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7/28/2019 Cell Culture Bioreactors
1/23
Cell Culture Bioreactors
Cell Culture Bioreactors
Basic Types of Bioreactors 1
Stirred ank (Well Mixed) vs. ubular Reactor (Plug Flow) . . . . . . . . . . . . . . . . . 3
Segregated Bioreactors (Dead Zone Present) Compartmentalized Bioreactors . . 4
Implication When Growth or Reaction Occurs in the Reactor . . . . . . . . . . . . . . . 4
Homogenous Reactor vs. Heterogeneous Reactor . . . . . . . . . . . . . . . . . . . . . . . . . 4
Operating Mode of Bioreactors 5Batch and Continuous Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Te Operating Mode o Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Batch Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Fedbatch Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Material Balance on Bioreactors 9
Material Balance Equation or Reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Tissue culture and disposable cell culture systems 11
issue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Disposable Culture Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Multiple Plate Culture System (Cell Cube and Cell Factory) . . . . . . . . . . . . . . . . 2
Cell Support Systems 13
Suspension Culture vs Adherent Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Microcarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Cell Aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Microsphere Induce Cell Aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Microencapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Cell Culture Bioreactors 18
Simple Stirred ank Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Airlif Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Membrane Stirred ank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Spin Filter Stirred ank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Vibromixer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Fluidized Bed Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Membrane Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Mammalian cell bioreactors are generally categorized
similarly to chemical reactors according to their mixingcharacteristics. It is instructive to review two ideal reactors
well-mixed stirred tank and plug-ow (tubular) reactor. In
an ideal well-mixed bioreactor, the mixing is assumed to be
intense enough that the uid is homogeneous through the
reactor. The mathematical description of ideal continuous
ow stirred tank reactor is described by the following rst-
order differential equation.
( )
Ai Ai o Ao A
d VCFC F C r V
dt= +
Basic Types of Bioreactors
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2/232 Cell Culture Bioreactors
V is the culture volume in the bioreactor, CA the concentration
of nutrient or product A, t is time, F is the ow rate and rA
is the volumetric consumption rate of nutrient or production
rate of product A.
In an ideal stirred tank reactor, there is no ow bypass and
no shunt of substrate from inlet to outlet, no dead zones or
clumps of undissolved solid substrate oating around. The
addition of a substrate through feeding is instantaneously
distributed throughout the entire reactor, and when gas
sparging is employed the agitator provides an intimately
mixed gas-liquid. It also follows from this assumption that th
e stream exiting the reactor will have the same composition
as the well mixed uid in the reactor.
The basic model for the tubular reactor (such as hollow ber
and ceramic systems to be described later in this chapter)
species that the liquid phase moves as a plug-ow, meaning
that there is no variation of axial velocity over the cross
section. The mass balance for component A in a volume
element S z that described an ideal plug-ow reactor is thefollowing:
A
AZ
A rz
Cv
t
C+
=
where vz is the linear velocity in the z direction along the
ow and S is the cross-sectional area. Note that we assume
there is no liquid dispersion or back mixing. All elements in
the uid move at the same velocity. At steady state (i.e., cel
concentration and cellular activities at a given position are
not changing with time), the equation becomes
F
Sr
z
c AA
=
which describes changes of concentration of A along the
direction of uid ow. It is clear that the nutrient concentration
will decrease from inlet to the distal end of the reactor,
while metabolite concentration increases. The length of the
reactor is limited because eventually nutrient depletion or
metabolite accumulation inhibits growth and metabolism
These ideal cases of completely mixed tanks or plug ow
tubular reactors are situations that can be approximated in
small-scale laboratory conditions. The conditions in large
scale process reactors deviate signicantly from these idea
conditions.
In a well-mixed bioreactor, there are no concentration
gradients in either the gas or the liquid phase. In othe
words, none of the chemical species or cells is segregated in
the reactor. The other extreme of mixing is total segregation
where there is no interaction between different volume
elements in the bioreactor. An ideal plug ow reactor i
assumed to be under conditions of total segregation. Mos
bioreactor systems have a mixing pattern between the two
extremes and are under partially segregated conditions.
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Stirred Tank (Well Mixed) vs. Tubular
Reactor (Plug Flow)
Cell Culture Bioreactors 3
In general, laboratory and small pilot plant bioreactors are used
for process development and optimization. The uid mixing
characteristics are rather sensitive to the scale of the reactor
Furthermore, plug-ow bioreactors are intrinsically more
difcult to scale up than mixing vessels, as the concentration
gradient of essential nutrients, oxygen in particular, wil
inevitably become limiting in the downstream region of
the reactor. In considering the selection of bioreactors for
mammalian cell cultures, the mixing characteristics and their
relationship to scale-up have to be kept in mind.
