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


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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.
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Journal of Integrative Agriculture 2015, 14(2): 208–216
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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
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,; Correspondence Mark J Post,
Tel: +31-43-3881200, Mobile: +31-6-46705558, Fax: +31-43-
3884166, E-mail:
© 2015, CAAS. All rights reserved. Published by Elsevier Ltd.
doi: 10.1016/S2095-3119(14)60889-3
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).
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
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
Human myoblasts
Boudreault et al. 2001
Oh et al. 2009
Goh et al. 2013
Sart et al. 2009
Packed bed bioreactor CHO (Chinese hamster ovary cells)
Cong et al. 2001
Chiou et al. 1991
Aggregated cells hiPSC (ROCKi-treated)
hESC (ROCKi-treated)
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
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
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
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)
... Currently, cellular agriculture is highly costly [50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66]. It is estimated that the current cost of laboratory meat is ~$40,000 per kg, making it a very exclusive product. ...
... Other studies have devised protocols to produce bone, skeletal muscle, fat, fibrous tissue, and cartilage [52][53][54][55][56][57][58][59]. Lab-grown meat, derived from the bovine stem cells, was first used to make a burger in 2013; however, the meat itself was very costly and requires around 10,000 individual muscle strips to mimic the natural product [60][61][62][63][64][65][66]. Even with the current progress, many puzzles still need to be solved to obtain the optimum meat substitutes for the general population using feasible methods [68][69][70][71][72][73][74][75][76][77][79][80][81][82][83][84][85][86]. ...
... [77,[80][81][82][83][84][85][86]. In the 1930s, Winston Churchill commented on "the absurdity of growing a whole chicken to eat the breast or wing by growing these parts separately under an appropriate medium" [57][58][59][60][61][62][63][64][65][66][68][69][70][71]. Since then, for the realization of the idea, two major technologies have been developed. ...
Full-text available
Induced pluripotent stem cell (iPSC) technology is an emerging technique to reprogram somatic cells into iPSCs that have revolutionary benefits in the fields of drug discovery, cellular therapy, and personalized medicine. However, these applications are just the tip of an iceberg. Recently, iPSC technology has been shown to be useful in not only conserving the endangered species, but also the revival of extinct species. With increasing consumer reliance on animal products, combined with an ever-growing population, there is a necessity to develop alternative approaches to conventional farming practices. One such approach involves the development of domestic farm animal iPSCs. This approach provides several benefits in the form of reduced animal death, pasture degradation, water consumption, and greenhouse gas emissions. Hence, it is essentially an environmentally-friendly alternative to conventional farming. Additionally, this approach ensures decreased zoonotic outbreaks and a constant food supply. Here, we discuss the iPSC technology in the form of a “Frozen Ark”, along with its potential impact on spreading awareness of factory farming, foodborne disease, and the ecological footprint of the meat industry.
... Cells attach and grow by apposition in microcarriers, which are beads ordinarily having a diameter of 100-200 µm [109]. Microcarriers differ in their physical properties such as size, porosity, rigidity, density and surface chemistry [110]. ...
... The disadvantages are the costs and potential inedibility [31]. For those that cannot become an integrated part of final product, the cells can be harvested from microcarriers by changing temperature, or through electronically induced shape change [109]. There is usually a significant cell/tissue yield lost independent of the dissociation process because the cell detachment is incomplete; this loss directly impacts the production efficiency and costs [110]. ...
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Cultivated meat (CM) technology has the potential to disrupt the food industry—indeed, it is already an inevitable reality. This new technology is an alternative to solve the environmental, health and ethical issues associated with the demand for meat products. The global market longs for biotechnological improvements for the CM production chain. CM, also known as cultured, cell-based, lab-grown, in vitro or clean meat, is obtained through cellular agriculture, which is based on applying tissue engineering principles. In practice, it is first necessary to choose the best cell source and type, and then to furnish the necessary nutrients, growth factors and signalling molecules via cultivation media. This procedure occurs in a controlled environment that provides the surfaces necessary for anchor-dependent cells and offers microcarriers and scaffolds that favour the three-dimensional (3D) organisation of multiple cell types. In this review, we discuss relevant information to CM production, including the cultivation process, cell sources, medium requirements, the main obstacles to CM production (consumer acceptance, scalability, safety and reproducibility), the technological aspects of 3D models (biomaterials, microcarriers and scaffolds) and assembly methods (cell layering, spinning and 3D bioprinting). We also provide an outlook on the global CM market. Our review brings a broad overview of the CM field, providing an update for everyone interested in the topic, which is especially important because CM is a multidisciplinary technology.
