Cell Culture Bioreactors

Basic Types of Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Stirred Tank (Well Mixed) vs. Tubular 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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Batch and Continuous Processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
The Operating Mode of Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Batch Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Fedbatch Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Material Balance on Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Material Balance Equation for Reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Tissue culture and disposable cell culture systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Tissue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Disposable Culture Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Multiple Plate Culture System (Cell Cube and Cell Factory) . . . . . . . . . . . . . . . . . 12
Cell Support Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Suspension Culture vs Adherent Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Microcarriers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Cell Aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Microsphere Induce Cell Aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Agarose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Microencapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Cell Culture Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
Simple Stirred Tank Bioreactor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Airlift Bioreactor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Membrane Stirred Tank. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Spin Filter Stirred Tank. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Vibromixer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Fluidized Bed Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Membrane Bioreactor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Basic Types of Bioreactors

Mammalian cell bioreactors are generally categorized
similarly to chemical reactors according to their mixing
characteristics. It is instructive to review two ideal reactors:
well-mixed stirred tank and plug-flow (tubular) reactor. In
an ideal well-mixed bioreactor, the mixing is assumed to be
intense enough that the fluid is homogeneous through the
reactor. The mathematical description of ideal continuous
flow stirred tank reactor is described by the following first-
order differential equation.

d (VC A )
= FC
i Ai − Fo C Ao + rAV
dt
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 flow 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 flow bypass and

no shunt of substrate from inlet to outlet, no dead zones or
clumps of undissolved solid substrate floating 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 fluid in the reactor.

The basic model for the tubular reactor (such as hollow fiber
and ceramic systems to be described later in this chapter)
specifies that the liquid phase moves as a plug-flow, 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-flow reactor is the
following:
∂C A ∂C A
= −v Z + rA
∂t ∂z
where vz is the linear velocity in the z direction along the
flow and S is the cross-sectional area. Note that we assume
there is no liquid dispersion or back mixing. All elements in
the fluid move at the same velocity. At steady state (i.e., cell
concentration and cellular activities at a given position are
not changing with time), the equation becomes
∂c A rA ⋅ S
=
∂z F
which describes changes of concentration of A along the
direction of fluid flow. 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 flow
tubular reactors are situations that can be approximated in
small-scale laboratory conditions. The conditions in larger
scale process reactors deviate significantly from these ideal
conditions.

In a well-mixed bioreactor, there are no concentration

gradients in either the gas or the liquid phase. In other
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 flow reactor is
assumed to be under conditions of total segregation. Most
bioreactor systems have a mixing pattern between the two
extremes and are under partially segregated conditions.

  Cell Culture Bioreactors

 In general, laboratory and small pilot plant bioreactors are used
for process development and optimization. The fluid mixing
characteristics are rather sensitive to the scale of the reactor.
Furthermore, plug-flow bioreactors are intrinsically more
difficult to scale up than mixing vessels, as the concentration
gradient of essential nutrients, oxygen in particular, will
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.

Stirred Tank (Well Mixed) vs. Tubular

Reactor (Plug Flow)
The distinction between a well mixed continuous stirred
tank reactor (CSTR) and plug flow 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 that
are equal in volumetric flow rate, the volume of the reactor is
thus constant. For the case that the feed stream is colorless,
but 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 will
be seen immediately in the effluent stream, since the color
is distributed instantaneously everywhere including the fluid
that is taken out in the effluent 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 will
take longer than the time needed to flow 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 (3t0) 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 color
until the “front” of the color feed solution reaches the outlet.
The time it will take will be a “hold time”, the exact time

Cell Culture Bioreactors 

 of flow a reactor volume into the reactor to displace all the
original clear solution in the reactor. As soon as the color
comes out in the outlet, the concentration will be equal as
in the feed.

If the reactor is not idealized, obviously the pattern of the

dye concentration will be different. For a tubular reactor, the
“front” may not be as sharp, rather the appearance in the exit
will be more gradual. Similarly in a stirred tank, there will
be deviation to the perfect mixing curve.

