Cell suspension culture

Cell suspension culture

A suspension culture consists of single cells, small cell groups and larger cell aggregates dispersed in a liquid media actively

growing under agitation and aeration.

Callus culture and suspension culture of Rauvolfia serpentina

• Under incubation conditions, cells divide and there is an increase in material. Such a suspension can be sub-cultured by pipetting out and adding to fresh medium when growth is resumed.

• The cultures are continuously propagated at regular intervals showing similar pattern of growth and yield of cell content.

Isolation of single cell

Single cells can be isolated from • plant organs, • by mechanical method • by enzymatic method. • From cultured tissue From plant organ The most suitable material is leaf tissue Mechanical isolation: leaves are cut into small (1 cm2) pieces, ground in a suitable culture medium using pestle and mortar.

Enzymatic method: lower epidermis of leaves is peeled off and the leaves are cut into moderate pieces (4 cm2), which are incubated in a macerozyme (0.5%) or pectinase solution.

• Partial vacuum may be used to facilitate the entry of enzyme solution into the tissue,

• The enzyme treatment tends to weaken cell walls; so a suitable osmoticum (e.g., 0.3 M mannitol) is added to the enzyme and culture medium solutions.

From cultured tissue:

Freshly cut pieces from surface sterilized plants are simply placed on culture medium consisting of a suitable proportions of cytokinins and auxins to initiate culture.

The callus is separated from an explant and transferred to a fresh medium of the same composition.

Types of suspension cultures:

There are two types of suspension culture. A-Batch culture B-Continous culture 3.1 Batch culture: • Slowly rotating culture • Shake culture • Spinning culture • Stirred culture 3.2-Continous culture • Chemostats • Turbidostats

Batch culture • Suspension culture in which cell material grows in a

finite volume of agitated liquid medium. • A small aliquot of inoculum in the moving liquid

medium and transferring it to fresh medium (5x dilution) at regular intervals.

• Batch suspension cultures maintained in conical flasks incubated on orbital platform shakers at the speed of 80-120 rpm.

• When the cell number in CSC. is plotted against the time of incubation, growth curve showing initially the culture passes through a

• Lag phase growth phase stationary phase

Batch culture

Slowly rotating cultures:

• Single cells and cell agg. are grown in a specially designed flask, the nipple flask.

• Each nipple flask possesses eight nipple-like projections. The capacity of each flask is 250 ml. Ten flasks are loaded in a circular manner on a large flat disc of a vertical shaker. When the flat disc rotates at the speed of 1-2 rpm, the cell within each nipple of the flask are alternatively bathed in a culture medium and exposed to air.

Slowly rotating culture

Shake culture

• Single cells and cell aggregates in fixed volume of liquid medium are placed in conical flask.

• Conical flasks are mounted with the help of clip on a horizontal large square plate of an orbital platform shaker. The square plate moves by a circular motion at 60-180 rpm.

Spinning culture

• Large volume of cell suspension may be cultured in 10L,bottles which are rotated in a culture spinner at 120 rpm at an angle of 450.

Stirred culture

• The cell suspension inside the vessel is kept dispersed continuously by bubbling sterile air through culture medium.

• The use of an internal magnetic stirrer is the most convenient way to agitate the culture medium safely.

• Magnetic stirrer revolves at 200-600 rpm. The culture vessel is a 5-10 litres round bottom flask

Agitation of the medium

• It can be achieved using a shaker or suitable flask.

• Muir (1953) was the first to introduce the orbital platform shaker for growing suspension cultures of tobacco and tagetes erecta.

• shaking speed of 30-150rpm is optimum for most of the tissues.

• Rotary shakers are also used which have a disc that can be rotated at slow speed (1-2 rpm) a shaft.

• When the disc rotates , the cells and tissue are alternately bathed in the culture medium and there by exposed to both nutrients and the culture air.

shaker

2-Continuous culture

• The old liquid medium is continuously replaced by the fresh liquid medium to stabilize the physiological stage of the growing cells. Normally, the liquid medium is not changed until the depletion of some nutrients in the medium .

