Subculture (biology)
In biology, a subculture is a new cell or microbiological culture made by transferring some or all cells from a previous culture to fresh growth medium. This action is called subculturing or passaging the cells. Subculture is used to prolong the life and/or expand the number of cells or microorganisms in the culture.
Role
Cell lines and microorganisms cannot be held in culture indefinitely due to the gradual rise in toxic metabolites, use of nutrients and increase in cell number due to growth. Subculture is therefore used to produce a new culture with a lower density of cells than the originating culture, fresh nutrients and no toxic metabolites allowing continued growth of the cells without risk of cell death. Subculture is important for both proliferating (e.g. a microorganism like E. coli) and non-proliferating (e.g. terminally differentiated white blood cells) cells.
Typically, subculture is from a culture of a certain volume into fresh growth medium of equal volume, this allows long-term maintenance of the cell line. Subculture into a larger volume of growth medium is used when wanting to increase the number of cells for, for example, use in an industrial process or scientific experiment.
Passage number
It is often important to record the approximate number of divisions cells have had in culture by recording the number of passages or subcultures. In the case of plant tissue cells somaclonal variation may arise over long periods in culture. Similarly in mammalian cell lines chromosomal aberrations have a tendency to increase over time. For microorganisms there is a tendency to adapt to culture conditions, which is rarely precisely like the microorganisms natural environment, which can alter their biology.
Protocols for passaging
The protocol for subculturing cells depends heavily on the properties of the cells involved.
Non-adherent cells
Many cell types, in particular many microorganisms, grow in solution and not attached to a surface. These cell types can be subcultured by simply taking a small volume of the parent culture and diluting it in fresh growth medium. Cell density in these cultures is normally measured in cells per millilitre for large eukaryotic cells, or as optical density for 600 nm light for smaller cells like bacteria. The cells will often have a preferred range of densities for optimal growth and subculture will normally try to keep the cells in this range.
Adherent cells
Adherent cells, for example many mammalian cell lines, grow attached to a surface such as the bottom of the culture flask. These cell types have to be detached from the surface before they can be subcultured. For adherent cells cell density is normally measured in terms of confluency, the percentage of the growth surface covered by cells. The cells will often have a preferred range of confluencies for optimal growth, for example a mammalian cell line like HeLa or Raw 264.7 generally prefer confluencies over 10% but under 100%, and subculture will normally try to keep the cells in this range. For subculture cells may be detached by one of several methods including trypsin treatment to break down the proteins responsible for surface adherence, chelating calcium ions with EDTA which disrupts some protein adherence mechanisms, or mechanical methods like repeated washing or use of a cell scraper. The detached cells are then resuspended in fresh growth medium and allowed to settle back onto their growth surface.