Types of Suspension Cultures
There are two types of suspension cultures, i) Bat culture ii) Continuous Culture
A) Batch Culture:
a. Slowly rotating culture
b. Shake culture
c. Spinning culture
d. Stirred culture
B) Continuous Culture:
A) Batch Culture:
These cultures are maintained continuously by propagating a small aliquot of inoculum in the moving liquid medium and transferring it to fresh medium ( 5 x dilution) at regular intervals. Generally cell suspensions are grown in flasks ( 100-250 ml) containing 25-75 ml of the culture medium. Batch suspension cultures are most commonly maintained in conical flasks incubated on orbital platform shakers at the speed of 80-120 rpm. The biomass growth in batch culture follows the fixed pattern. When the cell number in suspension cultures is plotted against the time of incubation, a growth curve is obtained depecting that initially the culture passes through a lag phase, followed by a brief exponential growth phase- the most fertile period for active cell division. The growth declines after three to four cell generations, signalling that the culture has entered the stationary phase.
For subculture, the flask containing suspension culture is allowed stand still for a few seconds to enable the large colonies to settle down. A pipette or syringe with orifice fine enough to hold aggregate of two to four cells or only single cells is used. The suspension is taken from the upper part of the culture and transferred to a fresh medium.
i) Slowly Rotating Cultures:
Single cells and cell aggregates 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 rp, the cell within each nipple of the flask are alternatively bathed in a culture medium and exposed to air.
ii) Shake Culture:
It is very simple and effective system of suspension culture. In this method, 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.
iii) 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 45 0.
iv) Stirred Culture:
This system is also used for large scale batch culture. In this method, the large culture vessel is not rotated but 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.
B) Continuous Culture System:
In this system, 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 and the cells are kept in the same medium for a certain period. As a result, the active growth phase of the cell declines the depletion of nutrient. The cells passing through out flowing medium are separated mechanically and reintroduced in the culture.
In this system, cultures vessels are generally cylindrical or circular in shape and posses inlet and outlet pores for aeration and for introduction of and removal of cells and medium. The liquid medium containing the cell is stirred by a magnetic stirrer. The introduction of fresh sterile medium, 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.
In this system, the input of medium is intermittent as it is mainly required to control the rise in turbidity due to cell growth. The turbidity of a suspension culture medium changes rapidly when cells increase in number due to their steady state growth. The changes in turbidity of the culture medium can be measured by the changes of optical density of the medium. In Turbostats 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.