... The observed effects of modular scaffold materials and sizes on the initial cell adhesions are essential to inform the adaptation of current microcarrier-based cell culture technologies for large-scale modular tissue cultures. In the microcarrier-based cell expansion systems, adherent mammalian cells are usually seeded on the microcarriers with suitable surface properties either continuously suspended in the media or precipitated statically in the bioreactors [27,[57][58][59]. The selection of either the dynamic or the static cell seeding methods is dependent on several issues including the size of the microcarriers. ...
... The selection of either the dynamic or the static cell seeding methods is dependent on several issues including the size of the microcarriers. It was revealed that the dynamic method is generally used for larger microcarriers (>100 µm) [28,57,58,60], while the static method is frequently selected for smaller microcarriers (56-100 µm) [61][62][63]. According to the size and surface properties of the modular scaffolds, these dynamic or static cell seeding methods designed for microcarrier-based cell expansion systems can be adapted for large-scale modular tissue cultures in DE. ...
Full-text available
Developmental engineering (DE) aims to culture mammalian cells on corresponding modular scaffolds (scale: micron to millimeter), then assemble these into functional tissues imitating natural developmental biology processes. This research intended to investigate the influences of polymeric particles on modular tissue cultures. When poly(methyl methacrylate) (PMMA), poly(lactic acid) (PLA) and polystyrene (PS) particles (diameter: 5–100 µm) were fabricated and submerged in culture medium in tissue culture plastics (TCPs) for modular tissue cultures, the majority of adjacent PMMA, some PLA but no PS particles aggregated. Human dermal fibroblasts (HDFs) could be directly seeded onto large (diameter: 30–100 µm) PMMA particles, but not small (diameter: 5–20 µm) PMMA, nor all the PLA and PS particles. During tissue cultures, HDFs migrated from the TCPs surfaces onto all the particles, while the clustered PMMA or PLA particles were colonized by HDFs into modular tissues with varying sizes. Further comparisons revealed that HDFs utilized the same cell bridging and stacking strategies to colonize single or clustered polymeric particles, and the finely controlled open pores, corners and gaps on 3D-printed PLA discs. These observed cell–scaffold interactions, which were then used to evaluate the adaptation of microcarrier-based cell expansion technologies for modular tissue manufacturing in DE.
... Additionally, cultured meat for the food industry requires large-scale cultivation, low resource consumption and the short production time of cell culture. Thus, it is important to explore the improvement and optimization of multiple cell culture technologies including microcarriers, suspension cultures, packed bed bioreactors, etc. [15], so as to achieve efficient and large-scale production of cultured meat. ...
Full-text available
As novel carrier biomaterials, decellularized scaffolds have promising potential in the development of cellular agriculture and edible cell-cultured meat applications. Decellularized scaffold biomaterials have characteristics of high biocompatibility, bio-degradation, biological safety and various bioactivities, which could potentially compensate for the shortcomings of synthetic bio-scaffold materials. They can provide suitable microstructure and mechanical support for cell adhesion, differentiation and proliferation. To our best knowledge, the preparation and application of plant and animal decellularized scaffolds have not been summarized. Herein, a comprehensive presentation of the principles, preparation methods and application progress of animal-derived and plant-derived decellularized scaffolds has been reported in detail. Additionally, their application in the culture of skeletal muscle, fat and connective tissue, which constitute the main components of edible cultured meat, have also been generally discussed. We also illustrate the potential applications and prospects of decellularized scaffold materials in future foods. This review of cultured meat and decellularized scaffold biomaterials provides new insight and great potential research prospects in food application and cellular agriculture.
