Scaling up animal cell culture by hcj


									    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
Determination of concentration-dependent
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
          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

           Choice of Bioreactor

Equipment designed to grow cells in culture

Type and design of bioreactor and mode of

- 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
   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
Parameter – pH control

Optimal pH - 7.4 – for maximum growth

Enriched Co2 atmosphere decreases pH

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
     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
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
 Fluid circulation is
  provided by stream of
 Less bubbling or
 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
 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
   This project is funded by a grant awarded under the President’s Community Based Job Training Grant as
    implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60).
    NCC is an equal opportunity employer and does not discriminate on the following basis:
   against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability,
    political affiliation or belief; and
   against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998
    (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in
    the United States, or his or her participation in any WIA Title I-financially assisted program or activity.
 This workforce solution was funded by a grant awarded under the
  President’s Community-Based Job Training Grants as implemented
  by the U.S. Department of Labor’s Employment and Training
  Administration. The solution was created by the grantee and does
  not necessarily reflect the official position of the U.S. Department of
  Labor. The Department of Labor makes no guarantees, warranties,
  or assurances of any kind, express or implied, with respect to such
  information, including any information on linked sites and including,
  but not limited to, accuracy of the information or its completeness,
  timeliness, usefulness, adequacy, continued availability, or
  ownership. This solution is copyrighted by the institution that
  created it. Internal use by an organization and/or personal use by
  an individual for non-commercial purposes is permissible. All other
  uses require the prior authorization of the copyright owner.

To top