The distinction between a well mixed continuous stirred
tank reactor (CSTR) and plug ow reactor (PFR) is best
illustrated by comparison of their response in the outlet to
a step change in feed concentration, consider a continuous
reactor that has an inlet stream (feed) and an outlet stream tha
are equal in volumetric ow rate, the volume of the reactor is
thus constant. For the case that the feed stream is colorlessbut at time 0 the stream is changed to a feed with red color
at a concentration of COIf the reactor is well mixed as in a
CSTR, as soon as the feed stream is switched, the color wil
be seen immediately in the efuent stream, since the color
is distributed instantaneously everywhere including the uid
that is taken out in the efuent stream. The plots shown
are the colors seen at the outlet. The red dye concentrate
will increase gradually. If the reactor volume is V, it wil
take longer than the time needed to ow through one reactor
volume to reach the same concentration as in the feed, since
the dye is also being taken out from the reactor from the
beginning. In fact, by solving the differentiation equation
tIcan be shown that it takes about three holding times (3t
0) to
reach almost the same concentration as in the feed.
Now examine the case of PFR. According to the model of
PFR, the red color dye will move downstream like a sharp
band, since there is no backmixing or diffusion to blur the
sharp boundary between the color and colorless streams. So
the detector at the exit will detect no color right after the
switch to dye solution in the feed. It will not see any colo
until the front of the color feed solution reaches the outlet
The time it will take will be a hold time, the exact time
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phases, liquid medium, gas bubbles and cell mass, they are
often treated as homogenous bioreactors. On the other hand
in addition to high cell density culture there are cases where
the bioreactor must be treated as heterogeneous. The solid
phase constitutes a large fraction of the culture volume. An
examples is the microcarrier culture. Microcarrier beads
often constitute 10-30% of the culture volume. In such cases
even cell concentration needs to be well dened, for example
whether 107per milliliter is referring to total culture volume
or liquid volume needs to be specied.
A reactor is called continuous when the feed and product
streams are continuously being fed and withdrawn from
the system. In principle, a reactor can have a continuou
recirculating ow, but no continuous feeding of nutrien
or product harvest; it is still a batch reactor. A fed-batch
bioreactor usually has intermittent feed. It may or may no
have medium withdrawal during the run.
Operating Mode of Bioreactors
Batch and Continuous Processes
Example: For instance, Yeast cells (saccharomycescereviciae) can metabolize glucose either to ethanol, or to
oxidize it to carbon dioxide, mammalian cells can convert
glucose mostly to lactate, or oxidize it to carbon dioxide
Cells in two such types of metabolism are in two differen
metabolic states. The two metabolic states are characterized
by different specic glucose consumption rates, lactate or
ethanol production and the yield coefcient for biomass, i.e
different stoichiometric ratio.
Example: For instance, a 1 l culture has 0.3 of solidmicrocarriers and 0.7 l of medium, with 109 cells in it
The cell concentration is 109 cells/L-culture or 1.43 x 10
cells/L-medium. If the glucose concentration in the culture
medium decreases from 2.10 g/L (medium) over one day
then the specic glucose consumption rate is (2.10-1.90)
g/L-medium (1.43 x 109 cells/L-medium) = 1.40 x 10-1
g/cell-hr. The specic rate calculated would have been very
different if one concentration is based on liquid volume and
the other is based on total culture volume.
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The Operating Mode of Reactors
Batch Cultures
Fedbatch Cultures
Intermittent Harvest
6 Cell Culture Bioreactors
Batch processes are simple and are widely used, especially
in the vaccine industry and in pre-production scales of rDNA
protein production. Fedbatch processes are widely used in
multi-purpose, multi-product facilities because of their
simplicity, scalability, and exibility. A variety of fedbatch
operations, ranging from very simple to highly complex andautomated, are seen in current production facilities.
In general, fedbatch processes do not deviate signicantly
from batch cultures. For both intermittent-harvest and
traditional fedbatch cultures, cells are inoculated at a
lower viable cell density in a medium that is usually very
similar in composition to a typical batch medium. Cells are
allowed to grow exponentially with essentially no external
manipulation until nutrients are somewhat depleted and cells
are approaching the stationary growth phase. At this point
for an intermittent-harvest fedbatch process, a portion of the
cells and product are harvested, and the removed culture uid
is replenished with fresh medium. This process is repeated
several times. This simple strategy is commonplace for theproduction of viral vaccines produced by persistent infection
as it allows for an extended production period. It is also used
in roller bottle processes with adherent cells.
