Cell Culture Bioreactors

7/28/2019 Cell Culture Bioreactors

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Cell Culture Bioreactors


Basic Types of Bioreactors 1

Stirred ank (Well Mixed) vs. ubular Reactor (Plug Flow) . . . . . . . . . . . . . . . . . 3

Segregated Bioreactors (Dead Zone Present) Compartmentalized Bioreactors . . 4

Implication When Growth or Reaction Occurs in the Reactor . . . . . . . . . . . . . . . 4

Homogenous Reactor vs. Heterogeneous Reactor . . . . . . . . . . . . . . . . . . . . . . . . . 4

Operating Mode of Bioreactors 5Batch and Continuous Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Te Operating Mode o Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Batch Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Fedbatch Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Material Balance on Bioreactors 9

Material Balance Equation or Reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Tissue culture and disposable cell culture systems 11

issue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Disposable Culture Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Multiple Plate Culture System (Cell Cube and Cell Factory) . . . . . . . . . . . . . . . . 2

Cell Support Systems 13

Suspension Culture vs Adherent Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Microcarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Cell Aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Microsphere Induce Cell Aggregates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Microencapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Cell Culture Bioreactors 18

Simple Stirred ank Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Airlif Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Membrane Stirred ank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Spin Filter Stirred ank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Vibromixer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Fluidized Bed Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Membrane Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Mammalian cell bioreactors are generally categorized

similarly to chemical reactors according to their mixingcharacteristics. It is instructive to review two ideal reactors

well-mixed stirred tank and plug-ow (tubular) reactor. In

an ideal well-mixed bioreactor, the mixing is assumed to be

intense enough that the uid is homogeneous through the

reactor. The mathematical description of ideal continuous

ow stirred tank reactor is described by the following rst-

order differential equation.

( )

Ai Ai o Ao A

d VCFC F C r V

dt= +

Basic Types of Bioreactors


2/232 Cell Culture Bioreactors

V is the culture volume in the bioreactor, CA the concentration

of nutrient or product A, t is time, F is the ow rate and rA

is the volumetric consumption rate of nutrient or production

rate of product A.

In an ideal stirred tank reactor, there is no ow bypass and

no shunt of substrate from inlet to outlet, no dead zones or

clumps of undissolved solid substrate oating around. The

addition of a substrate through feeding is instantaneously

distributed throughout the entire reactor, and when gas

sparging is employed the agitator provides an intimately

mixed gas-liquid. It also follows from this assumption that th

e stream exiting the reactor will have the same composition

as the well mixed uid in the reactor.

The basic model for the tubular reactor (such as hollow ber

and ceramic systems to be described later in this chapter)

species that the liquid phase moves as a plug-ow, meaning

that there is no variation of axial velocity over the cross

section. The mass balance for component A in a volume

element S z that described an ideal plug-ow reactor is thefollowing:

A

AZ

A rz

Cv

t

C+

=

where vz is the linear velocity in the z direction along the

ow and S is the cross-sectional area. Note that we assume

there is no liquid dispersion or back mixing. All elements in

the uid move at the same velocity. At steady state (i.e., cel

concentration and cellular activities at a given position are

not changing with time), the equation becomes

F

Sr

z

c AA

=

which describes changes of concentration of A along the

direction of uid ow. It is clear that the nutrient concentration

will decrease from inlet to the distal end of the reactor,

while metabolite concentration increases. The length of the

reactor is limited because eventually nutrient depletion or

metabolite accumulation inhibits growth and metabolism

These ideal cases of completely mixed tanks or plug ow

tubular reactors are situations that can be approximated in

small-scale laboratory conditions. The conditions in large

scale process reactors deviate signicantly from these idea

conditions.

In a well-mixed bioreactor, there are no concentration

gradients in either the gas or the liquid phase. In othe

words, none of the chemical species or cells is segregated in

the reactor. The other extreme of mixing is total segregation

where there is no interaction between different volume

elements in the bioreactor. An ideal plug ow reactor i

assumed to be under conditions of total segregation. Mos

bioreactor systems have a mixing pattern between the two

extremes and are under partially segregated conditions.


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Stirred Tank (Well Mixed) vs. Tubular

Reactor (Plug Flow)


In general, laboratory and small pilot plant bioreactors are used

for process development and optimization. The uid mixing

characteristics are rather sensitive to the scale of the reactor

Furthermore, plug-ow bioreactors are intrinsically more

difcult to scale up than mixing vessels, as the concentration

gradient of essential nutrients, oxygen in particular, wil

inevitably become limiting in the downstream region of

the reactor. In considering the selection of bioreactors for

mammalian cell cultures, the mixing characteristics and their

relationship to scale-up have to be kept in mind.