In more severe cases, a reactor may be compartmentalized

or segregated. Some channeling may occur to have some
portion of the feed stream passing right through, or some
portions of the reactor hardly see any feed stream.

Segregated Bioreactors (Dead

Zone Present) Compartmentalized
Bioreactors
Concentrations in different compartments may be different
Most reactors are not ideally all mixed or plug-flow;
segregated zone is not a completely dead zone

Implication When Growth or Reaction

Occurs in the Reactor
Flow and mixing behavior may have a profound effect on
the reaction or growth. Consider a nutrient stream entering
the reactor. If the reactor is a PFR, the cells in the upstream
will have abundant nutrient. As the fluid moves downstream
more nutrients get consumed and their concentration
decreases. The cells downstream may not have enough
nutrients or face starvation. One way to solve the problem
is of course to increase the supply rate by using a higher
nutrient concentration in the feed or by operating at a higher
flow rate. But there are limits on both nutrient concentration
and flow rate. Eventually the size of the reactor will be
restricted.

In a CSTR model, all cells in the reactor see the same

environment. The nutrients that feed into the reactor will be
distributed uniformly everywhere, either all have abundant
or suboptimal levels.

Homogenous Reactor vs.

Heterogeneous Reactor
Heterogeneous reactor—with a solid phase, e.g., microcarriers
in stirred tank, tubular reactor packed with foam. A typical
tissue has a cell concentration of about 5X108/ml. Unless a
rector have a very high cell concentration in the middle of 107/
ml, cell mass is only a small fraction of the culture volume.
So, even though almost all cell culture reactors have all three

  Cell Culture Bioreactors

 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 defined, for example,
whether 107 per milliliter is referring to total culture volume
or liquid volume needs to be specified.

Operating Mode of Bioreactors

Batch and Continuous Processes 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 continuous
recirculating flow, but no continuous feeding of nutrient
or product harvest; it is still a batch reactor. A fed-batch
bioreactor usually has intermittent feed. It may or may not
have medium withdrawal during the run.

Example: For instance, Yeast cells (saccharomyces

cereviciae) 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 different
metabolic states. The two metabolic states are characterized
by different specific glucose consumption rates, lactate or
ethanol production and the yield coefficient for biomass, i.e.
different stoichiometric ratio.

Example: For instance, a 1 l culture has 0.3 of solid

microcarriers and 0.7 l of medium, with 109 cells in it.
The cell concentration is 109 cells/L-culture or 1.43 x 109
cells/L-medium. If the glucose concentration in the culture
medium decreases from 2.10 g/L (medium) over one day,
then the specific glucose consumption rate is (2.10-1.90)
g/L-medium ÷ (1.43 x 109 cells/L-medium) = 1.40 x 10-10
g/cell-hr. The specific rate calculated would have been very
different if one concentration is based on liquid volume and
the other is based on total culture volume.

Cell Culture Bioreactors 

The Operating Mode of Reactors

Batch Cultures 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 flexibility. A variety of fedbatch
operations, ranging from very simple to highly complex and
automated, are seen in current production facilities.

Fedbatch Cultures

Intermittent Harvest In general, fedbatch processes do not deviate significantly

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 fluid
is replenished with fresh medium. This process is repeated
several times. This simple strategy is commonplace for the
production 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.

Fedbatch
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 the
length 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.

  Cell Culture Bioreactors

Fed-batch Culture with Metabolic Shift
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 growth,
productivity, 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 maintain
glucose at very low levels. After extended exposure to low
glucose concentrations, cell metabolism is directed to a
more efficient state, characterized by a dramatic reduction
in the amount of lactate produced. Such a change in cell
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, both
lactate 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.

Cell Culture Bioreactors 

Continuous Cultures

Simple Continuous Stirred Tank Reactor

(CSTR)
 Steady state
 Grow up the culture in batch mode. Then turn on
both in and out flow of medium. Cell and product
concentration reach steady state.
 Transient
 Same as that for steady state except that cell and
product nutrient concentration fluctuate.