For production of recombinant proteins and antibodies, a more
traditional fedbatch process is typically used. While cells
are still growing exponentially, but nutrients are becoming
depleted, concentrated feed medium (usually a 10-15 times
concentrated basal medium) is added either continuously
(as shown) or intermittently to supply additional nutrients
allowing for a further increase in cell concentration and thelength of the production phase. In contrast to an intermittent
harvest strategy, fresh medium is added proportionally to
cell concentration without any removal of culture broth. To
accommodate the addition of medium, a fedbatch culture
is started in a volume much lower than the full capacity of
the bioreactor (approximately 40% to 50% of the maximum
volume). The initial volume should be large enough to allow
the impeller to be submerged, but is kept as low as possible
to allow for a maximum extension of the cultivation period
Fedbatch
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In batch cultures and most fedbatch processes, lactate
ammonium, and other metabolites eventually accumulate in
the culture broth over time, inhibiting cell growth. Other
factors, such as high osmolarity and accumulation of reactive
oxygen species, are also likely to be growth inhibitory
and certainly contribute to the eventual loss of viability
and productivity. The effects of lactate and ammonia on
cultured cells are complex. Detectable changes in growthproductivity, and metabolism have all been documented
Additionally, metabolite accumulation has been found
to affect product quality. In recombinant erythropoietin
producing CHO cells, high ammonia concentration
has been reported to affect glycoform of the product
By minimizing metabolite accumulation, the duration of a
fedbatch culture can be even further extended and higher
cell and product concentrations can be achieved. Reduced
metabolite accumulation in fedbatch culture is traditionally
accomplished by limiting the availability of glucose and
glutamine using controlled feeding strategies that maintainglucose at very low levels. After extended exposure to low
glucose concentrations, cell metabolism is directed to a
more efcient state, characterized by a dramatic reduction
in the amount of lactate produced. Such a change in cel
metabolism from the normally observed high lactate
producing state to a much reduced lactate production state
is often referred to as metabolic shift. The observation
of such changes in metabolism was made more than two
decades ago, yet its application in fedbatch culture was not
realized until much later. Extending the methodology to
controlling both glucose and glutamine at low levels, bothlactate ammonium accumulations can be reduced. By
applying such a control scheme in fedbatch culture, lactate
concentration was reduced by more than three fold, and very
high cell concentrations and product titers were achieved in
hybridoma cells.
Fed-batch Culture with Metabolic Shift
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Continuous Cultures
Simple Continuous Stirred Tank Reactor
(CSTR)
Continuous Culture with a Metabolic Shift
8 Cell Culture Bioreactors
Steady state
Grow up the culture in batch mode. Then turn on
both in and out ow of medium. Cell and product
concentration reach steady state.Transient
Same as that for steady state except that cell and
product nutrient concentration uctuate.
TransientSame as CSTR, some cells are retained in bioreactor
to reach high cell concentration. Product throughput
is higher per reactor volume, but not the concentration.
Typically cell, nutrient and product concentrations
uctuate.
Steady state
Same as that for transient except that steady state is
achieved. This rarely happens.
This is the same as simple continuous culture except in the
start-up. Instead of starting from a batch culture, a fed-batch
culture with a metabolic shift is used. After cells reach a
high concentration and the metabolic shift is affected, the
culture is shifted to a continuous culture. Because no (or low)
lactate and ammonia is produced, the concentrations of cells
and products are substantially higher than in conventionalcontinuous cultures. In some cases, the cell concentration
approaches that of perfusion cultures. However, the medium
usage is substantially reduced, and the product concentration
is higher.
Continuous Culture with Cell Retention
(Recycle) Perfusion Culture
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( d(sv)( )
dt
vv s v
d x VVF t x V q x V
dt dt = = =
Cell Culture Bioreactors 9
Material Balance on Bioreactors
Material Balance Equation for Reactor
Batch Culture
Fed-batch Culture
Continuous Culture
at steady state,
vv
s v
dxV V xdt
dsV Vq x
dt
=
=
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If there exists one and only one limiting nutrient s,
meaning increasing its concentration increases the specic
growth rate, and if the specic nutrient consumption
rate q is constant, then the viable cell concentration atsteady state is dictated by the feed concentration. In
most studies, the Monod relationship was assumed
Therefore tor any given D, the cell concentration increases
with increasing feed nutrient concentration Until another
nutrient becomes limiting, or until the concentration of that
limiting nutrient becomes growth inhibitory.
Perfusion Culture
s
o
vvs
vv
q
ssDxssDxq
DxDx
dt
dx
dt
ds
)()(
0
0
==
==
==
0
v
- ( )
define dilution rate: D
( - )
vv v
s v
v v
s v o
dxV V x Fx
dt
dsV Vq x F s s
dt
F
V
dxx Dx
dt
dsq x D s s
dt
=
=
=
=
=
Continuous Culture
at steady state,
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Disposable Culture Systems
Roller Bottles
Multiple Plate Culture System (Cell
Cube and Cell Factory)
Cell Culture Bioreactors
Animal tissues (brain, egg etc.) are still used for viral
vaccine production. Automated process is available to
punch through chick eggshell to inoculate virus into
developing embryo. Virus is harvested from the tissue days
afterwards.