... However, additives may not be food grade, and can pose a safety hazard [69,70]. The marketing of PM products focuses on the improvement of color and a number of other attributes [23,56,71]. Consumers prefer natural additives to chemical additives in meat substitutes [72].These problems account for the reluctance of consumers to accept meat substitutes. ...
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The sustained growth of global meat consumption incentivized the development of the meat substitute industry. However, long-term global commercialization of meat substitutes faces challenges that arise from technological innovation, limited consumer awareness, and an imperfect regulatory environment. Many important questions require urgent answers. This paper presents a review of issues affecting meat substitute manufacturing and marketing, and helps to bridge important gaps which appear in the literature. To date, global research on meat substitutes focuses mainly on technology enhancement, cost reduction, and commercialization with a few studies fo-cused on a regulatory perspective. Furthermore, the studies on meat substitute effects on environmental pollution reduction, safety, and ethical risk perception are particularly important. A review of these trends leads to conclusions which anticipate the development of a much broader market for the meat substitute industry over the long term, the gradual discovery of solutions to technical obstacles, upgraded manufacturing, the persistent perception of ethical risk and its influence on consumer willingness to accept meat substitutes, and the urgent need for constructing an effective meat substitute regulatory system.
... The world is in search of systematized ways of protein production to assist the expanding world population while satisfying current challenges, such as environmental and animal welfare concerns (Aiking, 2014). Among the solutions, cultured meat is proposed as a viable substitute for consumers who do not wish to change the composition of their diet and a source for reducing the pressure on the livestock production system to ensure animal welfare (Kadim et al., 2015;Moritz et al., 2015;Post, 2012). ...
Full-text available
Rising environmental issues, animal welfare concerns and vulnerable food supply chain especially during pandemics, as COVID-19 demands an effective and long-term solution for food security in future. All of these challenges encourage the researchers to find more reliable and clean ways of food production such as cultured meat. This process involved the production of animal meat in lab using large bioreactors without raising animals. Cultured meat production is widely accepted among animal rights activists and it can solve the issues related to conventional farming such as excessive use of land resource, animal slaughter, foodborne diseases and antibiotic resistance. Despite of all these advantages, it is facing some serious challenges, which includes technical, social and ethical limitations. Extracting specific cell line, development of animal-free growth media, upgradation of bioreactors, development of desired scaffolds and changing the public perception towards lab grown meat are fundamental challenges that need to be discuss. This review intends to summarize both technical and social challenges that are halting the availability of cultured meat in market and suggests some feasible recommendations to overcome these obstacles.
Cultured meat is rapidly developing as an emerging meat production technology. Adipose tissue plays an essential role in the flavor of meat products. In this study, cultured fat was produced by cultured adipose-derived stem cells (ADSCs) based on collagen in vitro, with a 3D model. The research showed that ADSCs could attach to collagen hydrogels and differentiate into mature adipocytes. Texture analysis demonstrated that the springiness, cohesiveness, and resilience of cultured fat were consistent with porcine subcutaneous fat. Moreover, 28 volatile organic compounds (VOCs) were detected by headspace gas chromatography-ion mobility spectrometry. The relative contents of 17 VOCs in cultured fat were significantly higher than porcine subcutaneous fat and empty collagen hydrogels, and the relative contents of 5 VOCs in cultured fat were not significantly different from porcine subcutaneous fat. These findings assert the promising application of cultured fat in cultured meat production.
