Adherent cell lines will grow in vitro until they have covered the surface area available or the medium is depleted of nutrients. At this point the cell lines should be subcultured or passaged in order to prevent the culture dying. To subculture the cells they need to be brought into suspension. The degree of adhesion varies from cell line to cell line but in the majority of cases proteases, e.g. trypsin, are used to release the cells from the flask. However, this may not be appropriate for some lines where exposure to proteases is harmful or where the enzymes used remove membrane markers/receptors of interest. In these cases cells should be brought into suspension into a small volume of medium mechanically with the aid of cell scrapers.
Figure 1.Examples of an adherent cell line. NIH 3T3 cells 24 hours (A) and 72 hours (B) post freeze/thaw show typical adherent fibroblastic morphology. Confluence should carefully be monitored and passaged when cells reach ~80% confluency