2 Main Types of Suspension Culture | Biotechnology

The following points highlight the two main types of suspension culture. The types are: 1. Batch Culture 2. Continuous Culture. 

Type # 1. 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 cultural environment becomes limiting and the growth ceases. These cul­tures are maintained continuously by propagating a small aliquot of the inoculum in the moving medium and transferring it into fresh medium at regular intervals.

The biomass growth in batch cultures follows a fixed pattern, i.e., sigmoid pattern of growth curve. Batch cultures are not ideal system for studying the various aspects of cellular behaviour, as there is no steady state period in which relative concentration of metabolites or enzymes are constant.

This type of culture is generally used where the secondary meta­bolite is leached out in medium at the stationary phase or the recovery of drug is done by collecting the whole cell mass from the medium at a time.

Type # 2. Continuous Culture:

The large scale cultures are grown under steady-state condition for long periods by inflow of fresh medium and drawing out of the used medium keeping the culture volume constant. Continuous culture may be of two types: closed or open types.

In the open type the addition of fresh medium is balanced by the outflow of old medium including the harvest of cells, which maintains indefinitely a constant submaximal growth, i.e., majority of cells are in the similar metabolic state.

This open system may also be of two different types:

(i) Chemostats in which growth rate and cell density are held constant by a fixed rate of input of a growth limiting nutrient medium. The growth limiting sub­stance is so adjusted that its increase or decrease is reflected by increase or decrease in the growth rate of cells.

(ii) Turbidostats in which fresh medium flows in response to increase in the turbi­dity so as to maintain culture at a fixed optical density of suspension.

The closed type of continuous suspension culture is that where the cells are retained and inflow of fresh medium is balanced by outflow of corresponding volumes. The cells from the out flowing medium are separated mechanically and the same medium is added back to the culture. As a result in this type of culture cell biomass continues to increase as the growth proceeds.

Continuous Synchronous Culture:

In batch and continuous culture the cells divide randomly. In batch culture the dou­bling time of the successive generations is likely to be changing, but in the ‘open type’ continuous culture use of chemostat/turbidostat helps to bring all the cells averagely at the same stage of cell cycle.

Thus instead of this average condition, in the continuous syn­chronous culture there is a pattern of change which coincides with the cell cycle and repeats itself with each successive doubling of the cell population.

The cell thereby ampli­fied by the size of the synchronous populations may be examined at any stage of the cell cycle and at any desired growth rate. This has the advantage that enzymes or metabolites occurring only at certain stages of cell cycle can be obtained at maximum yields, a most useful criterion for consideration in a possible industrial production of such compounds.

Synchronization of Suspension Culture Cells:

A synchronous culture is defined as a culture in which the cell cycles of majority of cells or proportion of cells are synchronous, i.e., majority of cells proceeds through each cell cycle phase simultaneously.

In general the cells in suspension culture remain in a synchro­nous condition, hence it is essential to manipulate the growth conditions in such a way to achieve a high degree of synchronisation. It is expressed as percentage synchrony of cells in suspension. The effective procedures of synchronization differ from species to species.

The methods employed to achieve synchronization of suspension cultures may be grouped under two categories:

1. Physical and

2. Chemical.

Physical methods helpful in achieving synchrony are:

(i) Selection by volume:

Synchronization may be achieved by selecting a parti­cular size of cell aggregates by using the cell fractionation technique.

(ii) Temperature shock:

Low temperature shocks combined with nutrient starvation are reported to induce synchronization of suspension cultures.

Chemical methods used for achieving synchrony are:

In this method the cells are starved of an essential nutrient or the cell cycle is stopped by a biochemical inhibitor and then allowed to undergo simultaneous divisions either by supplementing the nutrient or withdrawing the inhibitor chemical.

(i) Starvation:

Cultures starved of nitrogen, phosphorus, or carbonate, result in the arrest of cell growth during the G₁ or G₂ phase of the Cell cycle. After a period of starvation when these growth limiting compounds are supplied to the medium, the stationary cell enters in divisional phase synchronously. Growth hormone starvation also can bring the synchronization.

(ii) Inhibition:

Inhibitors of DNA synthesis (5-amino-uracil, 5-fluorodeoxypurine, hydroxyurea, etc.) in cell culture help to accumulate the cells at the G₁/S boun­dary. Removal of the inhibitor is followed by synchronous division of cells.

(iii) Mitotic arrest:

Suspension cultures in exponential growth are supplied with 0.02% (w/v) colchicine for 4-8 hr inhibit the spindle formation. Then removal of colchicine will help to induce the synchrony among the cells in suspension culture.