Purpose The purpose of this article is (1) to carry out an ambivalent analysis of the determinants (benefits/risks) of the adoption of cultured meat, (2) to identify their impacts on consumers’ attitudes (cognitive, affective and conative) and (3) to propose a research agenda. Design/methodology/approach A systematic review of the relevant literature was conducted. The authors selected 86 articles that were coded using NVivo 12 software according to the theoretical framework chosen for this study: (1) consumer attitude ambivalence (benefit–risk) – conflicting presence of positive and negative attitudes in decision-making, (2) the consumer preference theory – choice of consumers based on utility maximisation or best characteristics/determinants and (3) the three-dimensional perspective of attitude – cognitive, affective and behavioural components. The authors followed the methodological steps (formulation of the research question, identification of relevant scientific studies, evaluation of the quality of studies, summary of evidence and interpretation of results) recommended by Lipsey and Wilson (2001) and Tranfield et al . (2003). Several keywords were drawn from a study by Bryant and Barnett (2019) on cultured meat (CM) nomenclature and its impact on consumer acceptance. Findings The identified articles were relatively recent (84/86 articles were published after 2010) and in the fields of agriculture and ethical agriculture (22/86), policy and regulations (12/86) and psychology (11/86). Content analysis helped identify four types of ambivalent determinants for the adoption of cultured meat: ethics, intrinsic, informational and belief. The results suggest the existence of a group of “dominant” determinants for each attitude component. Thus, the dominant determinants of cognitive, affective and conative components are informational, ethical and intrinsic determinants, respectively. Research limitations/implications This research is based on a systematic review of literature and is a review of the narrative literature that provides an overview of what is known about cultured meat adoption. The main weakness of this type of method is the feasibility generally associated with the existence (and a sufficient number) of studies that can be included. Other types of the meta-analytic method could have been used and could have explored different measures and biases (e.g. effect sizes, statistical power, sampling error, measurement error and publication bias). Also, as a food technology whose social acceptability would be influenced by all stakeholders, it would be relevant to expand the analysis to other types of stakeholders. Practical implications Little is still known to the public about the adoption mechanisms of this technology. In terms of behaviour, Siegrist et al . (2018) suggest that new studies should focus on factors that influence the individual differences in the willingness of consumers to eat or purchase cultured meat. By identifying the dominant target influence of informational determinants on cognitive components, that of ethical determinants on affective components and finally that of intrinsic determinants on conative attitudes, this article offers a first avenue of solution to businesses operating in this new industry, as well as to public authorities, to improve the acceptance of cultured meat. Private businesses will benefit from the results of this research by understanding the underlying motivations of consumers to adopt this type of innovation in order to adjust future marketing. Social implications This article, through better understanding of the psychological mechanisms that contribute to its social acceptability amongst the population, has the potential to improve educational campaigns for this technology. The results could thus guide both public policies as well as the regulation of activities related to cultured meat in the coming years, professional orders, private businesses and the general public. It thus provides initial insight needed to understand this public debate. Originality/value Research addressing cultured meat has come primarily from agribusiness and environmental and biological sciences. The authors highlighted the need for interdisciplinary collaboration between biological and social sciences to address ethical issues. This article, via multidisciplinary systematic reviews, links environmental/biological sciences and social sciences, and management.
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Plants were the primary source of food for early humans. Hinduism and Jainism have consistently urged their devotees to continue with a vegan lifestyle. Supporters of the Orphic secrets were the principal individuals to expound on veggie-lover food in the 6th century BC. Pythagoras, a Greek rationalist, is believed to be the organizer behind moral vegetarianism. Several famous people followed the Pythagorean way of life, which affected vegetarian diets until the 19th century. During the Middle Ages, vegan food essentially quit being eaten in India. Various individuals decided to be veggie lovers during the Renaissance and the Age of Enlightenment. In 1847, India was where the primary vegan culture was framed. In 1908, the International Vegetarian Society was begun, and in 1944, the leading veggie lover society was shaped. Sylvester Graham, John Harvey Kellogg, and Maximilian Bircher-Benner were all notable vegans during this time. Toward the beginning of the 21st hundred years, something changed. Scientists have disproved the old belief that vegetarianism leads to poor nutrition. Instead, they have shown that a vegetarian diet lowers the risk of most modern diseases. Today, vegetarian diets are becoming more popular and accepted all over the world.
