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Cell Culture Bioreactors

 

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Basic Types of Bioreactors Stirred Tank (Well Mixed) vs. Tubular Reactor (Plug Flow)

. Segregated Bioreactors (Dead Zone Present) Compartmentalized

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Implication When Growth or Reaction Occurs in the Reactor

 

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Homogenous Reactor vs. Heterogeneous Reactor

Batch Cultures

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4

Operating Mode of Bioreactors

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Batch and Continuous

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The Operating Mode of Reactors

Fedbatch Cultures

Material Balance on Bioreactors

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9

Material Balance Equation for Reactor

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9

Tissue culture and disposable cell culture systems

11

. Disposable Culture Systems

Tissue Culture

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Multiple Plate Culture System (Cell Cube and Cell Factory)

2

Cell Support Systems

13

Suspension Culture vs Adherent Cultures

3

Microcarriers

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3

. Microsphere Induce Cell Aggregates

Agarose

Cell Aggregates

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. Microencapsulation

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7

Cell Culture Bioreactors

18

Simple Stirred Tank Bioreactor

8

. Membrane Stirred Tank

Airlift Bioreactor

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9

Spin Filter Stirred

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20

Vibromixer

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. 2

Fluidized Bed Bioreactor

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22

Membrane Bioreactor

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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

)

dt

=

FC

i

Ai

F C

o

Ao

+

r V

A

Cell Culture Bioreactors

V is the culture volume in the bioreactor, CAthe concentration of nutrient or product A,
V is the culture volume in the bioreactor, CAthe concentration of nutrient or product A,

V is the culture volume in the bioreactor, CAthe 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

t

=− v

Z

C

A

z

+ r

A

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

r A

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.

Stirred Tank (Well Mixed) vs. Tubular Reactor (Plug Flow)

Stirred Tank (Well Mixed) vs. Tubular Reactor (Plug Flow) In general, laboratory and small pilot plant

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.

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 C O If 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, t I can 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 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

solution reaches the outlet. The time it will take will be a “hold time”, the exact

Segregated Bioreactors (Dead Zone Present) Compartmentalized Bioreactors

Implication When Growth or Reaction Occurs in the Reactor

Implication When Growth or Reaction Occurs in the Reactor Homogenous Reactor vs. Heterogeneous Reactor of flow

Homogenous Reactor vs. Heterogeneous Reactor

in the Reactor Homogenous Reactor vs. Heterogeneous Reactor of flow a reactor volume into the reactor

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.

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

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.

Heterogeneousreactor—withasolidphase,e.g.,microcarriers in stirred tank, tubular reactor packed with foam. A typical tissue has a cell concentration of about 5X10 8 /ml. Unless a rector have a very high cell concentration in the middle of 10 7 / ml, cell mass is only a small fraction of the culture volume. So, even though almost all cell culture reactors have all three

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 10 7 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.

It may or may not have medium withdrawal during the run. Example: For instance, Yeast cells

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 10 9 cells in it. The cell concentration is 109 cells/L-culture or 1.43 x 10 9 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 10 9 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.

The Operating Mode of Reactors

Batch Cultures

The Operating Mode of Reactors Batch Cultures Fedbatch Cultures Intermittent Harvest Fedbatch Batch processes are simple

Fedbatch Cultures

Intermittent Harvest

Batch Cultures Fedbatch Cultures Intermittent Harvest Fedbatch Batch processes are simple and are widely used,

Fedbatch

Cultures Fedbatch Cultures Intermittent Harvest Fedbatch Batch processes are simple and are widely used, especially

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.

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.

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.

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.

fold, and very high cell concentrations and product titers were achieved in hybridoma cells. Cell Culture

Continuous Cultures

Simple Continuous Stirred Tank Reactor (CSTR)

Cultures Simple Continuous Stirred Tank Reactor (CSTR) Continuous Culture with Cell Retention (Recycle) –

Continuous Culture with Cell Retention (Recycle) – Perfusion Culture

Culture with Cell Retention (Recycle) – Perfusion Culture Continuous Culture with a Metabolic Shift  Steady

Continuous Culture with a Metabolic Shift

Perfusion Culture Continuous Culture with a Metabolic Shift  Steady state  Grow up the culture

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.

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.

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.

