Large Scale production of animal Cell

1. Scaling up animal cell culture
Chapter 9 from ‘The Basics’ = Chapter 26 from
‘Culture of Animal Cell Culture’

2. 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

3. 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

4. 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

5. Factors to control in Fermenters/Bioreactors?
Commercial Cultures 1 – 5 liters
Oxygen supply, temperature control, pH
control and culture mixing
Electrodes

6. 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

7. 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

8. Bioreactors

9. The Stirred tank reactor (STR)
Large-scale animal cell culture processes –
10,000 liters
- Stainless steel
Bench-top STRs (1-5 liters)
- Glass

11. 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

12. Parameter - Agitation
Maximum stirring rates for suspension – 100-
150 rpm
Microcarrier - < 40 rpm (suspension and
anchorage dependent cells)
Round bottom (animal cells) and flat
bottomed vessels (bacterial cells)

13. 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

14. 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

16. 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)

17. 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)

18. 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

19. 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

20. 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)

21. 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

22. 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

23. 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 fluidized-
bed bioreactors

24. 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

25. 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

26. 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

27. 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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