Cell Dissociation

Trypsin may be used to remove adherent cells from a culture surface. Cells are most commonly removed from the culture substrate by treatment with trypsin, or trypsin•EDTA. Trypsin 1X solutions can range from 0.025% to 0.5%. The reasons for the range of concentrations are as follows: (1) Differences in trypsin activity or potency; (2) Different incubation times; (3) Different cell lines.

Incubating cells with too high a trypsin concentration for too long a time period will damage cell membranes and kill the cells. If unsure about the concentration of trypsin to use, use a low concentration. There can be lot-to-lot variation in dissociation times which is to be expected since the enzymatic activity of each lot will differ. If trypsin is being solubilized or diluted from a concentrated solution, it is important to use a buffered salt solution that contains no Ca²⁺ or Mg²⁺, such as Hank's Balanced Salt Solution, Modified (Product No. H9394). Adjust the pH of trypsin solution to 7.4-7.6.

1. Remove medium from culture vessel by aspiration and wash the monolayer with Ca⁺² and Mg⁺² - free salt solution to remove all traces of serum. Remove salt solution by aspiration.
2. Dispense enough trypsin or trypsin•EDTA solution into culture vessel(s) to completely cover the monolayer of cells and place in 37 °C incubator for approximately 2 minutes.
3. Remove the trypsin or trypsin•EDTA solution by aspiration and return closed culture vessel(s) to incubator. The coated cells are allowed to incubate until cells detach from the surface. Progress can be checked by examination with an inverted microscope. Note: The time required to remove cells from the culture surface is dependent on cell type, population density, serum concentration in the growth medium, potency of trypsin and time since last subculture. Trypsin causes cellular damage and time of exposure should be kept to a minimum.
4. When trypsinization process is complete the cells will be in suspension and appear rounded.
5. It is advisable to add serum or medium containing serum to the cell suspension as soon as possible to inhibit further tryptic activity which may damage cells. Soybean trypsin inhibitor (Product No. T9008) can also be added at an equimolar concentration to inhibit the trypsin that is present. Soybean trypsin inhibitor is used when culturing in serum-free conditions.

Cells can be resuspended by gently pipetting the cell suspension to break up the clumps. Further dilution can be made, if required, for cell counts and/or subculturing.