Proteolysis with the use of trypsin – or trypsinization – is a process where you expose cells to trypsin in oder to digest intercellular and cell-to-substrate linking proteins. The cells detach from the growth vessels and from each other. The cells are said to be trypsinized.
The number of cells adhering to the in vitro culture vessel will increase until they have covered the available surface or the nutrients in the medium are depleted. Before this happens (read here for tips) the cells should be subcultured – or passed – to prevent the culture from dying.
Although there are different methods of subculture cells, trypsinization is certainly one of the best known, and used.
- The protocol: trysinization
- Trypsinization: tips and tricks
- Trypsinization: troubles
- RNA degradation
- removal or damage of membrane surface proteins
- Trypsin induced cell clumping
The protocol: trypsinization
Step 1: observe the cell under a microscope
Look at the cell culture you think should be trypsinized.
To best visualize live, unlabeled cells, it is best to use a phase contrast microscope
If it has reached the correct degree of confluency or if you need to plate the cells for other purposes and the cells are healthy (no contamination), you can proceed with trypsinization.
Return the cells to the incubator as soon as possible!
Step 2: prepare the equipment, materials and solutions
Open the sterile hood, turn it on and spray the countertop with 70% alcohol. Leave the hood on for about twenty minutes before starting.
In the meantime put under the hood the reagents and materials you need (remember to spray everything with a 70% alcohol solution (even your gloved hands! Read here some tips for working under aseptic conditions).
Heat an aliquot of medium to 37 ° C (heat only a little more than the amount you need for cell subculture). See tip 1
The same must be done with 1 aliquot of PBS (calcium and magnesium free).
Trypsin, on the other hand, should not be placed at 37 ° C, since its autolysis – with loss of activity – is faster at this temperature, but keep in inside the hood at room temperature. See tip 2
Step 3: remove cell medium
When the growing medium has reached 37 ° C, trypsinization can be carried out.
Remember to trypsinize each culture separately to avoid the risk of cross-contamination between different cultures
Bring the culture under the sterile hood and remove the culture medium with a sterile serological pipette with cotton swab to avoid overfilling and contamination from small spray droplets that could eventually reach the filter inside the pipettor.
Step 4: add PBS
Add sterile PBS (without calcium and magnesium, here the protocol for the preparation).
The total volume of PBS to add varies according to the size of the culture vessels and in general you should add at least 100 μl / cm2 (so, for example, 2.5 – 3 ml in a T25 flask).
The addition should be made by placing the tip of the pipette on the side of the culture vessels without cells and letting the trypsin solution descend by gently sliding it on the wall to avoid detaching the cells.
This step allows you to rinse away any residual medium containing serum, which would inhibit the action of trypsin.
If the cell culture is grown in serum-free medium, you can avoid this step.
Step 5: Remove PBS
Step 6: Add trypsin
Add 40 ul / cm2 of trypsin solution (then 1 ml in a T25 flask) and return the flask to the incubator at 37 ° C for the minimum time necessary to detach the cells (see Tips 2 and 3).
The concentrationo of trypsin varies according to the cell type and its strength of adhesion to the substrate, but usually the concentration is 0.25% (2.5 g / L). However, in particular cases, for example to guarantee the integrity of the cell surface receptors, a lower concentration (down to 0.05%) can be used.
EDTA is sometimes present in the trypsin solution (see Tip 5 for concentration and function of EDTA)
The time of exposure to trypsin can vary according to the type of cells, the time since last subculture, and their degree of confluence.
Progress in cell detachment can be checked by examination with an inverted microscope.
It must be determined empirically and on the basis of what is suggested by the datasheet supplied with the cells (see Tip 3).
In general, however, it is better not to exceed 10 minutes in order not to irreparably damage the cells, since prolonged exposure could damage cell surface receptors.
If the cells do not detach within this time, you can try another method or TrypLE (see tip 6)
Step 7: Add new medium or trypsin inhibitor
When the cells have detached from the bottom of the vessel, they will be in suspension and appear rounded.
Add new medium with serum (200 ul / cm2, then 5 ml in a T25 flask) and, placing the tip of the sterile serological on the bottom of the vessel, pipette the cell suspension up and down in order to obtain single cells.
Serum inhibits further tryptic activity which may damage cells (see tip 4)
Avoid foaming and pipetting too intensely as you may damage the cells.
When culturing in serum-free conditions, soybean trypsin inhibitor can be added at an equimolar concentration to inhibit the trypsin that is present.
Trypsin and soybean trypsin inhibitor should be removed by centrifuging the cell suspension at 1000 rpm, followed by re-suspension in new medium.
Step 8: Count cell and replate
Count the cells with a hemacytometer or electronic coulter counter.
Based on the cell density needed for your experiment, calculate the necessary dilution.
Alternatively, if you already know the cell culture and have determined its growth curve, you can divide the cells based on the splitting ratio for routine subculture.
step 9: put the cells back into the incubator
As Soon As Possible!
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Trypsinization: tips and tricks
Work Fast! It is essential to minimize the time that crops spend outside the perfectly fitting environment of the incubator.
Tip 1
The culture medium can be pre-heated by placing one aliquot in a thermostated bath. However, this can cause problems because it is often a source of contamination.