The distinction between a well mixed continuous stirred

tank reactor (CSTR) and plug ow reactor (PFR) is best

illustrated by comparison of their response in the outlet to

a step change in feed concentration, consider a continuous

reactor that has an inlet stream (feed) and an outlet stream tha

are equal in volumetric ow rate, the volume of the reactor is

thus constant. For the case that the feed stream is colorlessbut at time 0 the stream is changed to a feed with red color

at a concentration of COIf the reactor is well mixed as in a

CSTR, as soon as the feed stream is switched, the color wil

be seen immediately in the efuent stream, since the color

is distributed instantaneously everywhere including the uid

that is taken out in the efuent stream. The plots shown

are the colors seen at the outlet. The red dye concentrate

will increase gradually. If the reactor volume is V, it wil

take longer than the time needed to ow through one reactor

volume to reach the same concentration as in the feed, since

the dye is also being taken out from the reactor from the

beginning. In fact, by solving the differentiation equation

tIcan be shown that it takes about three holding times (3t

0) to

reach almost the same concentration as in the feed.

Now examine the case of PFR. According to the model of

PFR, the red color dye will move downstream like a sharp

band, since there is no backmixing or diffusion to blur the

sharp boundary between the color and colorless streams. So

the detector at the exit will detect no color right after the

switch to dye solution in the feed. It will not see any colo

until the front of the color feed solution reaches the outlet

The time it will take will be a hold time, the exact time


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phases, liquid medium, gas bubbles and cell mass, they are

often treated as homogenous bioreactors. On the other hand

in addition to high cell density culture there are cases where

the bioreactor must be treated as heterogeneous. The solid

phase constitutes a large fraction of the culture volume. An

examples is the microcarrier culture. Microcarrier beads

often constitute 10-30% of the culture volume. In such cases

even cell concentration needs to be well dened, for example

whether 107per milliliter is referring to total culture volume

or liquid volume needs to be specied.

A reactor is called continuous when the feed and product

streams are continuously being fed and withdrawn from

the system. In principle, a reactor can have a continuou

recirculating ow, but no continuous feeding of nutrien

or product harvest; it is still a batch reactor. A fed-batch

bioreactor usually has intermittent feed. It may or may no

have medium withdrawal during the run.

Operating Mode of Bioreactors

Batch and Continuous Processes

Example: For instance, Yeast cells (saccharomycescereviciae) can metabolize glucose either to ethanol, or to

oxidize it to carbon dioxide, mammalian cells can convert

glucose mostly to lactate, or oxidize it to carbon dioxide

Cells in two such types of metabolism are in two differen

metabolic states. The two metabolic states are characterized

by different specic glucose consumption rates, lactate or

ethanol production and the yield coefcient for biomass, i.e

different stoichiometric ratio.

Example: For instance, a 1 l culture has 0.3 of solidmicrocarriers and 0.7 l of medium, with 109 cells in it

The cell concentration is 109 cells/L-culture or 1.43 x 10

cells/L-medium. If the glucose concentration in the culture

medium decreases from 2.10 g/L (medium) over one day

then the specic glucose consumption rate is (2.10-1.90)

g/L-medium (1.43 x 109 cells/L-medium) = 1.40 x 10-1

g/cell-hr. The specic rate calculated would have been very

different if one concentration is based on liquid volume and

the other is based on total culture volume.


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The Operating Mode of Reactors

Batch Cultures

Fedbatch Cultures

Intermittent Harvest

6 Cell Culture Bioreactors

Batch processes are simple and are widely used, especially

in the vaccine industry and in pre-production scales of rDNA

protein production. Fedbatch processes are widely used in

multi-purpose, multi-product facilities because of their

simplicity, scalability, and exibility. A variety of fedbatch

operations, ranging from very simple to highly complex andautomated, are seen in current production facilities.

In general, fedbatch processes do not deviate signicantly

from batch cultures. For both intermittent-harvest and

traditional fedbatch cultures, cells are inoculated at a

lower viable cell density in a medium that is usually very

similar in composition to a typical batch medium. Cells are

allowed to grow exponentially with essentially no external

manipulation until nutrients are somewhat depleted and cells

are approaching the stationary growth phase. At this point

for an intermittent-harvest fedbatch process, a portion of the

cells and product are harvested, and the removed culture uid

is replenished with fresh medium. This process is repeated

several times. This simple strategy is commonplace for theproduction of viral vaccines produced by persistent infection

as it allows for an extended production period. It is also used

in roller bottle processes with adherent cells.

