to use added
microcarriers for further tissue development after aggregate
culture. Since aggregated cells can attach to microcarriers,
the cells could still stay in the same bioreactor by just chang-
ing the medium and adding microcarriers. Another option
could be to add a scaffolds wherein the cells can organize
and mature (Neumann et al. 2003). Both procedures would
result in less operational handling of the cells and therefore
less risk of contamination.
2.3. Packed bed bioreactors
Packed bed bioreactor (PBR) is a bioreactor with a bed
of microcarriers on which the cells are immobilized. This
type of reactor has a ow of growth medium down-stream,
up-stream, or radially across cells in a static position within
the packed bed while the nutrients and gases are evenly
distributed.
PBRs have proved to increase viability of the cells be-
cause of the static immobilization and the ow of nutrients
and oxygen that can reach the cells (Park and Stephanopou-
los 1993; Cong et al. 2001). A packed bed with a ow of
medium has the advantage that the medium is oxygenated
before entering the bioreactor which improves oxygen dis-
tribution to the cells (Chiou et al. 1991). The system of a
continuous radial ow of growth medium seems to be the
most promising type of PBR (Bohmann et al. 1992). PBR
for use in mammalian cell culture has earlier been reviewed
and proven to achieve high cell densities (Table 1) but is
not common for large volumes: 30 L is the largest reported
PBR (Meuwly et al. 2007).
Since the packed bed in a PBR can serve as a scaffold,
further tissue development might be combined in one sys-
tem. Both proliferation and differentiation on a scaffold in a
PBR has shown to work efciently (de Peppo et al. 2013).
High cell density during the proliferation phase is an
important parameter that determines efciency of the pro-
duction system. Optimal conditions for each type of system
need to be dened for bovine satellite cells as these are not
well studied for large-scale cell production. A comparison
between the three possible scale-up strategies can be
seen that it is possible to recycle culture medium through
replenishment of utilized nutrients, such as glucose and
glutamine, and removal of waste-products, such as lactate
and ammonia. It might be benecial to reuse part of the
medium as growth factors and cytokines produced by the
Table 1 Cell densities previously reported for 3-dimensional (3D) suspension cultures
System Cell type1) Cell density per mL medium Reference
Microcarriers in
suspension
Human myoblasts
hESC
hfMSCs
Ear-MSC
1.5×106
3.5×106
8.3×105
1.7×106
Boudreault et al. 2001
Oh et al. 2009
Goh et al. 2013
Sart et al. 2009
Packed bed bioreactor CHO (Chinese hamster ovary cells)
g-CHO
2×107
6.8×107
Cong et al. 2001
Chiou et al. 1991
Aggregated cells hiPSC (ROCKi-treated)
hESC (ROCKi-treated)
1.23×107
1.27×107
Abbasalizadeh et al. 2012
Abbasalizadeh et al. 2012
1) hESC, human embryonic stem cells; MSC, mesenchymal stem cells; hiPSC, human induced pluripotent stem cells; ROCKi, Rho-
associated protein kinase-inhibitor.

212 Matilda S M Moritz et al. Journal of Integrative Agriculture 2015, 14(2): 208–216
cells can stimulate subsequent cell growth. It has been
reported that recycling of growth medium when culturing
algae was not efcient since centrifugation was needed to
separate the biomass. Other problems were the inhibitory
factors in the medium that were still present during further
cell culture (Rodol et al. 2003). For stem cell culture, these
reported problems can be solved. Recycling of media can
be done in a similar way as for PBRs meaning that the cells
stay in the reactor and the medium is recycled “on-line”.
A purication step can be added to get rid of unwanted
waste-products, for example by chromatography. If nutrients
needed for growth of satellite cells can be established, an
optimization of nutrients supplements can be calculated or
monitored during recycling. The reuse of other materials,
for example microcarriers, will be important. For instance,
it has been reported that washed cytodex-3 microcarriers
are reusable for mammalian cell culture without affecting cell
growth (Wang and Ouyang 1999). If necessary, microcarrier
beads can be recoated before usage.
Cell culture is a technology where many variables
determine its efciency, and most of these variables can
be optimized. This is a time consuming effort, but there it
is fair to assume that a resource efcient method can be
developed (Table 2).
3. Efciency
Efcient production is of utmost importance for large-
scale production of cultured beef as it drives the potential
food-security and environmental benets over livestock
beef. Resource efciency is also important to keep the
cost of production low as materials are by far the largest
cost component. Production cost will translate in consumer
price and this is the most important criterion in consumer
preference. To reach a high efciency, culture conditions
need to be optimized for culture medium utilization. Met-
abolic monitoring during cell growth is an integral part of
most large-scale cell culture systems, but monitoring may
need to be rened to optimize metabolism so that most
nutrients are being converted to animal edible proteins.
