Introduction
 Animal cell bioreactor depends on cell itself whether:
 The cell exhibit anchorage- dependent growth characteristics that it
must be attached to some form of solid matrix support.
 Cells can grow and produce when in suspended culture.
 Cells are completely surface-dependent.
 Animal cell bioreactor deals with cells that are larger, more
fragile and more complex than bacteria. The cells are more
subject to damage by osmotic, shear and other cytotoxic
effects.
 Furthermore, cell productivities are low and the cultures
more liable to contamination by faster growing bacteria or
fungal cells.
Bioreactor design for animal cell should consider :
• Surface adherence properties of the cells
• Increased demand on asceptic procedures
• The shear sensitivity of the cells
• Potential foaming in the medium
• Potential mass transfer limitations
• Control and automation
• Ease of scale up
• Removal of toxic waste products
Batch, Fed-batch, Repeated Fedbatch,Continuous, and Perfusion system.
Batch
 Condition in the medium change continuously with
respect to time, owing to the depletion of substrates
and the build-up of the waste products.
 Provides the cells with a highly fluctuating and
uncontrolled environment which can therefore never
be optimal.
Fed-batch and Repeated fed-batch
Advantages
• Supply of nutrients to the cells can be controlled to
some extent by the feeding policy, and also have
special application in processes where products are
obtained during the stationary stages of growth.
Continuos Operation (Chemostat & Turbidostat)
Advantages
 More constant and controlled condition.
 Steady state operation at controlled specific growth
rate or cell density conditions.
 Specific nutrient limitations may be easily effected
 Deliberate manipulation of the cell physiology for
optimal productivity can be obtained.
 Reduce capital cost, increase media utilization,
reduced downtime and removal of waste products of
reactor environment is continuously.
 However, it requires good control facilities and high
standards of plant containment to avoid
contamination.
Perfusion system.
 Cells provided with a continuous supply of fresh
medium and continuous withdrawal of waste
products.
 Closely approach the in vivo system and appear
particularly to be advantages in cases in which the
kinetics of product formation are related to growth.
 Often used in conjunction with an external loop,
which is used to recirculate medium to the cells at
controlled temp, dissolved O2, pH and etc.
 Fine mesh rotating metal filters mounted on the
agitator shaft, give high cell retention rates and good
scale-up potential for continuous operation.
 Figure 11.11(a) semi-batch reactor with control of pH,
Po2 and rate of medium additional.
 Fig 11.11(b) perfused chemostat operation with cell
retention filter.
 Fig 11.11(c) Perfused fluid lift microcarrier system.
Attached Growth Systems
Attached growth systems
 PROBLEMS OF SCALE-UP:
-need to maintain a uniform cell & medium distribution
-hard to control cell environment
-hard to monitor growth processes (difficult to do direct
measurement of cell concentration)
 Types of bioreactor considered based on:
a) surface anchorage cells,
b) cell density values,
c) surface area/medium volume ratios employed
 Microcarrier, immobilized bed, and plate heat exchanger
reactors are really efficient respect to high scale up potential
 Surface dependent cells required:
- large surface area to volume ratio
- enough nutrient supply
- removal of waste
- ease of harvesting
Reactor systems for surface anchorage dependent cell
1) Incubator Flasks
2) Roller Flasks
Provide about 200cm2/L
Used to increase surface area up
surface area
to 600-1600cm2/L
Starting culture for large scale
suspension fermentation.
Continue…
3) Multitray unit
4) Plastic bag units
Have sets horizontal trays
fixed vertically above one
another
Connected by channel for
nutrient and gas flow
Cell growth on the surface of
biologically inert gas permeable
bags
Continue…
5) Stacked Plate Unit
 Plates maybe used in a static or rotational mode
 Poorly it need very large quantity of medium volume
per unit surface area (V/A)
Continue…
6) Packed Bed Reactor
7) Plate Heat Exchanger System
Have added advantages of simplicity,
reliability, ease of scale-up
Area for growth increased if there is
Use wide variety of packing materials
Operated as circulating perfusion
system
installation of more plates (into 1 unit)
Factor increasing of growth:
(a) circulation of medium around
unit
(b) Temperature control
(c) process sterilization
Continue…
8) Hollow Fibre System
Conventional
Expensive
Suffer diffusional substrate limitation
(because of multi-layered nature of cell
growth)
Axial flow lumen configuration (limit scale
up, cell growth not uniform; diffusion supply
limitation)
combination of very high (A/V) and
high cell densities
Upgrade
Utilize expansion chamber
Connect to extra-capillary space (media can
flow uniformly across cartridge for better
distribution flow in opposite direction across
membrane)
Use ceramic modules-to automobile waste
gas catalyser (provide high {A/V} ratio & high
cell densities
Cell grow externally on tube surface
and raise by medium diffusing (via
porous tubing)
Product removed by flushing cell or
diffused it back to the opposite
direction to nutrients
Separate fibre systems may use to
supply medium&O2 to cell
Suspended Growth Systems
 Advantage of suspension system:
Easy to scale up.
rate of cell growth is easy to monitor.
the environment condition is easy to obtained.