Continuous Culture with Cell Retention

(Recycle) – Perfusion Culture  Transient
 Same 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
fluctuate.
 Steady state
 Same as that for transient except that steady state is
achieved. This rarely happens.

Continuous Culture with a Metabolic Shift

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 conventional
continuous 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.

  Cell Culture Bioreactors

Material Balance on Bioreactors
Material Balance Equation for Reactor

Batch Culture
dxv
V = V µ xv
dt
ds
V = −Vqs xv
dt

Fed-batch Culture

V d ( xvV d(sv)
= F (t ) = µ xvV = −qs xvV
dt dt dt

Continuous Culture

at steady state,

Cell Culture Bioreactors 

Continuous Culture

dxv
V = V µ xv − Fxv
dt
ds If there exists one and only one limiting nutrient s,
V = −Vqs xv - F ( s0 − s )
dt meaning increasing its concentration increases the specific
F growth rate, and if the specific nutrient consumption
define dilution rate: D = rate q is constant, then the viable cell concentration at
V
steady state is dictated by the feed concentration. In
dxv most studies, the Monod relationship was assumed.
= µ xv − Dxv
dt
ds Therefore tor any given D, the cell concentration increases
= − qs xv − D ( so - s )
dt with increasing feed nutrient concentration Until another
nutrient becomes limiting, or until the concentration of that
at steady state, limiting nutrient becomes growth inhibitory.

ds dx
= =0
dt dt
Dxv = µxv ⇒ D = µ
D(so − s )
qs xv = D(s0 − s ) → xv =
qs

Perfusion Culture

10  Cell Culture Bioreactors

Tissue culture and disposable cell
culture systems
Tissue Culture 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.

Disposable Culture Systems

Roller Bottles 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 filled 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 suspension
cells 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
Multiple Plate Culture System (Cell surface of the inner wall after inoculation. The cell layer
Cube and Cell Factory) 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 confluence.
Normal 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 confluence, if sufficient nutrients are provided.
Some variations of roller bottles are available to increase the
cell growth surface area in a bottle. Spiral films and waffles
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.

Cell Culture Bioreactors 11

 Each batch of manufacturing can easily involve hundreds or
even thousands of bottles, and the large number of manual
steps involved makes the risk of microbial contamination
rather high. Despite these significant 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 visual
or microscopic examination of the culture status. Microbial
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-scale
production 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, it
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 flat surface in a
single container.

Bag System
 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 inside
CO2 incubators. Newer arrivals come with a tilting
platform to provide some mixing.

12  Cell Culture Bioreactors

Cell Support Systems
Suspension Culture vs Adherent
Cultures
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 viral
vaccines, 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.

Microcarriers
Most normal diploid cell strains or primary cells are
anchorage-dependent. For large scale operations,
either surface cultures or microcarrier cultures are used.
Conventional 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
fibroblasts.

Solid Microcarriers
The use of microcarriers for cell culture was first
demonstrated by Van Wezel (1967). The basic concept is
to allow cells to attach to the surface of small suspended
beads 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 100–300 μm and 1.02–1.05 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 8–15% culture volume being
occupied microcarriers, a significantly larger surface area per
reactor volume can be achieved than that in roller bottles.

Cell Culture Bioreactors 13

 Although even smaller microcarriers, less than 100 μm 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 μm,
serve as nuclei for the initiation of aggregate formation.
Desired characteristics of microcarriers
Density ~1.02 g/cm3 Only slightly higher than water for easy
suspension and setting
Diameter 150-200 μm 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, slightly Positive charge enhances initial attachment
positively charged
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 fibroblasts, kidney,
epithelial, hepatocyte, neuroblastoma, and endothelial
cells 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.modified to improve cell
attachment. One of the most widely used microcarriers, the
dextran based ones, are derivatized with charged molecules
or denatured collagen.

