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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