Suspension Culture

Cells in suspension cultures receive more homogeneous stimuli in a defined medium supplemented with the requisite
amount of inducers such as sugar or auxin. Theoretically, the percentage and synchrony of differentiation may be expected
to be higher in cells of a suspension than in a callus culture system. Also, it may be possible to analyse biochemical
pathways related to differentiation of cells in suspension cultures. For example, enzymes regulating lignin synthesis in
tobacco suspension cultures showed that enzymes in the shikimate and cinnamate pathways were co-ordinately enhanced
during tracheary element differentiation (Kuboi and Yamada 1978a,b). Similarly, phenylalanine ammonia lyase and
peroxidase were found associated with differentiation in bean and pea cultures.

CELL SUSPENSION CULTURE

Cell Suspension Culture
Introduction

Cell suspension culture is an advanced form of plant tissue culture where plant cells are
maintained and grown in a liquid medium under continuous agitation. The technique is
usually initiated from friable callus tissue and is considered a vital method for studying cell
biology, large-scale metabolite production, and genetic manipulations in plants (Razdan,
2003).

Initiation of Suspension Cultures

Suspension cultures are initiated by transferring small friable pieces of callus into a liquid
medium. Since friable callus disintegrates easily into single cells and small aggregates, it
serves as an ideal explant source. The culture is then kept under agitation on a rotary shaker
at a speed of 80–120 RPM to ensure aeration, nutrient distribution, and to prevent cell
settling.

Growth Pattern of Suspension Cultures

According to Razdan, suspension cultures show a sigmoid growth curve similar to microbial
cultures, consisting of:

1. Lag Phase – cells adapt to the medium.

2. Exponential/Log Phase – active cell division occurs; maximum viability.

3. Stationary Phase – nutrient depletion and metabolite accumulation slow down cell
division.

4. Decline Phase – cell death begins due to toxic product accumulation and nutrient
exhaustion.

Subculture and Maintenance

Suspension cultures require regular subculturing to maintain viability. Typically, an actively

growing suspension is transferred to fresh liquid medium at defined intervals (7–14 days
depending on species). A suitable inoculum density is essential for continuous growth, as
too low density may cause culture failure.

Advantages of Cell Suspension Culture

1. Provides a homogeneous population of cells for experimental studies.

 2. Enables large-scale production of secondary metabolites, alkaloids, pigments, and
pharmaceuticals (e.g., shikonin, berberine, taxol).

3. Facilitates isolation of protoplasts and subsequent somatic hybridization.

4. Offers a suitable system for cell selection under stress or mutagenic agents.

5. Useful in studying cellular totipotency and differentiation under controlled conditions.

Factors Affecting Suspension Culture Growth

● Medium composition – requires macronutrients, micronutrients, vitamins, and plant

growth regulators (auxins and cytokinins).

● Shaker speed – optimum 80–120 RPM for proper aeration.

● Inoculum size and density – essential for sustained growth.

● Light conditions – most cultures are maintained in the dark to prevent differentiation,
though some may require light.

● pH of the medium – typically adjusted to 5.6–5.8 before sterilization.

Commercial Applications

Cell suspension cultures are extensively used in biotechnology for:

● Production of secondary metabolites (e.g., quinine, ajmalicine, vincristine, and

vinblastine).

● Biotransformation – converting precursors into valuable compounds.

● Genetic manipulation studies including transformation, mutagenesis, and selection.

● Synthetic seed production through somatic embryogenesis in suspensions.

Conclusion

Cell suspension culture represents a powerful technique in plant biotechnology, bridging

fundamental research with industrial applications. By providing a uniform system of single
cells in liquid medium, it not only advances our understanding of plant cell physiology but
also serves as a foundation for commercial metabolite production and crop improvement
programs.
 DEFINITION

🞿Suspension culture is a type of culture in which single cell or

small aggregates of cells multiply while suspended in agitated
liquid medium .
🞿It is also referred to as cell culture or cell suspension culture

🞿The cell suspension culture requires optimization of the cell

line, the cultivation media and the bioreactor system.
 SHORT HISTORY
• W H Muir 1953---First Reported Fragments of
Callus could be cultured in the form of Cell
Suspension.
• Nickell – 1956---First report of a continuously
maintained cell suspension culture, Phaseolus
vulgaris
• F C Steward & E M Shantz 1956--- Suspension
Culture from Root & obtained very large number
of Plantlets.
 TYPE OF CELL SUSPENSION CULTURES

• There are two type of cell suspension cultures :

1. Batch Culture

2. Continuous culture
 BATCH CULTURE

🞿A batch culture is a cell suspension culture grown in a fixed

volume of nutrient culture medium.
🞿Cell suspension increases in biomass by cell division and
cell growth until a factor in the culture environnent (nutrient
or oxygen availability) becomes limiting and the growth
ceases.
The cells in culture exhibit the following five phases of a growth
cycle
1. Lag phase : where cells prepare to divide.
2. Exponential phase : where the rate of cell division is highest.
3. Linear phase : where cell division slows but the rate of cells
expansion increases.
4. Deceleration phase : where the rates of cell division and
elongation decreases.
5. Stationary phase : where the number and size of cells remain
constant.
 CONTINUOUS CULTURE

• A culture is continuously supplied with nutrients by the inflow

of fresh medium but the culture volume is normally constant.
• Continuous culture is further divided into two types :
1. Close continuous culture
2. Open continuous culture
 Open continuous culture