Roller bottles are cylindrical screw-capped bottles mostly
made of disposable plastic; reusable glass ones are still used
occasionally. Each bottle is typically about 1 to 1.5 liters in
total volume. Typically, a bottle is lled with 0.1 to 0.3 liters
of culture medium for cell cultivation. A stack of bottles is
placed on a roller, the bottles rotate on the roller rack at 1 to 4
rpm and are incubated in an incubator or an incubation room
Roller bottles are used for the cultivation of both suspensioncells and anchored cells. However, for suspension cells
it is usually more convenient to grow the cells in a stirred
vessel, and roller bottles are only used in small scale when
convenience dictates this selection of cultivation methods
Anchorage-dependent or anchorage-preferred cells are
frequently cultivated in roller bottles, both in the laboratory
and in manufacturing of biologics. These cells attach to the
inner wall of the bottle. Initially, they cover only part of the
surface of the inner wall after inoculation. The cell laye
is bathed in a liquid medium and is alternately exposed to
medium and gaseous nutrient as the bottle rotates. As cells
grow, they cover the entire surface and reach conuenceNormal diploid cells then reach contact inhibition and stop
growing until they are detached by protease treatment and
inoculated into a larger number of roller bottles. Many
continuous cell lines or tumorous (transformed) cells can
continue to grow to form multiple layers of cells after
reaching conuence, if sufcient nutrients are provided
Some variations of roller bottles are available to increase the
cell growth surface area in a bottle. Spiral lms and wafes
on the inner wall of roller bottles have been introduced and
are sometimes used.
For small-scale operations, roller bottles provide many
advantages for the cultivation of adherent cells. It is relatively
inexpensive to set up. Often it allows for a rapid change
of throughput in response to need. Furthermore, replacing
medium from cell growth medium to one designed for
product formation is rather straightforward. It is particularly
useful in the case that the serum-containing medium needs
to be replaced by a serum-free or protein detachment and
expansion, medium exchange and product harvest, requires
extensive well-trained labor to ensure a high success rate
Tissue culture and disposable cell
culture systems
Tissue Culture
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Bag System
2 Cell Culture Bioreactors
Each batch of manufacturing can easily involve hundreds or
even thousands of bottles, and the large number of manua
steps involved makes the risk of microbial contamination
rather high. Despite these signicant drawbacks, roller
bottles are still widely used in the production of viral vaccines
often because the products involved have been approved
by regulatory agencies before more recent cultivation
technologies became more robust and widely accepted. In
some cases, roller bottles were selected over other bioreactors
because the process was easy to implement compared to
those based on other bioreactors. One notable example is
the production of erythroprotein (EPO) using recombinant
Chinese hamster ovary cells. Recent improvements include
developing a robotic handling system for handling a large
number of roller bottles to make industrial scale production.
free medium for the production of secreted proteins or
viruses. The transparent glass or plastic wall allows visua
or microscopic examination of the culture status. Microbia
contaminated bottles can be readily spotted and discarded
before they pool together with contaminated ones. However
the drawbacks of roller bottles are numerous for large-scaleproduction of biologics. On-line environmental monitoring
and control is virtually impossible, or at least impractical
The aspectic bottle handling for inoculation, trypsinization
for cell
The Roller bottle system has a couple of drawbacks. The
medium to cell ratio is unfavorable; it is not easy to be
operated in a continuous or fed-batch mode. Furthermore, i
is often desirable to have a large culture volume contained
in a reactor For suspension cells the alternative to
overcome these is is either using bags for a disposable
system or employing a fermentor. For adherent cells, the
disposable solution is to contain a large at surface in a
single container.
Blood bags are used extensively in cellular therapy
Wave bioreactor system
Blood bags have long been used in culture of blood
cells especially for adoptive cell therapy culturing NK
or MTL cells. Those are small bags just sitting insideCO2 incubators. Newer arrivals come with a tilting
platform to provide some mixing.
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Many cell lines used in the production of vaccines and
other conventional biologies are suspension cells. While
disposable systems minimize in plant validations required
for cGMP operations they are limited in scale. For viralvaccines, for which the number of cells required for
producing a dose is relatively small, a disposable system
provides some advantages. For antibody products, the
use of disposable systems may be limited to inoculum
preparation. Large scale operations thus still resorts to
fermentors or other bioreactors. The majority of cells used
for rDNA protein production are suspension cells, they
are simply suspended in the bioreactors. For cells grown
adherently cell supports are needed to provide adherent
surface while the reactor provides a mechanism to keep the
cell support in suspension. In some cases cell support is
also used for suspension cells. The main advantage is that
cells can be kept in the reactor while the medicine is being
perfused or exchanged. These systems are hardly used
for suspension cells any more as other simple methods of
retaining cells prevail.
Most normal diploid cell strains or primary cells are
anchorage-dependent. For large scale operations
either surface cultures or microcarrier cultures are usedConventional microcarriers are suitable for normal diploid
cells which require attachment and spreading. Macroporous
microcarriers can be used for a wide variety of continuous
cell lines, by may not be suitable for large normal diploid
broblasts.
The use of microcarriers for cell culture was rst
demonstrated by Van Wezel (1967). The basic concept is
to allow cells to attach to the surface of small suspendedbeads so that conventional stirred tank bioreactors can be
used for cell cultivation. To ease suspending these cell
laden microcarriers, their diameter and density are usually in
the range of 100300 m and 1.021.05 g/cm3 respectively
This diameter range also gives a good growth surface
area per reactor volume. Even at a moderate microcarrier
concentration, in the range of 815% culture volume being
occupied microcarriers, a signicantly larger surface area per
reactor volume can be achieved than that in roller bottles.
