Subculture of Adherent Cell Lines

Aim

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.

Materials

• Cell Culture Media: pre-warmed to the appropriate temperature (refer to the ECACC cell line data sheet for the correct medium and temperature.)
• 70% (v/v) alcohol in sterile water (793213)
• PBS without Ca²⁺/Mg²⁺ (D8537)
• 0.05% trypsin/EDTA in HBSS, without Ca²⁺/Mg²⁺ (T3924)
• Soybean Trypsin Inhibitor (T6522) or FBS
• Trypan Blue Solution (T8154)

Equipment

• Personal protective equipment (sterile gloves, laboratory coat, safety visor)
• Waterbath set to appropriate temperature
• Microbiological safety cabinet at appropriate containment level
• Incubator
• Pre-labelled flasks
• Inverted phase contrast microscope
• Centrifuge
• Hemocytometer or automated cell counter like a Scepter™ Cell Counter
• Marker Pen
• Pipettes
• Ampoule rack
• Tissue

Procedure

Key Points

1. Some cultures, whilst growing as attached lines, adhere only lightly to the flask, thus it is important to ensure that the culture medium is retained, and the flasks are handled with care to prevent the cells detaching prematurely.
2. Although most cells will detach in the presence of trypsin alone EDTA enhances the activity of the enzyme by removing inhibitory cations.
3. Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all traces of serum from the culture medium by washing the monolayer of cells with PBS without Ca²⁺/Mg²⁺.
4. Cells should only be exposed to trypsin/EDTA long enough to detach cells. Prolonged exposure could damage cell surface receptors.
5. Trypsin should be neutralized with serum prior to seeding cells into new flasks otherwise cells will not attach.
6. Trypsin may also be neutralized by the addition of soybean trypsin inhibitor, where an equal volume of inhibitor at a concentration of 1mg/ml is added to the trypsinized cells. The cells are then centrifuged, resuspended in fresh culture medium and counted as above. This is especially necessary for serum-free cell cultures.
7. If a CO₂ incubator is not available gas the flasks for 1-2 minutes with 5% CO₂ in 95% air filtered through a 0.2 μm filter.
8. If the cells harvested are at too low a cell density to re-seed at the appropriate cell density into fresh flasks it may be necessary to centrifuge the cells e.g. 5 mins at 150 x g, and resuspend in a smaller volume of medium.