Removal of Adherent Cells from a Culture Surface Using Trypsin


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 solutions. Trypsin concentration in 1x working solutions can range from 0.025–0.5%, depending on trypsin activity or potency, incubation times; and 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, start with a low concentration.


Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents and Equipment Required

Hank's Balanced Salt Solution, Modified (Catalog No. H9394)
Soybean trypsin inhibitor (Catalog No. T9008)

Preparation Instructions

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 (Catalog 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 a salt solution free of Ca2+ and Mg2+ 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 ~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 (Catalog 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.

  6. 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.




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