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Trypsin

By Corning® cellgro®

Enzymatic cell dissociation agents

Porcine paravirus tested.

Most cell cultures grow as a single thickness cell layer or sheet attached to a substrate. When subculturing adherent cells, these intercellular and cell-to-substrate connections must be broken. Proteolytic enzymes, such as trypsin, break these bonds, creating a single-cell suspension from which new subcultures are split. Trypsin is available in several formulations1, including solutions with or without EDTA, a chelator that binds divalent cations such as calcium and magnesium present between the cells allowing trypsin access to the cell-cell and cell-substrate bonds.

Mediatech trypsin products undergo a a variety of test and control procedures to assure conformity to standards.

Physio-Chemical Properties. Trypsin products undergo testing to determine pH and osmolality according to specific protocols in which all equipment is calibrated daily using standards which are traceable to the National Institute of Standards and Technology.

Biological Tests. Trypsin solutions are formulated under controlled manufacturing conditions to avoid loss of enzymatic activity by denaturation.

  • Sterility: Mediatech selects a representative number of samples from each production lot to test using the membrane filtration technique for 14 days, in accordance with USP current edition.
  • Mycoplasma: Media products are tested for mycoplasma off-site using a large-volume method described by Kern and Barile in "Isolation of Mycoplasmas from Cell Culture by Agar and Broth Techniques," M.F. Barile and G.J. McGarrity (1983) in Methods of Mycoplasmology, Vol. 11,5. Razin and J.G. Tulley, eds., New York: Academic Press, 159-165.
  • Porcine Parvovirus: Powder must test negative by the Code of Federal Regulations, Title 9; 113.53-d.
  • Activity: Finished product is tested according to predetermined specifications for its ability to remove adherent cultures without damaging cellular integrity. In vitro assays are performed on HEP-2/MRC-5 and VERO cultures to verify compliance with product specifications.
Cat.No. Description Pack Size Shipped Storage Intended Use
Trypsin, 1X (Formulation below)
23-25-050-CI 0.25% Trypsin in HBSS
without calcium & magnesium
Porcine Parvovirus Tested
6 x 100 mL F F I
23-25-051-CI 0.05% Trypsin/0.53 mM EDTA in HBSS
without calcium & magnesium
Porcine Parvovirus Tested
6 x 100 mL F F I
Trypsin EDTA, 1X (Formulation below)
23-25-052-CI 0.05% Trypsin/0.53 mM EDTA in HBSS
without sodium bicarbonate, calcium, & magnesium
Porcine Parvovirus Tested
6 x 100 mL F F I
23-25-052-CV 0.05% Trypsin/0.53 mM EDTA in HBSS
without sodium bicarbonate, calcium, & magnesium
Porcine Parvovirus Tested
6 x 500 mL F F I
23-25-053-CI 0.25% Trypsin/2.21 mM EDTA in HBSS
without sodium bicarbonate, calcium, & magnesium
Porcine Parvoviurs Tested
6 x 100 mL F F I
Trypsin, 10X (Formulation below)
23-25-054-CI 2.5% Trypsin in HBSS
without calcium, magnesium, & phenol red
Porcine Parvovirus Tested
6 x 100 mL F F I

Trypsin: Formulations

Catalog No. 23-25-050
1X, Liquid
0.25% Trypsin
in HBSS
23-25-051
1X, Liquid
0.05% Trypsin/
0.53 mM EDTA
in HBSS
23-25-052
1X, Liquid
0.05% Trypsin/
0.53 mM EDTA
in HBSS
23-25-053
1X, Liquid
0.25% Trypsin/
2.21 mM EDTA
in HBSS
23-25-054
10X, Liquid
2.5% Trypsin
in HBSS
Components
D-Glucose 1000.00mg/L 1000.00mg/L 1000.00mg/L 1000.00mg/L 1000.00mg/L
EDTA•4Na•4H2O - 240.00 240.00 1000.00 -
KCl 400.00 400.00 400.00 400.00 400.00
KH2PO4 60.00 60.00 60.00 60.00 60.00
NaCl 8000.00 8000.00 8000.00 8000.00 8000.00
NaHCO3 350.00 350.00 - - 350.00
Na2HPO4 (anhydrous) 47.70 47.70 47.70 47.70 47.50
Phenol red, Na 10.00 10.00 10.00 10.00 -
Trypsin 1:250 2500.00 500.00 500.00 2500.00 25000.00
Specifications
pH 7.2 - 8.0 7.2 - 8.0 7.2 - 8.0 7.2 - 8.0 5.4 ± 0.3
Osmolality (mOsm) 280 - 320 270 - 320 270 - 320 270 - 320 320 - 475

See below for more information on the dissociation of cell monolayers.

F = -5 to -20°C
R = 2 to 8°C
A = 15 to 30°C
* = Glass
** = Flexible Media Bag
I = Sterile, IVD Product
NI = Sterile, Research Use Only, Not for IVD Use
IVD = In Vitro Diagnostic
Res = Research Use Only, Not for IVD Use

Dissociation of Cell Monolayers

The following instructions are applicable to any cell line. Actual procedures and concentrations should be determined by experience with individual cell lines. Researchers should regularly monitor cell viability at subculturing to determine most suitable conditions and procedures.