For production of recombinant proteins and antibodies, a more

traditional fedbatch process is typically used. While cells

are still growing exponentially, but nutrients are becoming

depleted, concentrated feed medium (usually a 10-15 times

concentrated basal medium) is added either continuously

(as shown) or intermittently to supply additional nutrients

allowing for a further increase in cell concentration and thelength of the production phase. In contrast to an intermittent

harvest strategy, fresh medium is added proportionally to

cell concentration without any removal of culture broth. To

accommodate the addition of medium, a fedbatch culture

is started in a volume much lower than the full capacity of

the bioreactor (approximately 40% to 50% of the maximum

volume). The initial volume should be large enough to allow

the impeller to be submerged, but is kept as low as possible

to allow for a maximum extension of the cultivation period

Fedbatch



In batch cultures and most fedbatch processes, lactate

ammonium, and other metabolites eventually accumulate in

the culture broth over time, inhibiting cell growth. Other

factors, such as high osmolarity and accumulation of reactive

oxygen species, are also likely to be growth inhibitory

and certainly contribute to the eventual loss of viability

and productivity. The effects of lactate and ammonia on

cultured cells are complex. Detectable changes in growthproductivity, and metabolism have all been documented

Additionally, metabolite accumulation has been found

to affect product quality. In recombinant erythropoietin

producing CHO cells, high ammonia concentration

has been reported to affect glycoform of the product

By minimizing metabolite accumulation, the duration of a

fedbatch culture can be even further extended and higher

cell and product concentrations can be achieved. Reduced

metabolite accumulation in fedbatch culture is traditionally

accomplished by limiting the availability of glucose and

glutamine using controlled feeding strategies that maintainglucose at very low levels. After extended exposure to low

glucose concentrations, cell metabolism is directed to a

more efcient state, characterized by a dramatic reduction

in the amount of lactate produced. Such a change in cel

metabolism from the normally observed high lactate

producing state to a much reduced lactate production state

is often referred to as metabolic shift. The observation

of such changes in metabolism was made more than two

decades ago, yet its application in fedbatch culture was not

realized until much later. Extending the methodology to

controlling both glucose and glutamine at low levels, bothlactate ammonium accumulations can be reduced. By

applying such a control scheme in fedbatch culture, lactate

concentration was reduced by more than three fold, and very

high cell concentrations and product titers were achieved in

hybridoma cells.

Fed-batch Culture with Metabolic Shift


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Continuous Cultures

Simple Continuous Stirred Tank Reactor

(CSTR)

Continuous Culture with a Metabolic Shift


Steady state

Grow up the culture in batch mode. Then turn on

both in and out ow of medium. Cell and product

concentration reach steady state.Transient

Same as that for steady state except that cell and

product nutrient concentration uctuate.

TransientSame as CSTR, some cells are retained in bioreactor

to reach high cell concentration. Product throughput

is higher per reactor volume, but not the concentration.

Typically cell, nutrient and product concentrations

uctuate.

Steady state

Same as that for transient except that steady state is

achieved. This rarely happens.

This is the same as simple continuous culture except in the

start-up. Instead of starting from a batch culture, a fed-batch

culture with a metabolic shift is used. After cells reach a

high concentration and the metabolic shift is affected, the

culture is shifted to a continuous culture. Because no (or low)

lactate and ammonia is produced, the concentrations of cells

and products are substantially higher than in conventionalcontinuous cultures. In some cases, the cell concentration

approaches that of perfusion cultures. However, the medium

usage is substantially reduced, and the product concentration

is higher.

Continuous Culture with Cell Retention

(Recycle) Perfusion Culture


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( d(sv)( )

dt

vv s v

d x VVF t x V q x V

dt dt = = =


Material Balance on Bioreactors

Material Balance Equation for Reactor

Batch Culture

Fed-batch Culture

Continuous Culture

at steady state,

vv

s v

dxV V xdt

dsV Vq x

dt

=

=



If there exists one and only one limiting nutrient s,

meaning increasing its concentration increases the specic

growth rate, and if the specic nutrient consumption

rate q is constant, then the viable cell concentration atsteady state is dictated by the feed concentration. In

most studies, the Monod relationship was assumed

Therefore tor any given D, the cell concentration increases

with increasing feed nutrient concentration Until another

nutrient becomes limiting, or until the concentration of that

limiting nutrient becomes growth inhibitory.

Perfusion Culture

s

o

vvs

vv

q

ssDxssDxq

DxDx

dt

dx

dt

ds

)()(

0

0

==

==

==

0

v

- ( )

define dilution rate: D

( - )

vv v

s v

v v

s v o

dxV V x Fx

dt

dsV Vq x F s s

dt

F

V

dxx Dx

dt

dsq x D s s

dt

=

=

=

=

=

Continuous Culture

at steady state,


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Disposable Culture Systems

Roller Bottles

Multiple Plate Culture System (Cell

Cube and Cell Factory)


Animal tissues (brain, egg etc.) are still used for viral

vaccine production. Automated process is available to

punch through chick eggshell to inoculate virus into

developing embryo. Virus is harvested from the tissue days

afterwards.

