Trypsinization

Trypsinization is the process of treating cells or tissue with the proteolytic enzyme trypsin to dissociate adherent cells from each other and from the culture surface, creating a single-cell suspension. Trypsin, a serine protease, cleaves peptide bonds on the carboxyl side of lysine and arginine residues. In culture, trypsin is typically used with an agent such as EDTA to chelate calcium and magnesium and disrupt cell–cell and cell–matrix adhesions. Digestion is halted by transferring cells to serum-containing medium or by adding a trypsin inhibitor, which neutralizes enzymatic activity.

Applications of trypsinization include routine passage of adherent cell lines, preparation of single-cell suspensions for flow cytometry, cloning, genetic or proteomic analyses, and tissue dissociation for downstream assays. For tissue processing, trypsinization can aid in generating single-cell populations from dissociated organs or tumors.

Procedural overview: When cells reach appropriate confluence, remove medium, rinse with PBS, and add pre-warmed trypsin–EDTA. Incubate briefly at 37°C while gently tapping or pipetting to help detachment. Monitor under a microscope to avoid over-digestion. Neutralize digestion with serum-containing medium or a trypsin inhibitor, collect cells, centrifuge, and resuspend for subculture or analysis.

Important considerations include minimizing exposure time to preserve viability and surface proteins, as proteolysis can alter adhesion molecules and receptors. Cell type, confluence, and enzyme form affect outcomes. Alternatives such as Accutase, collagenase, or non-enzymatic dissociation buffers may be used for gentler detachment when needed. Safety protocols should be followed to handle enzymatic reagents and dispose of waste properly.