14  Cell Culture Bioreactors

Macroporous Microcarriers
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 interior
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 since
most macroporous microcarriers have a larger diameter (500
μm 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 final 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

Cell Culture Bioreactors 15

Cell Aggregates
Some transformed cells which normally attach to or
spread and grow on a surface can form aggregates when
cultivated in shaker flasks or in stirred vessels. Different
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 tank
reactors with environmental control. They can be allowed
to settle relatively rapidly by stopping agitation. Permitting
easy medium replenishment.

Microsphere Induce Cell Aggregates

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 influence, i.e., to the viral
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 μm. ,without the formation of necrotic centers (9, 11).

Agarose 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

16  Cell Culture Bioreactors

 in diameter. This causes oxygen transfer limitation at a
high cell concentration. Further, the agarose beads do not
have sufficient mechanical strength to sustain mechanical
optimization even in a moderately small scale (tens of liters)
bioreactor.

Microencapsulation
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 film to
control the crossing of molecules according to their size—
commonly referred to as microencapsulation—or 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 entrapment
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 containing
entrapped 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 liquefied through treatment
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 “artificial” cells) and
could be suitable for some tissue engineering applications.
Cell entrapment has been applied to hybridomas and small
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 sufficient
diffusion and transport of low-molecular weight molecules
important 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

Cell Culture Bioreactors 17

 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,
fixed-bed and fluidized-bed reactors. An airlift reactor
could be used as well, provided that the beads are small
and not significantly denser than the medium.

Cell Culture Bioreactors

Simple Stirred Tank Bioreactor
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 cell
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 configuration of stirred tank bioreactors for

mammalian cell culture is similar to that of microbial
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 cell
culture bioreactors. While the Rushton type impeller is
the norm in microbial fermentors, mammalian cell culture
fermentors mostly employ marine type impellers. As in
microbial fermentors, an axial impeller is often mounted at
the top of the bioreactor to drive the liquid downward in
large-scale cell culture bioreactors. These differences reflect
the 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 efficiency, 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 transfer
capacity in a cell culture bioreactor is also substantially lower
than that in a microbial fermentor. However, the typical
oxygen demand in a mammalian cell culture is also much
lower (10 to 50 times) than that in microbial fermentation.

Airlift Bioreactor
A variant of the bubble column reactor with internal
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

18  Cell Culture Bioreactors

 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 benefit
is the low energy requirement compared with stirred-tank
reactors. Although simple in construction, sound design is
critical for optimal hydrodynamic behavior. Nevertheless,
the flow in bubble column reactors is relatively well defined,
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
flow regime depends on the sparger used and the flow 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 the
advantages of permitting high efficiency mass transfer and
improving the flow and mixing properties in the vessel.
These reactors are characterized by low capital costs
mainly because of their simple mechanical configuration.
Considerable backmixing in both gas and liquid phases, high
pressure drop and bubble coalescence can be disadvantageous,
in some cases.

Membrane Stirred Tank The membrane stirred tank was developed by Professor
Jϋrgen 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.

Cell Culture Bioreactors 19

Spin Filter Stirred Tank
The rotating wire cage bioreactor, in contrast to a
microfiltration device, does not rely on filtration 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 μm.
The average diameter of cells is approximately 10 to 15 μm.
Typically, fresh medium is added continuously outside the
draft tube and the culture fluid 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 cell
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 filter
and no cake is formed. The retention of the cells does not
appear 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 fluid velocity across the screen due
to perfusion. The electrostatic effect is also unlikely to be
responsible, since the ionic strength of the culture fluid is
relatively high and the thickness of the Debye layer is only
in the order of 1 nm.

If we assume that the fluid flowing through the wire cage

carries only a fraction, δ, of particles from the outside region
into the wire cage, then the material balance equation can be
written as

VO (dCO / dt ) = − Fδ CO + µ COV
Vi (dCi / dt ) = µ CiVi − FCi

The discharge factor, δ, is a measure of the effectiveness

of particle retention. A discharge factor of 1 represents no
retention.