Roller bottles are cylindrical screw-capped bottles mostly

made of disposable plastic; reusable glass ones are still used

occasionally. Each bottle is typically about 1 to 1.5 liters in

total volume. Typically, a bottle is lled with 0.1 to 0.3 liters

of culture medium for cell cultivation. A stack of bottles is

placed on a roller, the bottles rotate on the roller rack at 1 to 4

rpm and are incubated in an incubator or an incubation room

Roller bottles are used for the cultivation of both suspensioncells and anchored cells. However, for suspension cells

it is usually more convenient to grow the cells in a stirred

vessel, and roller bottles are only used in small scale when

convenience dictates this selection of cultivation methods

Anchorage-dependent or anchorage-preferred cells are

frequently cultivated in roller bottles, both in the laboratory

and in manufacturing of biologics. These cells attach to the

inner wall of the bottle. Initially, they cover only part of the

surface of the inner wall after inoculation. The cell laye

is bathed in a liquid medium and is alternately exposed to

medium and gaseous nutrient as the bottle rotates. As cells

grow, they cover the entire surface and reach conuenceNormal diploid cells then reach contact inhibition and stop

growing until they are detached by protease treatment and

inoculated into a larger number of roller bottles. Many

continuous cell lines or tumorous (transformed) cells can

continue to grow to form multiple layers of cells after

reaching conuence, if sufcient nutrients are provided

Some variations of roller bottles are available to increase the

cell growth surface area in a bottle. Spiral lms and wafes

on the inner wall of roller bottles have been introduced and

are sometimes used.

For small-scale operations, roller bottles provide many

advantages for the cultivation of adherent cells. It is relatively

inexpensive to set up. Often it allows for a rapid change

of throughput in response to need. Furthermore, replacing

medium from cell growth medium to one designed for

product formation is rather straightforward. It is particularly

useful in the case that the serum-containing medium needs

to be replaced by a serum-free or protein detachment and

expansion, medium exchange and product harvest, requires

extensive well-trained labor to ensure a high success rate

Tissue culture and disposable cell

culture systems

Tissue Culture


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Bag System


Each batch of manufacturing can easily involve hundreds or

even thousands of bottles, and the large number of manua

steps involved makes the risk of microbial contamination

rather high. Despite these signicant drawbacks, roller

bottles are still widely used in the production of viral vaccines

often because the products involved have been approved

by regulatory agencies before more recent cultivation

technologies became more robust and widely accepted. In

some cases, roller bottles were selected over other bioreactors

because the process was easy to implement compared to

those based on other bioreactors. One notable example is

the production of erythroprotein (EPO) using recombinant

Chinese hamster ovary cells. Recent improvements include

developing a robotic handling system for handling a large

number of roller bottles to make industrial scale production.

free medium for the production of secreted proteins or

viruses. The transparent glass or plastic wall allows visua

or microscopic examination of the culture status. Microbia

contaminated bottles can be readily spotted and discarded

before they pool together with contaminated ones. However

the drawbacks of roller bottles are numerous for large-scaleproduction of biologics. On-line environmental monitoring

and control is virtually impossible, or at least impractical

The aspectic bottle handling for inoculation, trypsinization

for cell

The Roller bottle system has a couple of drawbacks. The

medium to cell ratio is unfavorable; it is not easy to be

operated in a continuous or fed-batch mode. Furthermore, i

is often desirable to have a large culture volume contained

in a reactor For suspension cells the alternative to

overcome these is is either using bags for a disposable

system or employing a fermentor. For adherent cells, the

disposable solution is to contain a large at surface in a

single container.

Blood bags are used extensively in cellular therapy

Wave bioreactor system

Blood bags have long been used in culture of blood

cells especially for adoptive cell therapy culturing NK

or MTL cells. Those are small bags just sitting insideCO2 incubators. Newer arrivals come with a tilting

platform to provide some mixing.



Many cell lines used in the production of vaccines and

other conventional biologies are suspension cells. While

disposable systems minimize in plant validations required

for cGMP operations they are limited in scale. For viralvaccines, for which the number of cells required for

producing a dose is relatively small, a disposable system

provides some advantages. For antibody products, the

use of disposable systems may be limited to inoculum

preparation. Large scale operations thus still resorts to

fermentors or other bioreactors. The majority of cells used

for rDNA protein production are suspension cells, they

are simply suspended in the bioreactors. For cells grown

adherently cell supports are needed to provide adherent

surface while the reactor provides a mechanism to keep the

cell support in suspension. In some cases cell support is

also used for suspension cells. The main advantage is that

cells can be kept in the reactor while the medicine is being

perfused or exchanged. These systems are hardly used

for suspension cells any more as other simple methods of

retaining cells prevail.

Most normal diploid cell strains or primary cells are

anchorage-dependent. For large scale operations

either surface cultures or microcarrier cultures are usedConventional microcarriers are suitable for normal diploid

cells which require attachment and spreading. Macroporous

microcarriers can be used for a wide variety of continuous

cell lines, by may not be suitable for large normal diploid

broblasts.

The use of microcarriers for cell culture was rst

demonstrated by Van Wezel (1967). The basic concept is

to allow cells to attach to the surface of small suspendedbeads so that conventional stirred tank bioreactors can be

used for cell cultivation. To ease suspending these cell

laden microcarriers, their diameter and density are usually in

the range of 100300 m and 1.021.05 g/cm3 respectively

This diameter range also gives a good growth surface

area per reactor volume. Even at a moderate microcarrier

concentration, in the range of 815% culture volume being

occupied microcarriers, a signicantly larger surface area per

reactor volume can be achieved than that in roller bottles.