• As a result, the active growth phase of the cell declines the depletion of nutrient. The cells passing through out flowing medium separated mechanically and reintroduced in the culture.

Types of continous culture:

• chemostats

• turbidostats

Chemo stats • Cultures vessels are generally cylindrical or circular in shape

and posses inlet and outlet pores.

• The liquid medium contains the cell is stirred by a magnetic stirrer. The introduction of fresh sterile med. which is pumped in at a constant rate into the vessel is balanced by the displacement of an equal volume of spent or old medium and cells.

• Such a system can be maintained in a steady state so that new cells are produced by division at a rate which compensate the number lost in outflow of spent medium.

Turbidostats

• A turbidostat is a continuous microbiological culture device, similar to a chemostat, which has feedback between the turbidity of the culture vessel and the dilution rate.

• The changes in turbidity of the culture medium can be measured by the changes of optical density of the medium.

• In Turbidostats an automatic monitoring unit is connected with the culture vessel and such unit adjusts the medium flow in such a way as to maintain the optical density or PH at chosen, present level.

Media for suspension culture • Manipulations in the media constitutes and subculture

routine may help in tissue dissociation although the addition to the medium of 2,4-D,small amounts of hydrolytic enzymes (cellolase and pectinase),or substances such as yeast extract.

• Good cell dissociation may also achieved…… the late lag phase by adding fresh medium every other day.

• Culture medium for tobacco cell suspension requires an increase in concentration of 2,4-D from 0.3 to 2mg l-1 ,followed by supplementing with additional vitamins and casein hydrolysate. Sometimes inorganic phosphate rapidly utilized.

Synchronization of culture

• The cell cycle time varies considerably with in individual cells. Therefore cell cultures are mostly asynchronous.

• Hence it is essential to manipulate the growth conditions in asynchronous suspension cultures in order to achieve high degree of synchronization.

• Synchronous culture: A synchronous culture is one in which the majority of cells proceed through each cell cycle phase (G1 ,S, G2 and M ) simultaneously

Measurement of growth

Assessment of the growth in suspension cultures can be accomplished by following,these include,

• Cell counting

• Packed cell volume

• Fresh/dry weight increase of cells and cell colonies

Cell counting

• Before cell counting, the cell clusters are dissociated into individual cell components by gentle treatment with chromic acid heated to 70 C for 5-10 min, cooled and vigorously shaken for effective cell separation.

• The suspension is now centrifuged, the chromic acid poured off and the pallet resuspended in 8% saline (NaCl) solution. After 10-15 minutes free cells are counted on a haemocytometer. Heating is avoided if an enzyme is used to disrupt cell aggregates.

Packed cell volume: transferred to a 15 ml conical graduated centrifuge tube and spinned at 1000 x g for 5 minutes. The PCV is expressed as ml cell pellet per culture. Cell fresh weigh A large sample of cells is collected in a wet condition on pre-weighed nylon fabric in a funnel and washed with sterile distilled water to eliminate the medium, then drained under vacuum and then reweighed along the cells. dry cell weight: A procedure similar to that for fresh weights is followed for determining cell dry weight except that the filter discs are dried in an oven for 12 hr at 60oC. After cooling in a desiccator containing silica gel, the dried filter is reweighed and the cell weight expressed as g ml-1 of culture or per 106 cells

Applications of cell suspension culture

• Ideal to study various factors and compounds that affect growth and differentiation.

• Most of the cells are in direct contact with the medium and therefore the effects of concentration gradients are avoided.

• Rapid preparation of protoplast in high yield. • The formation of somatic embryos in suspension cultures is ideal

for the large scale production of commercial plants. • As a production system for plant derived chemicals and

recombinant pharmaceutical proteins. • In vitro mutation studies • As a source for extraction of cellular enzymes and many secondary

metabolites. • Any Useful compounds could be produced under controlled

conditions and geared more accurately to meet market demand