In vitro cultured meat is an emerging area of research focus with an innovative approach through tissue engineering (i.e., cellular engineering) to meet the global food demand. The manufacturing of lab-cultivated meat is an innovative business that alleviates life-threatening environmental issues concerning public health and animal well-being on the global platform. There has been a noteworthy advancement in cultivating artificial meat, but still, there are numerous challenges that impede the swift headway of lab-grown meat production at a commercially large scale. In this review, we focus on the manufacturing of edible scaffolds for cultured meat production. In brief, first an introduction to cultivating artificial meat and its current scenario in the market is provided. Further, a discussion on the understanding of composition, cellular, and molecular communications in muscle tissue is presented, which are vital to scaling up the production of lab-grown meat. In continuation, the major components (e.g., cells, biomaterial scaffolds, and their manufacturing technologies, media, and potential bioreactors) for cultured meat production are conferred followed by a comprehensive discussion on the most recent advances in lab-cultured meat. Finally, existing challenges and opportunities including future research perspectives for scaling-up cultured meat production are discussed with conclusive interpretations.
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Stirred microcarrier (MC) culture has been suggested as the method of choice for supplying large volumes of mesenchymal stem cells (MSCs) for bone tissue engineering. In this study, we show that in addition to the improvement in cell expansion capacity, MSCs propagated and harvested from MC culture also demonstrate higher osteogenic potency when differentiated in vivo or in vitro in three-dimensional (3D) scaffold cultures as compared with traditional monolayer (MNL) cultures. Cytodex 3 microcarrier-expanded human fetal MSC (hfMSC) cultures (MC-hfMSCs) achieved 12- to 16-fold expansion efficiency (6×10(5)-8×10(5) cells/mL) compared to 4- to 6-fold (1.2×10(5)-1.8×10(5) cells/mL) achieved by traditional MNL-expanded hfMSC culture (MNL-hfMSCs; p<0.05). Both MC-hfMSCs and MNL-hfMSCs maintained similar colony-forming capacity, doubling times, and immunophenotype postexpansion. However, when differentiated under in vitro two-dimensional (2D) osteogenic conditions, MC-hfMSCs exhibited a 45-fold reduction in alkaline phosphatase level and a 37.5% decrease in calcium deposition compared with MNL-hfMSCs (p<0.05). Surprisingly, when MC-hfMSCs and MNL-hfMSCs were seeded on 3D macroporous scaffold culture or subcutaneously implanted into nonobese diabetic/severe combined immunodeficient mice, MC-hfMSCs deposited 63.5% (p<0.05) more calcium and formed 47.2% (p<0.05) more bone volume, respectively. These results suggest that the mode of hfMSC growth in the expansion phase affects the osteogenic potential of hfMSCs differently in various differentiation platforms. In conclusion, MC cultures are advantageous over MNL cultures in bone tissue engineering because MC-hfMSCs have improved cell expansion capacity and exhibit higher osteogenic potential than MNL-hfMSCs when seeded in vitro into 3D scaffolds or implanted in vivo.
The food scientists are giving priorities to the specific end-product uses rather than on generalities, for developing palatable meat analogs. Despite soy's position being the most common protein used in analogs, several different proteins can be used in making meat-mimickers. Innovations will prove to be a vital element in the future years to move analogs and soy to the next plateau. Fresh thinking, continued progress, and eyeing market opportunities will be essential in realizing this technology's full potential.
Microcarriers have been widely used for various biotechnology applications because of their high scale-up potential, high reproducibility in regulating cellular behavior, and well-documented compliance with current Good Manufacturing Practices (cGMP). Recently, microcarriers have been emerging as a novel approach for stem cell expansion and differentiation, enabling potential scale-up of stem cell-derived products in large bioreactors. This review summarizes recent advances of using microcarriers in mesenchymal stem cell (MSC) and pluripotent stem cell (PSC) cultures. From the reported data, efficient expansion and differentiation of stem cells on microcarriers rely on their ability to modulate cell shape (i.e. round or spreading) and cell organization (i.e. aggregate size). Nonetheless, current screening of microcarriers remains empirical, and accurate understanding of how stem cells interact with microcarriers still remains unknown. This review suggests that accurate characterization of biochemical and biomechanical properties of microcarriers is required to fully exploit their potential in regulating stem cell fate decision. Due to the variety of microcarriers, such detailed analyses should lead to the rational design of application-specific microcarriers, enabling the exploitation of reproducible effects for large scale biomedical applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013.