Cell Support Systems

Suspension Culture vs Adherent

Cultures

Microcarriers

Solid Microcarriers



Although even smaller microcarriers, less than 100 m in

diameter, can provide even more surface area, they are not

used often. Most anchorage-dependent cells do not develop

their normal morphology, and in many cases, do not multiply

well on surfaces with an excessively high curvature. These

cells do not grow well on small microcarriers. On the other

hand, some cells, especially transformed cells, do multiply

well even though they are attached to small microcarriers

and do not develop a well spread-out morphology. These

cells, after attaching to the small microcarriers, agglomerate

to form aggregates and continue to grow to high density. The

small microcarriers, usually with a diameter of about 50 m

serve as nuclei for the initiation of aggregate formation.

Desired characteristics of microcarriersDensity ~1.02 g/cm3 Only slightly higher than water for easy

suspension and settingDiameter 150-200 m Should be the smallest possible and yet

allow for cell spreading

Porosity From solid to nearly 90% Prefer solid, for use as inoculum bead to

bead transfer

Surface Properties ECM coating, slightlypositively charged Positive charge enhances initial attachment

The microcarriers can be made of many different materials

including dextran, gelatin, polystyrene, glass and cellulose

Not all of these are commercially available. In general, in

addition to having a wettable surface, the backbone materials

of microcarriers often need to be chemically collagen

Polystyrene microcarriers have also been coated with collagen

or other adhesion molecules for better performance.

An advantage for the industrial-scale culture of anchorage-

dependent cells is the ease of separating cells from culture

medium. Many microcarrier cultures require medium

exchanges during cultivation to remove accumulating

lactate, ammonia and other metabolites and to replenish

nutrients. In many cases, the cell-laden microcarriers are

simply allowed to settle and the spent medium be withdrawn

and replenished. In large-scale operations, continuous or

semi-continuous perfusion is more frequently used. This can

be accomplished by withdrawing medium through a coarse

screen which allows medium to pass through but retains

microcarriers in the reactor. A large number of cells have

been grown on microcarriers, including broblasts, kidney

epithelial, hepatocyte, neuroblastoma, and endotheliacells from various species. Overall, microcarrier culture

is a convenient laboratory and research tool, and it has the

advantage of being amenable to large-scale production if a

large quantity of product is needed.modied to improve cel

attachment. One of the most widely used microcarriers, the

dextran based ones, are derivatized with charged molecules

or denatured collagen.


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Macroporous Microcarriers


A variant of microcarrier culture, macroporous microcarriers

contain large internal pores, tens of micrometers across. The

void space inside allows cells to be cultivated not only on

the external surface but also internally. Cells in the interio

are less susceptible to mechanical damage caused by

agitation and gas sparging. Of course, being in the interior

of microcarriers, cells are more likely to be subject to oxygen

limitation due to the long diffusional distance, especially sincemost macroporous microcarriers have a larger diameter (500

m to a couple millimeters). Macroporous microcarriers are

made of different materials, including gelatin, collagen and

plastic. Many cell lines have been successfully grown on

macroporous microcarriers including Vero, HepG2, CHO

and 293 cells. The nal cell concentration achieved tends to

be higher than that obtained with an equivalent volumetric

concentration of conventional microcarriers. But in some

cases the growth kinetics are slower because the penetration

of cells into the interior may be slowed or even retarded by

restrictive opening of these pores


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Cell Aggregates


Microsphere Induce Cell Aggregates

Agarose

Some transformed cells which normally attach to or

spread and grow on a surface can form aggregates when

cultivated in shaker asks or in stirred vessels. Differen

methods have been used to induce aggregate formation for

cells. Aggregation can be promoted by manipulating the

calcium concentration in conjunction with the agitation rate

Aggregate cultures have advantages similar to microcarrier

cultures. They can be cultivated in conventional stirred tankreactors with environmental control. They can be allowed

to settle relatively rapidly by stopping agitation. Permitting

easy medium replenishment.

For many cell types, a limitation on the use of the aggregate

culture is that the rate of aggregate formation is slow. One

way to induce aggregate formation is to add microspheres

to cell suspension to allow for rapid agglomeration to the

microspheres (9). If the aggregate diameters become

too large, necrotic centers can be formed due to transport

limitation. The aggregate size may inuence, i.e., to the vira

infection kinetic and yield for vaccine formation (10). Many

cell lines, including BHK, CHO, 293 and ST cells, have been

cultivated as aggregates with sizes ranging between 90 and

400 m. ,without the formation of necrotic centers (9, 11).

