Cell suspension culture involves growing single plant cells or small cell aggregates in agitated liquid medium. It allows studying cellular events during growth and development without the limitations of callus culture. An ideal suspension culture consists of only uniformly growing single cells. It is established by transferring friable callus pieces to agitated medium, then filtering and subculturing the dispersed cells. Suspension culture offers insights into cell physiology and is useful for cloning, secondary metabolite production, and mutagenesis studies. While it addresses issues with callus culture, cell suspension cultures can have decreasing productivity over time and slow growth.

1. CELL SUSPENSION CULTURE

2. 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.

3. 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.

4. TYPE OF CELL SUSPENSION CULTURES
• There are two type of cell suspension cultures :
1. Batch Culture
2. Continuous culture

5. 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.

6. 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.

8. 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

10. 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

11. 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.

12. TURBIDOSTATS
• A turbidostat is a continuous
culturing method where the
turbidity of the culture is held
constant by manipulating the
rate at which medium is fed.
• In this system , the cells are
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.

13. Comparison of CHEMOSTAT AND TURBIDOSTAT

17. 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.

18. PROTOCOL
1. Take 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.

19. 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.

21. 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
formation starting from single cell or small cell aggregates.
 The technique of plating out cell suspension on agar plates is of particular value
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
 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.

23. 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.

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