Trypsin Cell Dissociation Protocol

Trypsin in Cell Culture
Trypsin Attributes
Trypsinization Protocol

Trypsin in Cell Culture

Cell dissociation is the process during cell passaging where cells are detached from the treated surface to create suspensions. These suspensions are important for reseeding for subculture, cell counting for analysis, and cell propagation. There are various proteolytic enzymes that are used to detach cells from the adherent substrate, including trypsin.

Trypsin is a member of serine protease family frequently used to detach cells. The optimum activity for trypsin occurs at 37 °C, so pre-warmed trypsin is often used to speed up cell detachment. However, long-term incubation with high concentrations of trypsin can damage cells by striping surface proteins and killing the cells. 

Cell Dissociation Products

Trypsin Attributes

Trypsin is tolerated by many cell types. However, proteomic studies and experiments requiring serum-free cultures often avoid using trypsin. Based on the application and cell type, various constituents and concentrations of trypsin can be employed. Some of the attributes are discussed below.

• EDTA (disodium ethylenediaminetetraacetic acid): Adhesion molecules in the presence of calcium determine cell-cell and cell-matrix interactions. EDTA is frequently included with trypsin to chelate with the divalent cations (Ca2+, Mg 2+) and weaken these interactions.
• Phenol red: Trypsin has optimum activity at the pH range from 7 to 9. At this range, phenol red inclusion gives the solution a pink color. Due to environmental conditions the pH of trypsin may turn acidic, rendering it less effective and producing an orange color. By adjusting the pH to 7.4 – 7.6 with NaOH the trypsin activity could be restored.
• Diluent: To help maintain pH and osmotic balance, concentrated trypsin can be diluted with or solubilized a buffered salt solution that contains no Ca2+ or Mg2+, such as Hank's Balanced Salt Solution . Some trypsin products can also be diluted in 0.9% NaCl.
• Source and form: The major source of trypsin is porcine (pig) and is available either in lyophilized powder or as a solution. To avoid animal or microbial products, recombinant bovine trypsin, Trypzean solution, is expressed in corn.
• Concentration: Based on the cell type and application trypsin is used in various concentrations. For strongly adherent cell lines, trypsin of 2.5 % to 0.25% (10X to 1X power) is used. Lower concentrations of trypsin, such as 0.05%, can be used for studies that require the cell surface proteins maintain integrity.

Trypsinization Protocol

This protocol is performed while maintaining aseptic environment, such as a laminar bio monitored hood.

1. Pre-warm the trypsin solution, balanced salt solution (Ca+2 and Mg+2-free solution) and growth medium to 37 °C
   
2. Examine the cells to ensure the cells are healthy and free of contamination
   
3. Remove and discard the culture media from flask
   
4. Gently rinse the cells with balanced salt solution without Ca+2 and Mg+2 ions and remove the solution. Firmly adherent cells could also be washed with tryspin solution. However, for sensitive cells balanced salt solution is preferred.
   
5. Add appropriate quantity (0.5 mL/10 cm2) of pre-warmed trypsin solution to the side wall of the flask. Gently swirl the contents to cover the cell layer.
   
6. Incubate the vessel in room temperate for 2-3 minutes (incubation time varies according to the cell line used). Firmly adherent cells can be detached quickly at 37 °C. Observe the cells under microscope. The detached cells appear rounded and refractile under microscope. If less than 90% of cells are detached incubate the flask for another 2 minutes and observe the cells under microscope for every 30 seconds.
   Note: Prevent cell exposure to trypsin solution for longer periods (>=10 min)
   
7. Once cells appear detached add two volumes of pre-warmed complete growth media to inactivate trypsin. Gently disperse the medium by pippeting over the cell layer surface several times to ensure recovery of >95% of cells. For serum free cultures, Soybean trypsin inhibitor is added at an equimolar concentration to inhibit the trypsin.
   Note: Vigorous pipetting can cause cell damage.
   
8. Transfer the cell suspension to the tube and gently centrifuge at 100-300g for 5-10 min. After removing the supernatant, gently resuspend the cell pellet in pre-warmed complete growth medium. Remove required sample to determine the cell density of viable cells by using hemocytometer and trypan blue exclusion or automated cell counter.  
   
9. Dilute the remaining solution to the seeding density and pipet appropriate volume to the culture flask and place it in the incubator.