Agarose entrapment of cells is usually accomplished by

passing cell-agarose suspension through a small oriface

opening into an air jet stream. The droplets of agarose

containing entrapped cells are collected in a chilled oil phase

to allow the agarose to gel. Alternatively, the cell-agarose

suspension is allowed to drop into the center of a fast rotating

disk. The centrifugal spinning force causes the droplets to

form and be dispensed outside the disk. The agarose beads

tend to be rather large, at least hundreds of micrometers



in diameter. This causes oxygen transfer limitation at a

high cell concentration. Further, the agarose beads do no

have sufcient mechanical strength to sustain mechanica

optimization even in a moderately small scale (tens of liters)

bioreactor.

Another method of cell cultivation that enables the use of a

stirred tank reactor is cell entrapment. This technique entails

entrapping cells in polymeric matrix to form spheres. The

spheres are then either coated with another polymeric lm to

control the crossing of molecules according to their size

commonly referred to as microencapsulationor they are

cultivated as they are. These beads are suspended in medium

to allow cell growth inside. One of the polymers most used

for cell entrapment is calcium alginate. Cell entrapmen

in calcium alginate is accomplished by preparing a cell

suspension in sodium alginate and adding it, in a dropwise

fashion, into a solution of calcium chloride. Calcium cross

links alginate, instantaneously forming beads containingentrapped cells. Alginate may be coated with polylysine

for increased mechanical and chemical stability, but such

treatment decreases the molecular weight cut-off and prohibits

large-molecular weight proteins in the medium from reaching

the cells. To prepare hollow spheres, the alginate gel inside a

bead coated with a polylysine is liqueed through treatmen

with a calcium chelator such as EDTA or citrate.

The diameter of these beads is often in the order of millimeters

The mechanical strength of the gel which constitutes the

beads is relatively weak. Large-scale application using such

techniques is not easy. On the other hand, the mirocapsule

provides a means of immunoisolation of transplanted cells

or tissues (sometimes referred to as articial cells) and

could be suitable for some tissue engineering applications

Cell entrapment has been applied to hybridomas and smal

clusters of adherent cells. It has also found application in the

cultivation of differentiated cells, such as insulin-secreting

cells. For tissue engineering applications using differentiated

cells, the enclosing membrane around the cells and the

hydrogel must be biocompatible. The membrane used in

microencapsulation is semipermeable to allow sufcien

diffusion and transport of low-molecular weight moleculesimportant for cell survival, such as oxygen, nutrient and

metabolites. For optimum transport across the membrane

the microcapsules should have a uniform wall thickness

Ideally, the semipermeable membrane prevents the passage

of high-molecular weight proteins, such as immunoglobulins

to allow for product to accumulate inside the microcapsule.

Since the cells are protected inside the capsules, they

are protected from hydrodynamic damage. The culture

Microencapsulation



can be stirred at faster rates than conventional culture,

which improves nutrient, metabolite and gas exchange.

Entrapped cells may be cultured in stirred tank bioreactors,

xed-bed and uidized-bed reactors. An airlift reactor

could be used as well, provided that the beads are small

and not signicantly denser than the medium.

Stirred tanks, or conventional fermentors, have been widely

used for culturing suspension cells since the 1960s. With

the use of microcarriers, conventional or macroporous ones

adherent cells can also be cultured in a stirred tank. For cel

entrapment in hydrogel, the use of stirred tanks is limited

to laboratory scale, as the preparation of a large quantity of

cell-entrapped beads can be a daunting task, and the risk

of mechanical damage caused by agitation increases with

scale.

The basic conguration of stirred tank bioreactors for

mammalian cell culture is similar to that of microbia

fermentors. A major difference is that the aspect ratio (the

height to the diameter ratio) is usually smaller in mammalian

cell culture bioreactors. The power input per unit volume

of bioreactor is also substantially lower in mammalian cel

culture bioreactors. While the Rushton type impeller i

the norm in microbial fermentors, mammalian cell culture

fermentors mostly employ marine type impellers. As in

microbial fermentors, an axial impeller is often mounted a

the top of the bioreactor to drive the liquid downward in

large-scale cell culture bioreactors. These differences reecthe different purposes of agitation in microbial fermentation

and in cell culture. In microbial fermentation, agitation is

needed at a higher power input to disperse air bubbles and to

increase oxygen transfer efciency, whereas in mammalian

cell culture, the primary purpose of agitation is to keep cells

or microcarriers suspended in nutrient medium relatively

uniformly. In general, the mixing time in a mammalian

cell culture bioreactor is substantially higher than that in a

microbial fermentor with similar scale. The oxygen transfe

capacity in a cell culture bioreactor is also substantially lower

than that in a microbial fermentor. However, the typica

oxygen demand in a mammalian cell culture is also much

lower (10 to 50 times) than that in microbial fermentation.

A variant of the bubble column reactor with interna

circulation loops is used to improve the performance of

conventional bubble column reactors. In airlift column

reactors, internal liquid circulation is achieved by sparging

only part of the reactor with air. The sparged section has a


Simple Stirred Tank Bioreactor

Airlift Bioreactor



lower effective density than the bubble-free section, and the

difference in hydrostatic pressure between the two sections

induces the liquid circulation upward in the additional bene

is the low energy requirement compared with stirred-tank

reactors. Although simple in construction, sound design is

critical for optimal hydrodynamic behavior. Nevertheless

the ow in bubble column reactors is relatively well dened

compared with that in stirred tank reactors.