Cell Support Systems
Suspension Culture vs Adherent
Cultures
Microcarriers
Solid Microcarriers
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Although even smaller microcarriers, less than 100 m in
diameter, can provide even more surface area, they are not
used often. Most anchorage-dependent cells do not develop
their normal morphology, and in many cases, do not multiply
well on surfaces with an excessively high curvature. These
cells do not grow well on small microcarriers. On the other
hand, some cells, especially transformed cells, do multiply
well even though they are attached to small microcarriers
and do not develop a well spread-out morphology. These
cells, after attaching to the small microcarriers, agglomerate
to form aggregates and continue to grow to high density. The
small microcarriers, usually with a diameter of about 50 m
serve as nuclei for the initiation of aggregate formation.
Desired characteristics of microcarriersDensity ~1.02 g/cm3 Only slightly higher than water for easy
suspension and settingDiameter 150-200 m Should be the smallest possible and yet
allow for cell spreading
Porosity From solid to nearly 90% Prefer solid, for use as inoculum bead to
bead transfer
Surface Properties ECM coating, slightlypositively charged Positive charge enhances initial attachment
The microcarriers can be made of many different materials
including dextran, gelatin, polystyrene, glass and cellulose
Not all of these are commercially available. In general, in
addition to having a wettable surface, the backbone materials
of microcarriers often need to be chemically collagen
Polystyrene microcarriers have also been coated with collagen
or other adhesion molecules for better performance.
An advantage for the industrial-scale culture of anchorage-
dependent cells is the ease of separating cells from culture
medium. Many microcarrier cultures require medium
exchanges during cultivation to remove accumulating
lactate, ammonia and other metabolites and to replenish
nutrients. In many cases, the cell-laden microcarriers are
simply allowed to settle and the spent medium be withdrawn
and replenished. In large-scale operations, continuous or
semi-continuous perfusion is more frequently used. This can
be accomplished by withdrawing medium through a coarse
screen which allows medium to pass through but retains
microcarriers in the reactor. A large number of cells have
been grown on microcarriers, including broblasts, kidney
epithelial, hepatocyte, neuroblastoma, and endotheliacells from various species. Overall, microcarrier culture
is a convenient laboratory and research tool, and it has the
advantage of being amenable to large-scale production if a
large quantity of product is needed.modied to improve cel
attachment. One of the most widely used microcarriers, the
dextran based ones, are derivatized with charged molecules
or denatured collagen.
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Macroporous Microcarriers
Cell Culture Bioreactors 5
A variant of microcarrier culture, macroporous microcarriers
contain large internal pores, tens of micrometers across. The
void space inside allows cells to be cultivated not only on
the external surface but also internally. Cells in the interio
are less susceptible to mechanical damage caused by
agitation and gas sparging. Of course, being in the interior
of microcarriers, cells are more likely to be subject to oxygen
limitation due to the long diffusional distance, especially sincemost macroporous microcarriers have a larger diameter (500
m to a couple millimeters). Macroporous microcarriers are
made of different materials, including gelatin, collagen and
plastic. Many cell lines have been successfully grown on
macroporous microcarriers including Vero, HepG2, CHO
and 293 cells. The nal cell concentration achieved tends to
be higher than that obtained with an equivalent volumetric
concentration of conventional microcarriers. But in some
cases the growth kinetics are slower because the penetration
of cells into the interior may be slowed or even retarded by
restrictive opening of these pores
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Cell Aggregates
6 Cell Culture Bioreactors
Microsphere Induce Cell Aggregates
Agarose
Some transformed cells which normally attach to or
spread and grow on a surface can form aggregates when
cultivated in shaker asks or in stirred vessels. Differen
methods have been used to induce aggregate formation for
cells. Aggregation can be promoted by manipulating the
calcium concentration in conjunction with the agitation rate
Aggregate cultures have advantages similar to microcarrier
cultures. They can be cultivated in conventional stirred tankreactors with environmental control. They can be allowed
to settle relatively rapidly by stopping agitation. Permitting
easy medium replenishment.
For many cell types, a limitation on the use of the aggregate
culture is that the rate of aggregate formation is slow. One
way to induce aggregate formation is to add microspheres
to cell suspension to allow for rapid agglomeration to the
microspheres (9). If the aggregate diameters become
too large, necrotic centers can be formed due to transport
limitation. The aggregate size may inuence, i.e., to the vira
infection kinetic and yield for vaccine formation (10). Many
cell lines, including BHK, CHO, 293 and ST cells, have been
cultivated as aggregates with sizes ranging between 90 and
400 m. ,without the formation of necrotic centers (9, 11).
