Synchronous Cell Cultures: Synchrony by Induction and Selection (With Graph)

The cells that are growing in a random culture repre­sent a heterogeneous collection at various stages of the growth-division cycle or cell cycle.

Some cells are dividing, some have just completed division, some are about to begin division, and so on.

Any study of the progressive changes in either the chemical composi­tion of the cells or cell morphology is difficult, if not impossible, when the cells are randomly distributed with respect to age.

This problem could be avoided by studying the cycle of individual cells, but cells are usu­ally too small for individual examination and analysis.

To resolve the problem, some clever methods have been devised in which the cells in a culture are brought to the same stage of the cell cycle. Conse­quently, the entire population can be studied as though it were a single cell. This condition is known as synchronous cell growth. The degree of synchrony that exists in a cell culture can be assessed by following changes in the culture’s mitotic index, which is a measure of the fraction of cells undergoing division at any instant and is given by the formula:

I_M =N_M/N …(2-7)

Where I_M is the mitotic index, N_M is the total number of cells visibly in mitosis, and N is the total number of cells present. During the exponential expansion of a cell culture, I_M remains constant; however, during synchronous growth, I_M changes from some minimal value (ideally 0) to some maximum value during a short time interval.

The efficiency of the synchronization procedure can be evaluated from a comparison of:

(1) The mitotic index,

(2) The time required for the popula­tion to double during synchronous division, and

(3) The doubling time observed in a corresponding asynchron­ous (i.e., random) culture of the cells. A comparison of the growth curves for a random culture and a synchro­nous culture is shown in Figure 2-10, and the corre­sponding mitotic indices are shown in Figure 2-11.

A variety of methods have been devised for inducing the synchronous growth of a cell culture; these fall into two major categories: synchrony by induction and synchrony by selection.

Synchrony by Induction:

The most frequently employed methods for inducing synchrony involve temperature cycles (temperature shocks), light cycles, and chemical manipulations. When temperature is used to induce synchrony, the cells are subjected to alternating cold and warm peri­ods.

Little cell division occurs during the cold periods, but on entry into the warm periods, cell division com­mences. For example, cultures of the flagellate proto­zoan Polytomella agilis can be synchronized by a re­petitive temperature cycle of 22 hours at 9°C followed by 2 hours at 25°C. Similar procedures have been suc­cessful with other protozoa, including Astasia longa and T. pyriformis.

Synchrony may also be induced by a rapid succession of very short cold and warm peri­ods. Following this sequence, the cell population en­ters synchronous division.Certain cultures of photosynthetic cells can be in­duced to divide synchronously by exposing them to al­ternating periods of dark and light, the population doubling with each light cycle.

If a particular chemical intermediate required for cell division is withheld from the culture medium for some time, the onset of division for all cells is delayed; the division of the population then follows the addition of the substrate to the culture medium.

Agents that chemically inhibit cell division can be added to the cul­ture. Cell growth continues but cell division is halted. An example of the latter is the action of colchicine, which halts the division of cells at the metaphase of mitosis. If colchicine-treated cells are then transfer­red to a colchicine-free medium, inhibition is soon re­versed and the cells all enter the G₁ phase. A general drawback to the use of colchicine and other drugs is the possibility of inducing abnormal behavior in the cells.

The “Double Thymidine Block”:

A popular procedure for inducing synchrony in cells is to cause the random population to accumulate at the beginning of the S phase by preventing the synthesis of DNA. One way to achieve this is through the addition of high concentra­tions of the nucleoside thymidine to the culture me­dium.

The addition of the thymidine acts through a feedback mechanism within the cells to prevent the production of other nucleosides that are needed for DNA synthesis. When the thymidine is first added to the culture, any cells that are in the S phase stop DNA synthesis immediately, thereby pro­ducing a culture in which some cells are halted at vari­ous stages of the S phase while the remaining cells are blocked at the entry into the S phase.

After this, the block is lifted for an interval of time greater than the length of the S phase. This allows all cells to complete the S phase regardless of the point in the cell cycle where they were halted after the first application of the thymidine.

Most of the cells in the population would now be at various stages of the G₂ phase. Thy­midine is then applied again, and during this second exposure the cells continue through the cycle until they finish the G₁ phase. Therefore, as a result of the “double thymidine block” all the cells in the culture are arrested at the start of the S phase.These are but a few examples of the variety of chemical procedures in which cyclic manipulation of the culture medium’s chemistry results in cell syn­chrony.

Synchrony by Selection:

Selection techniques involve the mechanical isolation of cells of similar age from a random culture; these cells are then inoculated into fresh medium, where they grow and divide synchronously for some time. Among the methods frequently employed for mechan­ically isolating cells of similar age is filtration.

In this procedure, a cell culture is passed through a filter that separates the larger cells from the smaller ones. The isolated smaller cells (which are also the younger cells of the population) are then inoculated into fresh me­dium to start a new culture.

Cells of similar age may also be isolated from a ran­dom culture by sedimentation. Because young cells are generally smaller than older cells, they sediment more slowly and may be isolated for subculture.

Another technique that is used in tissue culture is the “grow off’ method in which the cells are adsorbed onto some surface (such as filter paper); during cell di­vision one of the two daughter cells that is produced detaches from the surface and can be collected for sub-culturing.

Regardless of the procedure that is employed, the ultimate effect is to alter the random age distribution of the cells so that for several subsequent generations all divisions occur over a short interval of time and all cells are at a similar stage of their cell cycle.

Of the various procedures employed, synchrony by selection is to be preferred, because chemical or temperature variations are unnatural and may have undesirable ef­fects on the cell population. The synchrony achieved by any one of these methods is usually observed to de­cay after several generations of synchronous growth.