How Does Trypsin Work in Cell Culture

Trypsin is a serine protease that cleaves lysine and arginine residues at the C-terminal of peptides. It is the most widely used enzyme in cell culture to release the adherent cells from culture vessel surfaces. Trypsin is easily tolerated by most of the cell types grown in cultures. Its activity can be easily neutralized with the addition of serum into the culture medium. These two features of trypsin facilitate their use in cell cultures. Generally, trypsin is added to the cell cultures with EDTA. The role of trypsin in cell cultures is described in this article.

Key Areas Covered

1. What is Trypsin
     – Definition, Features, Function
2. How Does Trypsin Work in Cell Culture
     – Role of Trypsin in Cell Culture

Key Terms: Adhesive Proteins, Cell Adhesion, Digestive Enzyme, Trypsin, Trypsinization

What is Trypsin

Trypsin is a digestive enzyme that breaks down proteins in the digestive system of many vertebrates. It is found in the pancreatic juice. Trypsin cleaves lysine and arginine residues of the carboxy-terminal of peptides. However, it does not cleave them when these two amino acids are followed by praline. The activity of trypsin is used in many biotechnological processes, and the action of trypsin is known as trypsinization or trypsin proteolysis. The crystal structure of trypsin is shown in figure 1.

Figure 1: Trypsin

The optimal pH for the action of trypsin is 7.6-8.5. Generally, phenol red is used in trypsin assays to monitor the above pH range. Phenol red gives a pinkish color at this pH range. Trypsin assays are carried out on dry ice. 

How Does Trypsin Work in Cell Culture

The proteins on the plasma membrane are responsible for a wide variety of functions that are essential for the maintenance of the normal physiological activity of the cell. Some plasma membrane proteins such as cadherin families are adhesive proteins that serve as anchors, linking the cytoskeleton proteins to the extracellular matrix. This aids in the cell adhesion and cell migration. The adhesive proteins within the plasma membrane are shown in figure 2.

Figure 2: Cell Adhesive Proteins

In cell cultures, trypsin can be added to the medium to release the adherent cells from culture vessel surface by digesting the adhesive proteins. Trypsin also releases cells from aggregates through the digestion of adhesive proteins. EDTA is also added to cell cultures along with trypsin to chelate divalent ions in the medium. Calcium and magnesium ions may inhibit the action of trypsin. Ultimately, trypsin helps to obtain individual cells from the cell cultures, facilitating the downstream processing of the cells.

Conclusion

Trypsin is a photolytic enzyme that digest peptides. Trypsin is widely used in cell culture in order to obtain individual cells as trypsin digests the adhesive proteins and releases the cells into the medium.

Reference:

1. “Trypsin-Induced Proteome Alteration during Cell Subculture in Mammalian Cells.” Journal of Biomedical Science, BioMed Central, 11 May 2010, Available here.

Image Courtesy:

1. “1UTN” By user:DrKjaergaard – Own work: From PDB file 1UTN. Created with PyMol (Public Domain) via Commons Wikimedia
2. “Cell Adhesion” By ZabMilenko at English Wikipedia – Transferred from en.wikipedia to Commons by Premeditated Chaos using CommonsHelper (Public Domain) via Commons Wikimedia