2. CELL SUSPENSION
– A cell suspension culture consists of cell aggregates dispersed and growing in
moving liquid media.
– It is normally initiated by transferring pieces of undifferentiated and friable
calluses to a liquid medium, which is continuously agitated by a suitable device.
– Suspension cultures have also been started from sterile seedlings or imbibed
embryos or leaves by mechanical method.
Method;
– Leaves or the other tissue can be gently grinded or soft tissues can be broken up
in a hand operated glass homogenizer.
– This homogenate, containing intact living cells, dead cells and cell debris is cleared
by filtration and centrifugation and then transferred to moving liquid medium.
– An enzymatic method for isolation of single cells by the use of pectinases, which
digest the pectin wall, has also been employed.
3. • . On a medium the explant exhibits callusing, which is separated from the parent
explant and transferred to a fresh medium to build up reasonable amount of callus
tissue.
• This callus is transferred to a liquid medium and agitated to raise fine suspension of
cells. Successful establishment of the suspension culture depends upon the initial
callus.
• A wide variety of explants and media compositions have been used with success for
callus induction ---MS, B5, LS .
• . To these media are added vitamins, inositol, sucrose and growth regulators, especially
auxin (2,4-D at ~1–5 μM concentration), for cells to divide.
• The addition of 1 μM kinetin may be beneficial
• The culture medium for suspension culture should be such that it maintains good
growth of callus.
• Platform (orbital) shakers are widely used for the initiation and serial propagation of
plant cell suspension culture.
• They should have a variable speed control (30–150 rpm) and the stroke should be in
the range of 4–8 cm orbital motion. Agitation of medium on a shaker serves
4. • two purposes.
• First it exerts a mild pressure on cell aggregates, breaking them in to smaller clumps
and single cells and
• secondly agitation maintains uniform distribution of cell and cell clumps in the
medium.
• Movement of the medium also provides good gaseous exchange between the culture
medium and air.
• The shaker should be kept in the air-conditioned room with good temperature
control.
• Wide-mouthed Erlenmeyer flasks are widely used as culture vessels
5. TYPES OF SUSPENSION
CULTURES
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 (nutrient or oxygen availability) becomes limiting and the growth ceases. The cells
in culture exhibit the following five phases of a growth cycle).
i. Lag phase, where cells prepare to divide.
ii. Exponential phase, where the rate of cell
division is highest.
iii. Linear phase, where cell division slows but
the rate of cells expansion increases.
iv. Deceleration phase, where the rates of cell
division and elongation decreases.
v. Stationary phase, where the number and
size of cells remain constant.
6. Continuous culture
A culture is continuously supplied with nutrients by the inflow of fresh medium but the
culture volume is normally constant. It is of two types:
(i) Open continuous culture: A continuous culture is one in which inflow of fresh
medium
is balanced by outflow of corresponding volumes of culture including harvest of cells.
• Cells are constantly washed out with the outflowing liquid.
• The rate of inflow of medium and culture harvest are adjusted so that the cultures are
maintained indefinitely at a constant sub-maximal growth rate.
• In a steady state, the rate of cells washout equals the rate of formation of new cells in
the system.
• These open continuous systems may be:
(a) chemostats in which growth rate and cell density are held constant by a fixed rate of
input of a growth limiting nutrient medium (nitrogen, phosphorus or glucose).
7. (b) turbidostats in which fresh medium flows in response to increase in the turbidity
so as to maintain the culture at a fixed optical density of suspension. A pre-selected
biomass density
is maintained by the washout of cells.
(ii) Closed continuous culture: A closed culture is one in which cells are retained and
inflow of fresh medium is balanced by outflow of corresponding volumes of spent
medium only.
(iii) Semi Continuous Culture; In this type of culture the inflow of fresh medium is
manually controlled at infrequent intervals by “drain and refill” process, such that the
volume of
culture removed is always replaced by an equivalent volume of fresh medium.
8. GROWTH MEASUREMENTS
1. Cell number: This is an indispensable growth parameter for suspension cultures.
To count the number of cells in aggregates of cells, these are treated with chromium
trioxide.
2. Fresh weight: This can be determined by collecting cells on pre-weighed (in wet
condition) nylon fabric filters which can be supported in a funnel.
3. Dry weight/Packed cell volume (PCV): An appropriate volume of suspension culture is
centrifuged in a graduated conical centrifuge tube at 2000 × g for 5 min and pellet
volume
or packed cell volume is noted. PCV is expressed as ml pellet/ml culture
9. SYNCHRONIZATION OF SUSPENSION CULTURE CELLS
A synchronous culture is defined as a culture in which the cell cycles (or a specific
phase of the cycles) of a proportion of cells (often a majority) are synchronous, i.e.
majority of cells proceed through each cell cycle phase simultaneously.
The various methods employed to bring this synchronization have been listed
below.
1. Cold treatment: Temperature shocks at 4°C for few days can be used.
2. Starvation method: Deprivation of an essential growth compound from suspension
cultures leads to a stationary growth phase.
• Resupplying the missing compound or subculturing to a fresh complete medium will
allow growth to resume and may result in synchronization of cell growth.
3. Use of inhibitors: Synchronization is achieved by temporarily blocking the
progression of events in the cell cycle by addition of DNA synthesis inhibitors and
accumulating cells in a specific stage.
10. 4. Colchicine method: Colchicine is one of the most effective spindle fiber inhibitors,
which
arrests the cells at metaphase stage of cell division.
BERGMANN CELL PLATING TECHNIQUE
• Bergmann (1960) first used the most populartechnique of plating out cell
on agar plates.
• This technique is particularly useful where attempts are being made to obtain
cell
• clones. The objective is to establish evenly distributed suspension in a thin layer
culture
• medium with as low a cellular density as is compatible with the growth of
from a high proportion of the cellular units of single cells and cell aggregates .
• The basic technique of plating is to first count the cell number measurements.
• This will enable a known number of cell units to be established per unit
11. • volume of plating media. The counted suspension is adjusted by dilution or
concentrated by low speed centrifugation.
• Method;
• Both the suspension and nutrient medium containing agar are prepared in double the
concentration separately.
• The equal volumes of suspension and the agar medium cooled at 35°C are mixed and
then dispensed rapidly into petri dishes in such a manner that cells are evenly
distributed in a thin layer (~1 mm thick).
• The suspension culture, which carries cell aggregates should be filtered through a
sieve and only the fine suspension should be plated.
• The number of cellular units in per square mm are directly counted in situ under
inverted microscope.
• The dishes are then sealed and incubated at 25°C for 21 days.
• After incubation, the number of colonies per plate are counted by making
shadowgraph prints of each plate by resting the plate on a sheet of photographic
document paper under a photographic enlarger as the light source.
