Suspension Culture

Content

• Suspension Culture

• Definition

• Types

• Apparatus used

Objective

At the end of the lecture the student will able to:

• Discuss the technique of suspension culture

• Discuss the various suspension culture apparatus

Suspension
Culture

• Type of culture –
single cell or small aggregates of cells multiply while suspended in agitated
liquid medium under aseptic conditions

• Cell suspension –
friable callus cells – liquid medium – agiated – rotary shaker

• Agitation of medium
is very important, it helps in two ways

i. Prevent the clumping or settling of cells – dispersion –
auxins or pectinase or cellulase enzyme

ii. Gaseous exchange, aeration

• Mainly of two types

Batch suspension culture and Continuous suspension culture

Batch suspension culture:

• Cell material or inoculums grow – finite volume of
agitated liquid medium

Continuous suspension
culture:

• Process in which spent medium is continuously replaced by
fresh medium in order to maintain steady growth

• Depletion of nutrients does not occur due to continuous
flow of fresh medium

Suspension culture apparatus:

i) Agitated liquid
culture apparatus

a. Shake
culture                   

b. Slowly
rotating culture (Auxophytons) – Tumble tube Nipple flask Spin culture

ii) Stationary liquid
culture apparatus – Chemostat and Turbidostat

Shake Culture:

• Agitation is provided by moving the liquid medium

• Consists a metal platform and number of clamps

• Clamps are used to hold container

• When platform shakes, it provides circular motion to
medium and leads to gaseous exchange or aeration

• Shaker speed – 30 – 150 rpm

Advantages: Large
amount of cells can be grown, various sized flasks can be used

Disadvantage: Sometimes
evaporation of medium due to high speed

Tumble tube:

• Consists of glass tube of capacity 10 ml with a neck at
the centre

• Neck is plugged with cotton (gaseous exchange)

• 24 such tubes are placed on a circular disc, 5-6 circular
discs are placed on a shaft

• When shaft rotates, tubes tumble, medium flows from one
end to another

• As the tube tumbles, explant alternatively exposed to
medium and air, which helps in gaseous exchange

• Large amount of cells cannot be grown

Nipple flask:

• Modified form of tumble tube

• Available in 2 sizes, 250 ml with 8 nipples and 1000 ml
with 10 nipples

• About 10 nipple flasks are placed on a circular disc

• When disc rotates, medium enters and leaves the
projection, explant alternatively exposed to medium and air Speed of rotation –
1-2 rpm

• Large amount of cells can be grown

Spin culture:

• Consists of bottle of capacity 10 Liter

• Mounted at an angle 450 in the spin

• Bottle spins because of the inclined angle

• As a result there is a movement of medium

• Movement leads to gaseous exchange

• Large amount of cells can be grown compare to tumble tube
and nipple flask

Chemostat:

• It is a continuous suspension culture

• Consists air inlet, air outlet, provision for continuous supply
of medium and removal of spent medium, and magnetic stirrer

• There is a fixed rate of media input and it is time
programmed

• A steady state is achieved by the addition of the medium

Turbidostat:

• It is also a continuous suspension culture

• Consists similar apparatus as that of chemostat along with
densitometer

• Turbidity of suspension culture medium increases with
increase in number of cells

• Turbidity is measured by measuring optical density using
densitometer

• Once turbidity reaches maximum level spent medium is
removed and fresh medium enters

Growth measurement:

• Cell count/cell number

• Cell viability

Packed cell volume:

• Suspension culture medium with cells is subjected to Centrifugation
at 2000 rpm for 10-15 min

• Cells settled down at the bottom         

• Volume of these settled cells is measured – Packed cell
volume           

Summary

• Tissue culture is growing of tissues or cells on a
suitable nutrient medium under aseptic conditions in vitro, may be plant or
animal tissue culture

• Types include callus, suspension, hairy root, single cell,
haploid, organogenesis, embryo culture etc

• Suspension culture is growing of cells/cell aggregates in
liquid media, batch or continuous

• Agitation is essential, helps in gaseous exchange

• Initiated using friable callus cells, sterile seedling,
embryo etc

• Agitated liquid apparatus like shake culture, tumble tube,
nipple flask, spin culture and stationary liquid culture like chemostat and
turbidostat

• Large amount of cells can be grown compare to callus
culture