What Makes StableCell™ Trypsin Different?

Trypsin is a proteolytic enzyme secreted as trypsinogen by the pancreas, and activated to aid in degradation of food proteins in the proximal intestine. In the life sciences lab, trypsin is often used to dissociate adherent cells from culture plates, flasks, etc. Traditional trypsin solutions must be stored frozen at -20 °C to prevent autodigestion, which can lead to reduced enzyme activity (Figure 1), resulting in ineffective cell detachment. StableCell™ Trypsin solutions are specifically designed to perform cell detachment as standard trypsin solutions do, without the need to aliquot, freeze, and thaw the trypsin. This saves significant time by eliminating time-consuming aliquoting and waiting for trypsin to thaw for cell passaging or experimentation, not to mention freeing space in your freezer. Our StableCell™ Trypsin solutions are recommended for storage at 2-8 °C, however, our studies indicate, with a high degree of confidence, that StableCell™ Trypsin solutions retain >90% of their activity and performance even when stored at 37 °C.

StableCell™ Trypsin has been validated using a variety of cell lines. Our studies have included SKOV-3, DU145, HEK293, 16HBE41o-, HL-1 and MDCK cell lines. We’ve compared detachment and enzyme activity of StableCell™ Trypsin to our standard Trypsin solutions, and found similar performance throughout for each cell line we observed. StableCell™ Trypsin’s unique formulation, however, allows it to retain its enzyme activity over time, at 2-8 °C, whereas our standard trypsin solutions degrade quickly when not kept frozen. See Figure 1 and Figure 2 for details (StableCell™ in purple, standard trypsin in blue).

Enzyme activity over time

Figure 1. Enzyme activity measured over time. StableCell™ Trypsin Solution compared to normal, research grade trypsin – both were stored at 2-8 °C. Results show that while normal trypsin activity tapers off over time, StableCell™ Trypsin retains >90% of it's activity.

Cell detachment and viability

Figure 2. Average number of cells detached using both our standard trypsin solutions (Product No. T3924, T4049, T4174) compared with StableCell™ Trypsin solutions (Product No. T2601, T2605, T2610).

Our studies conclusively suggest that StableCell™ Trypsin solutions demonstrate identical cell detachment performance when compared with standard trypsin solutions. (Figure 2).

Note:

  • SK-OV3 cells, plated in 6-well plates at ~90% confluence
  • Cell detachment and viability were measured for three different concentrations (1x, 5x, 10x) of standard or StableCell™ Trypsin solutions.
  • All solutions were diluted in HBSS to 1x equivalent
  • Negative controls: stabilizer alone in HBSS and untreated cells (t=0)
  • Experiment was performed in triplicate, 37 °C for 10min
"There are other solutions for cell detachment which similarly allow for storage at 2-8 °C, or even room temperature. It’s nice not having to wait for it to thaw, but what I like most about StableCell™ is that it’s actually trypsin, so I know exactly what I’m putting in with my cells."

StableCell™ Trypsin vs Normal Trypsin

Figure 3. StableCell™ Trypsin vs Normal Trypsin

Procedure

StableCell™ Trypsin Solution may be used to remove adherent cells from a culture surface for passage, cryostorage, or experiments. Cells are most commonly removed from the culture substrate by treatment with trypsin or trypsin - EDTA. StableCell™ Trypsin Solutions can range from 0.05% 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 excessive trypsin concentration or for longer than needed will cause cell injury and death by damaging cell membranes. If unsure about the concentration of trypsin to use, start with the lowest expected effective concentration, and increase only if cells do not detach. Lot-to-lot variation in dissociation times can occur and are 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 StableCell™ Trypsin Solution into culture vessel(s) to completely cover the monolayer of cells and place in 37 °C incubator for ~2 minutes.
  3. Remove the StableCell™ Trypsin 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 can cause cellular damage, so exposure should be kept to a minimum.

  1. When trypsinization process is complete the cells will be in suspension and appear rounded.
  2. 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. T6414) 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.
  3. 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.

FAQ

What if I leave StableCell™ Trypsin Solution out at room temperature? How will that affect the enzyme activity?

Our studies show that StableCell™ retains >90% activity for up to 7 weeks at temperatures up to 37 °C.

Many non-freezer solutions for cell detachment are sold as a replacement for trypsin, and are not actual trypsin solutions. StableCell™ is an optimized trypsin solution, so you can be sure of what you are adding to your cells.

We’ve conducted tests on a small variety of cell lines, including commonly used lines, as well as a few cell lines which have been known to be difficult to dissociate. Our evidence suggests StableCell™ Trypsin works well against the same large variety of cell lines as any other trypsin.

First, try our general Troubleshooting Guide for detaching cells using trypsin. You can also give us a call for support or contact our technical service team online.

No, studies have been done cellular protein modifications using both StableCell and traditional trypsin and no difference was seen between the samples.

Materials
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