Material Balance on Bioreactors

Material Balance Equation for Reactor

Batch Culture

Material Balance Equation for Reactor Batch Culture Fed-batch Culture Continuous Culture at steady state, V dt

Fed-batch Culture

Continuous Culture at steady state,
Continuous Culture
at steady state,

V

dt

V

V

=

dx

v

dt

ds

dt

V x

= µ

v

=−

Vq x

s

v

F

( )

t

(

d x V

v

dt

=µ

x V

v

d(sv)

dt

=−

q x V

s

v

Continuous Culture

V

V

dx

v

dt

=

µ

V x

v

ds

dt

=−

Vq x

s

v

-

Fx

v

(

F s

0

s

)

define dilution rate: D =

dx

v =

dt

ds

dt

µ x

v

=−

q x

s

v

Dx

v

(

D s

o

-

s

)

at steady state,

F

V

ds

dt

Dx

v

dx

=

=

dt

µ

x

= 0

v

D

⇒ =

µ

q x

s

v

=

(

D s

0

− →

)

s

x

v

=

(

D s

o

s

)

q s

Perfusion Culture

If there exists one and only one limiting nutrient s,

meaning increasing its concentration increases the specific growth rate, and if the specific nutrient consumption rate q is constant, then the viable cell concentration at steady 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.

limiting, or until the concentration of that limiting nutrient becomes growth inhibitory. 0 Cell Culture Bioreactors

0

Cell Culture Bioreactors

Tissue culture and disposable cell culture systems

Tissue Culture

Disposable Culture Systems

Roller Bottles

Tissue Culture Disposable Culture Systems Roller Bottles Multiple Plate Culture System (Cell Cube and Cell Factory)

Multiple Plate Culture System (Cell Cube and Cell Factory)

Multiple Plate Culture System (Cell Cube and Cell Factory) Animal tissues (brain, egg etc.) are still

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 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 surface of the inner wall after inoculation. The cell layer 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

Bag System 2 Cell Culture Bioreactors Each batch of manufacturing can easily involve hundreds or

Bag System

Bag System 2 Cell Culture Bioreactors Each batch of manufacturing can easily involve hundreds or even

2

Cell Culture Bioreactors

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.

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.

Cell Support Systems

Suspension Culture vs Adherent Cultures

Microcarriers

Solid Microcarriers

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.

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.

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/cm 3 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

3

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/cm 3

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 positively charged

Positive charge enhances initial attachment

charged Positive charge enhances initial attachment 4 Cell Culture Bioreactors The microcarriers can be made of
charged Positive charge enhances initial attachment 4 Cell Culture Bioreactors The microcarriers can be made of
charged Positive charge enhances initial attachment 4 Cell Culture Bioreactors The microcarriers can be made of

4

Cell Culture Bioreactors

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.

Macroporous Microcarriers

Macroporous Microcarriers Avariant of microcarrier culture, macroporous microcarriers, contain large internal pores, tens

Avariant 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

cells into the interior may be slowed or even retarded by restrictive opening of these pores

Cell Culture Bioreactors

5

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

medium replenishment. Microsphere Induce Cell Aggregates For many cell types, a limitation on the use of

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

6

Cell Culture Bioreactors

Microencapsulation

Microencapsulation in diameter. This causes oxygen transfer limitation at a high cell concentration. Further, the agarose

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.

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

7

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

Airlift Bioreactor

8

Cell Culture Bioreactors

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.

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

Membrane Stirred Tank lower effective density than the bubble-free section, and the difference in hydrostatic

Membrane Stirred Tank

Membrane Stirred Tank lower effective density than the bubble-free section, and the difference in hydrostatic pressure

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 canbe disadvantageous, in some cases.

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.

microcarriers or suspended cells. Even at a high serum concentration, foaming can be avoided. Cell Culture

Cell Culture Bioreactors

9

Spin Filter Stirred Tank

Spin Filter Stirred Tank The rotating wire cage bioreactor, in contrast to a microfiltration device, does

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

V

O

(

dC

O

/

dt

) =−

F C

δ

O

+

µ

V dC dt

i

i

(

/

) =

µ

CV

i

i

FC

i

C V

O

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.

Vibromixer

Vibromixer In addition to its use for simple suspension cultures, the rotating wire cage bioreactor has

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.

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.

Fluidized Bed Bioreactor

Fluidized Bed Bioreactor Membrane Bioreactor Hollow Fiber Bioreactor Fluidized bed has long been used in chemical

Membrane Bioreactor

Hollow Fiber Bioreactor

Bed Bioreactor Membrane Bioreactor Hollow Fiber Bioreactor Fluidized bed has long been used in chemical catalysis.

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.

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.

Multiple Membrane Plate Bioreactor

Multiple Membrane Plate Bioreactor Ceramic System Scaling-up of a hollow-fiber system eventually is limited by the

Ceramic System

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.

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.