Guidelines for Maintaining Cultured Cells
What is Subculture?
Subculturing, also referred to as passaging, is the removal of the medium and transfer of cells from a previous culture into fresh growth medium, a procedure that enables the further propagation of the cell line or cell strain.
Characteristic Growth Pattern of Cultured Cells
|Figure 4.1: Characteristic Growth Pattern of Cultured Cells. The semi-logarithmic plot shows the cell density versus the time spent in culture. Cells in culture usually proliferate following a standard growth pattern. The first phase of growth after the culture is seeded is the lag phase, which is a period of slow growth when the cells are adapting to the culture environment and preparing for fast growth. The lag phase is followed by the log phase (i.e., “logarithmic” phase), a period where the cells proliferate exponentially and consume the nutrients in the growth medium. When all the growth medium is spent (i.e., one or more of the nutrients is depleted) or when the cells occupy all of the available substrate, the cells enter the stationary phase (i.e., plateau phase), where the proliferation is greatly reduced or ceases entirely.|
When to Subculture?
Exhaustion of Medium
It is best to perform experiments and other non-routine procedures (e.g., changing type of media) according to your subculture schedule. If your experimental schedule does not fit the routine subculture schedule, make sure that you do not passage your cells while they are still in the lag period or when they have reached confluency and ceased growing.
Media Recommendations for Common Cell Lines
If there is no information available on the appropriate medium for your cell type, choose the growth medium and serum empirically or test several different media for best results. In general, a good place to start is MEM for adherent cells and RPMI-1640 for suspension cells.
Insect cells are cultured in growth media that are usually more acidic than those used for mammalian cells such as TNM-FH and Grace’s medium.
The conditions listed below can be used as a guide when setting up a new mammalian cell culture:
Dissociating Adherent Cells
|Shake-off||Gentle shaking or rocking of culture vessel, or vigorous pipetting.||Loosely adherent cells, mitotic cells|
|Scraping||Cell scraper||Cell lines sensitive to proteases; may damage some cells|
|Enzymatic dissociation||Trypsin||Strongly adherent cells|
|Enzymatic dissociation||Trypsin + Collagenase||High density cultures, cultures that have formed multiple layers, especially fibroblasts|
|Enzymatic dissociation||Dispase||Detaching epidermal cells as confluent, intact sheets from the surface of culture dishes without dissociating the cells|
|Enzymatic dissociation||TrypLE™ dissociation enzyme||Strongly adherent cells; direct substitute for trypsin; applications that require animal origin-free reagents|
TrypLE™ Dissociation Enzymes
|TrypLE™ Express and TrypLE™ Select||Trypsin|
|Completely free of animal- and human derived components||Porcine- or bovine-derived|
|Stable at room temperature for at least six months||Not stable at room temperature|
|Does not require inactivation||Requires inactivation with serum or other inhibitors|