Procedure: Dissociation of Cell Monolayers

  1. Observe cells as suggested in Guide to Subculturing Cell Cultures on page 109.
  2. Pre-warm the trypsin or trypsin/EDTA solution to 37°C using a water bath or by placing the bottle in an incubator for a few minutes.
  3. Remove and discard the culture medium from the culture vessel (flask, plate, etc.).
  4. Carefully rinse the cell sheet with the appropriate amount of a balanced salt solution or the trypsin solution and discard. (Use approximately 3 mL for 25 cm2 flasks and 5 mL for 75 cm2 flasks. These amounts may be reduced or increased when needed.) It is imperative to remove all traces of serum, which contains trypsin inhibitors. The monolayer must be washed with either a balanced salt solution free of calcium and magnesium (MT Catalog Number 21-021 or 21-031) or the trypsin solution. Testing the effects of each on a particular cell line will help determine the appropriate wash solution to use. For sensitive cells, washing with a balanced salt solution may be best to avoid damage to the cells.
  5. Add the trypsin solution to the side of the vessel opposite the cells and gently swirl the vessel to cover the monolayer completely. Allow cells to incubate for 1-3 minutes and check for dissociation. Cells will begin to round up and tilting the vessel will cause the monolayer to slide down the surface. Gently tapping the flask with your hands may facilitate dissociation, but may lead to clumping. For monolayers that are particularly difficult to detach, the flask may be placed in a 37°C incubator for a short time. Timing may vary depending on the cell type, age of monolayer, and other factors, but usually cells dissociate within 5 to 15 minutes.
  6. Once cells appear detached, add an appropriate amount of complete (i.e., containing serum) growth medium (approximately 0.1 to 0.2 mL per cm2). For serum-free conditions2, add a trypsin inhibitor to neutralize the action of the trypsin. Gently pipet up and down to disperse cells into suspension. Pipetting too vigorously may cause cell damage. If cells are too difficult to disperse without causing damage to the cells, a more aggressive dissociation solution3 may be needed.
  7. Proceed with counting and/or subculturing, as necessary.

Notes

1 Formulations: 0.25% Trypsin in HBSS, 1X Liquid (23-25-050)
0.05% Trypsin/0.53 mM EDTA in HBSS, 1X Liquid (23-25-052)
0.25% Trypsin/2.21 mM EDTA in HBSS, 1X Liquid (23-25-053)
2.5% Trypsin in HBSS, 10X Liquid (23-25-054)
2 For serum-free conditions, a non-enzymatic dissociation solution, such as Cellstripper™ (23-25-056), gently dislodges cells using a mixture of chelators and does not require deactivation with serum.
3 More aggressive dissociation solutions may contain higher concentrations of trypsin and EDTA, but may also increase the risk of enzymatic damage to the cells. It is recommended to test these higher concentration trypsin solutions on a test monolayer prior to use.

Troubleshooting Tips for Dissociation of Cell Monolayers

Problem Possible Causes Suggestions
Cells are difficult to detach Trypsin concentration is too low
  1. Increase the concentration of trypsin and/or add some EDTA.
Age of monolayer
  1. Increasing the volume of trypsin or trypsin/EDTA solution and placing the flask in the 37°C incubator may help. Subculture as the cells approach confluence to prevent this problem. If a culture reaches confluence and is not promptly subcultured, cells become densely packed preventing the trypsin from reaching the cell-substrate junction.
Serum is still present on the monolayer
  1. Repeat rinsing of the monolayer to remove all traces of serum (trypsin inhibitor).
Low Viability Trypsin concentration is too high
  1. An enzyme, trypsin may harm cell membranes at increased concentrations or when left on the cells for long periods of time. Lower the concentration of trypsin in the solution or decrease the reaction time.
pH or osmolality problems with trypsin solution
  1. Verify the expiration date and pH of the balanced salt solution used as a base for the trypsin or trypsin/EDTA.
Cells are in clumps, not a single-cell suspension Cell-to-cell junctions are very tight (caused by increased age of monolayers)
  1. Gently pipette the suspension up and down to encourage a single-cell suspension. (Subculture before the monolayer reaches 100% confluence.)
Cells will not reattach to the flask Trypsin still present in the media
  1. Be sure to add serum-containing medium to the cells once detachment is complete. Serum contains trypsin-inhibitors, which inactivate the enzyme. Isolation of the cells from the media by centrifugation may be necessary to remove trypsin, and then resuspend the cell pellet in medium. Addition of trypsin inhibitors is also an option.
Not enough serum or attachment factors in medium; flasks not cell culture treated
  1. Attachment factors may not be present in some serum-free media formulations. Add attachment factors such as fibronectin or laminin to the culture media. Note: Culture flasks are available pre-treated or coated. Coatings such as collagen, promote attachment to the flask surface.
Cell membrane damage
  1. Extensive incubation with trypsin may damage the cell membrane, specifically attachment proteins on the cell surface. Decrease the enzyme concentration, shorten exposure time, and/or pipette more gently.
             
Cat.No. Name Comments Pkg Size PK    
23-25-050-CI 0.25% Trypsin in HBSS w/o calcium, magnesium Porcine paravirus tested 6-pack/100mL bottles $55.37
1/PK
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23-25-051-CI 0.05% Trypsin/0.53 mM EDTA in HBSS w/o calcium, magnesium Porcine paravirus tested 6-pack/100mL bottles $54.43
1/PK
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23-25-052-CI 0.05% Trypsin/0.53 mM EDTA in HBSS w/o calcium, magnesium, sodium bicarbonate Porcine paravirus tested 6-pack/100mL bottles $54.12
1/PK
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23-25-052-CV 0.05% Trypsin/0.53 mM EDTA in HBSS w/o calcium, magnesium, sodium bicarbonate Porcine paravirus tested 6-pack/500mL bottles $181.39
1/PK
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23-25-053-CI 0.25% Trypsin/2.21 mM EDTA in HBSS w/o calcium, magnesium, sodium bicarbonate Porcine paravirus tested 6-pack/100mL bottles $54.04
1/PK
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23-25-054-CI 2.5% Trypsin in HBSS w/o calcium, magnesium, phenol red Porcine paravirus tested 6-pack/100mL bottles $144.21
1/PK
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