- Cell culture is not static. Cells acquire changes when maintained in culture for an extended period (Masters & Stacey, 2007). Additionally, stressful conditions accelerate these changes, resulting in inconsistencies in experimental outcomes. Therefore, it is important to maintain cultures under specified conditions to maintain the culture characteristics.
- Cells in culture grow and divide in the presence of nutrients and proper physiological conditions. As they grow, the cell density in culture increases, leading to confluence. With the increase in cell density, cell-cell contacts become more prominent, which affects cell physiology in various ways, leading to transient changes (such as the inhibition of cell division by contact inhibition in untransformed cells) or sometimes even permanent changes in cell characteristics (e.g., differentiation in cells).
- Moreover, a highly confluent culture consumes nutrients from the medium quickly, creating a nutrient-deprived state. This nutrient-deprived state results in poor proliferation and cell death, leading to the accumulation of toxic products in the medium. The accumulation of these toxic products further enhances cell death.
- To avoid complications and attain reproducibility in cell culture-based experiments, cultures must be maintained under conditions that allow for exponential growth. Therefore, it is necessary for cultures not to reach 100% confluency (in adherent cell lines) or become overcrowded (in suspension cultures). To reduce confluency or cell density, cells are regularly diluted by transferring a small number of cells from the existing culture to another dish containing fresh culture medium, where they can continue to grow. This process of transferring a small proportion of cells to a fresh tissue culture dish is called passaging or subculturing.
References:
Masters, J. R., and Stacey, G. N. (2007). Changing medium and passaging cell lines. Nat Protoc . 2(9), 2276-84. PMID-17853884, Full text – Researchgate, Nature protocols
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