In response to muscle injury, satellite cells activate the p38α/β MAPK pathway to exit quiescence, then proliferate, repair skeletal muscle, and self-renew, replenishing the quiescent satellite cell pool. Although satellite cells are capable of asymmetric division, the mechanisms regulating satellite cell self-renewal are not understood. We found that satellite cells, once activated, enter the cell cycle and a subset undergoes asymmetric division, renewing the satellite cell pool. Asymmetric localization of the Par complex activates p38α/β MAPK in only one daughter cell, inducing MyoD, which permits cell cycle entry and generates a proliferating myoblast. The absence of p38α/β MAPK signaling in the other daughter cell prevents MyoD induction, renewing the quiescent satellite cell. Thus, satellite cells employ a mechanism to generate distinct daughter cells, coupling the Par complex and p38α/β MAPK signaling to link the response to muscle injury with satellite cell self-renewal.
Bone tissue engineering represents a promising strategy to obviate bone deficiencies, allowing the ex vivo construction of bone substitutes with unprecedented potential in the clinical practice. Considering that in the human body cells are constantly stimulated by chemical and mechanical stimuli, the use of bioreactor is emerging as an essential factor for providing the proper environment for the reproducible and large-scale production of the engineered substitutes. Human mesenchymal stem cells (hMSCs) are experimentally relevant cells but, regardless the encouraging results reported after culture under dynamic conditions in bioreactors, show important limitations for tissue engineering applications, especially considering their limited proliferative potential, loss of functionality following protracted expansion, and decline in cellular fitness associated with aging. On the other hand, we previously demonstrated that human embryonic stem cell-derived mesodermal progenitors (hES-MPs) hold great potential to provide a homogenous and unlimited source of cells for bone engineering applications. Based on prior scientific evidence using different types of stem cells, in the present study we hypothesized that dynamic culture of hES-MPs in a packed bed/column bioreactor had the potential to affect proliferation, expression of genes involved in osteogenic differentiation, and matrix mineralization, therefore resulting in increased bone-like tissue formation. The reported findings suggest that hES-MPs constitute a suitable alternative cell source to hMSCs and hold great potential for the construction of bone substitutes for tissue engineering applications in clinical settings.
A concentric-cylinder airlift reactor, in which the annulus is a packed bed of glass fibers, has been developed in order to facilitate the scaleup and enhance the volumetric productivity of anchorage-dependent animal cell cultures. In this bio-reactor, oxygen-containing gas is sparged through the inner draft tube, causing bubble-free medium to flow through the fiber bed in the outer cylinder and providing both oxygenation and convective nutrient transfer to the cells. Several other desirable features for reactor operation are also provided by this design. Cell cultivations in this bioreactor have been successfully carried out and provide data for the feasibility of the large-scale cell cultivation.
A novel method for the scale-up culture of Chinese hamster ovary (CHO) cells in a packed-bed bioreactor is developed wherein microcarriers, attached with CHO cells in a microcarrier culture system, are inoculated directly into the packed-bed bioreactor. Cells continue to grow after inoculation and the maximum cell density reaches about 2107 cells ml–1. The method provides a new technique for the scale-up of a packed-bed culture while decreasing the labour cost and ensuring the safety of operation.
It is commonly considered not desirable to use microcarriers more than once in the cultivation of anchorage-dependent animal cells. However, our experiment contradicts this belief. The collagen-coated microcarriers, Cytodex-3, from a batch culture of Vero cells, were collected, cooled to 4, agitated in basic phosphate-buffered solution to detach the cells, and then fully washed to remove the cell debris. The microcarriers were then re-applied in cell culture. The rate of cell attachment, growth and metabolism on re-used carriers were found to be comparable to that of on new ones.