scaling up animal cell culture chapter 9 from ‘the basics’ = chapter 26 from ‘culture of...
TRANSCRIPT
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
replicatesDetermination of concentration-dependent
effectsMulti-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 aerationStirring speed is low – rotation of a
suspended bar by a magnetic stirrer
- are not suitable in larger volumesImpellers – vertical and horizontal movement
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)
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 additionNet acidic production (lactic acid) from
cellular metabolism – alkali (NaHCO3)HCl is added Computer-controlled pump or gas valve to a
pre-set pH valueRotameters 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 culturesCell damage is caused – bubble burstingLeads to foamingAlternative 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 fermenterConsists 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 mediumGlass bead column, ceramic and fluidized-
bed 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 bioreactorSeries 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 CubeStack 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 reactorUpward flow of
medium recirculated by pumping
Cells immobilized or entrapped in beads are held in suspension in column
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