Agarose entrapment of cells is usually accomplished by
passing cell-agarose suspension through a small oriface
opening into an air jet stream. The droplets of agarose
containing entrapped cells are collected in a chilled oil phase
to allow the agarose to gel. Alternatively, the cell-agarose
suspension is allowed to drop into the center of a fast rotating
disk. The centrifugal spinning force causes the droplets to
form and be dispensed outside the disk. The agarose beads
tend to be rather large, at least hundreds of micrometers
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in diameter. This causes oxygen transfer limitation at a
high cell concentration. Further, the agarose beads do no
have sufcient mechanical strength to sustain mechanica
optimization even in a moderately small scale (tens of liters)
bioreactor.
Another method of cell cultivation that enables the use of a
stirred tank reactor is cell entrapment. This technique entails
entrapping cells in polymeric matrix to form spheres. The
spheres are then either coated with another polymeric lm to
control the crossing of molecules according to their size
commonly referred to as microencapsulationor they are
cultivated as they are. These beads are suspended in medium
to allow cell growth inside. One of the polymers most used
for cell entrapment is calcium alginate. Cell entrapmen
in calcium alginate is accomplished by preparing a cell
suspension in sodium alginate and adding it, in a dropwise
fashion, into a solution of calcium chloride. Calcium cross
links alginate, instantaneously forming beads containingentrapped cells. Alginate may be coated with polylysine
for increased mechanical and chemical stability, but such
treatment decreases the molecular weight cut-off and prohibits
large-molecular weight proteins in the medium from reaching
the cells. To prepare hollow spheres, the alginate gel inside a
bead coated with a polylysine is liqueed through treatmen
with a calcium chelator such as EDTA or citrate.
The diameter of these beads is often in the order of millimeters
The mechanical strength of the gel which constitutes the
beads is relatively weak. Large-scale application using such
techniques is not easy. On the other hand, the mirocapsule
provides a means of immunoisolation of transplanted cells
or tissues (sometimes referred to as articial cells) and
could be suitable for some tissue engineering applications
Cell entrapment has been applied to hybridomas and smal
clusters of adherent cells. It has also found application in the
cultivation of differentiated cells, such as insulin-secreting
cells. For tissue engineering applications using differentiated
cells, the enclosing membrane around the cells and the
hydrogel must be biocompatible. The membrane used in
microencapsulation is semipermeable to allow sufcien
diffusion and transport of low-molecular weight moleculesimportant for cell survival, such as oxygen, nutrient and
metabolites. For optimum transport across the membrane
the microcapsules should have a uniform wall thickness
Ideally, the semipermeable membrane prevents the passage
of high-molecular weight proteins, such as immunoglobulins
to allow for product to accumulate inside the microcapsule.
Since the cells are protected inside the capsules, they
are protected from hydrodynamic damage. The culture
Microencapsulation
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can be stirred at faster rates than conventional culture,
which improves nutrient, metabolite and gas exchange.
Entrapped cells may be cultured in stirred tank bioreactors,
xed-bed and uidized-bed reactors. An airlift reactor
could be used as well, provided that the beads are small
and not signicantly denser than the medium.
Stirred tanks, or conventional fermentors, have been widely
used for culturing suspension cells since the 1960s. With
the use of microcarriers, conventional or macroporous ones
adherent cells can also be cultured in a stirred tank. For cel
entrapment in hydrogel, the use of stirred tanks is limited
to laboratory scale, as the preparation of a large quantity of
cell-entrapped beads can be a daunting task, and the risk
of mechanical damage caused by agitation increases with
scale.
The basic conguration of stirred tank bioreactors for
mammalian cell culture is similar to that of microbia
fermentors. A major difference is that the aspect ratio (the
height to the diameter ratio) is usually smaller in mammalian
cell culture bioreactors. The power input per unit volume
of bioreactor is also substantially lower in mammalian cel
culture bioreactors. While the Rushton type impeller i
the norm in microbial fermentors, mammalian cell culture
fermentors mostly employ marine type impellers. As in
microbial fermentors, an axial impeller is often mounted a
the top of the bioreactor to drive the liquid downward in
large-scale cell culture bioreactors. These differences reecthe different purposes of agitation in microbial fermentation
and in cell culture. In microbial fermentation, agitation is
needed at a higher power input to disperse air bubbles and to
increase oxygen transfer efciency, whereas in mammalian
cell culture, the primary purpose of agitation is to keep cells
or microcarriers suspended in nutrient medium relatively
uniformly. In general, the mixing time in a mammalian
cell culture bioreactor is substantially higher than that in a
microbial fermentor with similar scale. The oxygen transfe
capacity in a cell culture bioreactor is also substantially lower
than that in a microbial fermentor. However, the typica
oxygen demand in a mammalian cell culture is also much
lower (10 to 50 times) than that in microbial fermentation.
A variant of the bubble column reactor with interna
circulation loops is used to improve the performance of
conventional bubble column reactors. In airlift column
reactors, internal liquid circulation is achieved by sparging
only part of the reactor with air. The sparged section has a
Cell Culture Bioreactors
Simple Stirred Tank Bioreactor
Airlift Bioreactor
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lower effective density than the bubble-free section, and the
difference in hydrostatic pressure between the two sections
induces the liquid circulation upward in the additional bene
is the low energy requirement compared with stirred-tank
reactors. Although simple in construction, sound design is
critical for optimal hydrodynamic behavior. Nevertheless
the ow in bubble column reactors is relatively well dened
compared with that in stirred tank reactors.