To avoid contamination, antifungals and antibacterials are marketed to add to bath water.
Alternatively, metallic pearls are now being sold that heat up in a special bath and do not have the contamination problems caused by water, also making it possible to avoid using biocides.
The best method, however, is to preheat the medium in a sterile flask placed in an incubator (if possible different from the one in which the cell cultures are stored to avoid continuous opening and closing).
In this way, in addition to preheating, you can allow also the pre-equilibrium with the CO2 of the incubator.
Tip 2
Do not use trypsin cold at 4 ° C because at this temperature its proteolytic activity is very low.
This may be convenient to try only in the presence of particularly delicate cell lines and if you do not have other subculture methods available
Tip 3
The specific activity of trypsin can vary in different batches. For this reason, a different concentration may be needed to detach the cells at the same time of exposure.
To improve reproducibility it is therefore advisable to opt for batch reservation, similar to what happens for serum.
Tip 4
Serum in the growth medium contains alpha-1 antitrypsin, which is a potent inhibitor of is a potent inhibitor of trypsin, elastase and other serine proteases.
The addition of serum neutralize the trypsin (∼100 μM) present in the small volume of trypsin-EDTA solution.
A single cell suspension must be obtained to ensure accurate cell count and uniform cell growth (without the formation of “islets”) after re-seeding.
This is critical if you have to do experiments like FACS and it is essential if quantitative estimates of cell proliferation or plating efficiency are in progress and if single clones are to be isolated.
Tip 5
Depending on the cell type, incubation time and the subsequent experimental protocol, trypsin can be used in various concentrations.
For strongly adherent cell lines, generally from 0.025% to 0.5% (from 0.25 to 5 g / L) trypsin is used.
For studies where it is necessary to maintain the integrity of extracellular surface proteins, lower concentration solutions (0.05% trypsin) or other enzymes are used (see here).
Concentrated trypsin is solubilized or diluted in a buffered saline solution that does not contain Ca2+ or Mg2+ (such as PBS or HBSS).
If in doubt about the trypsin concentration to use, expose the cells starting with a low concentration.
In addition, EDTA is often included with trypsin (usually at a concentration of 0.02% (0.2 g / L). EDTA chelates divalent cations (Ca2+, Mg 2+) therefore promoting the weakening of cell-to-cell adhesion in tissues: cell to cell anchorage is mediated by cell adhesion molecules (CAM), some of which are calcium-dependent (cadherins). Integrins, which interact with the extracellular matrix, also have Ca2+ binding domains and are affected by Ca2+ depletion by EDTA.
EDTA is also used to reduce cell aggregation due to the formation of “Ca2+ bridges” between negatively charged molecules of the cell surface or cell debris.
Tip 6
A good substitute for trypsin is TrypLe manufactured and sold by several biopharmaceutical companies including gibco and sigma.
TrypLe solution contains an enzyme with a proteolytic activity similar to that of trypsin. The advantages provided by the use of this enzyme consists in the milder action, which allows less damage to the cell surface receptors, the complete absence of animal contaminats (it is derived and purified from a fungus) and, unlike trypsin , is stable at room temperature (does not require freezing).
See here for futher infos
Trypsinization: troubles
1. RNA DEGRADATION
Following trypsinization, extensive RNA degradation has been observed (Peter Vrtačnik et al. 2014).
RNA quality worsening resulting from trypsinization can be an issue during methods involving the use of this nucleic acid as a microarray, reverse transcription polymerase chain reaction, and whole transcriptome shotgun sequencing.
RNA integrity was instead preserved by other methods such as direct cell lysis (using 1 ml of QIAzol Lysis Reagent), cell scraping (followed by lysis in of QIAzol Lysis Reagent), the use of temperature-responsive Nunc UpCell cell culture dishes that allow for dissociation from the surface at room temperature and dissociation with TrypLE Express reagent
2. REMOVAL OR DAMAGE OF MEMBRANE SURFACE PROTEINS
Trypsinization can also eliminate or damage some receptors or surface proteins (see here, thus altering the results aimed at the characterization of these membrane proteins or experimental procedures such as FACS.
in this case other methods to detach the cells should be used such as using Citric saline buffer for cell detachment (Zhang et al. 2012)
3. TRYPSIN INDUCED CELL CLUMPING
Excessive trypsinization (exposure to trypsin that is too concentrated or excessively prolonged over time) can promote cell aggregation, because with too hard treatment some cells lysate, releasing sticky DNA and cellular debris that tend to create clusters of cells.
In experiments particularly sensitive to cell aggregation, such as FACS, or even more simply as a cell count or subculture, you should adopt some precautions to avoid cell aggregation:
- use the lowest possible trypsin times and concentrations
- add DNase I (0.02 mg/ml) to the sample to fragment DNA from ruptured cells (In this case, however, it is necessary not to add EDTA because it inhibits the activity of DNAse by removing calcium). Be careful as DNase I can affect cell health and physiology.
- You can use Pluronic F-68 to help eliminate clumping (Tharmalingam et al.)
- Clumps can form less frequently through proper handling and use of equipment.
- If you are using a centrifuge to sediment the cells, setting it to the correct speed can reduce the chance of clumping with higher speeds preventing the cells from clumping.