Synchronization of Suspension Culture

Synchronization of Suspension Culture

Cells in suspension cultures vary greatly in size, shape DNA and nuclear content. Moreover, the cell cycle time varies considerably within individual cells. Therefore, cell cultures are mostly asynchromous. This variation complicates studies of biochemical, genetic physiological and other aspects of cell metabolism. A synchronous culture is one in which the majority of cells proceed through each cell cycle phase (G,S ,G2 and M) simultaneously.

A) Physical Methods:

i) Selection by Volume:

Synchronization may be achieved on the basis of selecting the size of cell aggregates present even in the finest possible suspension cultures. Cell fractionation is employed for selection.

ii) Temperature Shock:

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

B) Chemical Methods:

i) Starvation:

The principle of starvation is based on depriving suspension cultures of an essential growth compound leading to a stationary growth phase. Resupplying the missing compounds is expected to induce resumption of cell growth synchronously. Growth hormone starvation is also reported to induce synchronization of cell cultures.

ii) Inhibtiion:

Synchronization is achieved by temporarily blocking the progression of events in the cell cycle and accumulating cells in a specific stage using a biochemical inhibitor. On release the block cells with synchronously enter the next stage. Inhibitors of DNA synthesis ( 5-aminourail, 5-flurodexypurine, hydroxyurea or excess thymidine) in cell cultures accumulate cells at the G1/S boundary.

iii) Mitotic Arrest:

Colchicine has been widely used to arrest cells at metaphase. Suspension cultures in exponential growth are supplied with 0.02% (w/v) colchicine for 4-8 hr in order to inhibit spindle formation.