Airlift bioreactors for cell cultivation are considered to be
low-shear devices because there is no mechanical agitation
Also, the direct air sparging may not cause excessive cell
damage, at least under normal cultivation conditions. The
ow regime depends on the sparger used and the ow rate.
Various type of spargers are used to provide different bubble
size.
Airlift reactors have been used successfully with suspension
cultures of BHK 21, human lyphoblastoid, CHO, hybridomas
and insect cells.sparged section (riser) and downward in
the bubble-free section (downcomer). The loop has theadvantages of permitting high efciency mass transfer and
improving the ow and mixing properties in the vessel.
These reactors are characterized by low capital costs
mainly because of their simple mechanical conguration
Considerable backmixing in both gas and liquid phases, high
pressure drop and bubble coalescence can be disadvantageous
in some cases.
The membrane stirred tank was developed by Professor
Jrgen Lehmann in the 1980s. It uses long microporous
polypeopylene tubing wrapped around rotating rods. By
adjusting the air pressure in the propylene tubing, the
micropore expands to allow gas to be in direct contact with
medium while not bursting to become gas spargers. The
rotation of those tubings also provides gentle agitation to
microcarriers or suspended cells. Even at a high serum
concentration, foaming can be avoided.
Membrane Stirred Tank
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Spin Filter Stirred Tank
20 Cell Culture Bioreactors
The rotating wire cage bioreactor, in contrast to a
microltration device, does not rely on ltration to achieve
cell retention in the bioreactor. The centerpiece of this
bioreactor is the wire cage, which is often mounted on
the agitation shaft. At times other designs may be seen in
which the wire cage is mounted to a shaft rotated by a top
motor. The bottom of the cage is solid wile the side is made
of wire screen with an opening ranging from 25 to 60 m.The average diameter of cells is approximately 10 to 15 m
Typically, fresh medium is added continuously outside the
draft tube and the culture uid is withdrawn from inside the
cage at the same rate.
Under certain operating conditions, the cell concentration
inside the wire cage is lower that that outside, thus
achieving cell retention in the bioreactor. The attainable cel
concentration in such a bioreactor has been reported to be as
high as ten times of that in a typical bioreactor. Since under
optimal conditions, the device does not appear to act as a lter
and no cake is formed. The retention of the cells does noappear to be due to centrifugal force exerted by the rotating
motion of the cage, because the terminal velocity of the cells
due to centrifugal force is two to three orders of magnitude
lower than that of the uid velocity across the screen due
to perfusion. The electrostatic effect is also unlikely to be
responsible, since the ionic strength of the culture uid is
relatively high and the thickness of the Debye layer is only
in the order of 1 nm.
If we assume that the uid owing through the wire cage
carries only a fraction, , of particles from the outside regioninto the wire cage, then the material balance equation can be
written as
( / )
( / )
O O O O
i i i i i
V dC dt F C C V
V dC dt C V FC
= +
= The discharge factor, , is a measure of the effectiveness
of particle retention. A discharge factor of 1 represents no
retention.
The values of the discharge factor , under different operatingconditions were investigated using polystyrene particles of
the same diameter and density as cells. It was found tha
the discharge factor was affected by the agitation rate: it
decreased as the agitation rate increased from 50 to 100 rpm
However, further increase in the agitation rate increased
the value of . There thus appears to an optimal range o
agitation rate for cell retention. The fact that the discharge
factor increases after the agitation rate increases beyond an
optimal range indicates that centrifugation may not be the
dominating mechanism for cell retention.
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In addition to its use for simple suspension cultures, the
rotating wire cage bioreactor has also been used in aggregate
or microcarrier cultures. In those cases the cell retention is
relatively straight forward; the opening of the pores on the
screen is smaller than the size of most aggregates. Since the
diameter of the microcarriers or the aggregates is in the range
of hundreds of micrometers, the underlying mechanism for
particle retention is mostly likely centrifugation. But the
most intriguing effect of a rotating wire cage is still its
ability to retain single suspension cells. It is likely that the
retention is caused by a uid mechanical effect, maybe one
similar to the behavior of particles of low Reynolds number
near the wall in a Poiseulle ow or a laminar boundary
layer ow along a at plate. However, the ow pattern
around the cage is complicated: a rotating ow due to the
rotation of the cage, an upward ow cause by agitation and a
perpendicular ow inward to the cage cause by perfusion. A
wire cage bioreactor is very effective for large scale animal
cell cultivation once the optimal operating conditions are
dened. However, because of our lack of understanding of
its mechanism, the design and operation of such a system isvery difcult and the effectiveness of the magnitude of the
discharge factor () under different operating conditions is
unpredictable.
Note: More recent application of centrofugal lter installs
spin lter outside the bioreactor. Many such spin lters are
operated at very high agitation rates. The mechanism of cel
separation is certainly different from the one described in
this section.
