BIOREACTORS FOR ANIMAL CELL

SUSPENSION CULTURE

GRACE FELCIYA S.J

FIRST YEAR
M.TECH - BIOPHARMACEUTICAL TECHNOLOGY

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 INTRODUCTION
 Animal cell culture is the process of culturing animal cells outside
the tissue (in vitro) .It will continue to grow if supplied with
appropriate conditions and nutrients

 The culture process allow single cells to act as independent units,

much like bacterium or fungus

 The cells are capable of dividing

 Animal cell culture was successfully established in 1907

 First, the development of antibiotics to avoid contamination that

plagued earlier cell culture attempts. Second, the development of
the technique. Third, development of culture media.
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 TYPES OF CULTURE
Primary culture
 explant culture
 enzymatic dissociation culture
Secondary culture
 monolayer culture/ anchorage dependent culture
 suspension culture/ non anchorage dependent culture

Primary culture – cells were directly taken from the tissues.

Secondary culture – derived from the primary culture

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 TECHNIQUE OF CULTIVATING ANIMAL CELLS
Excise tissues from specific organ of animals (lung, kidney) under aseptic conditions.

Transfer tissues into a growth medium containing serum and antibiotics in small T-flasks.

These cells form a primary culture that usually attach onto the glass surface of flask in
monolayer form.
The cells growing on support surfaces are known as anchorage-dependent cells.

Some cells grown in suspension culture and are known to be non anchorage-dependent
cells.
Then a cell line appear from the primary culture and known as secondary culture.

Remove cell from the surface of flasks using trypsin and add serum to the culture bottle.

The serum containing suspension is then use to inoculate secondary cultures.

Many secondary lines can be adapted to grow in suspension and are non anchorage
dependent.
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SUSPENSION CULTURE SYSTEM/NON ANCHORAGE
DEPENDENT CULTURE

The cells are grown either :

a)In magnetically rotated spinner flasks or shaken flasks where the

cells are kept actively suspended in the medium
b)In stationary culture vessels such as T-flasks and bottles where
unable to attach firmly to the substrate
c)In mass cultivation, animal cells are grown in bioreactors almost
similar to plant cells.

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 BIOREACTOR CONSIDERATIONS FOR
ANIMAL CELL SUSPENSION CULTURES

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Properties of animal cell that set constrains on design of animal cell bioreactor

- cell are large (10-20µm)

- more fragile

- grow more slowly than most bacteria and fungi

- toxic metabolites eg ammonium & lactate produced during growth

Common features of animal cell bioreactor:

1) Reactor should be gently agitated and aerated. Agitation speed ≈20rpm.Bubble-

column & airlift reactor operating at high aeration may cause damage of cells

2) Supply of CO2-enriched air

3) Removal of toxic products from metabolism eg lactic acid, ammonium

Require gentler culture condition and control systems that are optimized for lower
metabolic rates.
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REQUIREMENTS FOR A BIOREACTOR FOR ANIMAL CELL
CULTURE

1) well-controlled environment (T, pH, DO, nutrients, and wastes)

2) supply of nutrients

3) gentle mixing (avoid shear damage to cells)

4) gentle aeration (add oxygen slowly to the culture medium, but

avoid the formation of large bubbles which can damage cells on
contact).

5) removal of wastes

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REACTORS USED FOR SUSPENSION CULTURE
The reactors used for large-scale suspension cultures are of 3 main
types.
–Stirred bioreactors
–Continuous flow reactors
–Airlift fermentors
• Tissue flasks
– Easy to use for small scale
• Cell factories
– Production of large numbers of cells
– Labor intensive
• Roller bottles
– Good control of gas phase
– Labor intensive
• Hollow fiber systems
– High cell densities, good oxygenation
– Difficult to remove cells
• Spinner flasks
– Mimic a traditional stirred tank reactor
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STIRRED TANK REACTORS
 Single use
 Membrane bioreactors

CONTINUOUS FLOW REACTORS

Continuous flow reactor technology can produce a cheaper better quality product at
reduced energy and environmental cost through:
• Optimum mixing (efficient mass transfer)
• Optimum temperature control (efficient heat transfer)
• The application of the ‘scaling out’ approach (as opposed to the traditional ‘scale
up’ approach)

AIRLIFT FERMENTORS
 Air driven
 External and internal loop system
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 AIRLIFT FERMENTORS

STIRRED TANK BIOREACTORS

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 SPINNER FLASKS
McLimans' group developed the first "spinner flasks" in 1957.

Advantages of Spinner Flasks

 Easy
 Visible
 Cheap
 Depyrogenation feasible Original Model

Disadvantages of Spinner Flasks

 Poor aeration
 Impeller jams
 Requires cleaning siliconizing &
sterilization
 High space requirements in
incubator

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 DISPOSABLE BIOREACTOR
• Can be scaled to at least 500 liters

• A non-invasive agitation mechanism

• Easy to use

• Disposable, presterile, and biocompatible

• Well instrumented, and can be sampled

• Useful for suspension and adherent culture

• Suitable for GMP operation

WAVE BIOREACTOR

• Wave induced agitation is the principle

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Wave Bioreactor
 Wave-induced Agitation

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ADVANTAGE OF WAVE BIOREACTOR
Disposable bioreactor chamber-no cross-contamination, cleaning, sterilization or other
validation headaches.

Seed preparation-Seed culture can be prepared in the final system itself, i.E. Batch can be
started with 100ml and can go to 2000ml.

Maintain quality of cells- Lack of bubbles and mechanical devices

Scalable to 500 liters

Completely closed system-ideal for cell culture, GMP operations.

Operates with or without an incubator

Proven for gmp operations-used in the gmp production of human therapeutics. Closed system
is easy to validate. All contact materials are FDA approved.

Perfusion culture option-patented internal perfusion filters enable perfusion of media for high-
density cell culture.

Easy to operate-no complex piping or sterilization sequences. Simply place a new presterile
cellbag on the rocker; fill with media, and add your cells
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 PERFUSION CULTURE
– Characterized by the continuous addition of fresh nutrient
medium and the withdrawal of an equal volume of used
medium.

• Need of perfusion
– Product is unstable
– Product concentration is low
• Perfusion technologies
• Enhanced sedimentation
Conical settlers
Incline settlers
Lamellar settlers
• Centrifugation
• Spin filters
External
Internal
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 PERFUSION CULTURE

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 ADVANTAGES AND DISADVANTAGES

Advantages of Perfusion Disadvantages of Perfusion

Technology Technology
• Better economics • Contamination risk
• High cell density
• Equipment failure
• High productivity
• Longer operation duration • Increased analytical costs
• Small fermenter size • Long validation time
• flexibility
• Potential
• Fast start up in process
development regulatory/licensing issues
• Constant nutrient supply
• Better controlled culture
environment
• Steady state operation
• Ease of control
• Better product quality
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 Tissue culture flasks (T-flasks)

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 HOLLOW FIBER BIOREACTOR
• Intraluminal (Cells inside fibers )
• Extraluminal (Cells outside fibers)
• Fibers are made of a porous material (PTFE and others).
• Permits movement of small molecules (O2, glucose), but not cells

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 REFERENCES

“Bioprocess engineering: basic concepts” 2nd

edition by Shuler M.L and Kargi F
“Animal Biotechnology “ by M.M Ranga

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 Thank you

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