Passaging/subculturing cells

  • Subculturing (also called passaging) involves transferring a portion of cells from an existing culture to a new culture dish that contains fresh growth medium. This process is crucial for maintaining healthy cell populations and preventing overcrowding in the original culture. It allows for the expansion of cell lines and makes cells available for experiments.
  • Cells in culture typically follow a sigmoid growth curve, characterized by the lag phase, log phase (exponential growth phase), and stationary phase. Cells are usually harvested for subculturing during the late log phase or early stationary phase. This period is referred to as the harvesting time. Growth curve
  • The procedure for subculturing depends on the growth mode of the cell line, which can be adherent, semi-adherent, or suspension culture. Adherent and semi-adherent cultures must be detached from the substratum before being diluted in culture medium and transferred to new culture dishes. In contrast, suspension cultures can be directly diluted in culture medium and transferred to fresh dishes without additional detachment.
  • Detachment of adherent cells is typically achieved through enzymatic treatment, such as Trypsin-EDTA. In contrast, semi-adherent cells are usually detached by mechanical methods, including shaking, pipetting, or scraping with a rubber policeman.
  • Detached cells/cell suspensions are diluted in fresh culture medium and transferred to new culture dishes. It is crucial to seed these dishes with an appropriate number of cells, known as seeding density, which is expressed as cells per cm² for adherent cells or cells per ml for suspension cultures. Seeding below or above this optimal density can lead to slow or negligible growth, or cause the culture to become confluent earlier than anticipated.
  • Cells can be counted and seeded into fresh culture dishes to achieve the desired seeding density. However, since counting can be time-consuming, researchers often use a split ratio to prepare fresh cultures from existing ones. The split ratio indicates how many new culture dishes can be generated from a single existing culture dish.
  • We maintain a record of the number of times cells are subcultured, referred to as passage numbers. Each time cells are subcultured, the passage number is incremented by one. The passage number serves as an indicator of the cell’s age in culture.

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

Was this post helpful?

Author: admin

Leave a Reply

Your email address will not be published. Required fields are marked *