Passaging cells, also known as subculturing, is the process of transferring a small number of cells from a crowded culture vessel into a new, fresh vessel. This action maintains their growth and health by preventing overcrowding. Cells grown outside of a living organism cannot sustain themselves indefinitely in a single container, so this transfer provides the necessary room and renewed resources for continued multiplication, ensuring a continuous supply for research.
Why Cells Cannot Live Forever in a Flask
Cells in a culture flask are in a closed environment. As they grow and divide, they gradually deplete resources and alter the conditions of their surroundings. Nutrients in the culture medium, such as glucose and amino acids, are consumed rapidly, while metabolic waste products, like lactic acid, accumulate and acidify the medium. This combination of nutrient exhaustion and toxic waste buildup is detrimental to cell health and will eventually cause cell division to slow or stop entirely.
A physical limitation also dictates the need for transfer, especially for adherent cells that must attach to a surface to grow. These cells multiply until they completely cover the available surface area, a state referred to as “confluence.” Once confluence is reached, the cells trigger contact inhibition, which halts further division. If the cells are not split, they can enter senescence or begin to die, compromising the integrity of the cell line. To remain healthy and in its optimal growth phase, the population must be regularly thinned out and moved into a new environment.
The Physical Steps of Subculturing
The transfer process for adherent cells begins with removing the spent culture medium from the flask. The cells are then gently washed with a buffer solution, such as phosphate-buffered saline (PBS). This buffer lacks the calcium and magnesium ions that help cells stick to the surface, and the washing removes residual proteins that could interfere with the next step.
A specialized detaching agent, most commonly the enzyme trypsin, is then added to the flask. Trypsin is a protease that breaks the protein bonds anchoring the cells to the flask surface and to each other. The flask is incubated briefly, often for two to ten minutes, until the cells detach and round up into a suspension. Once detached, the trypsin must be neutralized, typically by adding fresh culture medium containing serum, as the proteins in the serum inhibit the enzyme’s activity.
The resulting cell suspension is then collected, and a small sample is taken to determine the cell concentration and viability. This counting is performed using a hemocytometer or an automated cell counter, often using a dye like Trypan blue to identify living cells. Based on the count, a precise volume of the cell suspension is calculated and transferred into a new culture flask containing fresh media. The flask is labeled and placed back into the incubator, where the cells will reattach and begin their growth cycle.
Calculating the Cell Dilution Factor
Standardizing the passaging process is accomplished through careful mathematical planning to ensure experimental consistency and predictable growth rates. The primary tool for this is the “split ratio,” which defines the cell dilution factor. For example, a 1:4 split ratio means that one-fourth of the cell suspension volume is transferred to a new flask. This ratio controls the initial number of cells plated, ensuring they are neither too sparse, which can prevent growth, nor too dense, which would cause them to quickly reach confluence.
The chosen split ratio is directly related to the cell line’s “Population Doubling Time,” which is the time it takes for a cell population to double. Cells with a short doubling time, like many immortalized cancer lines, can be split at a higher ratio, such as 1:10. Conversely, slower-growing cells, such as primary cells, require a lower split ratio, often 1:2 or 1:3, to maintain their proliferation rate. Consistently applying an appropriate split ratio ensures that the cells are harvested while they are in the logarithmic growth phase.