Cell passaging, also known as subculturing or splitting cells, is a foundational technique in cell culture laboratories. It involves transferring cells from one culture vessel to another to sustain their growth and health. This process ensures cell populations remain viable and proliferate under controlled laboratory conditions. Passaging is an essential aspect of biological research and biotechnology, enabling scientists to work with consistent and healthy cell models.
Understanding Cell Growth and Why Passaging is Needed
Cells grown in a laboratory setting multiply and divide, gradually filling the available space. This growth is supported by a nutrient-rich liquid called culture medium. As cells proliferate, they eventually reach “confluence,” which describes the percentage of the culture vessel surface covered by adherent cells. For many adherent cell types, passaging becomes necessary when they reach approximately 70% to 90% confluence.
If cells are not passaged at the appropriate time, problems can arise that compromise their health. The growing cell population consumes nutrients from the culture medium, leading to their depletion. Cells also produce metabolic waste products, which can accumulate to toxic levels. Overcrowding triggers contact inhibition, where cell division slows or stops due to physical contact with neighboring cells.
Prolonged overcrowding can result in cell stress, changes in cell behavior, or even widespread cell death. Passaging mitigates these issues by providing fresh nutrients, removing waste, and offering new space for continued growth.
The Steps of Cell Passaging
The process of cell passaging involves several stages to ensure the healthy transfer and continued growth of cells. The first step involves assessing the current cell culture, often by observing cell morphology and density under a microscope. For adherent cells, the old culture medium is first removed. The cells are then gently rinsed, usually with a balanced salt solution, to remove residual medium and serum that could interfere with the next step.
Next, an enzyme solution, most commonly trypsin-EDTA, is added to the vessel to detach the adherent cells from the surface. Trypsin is a protease that breaks down proteins responsible for cell adhesion, while EDTA helps by chelating calcium ions, further weakening cell connections. The vessel is incubated briefly, typically for a few minutes at 37°C, until the cells round up and detach. This detachment is carefully monitored under a microscope to prevent over-exposure to the enzyme, which can damage the cells.
Once detached, a fresh, complete growth medium is added to the cell suspension, which dilutes the cells and neutralizes the trypsin’s activity. The cells are then gently mixed, and a small portion of this cell suspension is transferred into new, sterile culture vessels containing fresh growth medium. This re-seeding is done at a lower density, providing ample space and resources for the cells to begin a new growth cycle. Strict aseptic technique is followed to prevent microbial contamination, which could otherwise ruin the cell culture.
The Broader Importance of Cell Passaging
Maintaining healthy, proliferating cell lines through regular passaging is significant across many scientific and medical disciplines. In biomedical research, passaged cells serve as valuable models for investigating disease mechanisms, such as cancer progression or viral infections. They are widely used for testing new drugs, allowing scientists to assess efficacy and toxicity in a controlled environment before moving to animal or human trials. This provides an important preliminary step in drug discovery and development.
Cell passaging also underpins various biotechnological applications, including the large-scale production of vaccines and therapeutic proteins. By continuously expanding cell populations, biotechnology companies can generate the quantities needed for commercial applications. Passaged cells are also utilized in diagnostic applications, aiding in the identification of pathogens or the analysis of cellular responses to specific stimuli. The ability to maintain cells for extended periods through passaging allows for long-term studies and consistent experimental material.
The practice of cell passaging contributes significantly to the reproducibility and reliability of experimental results in cell-based research. Consistent passaging ensures researchers work with cells that are in an optimal growth phase and exhibit predictable characteristics. This consistency minimizes variability between experiments and laboratories, which is necessary for validating scientific findings and advancing collective knowledge. Without proper passaging, cell cultures can become unstable, leading to unreliable data and hindering scientific progress.