Airlift bioreactors for cell cultivation are considered to be

low-shear devices because there is no mechanical agitation

Also, the direct air sparging may not cause excessive cell

damage, at least under normal cultivation conditions. The

ow regime depends on the sparger used and the ow rate.

Various type of spargers are used to provide different bubble

size.

Airlift reactors have been used successfully with suspension

cultures of BHK 21, human lyphoblastoid, CHO, hybridomas

and insect cells.sparged section (riser) and downward in

the bubble-free section (downcomer). The loop has theadvantages of permitting high efciency mass transfer and

improving the ow and mixing properties in the vessel.

These reactors are characterized by low capital costs

mainly because of their simple mechanical conguration

Considerable backmixing in both gas and liquid phases, high

pressure drop and bubble coalescence can be disadvantageous

in some cases.

The membrane stirred tank was developed by Professor

Jrgen Lehmann in the 1980s. It uses long microporous

polypeopylene tubing wrapped around rotating rods. By

adjusting the air pressure in the propylene tubing, the

micropore expands to allow gas to be in direct contact with

medium while not bursting to become gas spargers. The

rotation of those tubings also provides gentle agitation to

microcarriers or suspended cells. Even at a high serum

concentration, foaming can be avoided.

Membrane Stirred Tank


20/23

Spin Filter Stirred Tank


The rotating wire cage bioreactor, in contrast to a

microltration device, does not rely on ltration to achieve

cell retention in the bioreactor. The centerpiece of this

bioreactor is the wire cage, which is often mounted on

the agitation shaft. At times other designs may be seen in

which the wire cage is mounted to a shaft rotated by a top

motor. The bottom of the cage is solid wile the side is made

of wire screen with an opening ranging from 25 to 60 m.The average diameter of cells is approximately 10 to 15 m

Typically, fresh medium is added continuously outside the

draft tube and the culture uid is withdrawn from inside the

cage at the same rate.

Under certain operating conditions, the cell concentration

inside the wire cage is lower that that outside, thus

achieving cell retention in the bioreactor. The attainable cel

concentration in such a bioreactor has been reported to be as

high as ten times of that in a typical bioreactor. Since under

optimal conditions, the device does not appear to act as a lter

and no cake is formed. The retention of the cells does noappear to be due to centrifugal force exerted by the rotating

motion of the cage, because the terminal velocity of the cells

due to centrifugal force is two to three orders of magnitude

lower than that of the uid velocity across the screen due

to perfusion. The electrostatic effect is also unlikely to be

responsible, since the ionic strength of the culture uid is

relatively high and the thickness of the Debye layer is only

in the order of 1 nm.

If we assume that the uid owing through the wire cage

carries only a fraction, , of particles from the outside regioninto the wire cage, then the material balance equation can be

written as

( / )

( / )

O O O O

i i i i i

V dC dt F C C V

V dC dt C V FC

= +

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

of particle retention. A discharge factor of 1 represents no

retention.

The values of the discharge factor , under different operatingconditions were investigated using polystyrene particles of

the same diameter and density as cells. It was found tha

the discharge factor was affected by the agitation rate: it

decreased as the agitation rate increased from 50 to 100 rpm

However, further increase in the agitation rate increased

the value of . There thus appears to an optimal range o

agitation rate for cell retention. The fact that the discharge

factor increases after the agitation rate increases beyond an

optimal range indicates that centrifugation may not be the

dominating mechanism for cell retention.



In addition to its use for simple suspension cultures, the

rotating wire cage bioreactor has also been used in aggregate

or microcarrier cultures. In those cases the cell retention is

relatively straight forward; the opening of the pores on the

screen is smaller than the size of most aggregates. Since the

diameter of the microcarriers or the aggregates is in the range

of hundreds of micrometers, the underlying mechanism for

particle retention is mostly likely centrifugation. But the

most intriguing effect of a rotating wire cage is still its

ability to retain single suspension cells. It is likely that the

retention is caused by a uid mechanical effect, maybe one

similar to the behavior of particles of low Reynolds number

near the wall in a Poiseulle ow or a laminar boundary

layer ow along a at plate. However, the ow pattern

around the cage is complicated: a rotating ow due to the

rotation of the cage, an upward ow cause by agitation and a

perpendicular ow inward to the cage cause by perfusion. A

wire cage bioreactor is very effective for large scale animal

cell cultivation once the optimal operating conditions are

dened. However, because of our lack of understanding of

its mechanism, the design and operation of such a system isvery difcult and the effectiveness of the magnitude of the

discharge factor () under different operating conditions is

unpredictable.

Note: More recent application of centrofugal lter installs

spin lter outside the bioreactor. Many such spin lters are

operated at very high agitation rates. The mechanism of cel

separation is certainly different from the one described in

this section.

