Passaging/subculturing cells

  • Cell culture is not static. Cells acquire changes when maintained for a long time in culture (Masters & Stacey, 2007). In addition, stressful conditions accelerate such changes, which results in inconsistency in the experimental outcome. Therefore, it is important to maintain culture under specified culture conditions.
Subculturing/passaging of cell culture
Subculturing/passaging cell culture
  • Cells in culture grow and divide in the presence of nutrients and proper physiological conditions. As they grow, cell density in culture increases, and culture becomes confluent. With the increase in cell density, cell-cell contacts become more prominent, which affect the cell physiology in various ways, leading to transient (inhibition of cell division by contact inhibition in untransformed cells) or sometimes even permanent changes in cell characteristics (e.g., may induce differentiation in cells).
  • Moreover, a highly confluent culture consumes nutrients from the medium quickly, causing a nutrient-deprived state. A nutrient-deprived state results in poor proliferation and cell death, leading to the accumulation of toxic products in the medium. Accumulation of toxic products further enhances cell death.
  • In order to avoid complications and attain reproducibility in cell culture-based experiments, cell culture must be maintained under a condition, which allows exponential growth. Therefore, it is necessary that culture must not reach 100% confluency (adherent cell line) or get overcrowded (suspension culture). To reduce the confluency/cell density, cells are regularly diluted by transferring a small number of cells from the existing culture to another culture dish containing fresh culture medium, where cells further grow. The process of transferring a small proportion of cells to another fresh tissue culture dish is called passaging or subculturing.
  • The procedure of passaging is dependent on the growth mode of cells. Adherent cells need to be detached from the substratum for passaging. Many methods of detaching cells from the substratum have been developed. Enzymatic treatments (Trypsin, Collagenase) of cell detachment are the most common methods used in most laboratories for routine passaging of adherent cells. However, other methods, like mechanical means (using rubber policeman or vigorous shaking for semi-adherent cells) or treatment with chelators can also be used, which depend on the cell type or experimental requirement.
  • Enzymatic methods often aim to make single-cell suspension of cells that require both disrupting the cell-substratum as well as cell-cell contacts. However, the use of only mechanical means often results in small cell clumps.
  • Detached cells are further diluted with the fresh culture medium and placed into new culture dishes.
  • Non-adherent cell culture (suspension cell culture) is simply diluted with culture medium and placed in new culture dishes. However, sometimes cells are treated with the enzymatic solution to make single-cell suspension for the experiment.


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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