Even with optimized medium utilization it is still likely that
not all components of the medium are equally consumed,
suggesting that additional resource efciency can be
gained by recycling the medium and microcarriers (Wang
and Ouyang 1999). Recycling of medium or carriers is
not routinely practiced because there is no economical or
environmental need for it with small or intermediate scale
cell production for medical applications. For food appli-
cation however, recycling may be essential to cost- and
resource-efcient cell production.
3.1. Optimization of culture conditions
Culturing cells in large-scale can be done by step-wise in-
creasing the size of the cell culture, meaning that after cells
are isolated from a cow, they are transferred to 2D surface
plates and then to bioreactors, going from small to large
volume tanks. These transferring steps, i.e., passages, need
to be optimized. For cultivation of cells on microcarriers or
in aggregates, high density of cells can be achieved by step-
wise increasing the number of beads and cells per bead or
by splitting the aggregates during culture. This technique
involves minimal handling. Another condition that is required
for high cell density is efcient distribution of oxygen and
nutrients and here, culture medium agitation is a factor of
importance (Zhao et al. 2005).
Cells need to be temporarily dissociated from their envi-
ronment to assume a new growth promoting state. Different
methods for dissociation of cells have been developed,
such as enzymatic treatment, chemical treatment, and
mechanical disruption (Collins et al. 2005; Suemori et al.
2006; Amit et al. 2010). Each of these methods may affect
viability and genetic stability of the cells (Mitalipova et al.
2005), so a balance needs to be found between efcient
cell dispersion and potential side effects. For cells in aggre-
gated form the homogeneity of the aggregates can also be
affected by the splitting treatment (Amit et al. 2010). Even
though mild dissociation treatments exists, optimization of
Table 2 Challenges and prospects of the three scale-up systems, microcarrier suspension culture, aggregated cells in suspension, and
packed bed bioreactor (PBR)
System Prospects Challenges
Microcarriers Many different possibilities of characteristics on
microcarriers
Can aggregate and build clusters
Reuse of microcarriers possible Shear forces from microcarriers or clusters
Easy to scale-up by adding new microcarriers
Aggregates Cheap because of no extra material needed Could be difcult to achieve on satellite cells without any cell
modications
Simple harvest Hard to control aggregate size
PBR Protective for the cells from shear forces Difcult to scale up
Good oxygenation
Easy recycling of growth media possible

213
Matilda S M Moritz et al. Journal of Integrative Agriculture 2015, 14(2): 208–216
passaging is important and has previously been done for
human embryonic stem cells and human pluripotent stem
cells (Amit et al. 2011; Chen et al. 2012). They need to be
repeated for satellite cells.
Bead-to-bead transfer is affected by stirring conditions
and for efcient scale-up of cells on microcarriers, this has to
be optimized. Intermittent agitation compared to continuous
agitation has shown positive effect on bead-to-bead transfer
(Wang and Ouyang 1999). Different intermittent agitation
conditions have also been studied and showed that resting
time and stirring time were both important in addition to
the interaction between stirring time and resting time, and
stirrer speed and resting time (Luo et al. 2008). Experience
with myoblasts showed more efcient culture with contin-
uous agitation, however with a lag-phase during which no
increase in cell density occurred. In this study, different
initial cell densities were also tested and the conclusion was
that by changing initial cell density efcient cell culture with
continuous agitation is better than static 2D-layered culture
(Boudreault et al. 2001). Another study showed different
cell-attachment on different types of microcarriers. For the
microcarrier Cytodex-1, continuous agitation was preferable
for cell attachment while for the microcarrier Cultispher-G,
intermittent agitation was superior (Ng et al. 1996).
A good distribution of oxygen and CO2 is required to sup-
port high cell densities. Homogenous mixing and agitation
without harming the cells are difcult tasks to achieve for
large-scale mammalian cell culture, even though different
kind of impellers, and control systems are available (Marks
2003). Conventionally, stirred tank bioreactors are used
in which the uid is mixed by large impellers but recently
other mixing technologies have been designed including
orbital shaken disposal bioreactors, rotating wall vessels
and wave reactors (Pierce and Shabram 2004; Chen et al.
2006; Zhang et al. 2010). Only rotating wall vessel has been
tested for satellite cells so far (Molnar et al. 1997).
A scale-up strategy by step-wise optimization of pa-
rameters such as oxygen inux, agitation, and initial cell
density is necessary for transforming adherent cultures to
suspensions (Abbasalizadeh et al. 2012). Since earlier
mentioned studies have been on other type of cells, new
optimized systems have to be established for satellite cells
used for cultured beef.
