Scaling up animal cell culture

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					    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 – 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 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 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 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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