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BIOREACTORS FOR ANIMAL CELL SUSPENSION CULTURE

GRACE FELCIYA S.JFIRST 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 CELLSExcise 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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STIRRED TANK BIOREACTORS

AIRLIFT FERMENTORS

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

Present Model

Original Model

SPINNER FLASKS

Advantages of Spinner Flasks Easy Visible Cheap Depyrogenation feasible

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

Wave Bioreactor

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Wave-induced Agitation

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ADVANTAGE OF WAVE BIOREACTORDisposable 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 settlersIncline settlersLamellar settlers

• Centrifugation• Spin filters

ExternalInternal

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PERFUSION CULTURE

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ADVANTAGES AND DISADVANTAGES

Advantages of Perfusion Technology

• Better economics• High cell density• High productivity• Longer operation duration• Small fermenter size• flexibility• Fast start up in process

development• Constant nutrient supply• Better controlled culture

environment• Steady state operation• Ease of control• Better product quality

Disadvantages of Perfusion Technology

• Contamination risk• Equipment failure• Increased analytical costs• Long validation time• Potential

regulatory/licensing issues

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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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“Bioprocess engineering: basic concepts” 2nd

edition by Shuler M.L and Kargi F

“Animal Biotechnology “ by M.M Ranga

REFERENCES

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Thank you