 Traditional method of suspension culture-spinner
flask.
 Used for low volumes (200ml-20L).
 The conventional stirred tank bioreactor can also be
used for small to large scales of operation.
 Spinner
Flasks
Triangular Flasks
with Vent Cap
• Characteristics needed in animal cell bioreactors:
Combination of round-bottomed vessels with slow
moving propeller agitators are preferred.
Vibromix agitators may be used for uniform power
dissipation but are unsuitable for microcarrier
applications.
Fermenter wall surfaces must be smooth
The vessels have no baffles, to minimize shear.
 In stirred tank bioreactor operating at low densities,
sufficient oxygen transfer for dissolved oxygen was control
by regulating the oxygen partial pressure in the bioreactor
using a nitrogen and air gas mixture.
 As growth proceeds, this may be suplemented by
additional oxygen by the direct sparging. The head space
usually contain CO2 as a means of regulating pH.
 This means that control of dissolved oxygen and pH are
strongly important and can lead to contradictory
requirements.
 Increase in supply of CO2 at high pH value=lower partial
pressure of O2 and lower oxygen supply rate
Stirred tank unit
 Airlift bioreactor are attractive for animal cell culture:
Reduce the contamination-absence of any moving
parts or mechanical seals.
Shear conditions is low
Possibility to control dissolved oxygen and CO2 by
varying the rate and composition of the gas flow
through the bioreactor.
Immobilized cell systems
Immobilization Technique
 An immobilized enzyme is an enzyme which is attached to an
inert, insoluble material such as sodium alginate. This can
provide increased resistance to changes in conditions such as
pH or temperature. It also allows enzymes to be held in place
throughout the reaction, following which they are easily
separated from the products and may be used again
 It is well known that pure enzymes change their behavior, their
stability, when they are immobilized. In the past two decades
the immobilization of microorganisms, cells and parts of cells
was gradually introduced into microbiology and biotechnology.
 The immobilized whole cell system is an alternative to enzyme
immobilization. Unlike enzyme immobilization, where the
enzyme is attached to a substrate (such as calcium alginate), in
immobilized whole cell systems, the target cell is immobilized
 According to A.H. Scragg (1988) many of the cell
immobilization techniques are modifications of the
techniques developed for enzymes. However, the larger
size of microbes has influenced the techniques
 In industrial , methods for cell immobilization are
categories under two broad categories that is :
i) immobilized-“free” cell method
ii) modified “cell” method
 In the free-cell method, cells are immobilized by
confining them behind dialysis/ filtration membranes.
Examples are hollow fibre, membrane sheet reactor,
ceramic matrix, encapsulated and entrapped cell
system.
 In modified cell system, examples are show in table 1
Immobilized cell reactor develop for
 To provide systems of high cell density
 Continual harvesting of product can be obtained at high
productivity when combined high throughput perfusion
system
 Design to minimize problems of nutrient and waste
product diffusion.
Hollow
Fiber
Reactor
System
 Cells grow in the extracapillary space of a hollow
cartridge but with transfer of oxygen
 removal of waste products occurring via the combination
of a cell exclusion membrane
 product exclusion or ultrafiltration membrane with a cutoff chosen to exclude, but passing low-molecular weight
metabolites.
 The cell growth is perfused directly by medium.
 A highly oxygenated medium stream on the other side of
this membrane provides sufficient oxygen transfer.
 PH control is effected by a secondary loop of medium on
the product exclusion membrane side.
Stacked Membranes
With Support
System
 Similar principle was proposed but comprising
stacked membranes with support.
 Alternate microfiltration and ultrafiltration
membranes provide a system of a separate chamber,
which contain medium, cells and product respectively.
 The cells are immobilized in a wire net matrix
between the differing membrane sheets, from the
medium chamber, are fed by nutrient and oxygen
diffusing via the ultrafiltration membrane
High molecular- weight products similarly diffuse via
the microfiltration membrane into the product number
The factor need to consider when using cell
immobilization technique:
 Choice of matrix material
 Special sensitivity of the cells to environmental shock
 Natural material such as gelatine, alginate, hydrogel, and
agrose are generally preferred.
Crossflow
consumables
To remove mammalian
cells from liquid.