A vibromixer uses a perforated disk as the mixing

mechanism instead of conventional impeller. The disk

vibrates in the vertical direction at a very high frequency

causing liquid to circulate through the perforated holes

and provide mixing. It was used in the 1960s for the

cultivation of suspension cells and virus production.

Its use in cell cultivation has diminished in the past

couple of decades. However, in some cases it is used to

keep concentrated microcarriers in suspension for cell

detachment during the trypsinization step.

Vibromixer


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Membrane Bioreactor

Hollow Fiber Bioreactor


Fluidized bed has long been used in chemical catalysis.

It is also used in adsorption (chromatographic) column

in bioseparation. Typically the uid stream (often gas

in catalysis) that enters through a ow distributor at the

bottom at velocity is sufcient to blow up or suspend

the solid catalyst particles. The reactants enter the catalyst

and products diffuse out into the uid to be carried outthrough the top of the reactor where a separator prevents

any particles from being blown out. The main advantage

of a uidized bed is the high heat transfer between the

high velocity uid and the catalyst surface. When applied

to cells or microcarrers directly, the density difference

between solid phase and liquid phase is too small,

making particle retention difcult. In the 1980s collagen

macroporous beads were used in commercial uidized

beds offered by Verax. The carriers need to be weighted by

inclusion of iron particles to allow for particle retention at

the ow rate that is needed to supply enough oxygen for the

cells.

The use of hollow ber reactors for cultivation of mammalian

cells dates back to the early 1970s. A hollow ber system

can be used for anchorage-dependent and suspension cells

It consists of a bundle of capillary bers sealed inside a

cylindrical tube. The basic conguration is rather similar

to the hollow ber cartridge used in kidney dialysis. The

hollow-ber, in most cases, consists of supporting polymeric

porous materials for mechanical strength and a thin layer of

membrane which provides selective passage of molecules

depending on their size. In most cases, an ultraltration

membrane is used. The molecular weight cut-off (MWCO)

of the membrane differs according to applications, ranging

from a few thousand to a hundred thousand daltons. The

ultraltration membrane prevents free diffusion of secreted

product molecules from passing through the membrane and

allows them to accumulate in the extracapillary space to a high

concentration. The culture media is pumped usually through

the ber lumen, and cells grow in the extracapillary space

or the shell side. Supply of low-molecular weight nutrientsto the cells and the removal of waste products occur by

diffusive transport across the membrane between the lumen

and the shell spaces. Although the use of microltration

hollow ber membranes for cell culture is infrequent, i

does nd application in various research uses for studying

metabolism and for the cultivation of anchorage-dependent

or highly aggregated cells for which a convective ow of

medium through the extracapillary space to bathe cells in

medium is desired.

Fluidized Bed Bioreactor


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Multiple Membrane Plate BioreactorScaling-up of a hollow-ber system eventually is limited

by the ability to extend the axial length of the ber withou

incoming oxygen transfer limitation. The capacity of the

pump and the mechanical strength of the membrane limit the

maximum operable ow rate. Scale-up in cartridge diameter

eventually runs into ow distribution problems among

thousands of bers. Ideally, one can put different bers

some for gas ow for oxygen transfer, others for medium

ow. However, this approach poses a major challenge in

manufacturing. One approach (taken from the 1980s) was

to use a multiple at membrane. By keeping the heigh

(i.e. the clearance between membranes) of the cell chamber

small, one can avoid diffusion limitation. This seemingly

versatile reactor also suffers from practical manufacturing

complexity of designing a satisfactory sealing mechanism

that is needed for a long-term aseptic operation.

The ceramic system is a cylinder of porous ceramic with

square channels passing through the cylinder. Cells are

inoculated into the channels and either adhere to the surface

or are entrapped in the pores of the ceramic. Medium is

passed through the channels to provide nutrients and to

remove the metabolites. In a ceramic system, the cells

are directly bathed in the recirculating medium, whereas

in a hollow-ber system, cells populating the shell side

are exposed to a slow stream of permeate. The ceramicbioreactor, to some extent, can be considered a variant o

the hollow ber system. It consists of a cylindrical ceramic

core with many channels passing longitudinally through the

ceramic material. Cells inoculated in the channels adhere to

the material or become entrapped in the pores of the ceramic

As in a hollow ber system, ceramic reactors are supported

by medium perfusion loops. Cell culture medium is pumped

through the longitudinal channels in the ceramic cores from

a medium reservoir in a recirculating loop conguration

Fresh medium is fed into the system, and harvested culture

uid is removed to the medium reservoir. Unlike the hollow

ber system, there is no membrane separating the cells and

bulk medium. Product is secreted directly into the bulk

medium. Essentially, the ceramic bioreactor can be used

to conveniently replace a large number of roller bottles

As in hollow ber systems, oxygen concentration gradient

develops along the axial direction and limits the length, i.e.

the scale of the reactor.

Ceramic System

Cell Culture Bioreactors

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