• In open continuous culture both the cells and used medium

are replaced with fresh medium thus maintaining culture at
constant and sub maximal growth rate.
• Open continuous cell suspension culture is of two types

1. Chemostat
2. Turbidostats
 CHEMOSTAT
• In this system , culture vessels
are usually cylindrical or
circular in shape and possess
inlet and outlet pores for
aeration and the introduction
and removal of cells and
medium.
• Thus in a steady state condition
the density, growth rate ,
chemical composition and
metabolic activity of the cells
all remain constant.
 TURBIDOSTATS
• A turbidostat is a continuous
culturing method where the
turbidity of the culture is held
constant by manipulating the
• Inrate
thisatsystem
which ,medium
the cellsisare
fed.
allowed to grow upto a certain
turbidity, when the predetermine
d volume of culture is replaced
by fresh culture.
• the turbidity is measured by the
changes of optical density of
medium.
Comparison of CHEMOSTAT AND TURBIDOSTAT
 PRINCIPLE
🞿 Callus proliferates as an unorganized mass of cells. So it is very difficult to follow
many cellular events during its growth and development phases.
🞿 To overcome such limitations of callus culture, the cultivation of free cells as well
as small cell aggregates in a chemically defined liquid medium as a suspension was
initiated to study the morphological and biochemical changes during their growth and
development phases.
🞿 To achieve an ideal cell suspension, most commonly a friable callus is transferred
to agitated liquid medium where it breaks up and readily disperses.
🞿 After eliminating the large callus pieces, only single cells and small cell aggregates
are again transferred to fresh medium and after two or three weeks a suspension of
actively growing cells is produced.
🞿 This suspension can then be propagated by regular sub-culture of an aliquot to
fresh medium. Ideally suspension culture should consist of only single cells which
are physiologically and biochemically uniform.
PROTOCOL
 PROTOCOL
1. Ta ke 150/250 ml conical flask containing autoclaved 40-60 ml liquid medium.
2. Transfer 3-4 pieces of pre-established callus tissue (approx. wt. 1 gm. each) from
the culture tube using the spoon headed spatula to conical flasks.

3. Flame the neck of conical flask, close the mouth of the flask with a piece of
aluminum foil or a cotton plug. Cover the closure with a piece of brown paper.
4. Place the flasks within the clamps of a rotary shaker moving at the 80-120 rpm
(revolution per minute)
5. After 7 days, pour the contents of each flask through the sterilized sieve pore
diameter -60µ-100µ and collect the filtrate in a big sterilized container. The filtrate
contains only free cells and cell aggregates.

6. Allow the filtrate to settle for 10-15 min. or centrifuge the filtrate at 500 to 1,000
rpm and finally pour off the supernatant.
7. Re-suspend the residue cells in a requisite volume of fresh liquid medium and
dispense the cell suspension equally in several sterilized flasks (150-250 ml). Place the
flasks on shaker and allow the free cells and cell aggregates to grow.
8. At the next subculture, repeat the previous steps but take only one-fifth of the
residual cells as the inoculum and dispense equally in flasks and again place them on
shaker.

9. After 3-4 subcultures, transfer 10 ml of cell suspension from each flask into new
flask containing 30 ml fresh liquid medium.
10. To prepare a growth curve of cells in suspension, transfer a definite number of cells
measured accurately by a haemocytometer to a definite volume of liquid medium and
incubates on shaker.
• Pipette out very little aliquot of cell suspension at short intervals of time (1 or 2
days interval) and count the cell number. Plot the cell count data of a passage on a
graph paper and the curve will indicate the growth pattern of suspension culture.
 Synchronization of Culture
Synchronous Culture: Majority of cells proceed through each cell cycle event
simultaneously. Percentage synchrony- degree of synchrony
Depends upon :
[Link] of cells at a specific point in the cycle at one moment in time
[Link] of cells passing a specific point in the cycle during a brief specific
period
[Link] of total time required for all the cells to pass a specific point in the
cycle
Methods:
[Link]: Cells arrested at initial lag phase by starving them of a nutrient or
hormonal factor
[Link]: 5-aminouracil, hydroxyurea and thymidine – inhibitors of DNA
synthesis
Cell cycle proceeds only upto G1 phase and cells are collected at G1/S boundry.
Removal of inhibitor is followed by synchronous division of cells
Limited to only one cell cycle.
 IMPORTANCE OF CELL SUSPENSION CULTURE
This system is capable of contributing many significant information’s about cell
physiology, biochemistry, metabolic events at the level of individual cells and small
cell aggregates.
It is also important to build up an understanding of an organ formation or embryoid
where attempts are being made to obtain single cell clones.
Suspension culture derived from medicinally important plants can be studied for the
production of secondary metabolites such as alkaloids formation starting from single
cell or small cell aggregates.
The technique of plating out cell suspension on agar plates is of particular value
Mutagenesis studies may be facilitated by the use of cell suspension cultures to
produce mutant cell clones from which mutant plants can be raised. Cell population
in a suspension can be subjected to a range of mutagenic chemicals e.g. ethyl
methane-sulphonate (EMS), N-nitroso-N- methyl urea, etc.
 Disadvantages

• The productivity of suspension cultures decreases over

extended subculture periods.
• Slow growth and low productivity of plant cells.
• Cells may get damaged by shear conditions.