Scaling up animal cell culture

Chapter 9 from The Basics = Chapter 26 from

Culture of Animal Cell Culture

Why scale-up cultures?

What are small cultures?
Experiments requiring large number of
replicates
Determination of concentration-dependent
effects
Multi-well plates accommodate culture
volumes 2-3mls

What are small cultures?

Experiments require multiple samples
- To observe cell growth and to perform
substrate or product assays
- Culture volumes 100 ml
- Conducted in T-flasks or spinner flasks in
Incubator

Why scale-up cultures?

Required to produce substantial quantities of
a cell product such as virus or glycoprotein
or enzymes
Two approaches
- A Multiple process involving 1000 culture
flasks (100 ml)
- A Unit process involving 100-liter fermenter

Factors to control in Fermenters/Bioreactors?

Commercial Cultures 1 5 liters

Oxygen supply, temperature control, pH
control and culture mixing
Electrodes

Choice of Bioreactor
Equipment designed to grow cells in culture
Type and design of bioreactor and mode of
operation
- Depends on cell densities and productivity

The Stirred tank reactor (STR)

Simplest and most
widely used
Consists of cylindrical
vessel with a stirrer (a
pot and paddle)
Designed differently for
animal cells and for
bacterial or fungal
cultures

Bioreactors

The Stirred tank reactor (STR)

Large-scale animal cell culture processes
10,000 liters
- Stainless steel
Bench-top STRs (1-5 liters)
- Glass

What are the parameters that control

adequate culture growth?
Agitation
Bubble bursting on culture surface resulting
from culture aeration
Stirring speed is low rotation of a
suspended bar by a magnetic stirrer
- are not suitable in larger volumes
Impellers vertical and horizontal movement

Parameter - Agitation
Maximum stirring rates for suspension 100150 rpm
Microcarrier - < 40 rpm (suspension and
anchorage dependent cells)
Round bottom (animal cells) and flat
bottomed vessels (bacterial cells)

Parameter Temperature control

Thermocirculator pumps heated water
around an outer jacket
- Larger fermenters pumps water through
coiled pipes within culture
Circulating warm air
Low volume fermenters External heating
pads

Parameter pH control
Optimal pH - 7.4 for maximum growth
Enriched Co2 atmosphere decreases pH
fluctuations
1-litre culture @ 2x106 cells/ml @ gas flow
of 100 ml/min

Parameter pH control
Direct acid or alkali addition
Net acidic production (lactic acid) from
cellular metabolism alkali (NaHCO3)
HCl is added
Computer-controlled pump or gas valve to a
pre-set pH value
Rotameters indicate rate of gas flow
controlled by flow regulators (fig-9.6)

Parameter Oxygen supply

Major problem
Gas diffusion from head space through culture
surface decreases
Oxygen transfer rate (OTR) across liquid
surface > Oxygen utilization rate (OUR)

What is Sparging?

Common in bacterial cultures

Cell damage is caused bubble bursting
Leads to foaming
Alternative method Surround gas sparger by fine-mesh
cage

Parameter Oxygen supply

Control of oxygen supply by a sterilizable
oxygen probe
Solubility of oxygen in media can be
increased by addition of some liquid
perfluorocarbons

Indirect aeration Oxygen supply

Indirect aeration involves medium sparging
Suitable for media recirculation
Oxygen supplied by gas diffusion through
thin walled silicone tubing
1 meter of tubing/2 liters of culture
(adequate twining or binding)

Alternative types of Bioreactors

Airlift fermenter

Consists of tall column

with an inner draught
tube
Fluid circulation is
provided by stream of
air
Less bubbling or
foaming
Production of
monoclonal antibodies

Alternative types of Bioreactors

Hollow-fiber
Cartridge made up of
several thousand
minute capillary-like
plastic tubules (fibers)
with perfusable
membrane walls
Anchorage-dependent
and independent cells

Alternative types of Bioreactors

Packed-bed or fixed-bed bioreactor
Support matrix for attachment and growth of
anchorage dependent cells
Continuous flow of medium
Glass bead column, ceramic and fluidizedbed bioreactors

Alternative types of Bioreactors

Glass bead column
Glass column-glass
beads with a diameter
of 3-5 mm
Medium is
recirculated through
packed bed by pump
and oxygenated by air

Alternative types of Bioreactors

Ceramic bioreactor
Series of channels run
through ceramic
cylinder
Each channel is a square
with 1mm sides and an
inner surface area for
cell attachment
No longer in use

Alternative types of Bioreactors

Cell Cube
Stack of 20 cm2
polystrene plates
spaced 1mm apart by
rigid spacers
Cells attach to either
side of plate
Flow of culture
medium between plates
Production of vaccines

Alternative types of Bioreactors

Fluidized-bed reactor
Upward flow of
medium recirculated
by pumping
Cells immobilized or
entrapped in beads are
held in suspension in
column

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