The values of the discharge factor δ, under different operating

conditions were investigated using polystyrene particles of
the same diameter and density as cells. It was found that
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 δ. There thus appears to an optimal range of
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.

20  Cell Culture Bioreactors

 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 fluid mechanical effect, maybe one
similar to the behavior of particles of low Reynolds number
near the wall in a Poiseulle flow or a laminar boundary
layer flow along a flat plate. However, the flow pattern
around the cage is complicated: a rotating flow due to the
rotation of the cage, an upward flow cause by agitation and a
perpendicular flow 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
defined. However, because of our lack of understanding of
its mechanism, the design and operation of such a system is
very difficult and the effectiveness of the magnitude of the
discharge factor (δ) under different operating conditions is
unpredictable.

Note: More recent application of centrofugal filter installs

spin filter outside the bioreactor. Many such spin filters are
operated at very high agitation rates. The mechanism of cell
separation is certainly different from the one described in
this section.

Vibromixer
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 1960’s 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.

Cell Culture Bioreactors  21

Fluidized Bed Bioreactor
Fluidized bed has long been used in chemical catalysis.
It is also used in adsorption (chromatographic) column
in bioseparation. Typically the fluid stream (often gas
in catalysis) that enters through a flow distributor at the
bottom at velocity is sufficient to “blow up” or suspend
the solid catalyst particles. The reactants enter the catalyst
and products diffuse out into the fluid to be carried out
through the top of the reactor where a separator prevents
any particles from being blown out. The main advantage
of a fluidized bed is the high heat transfer between the
high velocity fluid 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 difficult. In the 1980’s collagen
macroporous beads were used in commercial fluidized
beds offered by Verax. The carriers need to be weighted by
inclusion of iron particles to allow for particle retention at
the flow rate that is needed to supply enough oxygen for the
cells.

Membrane Bioreactor

Hollow Fiber Bioreactor The use of hollow fiber reactors for cultivation of mammalian
cells dates back to the early 1970s. A hollow fiber system
can be used for anchorage-dependent and suspension cells.
It consists of a bundle of capillary fibers sealed inside a
cylindrical tube. The basic configuration is rather similar
to the hollow fiber cartridge used in kidney dialysis. The
hollow-fiber, 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 ultrafiltration
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
ultrafiltration 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 fiber lumen, and cells grow in the extracapillary space,
or the shell side. Supply of low-molecular weight nutrients
to 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 microfiltration
hollow fiber membranes for cell culture is infrequent, it
does find application in various research uses for studying
metabolism and for the cultivation of anchorage-dependent
or highly aggregated cells for which a convective flow of
medium through the extracapillary space to bathe cells in
medium is desired.

22  Cell Culture Bioreactors

Multiple Membrane Plate Bioreactor
Scaling-up of a hollow-fiber system eventually is limited
by the ability to extend the axial length of the fiber without
incoming oxygen transfer limitation. The capacity of the
pump and the mechanical strength of the membrane limit the
maximum operable flow rate. Scale-up in cartridge diameter
eventually runs into flow distribution problems among
thousands of fibers. Ideally, one can put different fibers,
some for gas flow for oxygen transfer, others for medium
flow. However, this approach poses a major challenge in
manufacturing. One approach (taken from the 1980’s) was
to use a multiple flat membrane. By keeping the height
(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.

Ceramic System
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-fiber system, cells populating the shell side
are exposed to a slow stream of permeate. The ceramic
bioreactor, to some extent, can be considered a variant of
the hollow fiber 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 fiber 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 configuration.
Fresh medium is fed into the system, and harvested culture
fluid is removed to the medium reservoir. Unlike the hollow
fiber 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 fiber systems, oxygen concentration gradient
develops along the axial direction and limits the length, i.e.,
the scale of the reactor.

Cell Culture Bioreactors  23