A vibromixer uses a perforated disk as the mixing
mechanism instead of conventional impeller. The disk
vibrates in the vertical direction at a very high frequency
causing liquid to circulate through the perforated holes
and provide mixing. It was used in the 1960s for the
cultivation of suspension cells and virus production.
Its use in cell cultivation has diminished in the past
couple of decades. However, in some cases it is used to
keep concentrated microcarriers in suspension for cell
detachment during the trypsinization step.
Vibromixer
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Membrane Bioreactor
Hollow Fiber Bioreactor
22 Cell Culture Bioreactors
Fluidized bed has long been used in chemical catalysis.
It is also used in adsorption (chromatographic) column
in bioseparation. Typically the uid stream (often gas
in catalysis) that enters through a ow distributor at the
bottom at velocity is sufcient to blow up or suspend
the solid catalyst particles. The reactants enter the catalyst
and products diffuse out into the uid to be carried outthrough the top of the reactor where a separator prevents
any particles from being blown out. The main advantage
of a uidized bed is the high heat transfer between the
high velocity uid and the catalyst surface. When applied
to cells or microcarrers directly, the density difference
between solid phase and liquid phase is too small,
making particle retention difcult. In the 1980s collagen
macroporous beads were used in commercial uidized
beds offered by Verax. The carriers need to be weighted by
inclusion of iron particles to allow for particle retention at
the ow rate that is needed to supply enough oxygen for the
cells.
The use of hollow ber reactors for cultivation of mammalian
cells dates back to the early 1970s. A hollow ber system
can be used for anchorage-dependent and suspension cells
It consists of a bundle of capillary bers sealed inside a
cylindrical tube. The basic conguration is rather similar
to the hollow ber cartridge used in kidney dialysis. The
hollow-ber, in most cases, consists of supporting polymeric
porous materials for mechanical strength and a thin layer of
membrane which provides selective passage of molecules
depending on their size. In most cases, an ultraltration
membrane is used. The molecular weight cut-off (MWCO)
of the membrane differs according to applications, ranging
from a few thousand to a hundred thousand daltons. The
ultraltration membrane prevents free diffusion of secreted
product molecules from passing through the membrane and
allows them to accumulate in the extracapillary space to a high
concentration. The culture media is pumped usually through
the ber lumen, and cells grow in the extracapillary space
or the shell side. Supply of low-molecular weight nutrientsto the cells and the removal of waste products occur by
diffusive transport across the membrane between the lumen
and the shell spaces. Although the use of microltration
hollow ber membranes for cell culture is infrequent, i
does nd application in various research uses for studying
metabolism and for the cultivation of anchorage-dependent
or highly aggregated cells for which a convective ow of
medium through the extracapillary space to bathe cells in
medium is desired.
Fluidized Bed Bioreactor
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Multiple Membrane Plate BioreactorScaling-up of a hollow-ber system eventually is limited
by the ability to extend the axial length of the ber withou
incoming oxygen transfer limitation. The capacity of the
pump and the mechanical strength of the membrane limit the
maximum operable ow rate. Scale-up in cartridge diameter
eventually runs into ow distribution problems among
thousands of bers. Ideally, one can put different bers
some for gas ow for oxygen transfer, others for medium
ow. However, this approach poses a major challenge in
manufacturing. One approach (taken from the 1980s) was
to use a multiple at membrane. By keeping the heigh
(i.e. the clearance between membranes) of the cell chamber
small, one can avoid diffusion limitation. This seemingly
versatile reactor also suffers from practical manufacturing
complexity of designing a satisfactory sealing mechanism
that is needed for a long-term aseptic operation.
The ceramic system is a cylinder of porous ceramic with
square channels passing through the cylinder. Cells are
inoculated into the channels and either adhere to the surface
or are entrapped in the pores of the ceramic. Medium is
passed through the channels to provide nutrients and to
remove the metabolites. In a ceramic system, the cells
are directly bathed in the recirculating medium, whereas
in a hollow-ber system, cells populating the shell side
are exposed to a slow stream of permeate. The ceramicbioreactor, to some extent, can be considered a variant o
the hollow ber system. It consists of a cylindrical ceramic
core with many channels passing longitudinally through the
ceramic material. Cells inoculated in the channels adhere to
the material or become entrapped in the pores of the ceramic
As in a hollow ber system, ceramic reactors are supported
by medium perfusion loops. Cell culture medium is pumped
through the longitudinal channels in the ceramic cores from
a medium reservoir in a recirculating loop conguration
Fresh medium is fed into the system, and harvested culture
uid is removed to the medium reservoir. Unlike the hollow
ber system, there is no membrane separating the cells and
bulk medium. Product is secreted directly into the bulk
medium. Essentially, the ceramic bioreactor can be used
to conveniently replace a large number of roller bottles
As in hollow ber systems, oxygen concentration gradient
develops along the axial direction and limits the length, i.e.
the scale of the reactor.
Ceramic System