3.2. Metabolic control
To increase efciency, controlling metabolites utilized and
produced during cell culture is also required. Eukaryotic
cells produce energy (ATP) by aerobic or anaerobic res-
piration. Through glycolysis, glucose is modied to ATP.
The up-take of glucose is transported by proteins in the
plasma membrane to mitochondria where the conversion
takes place. Different fatty acids and amino acids are also
converted in the cell.
In vivo, many stem cells are situated in areas where the
oxygen pressure is lower than the “normoxic” state (20
-
21% pO2) for culture practice, which might be an indication
that lower oxygen pressure could be preferable in cell
culture. In low oxygen concentrations, so-called hypoxia
state (2
-
6% pO2), muscle cells have shown to increase
glucose consumption. Myotubes in cell culture showed a
large increase in glucose utilization and increased lactic
acid production already after 24 h of culture compared to
normoxia (Bashan et al. 1992). Other studies have shown
an increase in both myoblasts and myotubes in hypoxic
conditions (Chakravarthy et al. 2001). For human mesen-
chymal stem cells (hMSC) cultured on a 3D scaffold hypoxia
proved to increased proliferation and enhanced tissue
formation (Grayson et al. 2006). Hypoxic culture of hMSC
have also resulted in increased cell density during hypoxic
culture, suggesting better efciency (Grayson et al. 2007).
Thus, oxygen regulation during stem cell cultivation is an
important aspect that needs to be considered and optimized
as previously reviewed (Csete 2005).
Lactic acid is a byproduct produced by the cells when
consuming glucose. When there is no glucose or very little
available, a metabolic switch occurs and lactate can serve
as a carbon source and produce energy for the cell, but
high concentrations lactate can also inhibit growth of cells.
By on-line monitoring of glucose and lactate content, the
feeding with new medium can be regulated to maximize
glucose consumption and minimize lactate production
(Ozturk et al. 1997).
Glutamine is an important amino acid for the cells
and ammonia is a byproduct produced when consuming
glutamine. Ammonia can inhibit cells already in small con-
centrations therefore regulation of glutamine is preferred
to decrease ammonia concentration. It also seem that
glutamine concentration can regulate lactate consumption,
when glutamate was decreased lactate degradation started
(Zagari et al. 2013). Both glucose, glutamine, lactate and
ammonia production/consumption can be regulated by op-
timizing refreshment of culture medium (Schop et al. 2008),
therefore a feeding prole for the cell system is required to
optimize metabolite utilization/inhibition.
3.3. Recycling
It is possible to recycle culture medium through replenish-
ment of utilized nutrients, such as glucose and glutamine,
and removal of waste-products, such as lactate and am-
monia. It might be benecial to reuse part of the medium
as growth factors and cytokines produced by the cells can
stimulate subsequent cell growth. It has been reported that

214 Matilda S M Moritz et al. Journal of Integrative Agriculture 2015, 14(2): 208–216
recycling of growth medium when culturing algae was not
efcient since centrifugation was needed to separate the
biomass. Other problems were the inhibitory factors in the
medium that were still present during further cell culture
(Rodol et al. 2003). For stem cell culture, these reported
problems can be solved. Recycling of media can be done in
a similar way as for PBRs meaning that the cells stay in the
reactor and the medium is recycled “on-line”. A purication
step can be added to get rid of unwanted waste-products,
for example by chromatography. If nutrients needed for
growth of satellite cells can be established, an optimization
of nutrients supplements can be calculated or monitored
during recycling. The reuse of other materials, for example
microcarriers, will be important. For instance, it has been
reported that washed cytodex-3 microcarriers are reusable
for mammalian cell culture without affecting cell growth
(Wang and Ouyang 1999). If necessary, microcarrier beads
can be recoated before usage.
Cell culture is a technology where many variables de-
termine its efciency, and most of these variables can be
optimized. This is a time-consuming effort, but there it is fair
to assume that a resource efcient method can be developed
4. Conclusion
Tissue engineering in large-scale is a difcult task and the
scale of cell and tissue culture needed for food applications
is orders of magnitude higher than for medical applications.
Commercially available systems, microcarrier or cell-aggre-
gate based are a good start but need to be optimized for
bovine satellite cells, including but not limited to, specialized
microcarriers. The highest cell densities and therefore
the highest efciencies have been reported for packed
bed bioreactors but they are still in an experimental stage.
Limitations in up-scaling systems discussed are costs
(microcarriers), apoptotic cells (aggregates) and lack of
commercial availability (PBR). It is anticipated however,
that the optimization of large-scale cell culture as performed
for other stem cells can be translated into successful proto-
cols for bovine satellite cells resulting in resource and cost
efcient cultured beef.
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(Managing editor ZHANG Juan)