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Cell Dissociation and Trypsin for Cell Culture
Detach adherent cells and tissues with Gibco cell dissociation reagents
To acquire a suitable number of cells for research, a group of cells must first be disaggregated from a larger culture. Enter: cell dissociation. Gibco trypsin and alternative cell dissociation products are ideal for use with tissues and cell monolayers. Available in a variety of formats, both enzymatic and chemical, to meet the varied needs of researchers performing adherent cell culture. Explore the selection guide below to choose the correct cell dissociation reagent for your cell culture type.
Choose the best cell dissociation reagent for your cell culture type
| Cell culture type | Wash solution | Dissociation method | Recommended product | Format | Contains phenol red |
|---|---|---|---|---|---|
| Most continuous and strongly adherent cell lines | HBSS or PBS w/o calcium and magnesium | Trypsin 0.25% in balanced salt solution w/o calcium and magnesium | 0.25% Trypsin (1X) | Liquid | Yes |
| 2.5% Trypsin (10X) | Liquid | No | |||
| Trypsin (1:250) | Powder | No | |||
| Many cells at early passage | D-PBS | TrypLE reagent | TrypLE Express | Liquid | Yes |
| Continuous cell lines where cell surface protein integrity is important | HBSS or PBS w/o calcium and magnesium | Trypsin 0.05% in 0.53 mM EDTA | 0.05% Trypsin 0.53 mM EDTA | Liquid | Yes |
| 0.05% Trypsin 0.53 mM EDTA (10X) | Liquid | No | |||
| TrypLE reagent | TrypLE Express | Liquid | Yes | ||
| Weakly adherent epithelial cells | HBSS or PBS w/o calcium and magnesium | EDTA, glycerol in sodium citrate | Cell Dissociation Buffer, Hanks'-based | Liquid | No |
| Cell Dissociation Buffer, PBS-based | Liquid | No | |||
| Transformed fibroblasts | HBSS or PBS w/o calcium and magnesium | TrypLE reagent | TrypLE Express (1X) | Liquid | Yes |
| Primary cells where cell surface protein integrity is important | HBSS or PBS w/o calcium and magnesium | TrypLE reagent | TrypLE Express (1X) | Liquid | Yes |
| Strongly adherent early-passage cell lines | HBSS or PBS w/o calcium and magnesium | Trypsin 0.25% in 1 mM EDTA | 0.25% Trypsin 1 mM EDTA | Liquid | Yes |
| Trypsin Powder (1:250) | Powder | No | |||
| Dispase 0.6–2.4 units/mL in PBS | Dispase II | Powder, Lyophilized | No | ||
| Epithelial cells | 0.5–1 mM EDTA | 0.5–1 mM EDTA | Versene solution 1:5,000 (0.53 mM EDTA in PBS) | Liquid | No |
| Dispase 0.6–2.4 units/mL in PBS | Dispase II | Powder, Lyophilized | No | ||
| Strongly adherent, epithelial and some tumor cell lines | 0.5–1 mM EDTA | 0.25% with 1 mM EDTA | 0.25% Trypsin 1 mM EDTA | Liquid | Yes |
| Versene solution 1:5,000 (0.53 mM EDTA in PBS) | Liquid | No | |||
| Trypsin Powder (1:250) | Powder | No | |||
| Dispase 0.6–2.4 units/mL in PBS w/o calcium and magnesium | Dispase II | Powder, Lyophilized | No | ||
| Thick cultures, multiple layers | 1 mM EDTA | Trypsin 0.25% | 0.25% Trypsin 1 mM EDTA | Liquid | Yes |
| Trypsin Powder (1:250) | Powder | No | |||
| Dense cultures that are collagen rich | 1 mM EDTA | Collagenase 200 units/mL, 1 mM EDTA in balanced salt solution w/o calcium and magnesium | Collagenase Type I | Powder, Lyophilized | No |
| Collagenase Type II | Powder, Lyophilized | No | |||
| Collagenase Type IV | Powder, Lyophilized | No | |||
| All adherent cell cultures | HBSS or PBS w/o calcium and magnesium | Physical/mechanical | Cell scrapers | – | – |
Trypsin and cell trypsinization
Trypsin, a proteolytic enzyme, is the standard way to detach adherent cell cultures and monolayers. This globular, pancreatic protease cleaves at the C-terminal side of lysine and arginine, breaking down vessel-adhering proteins and allowing easy resuspension during cell harvesting.
Thermo Fisher Scientific offers porcine-derived trypsin, EDTA-enhanced trypsin, as well as formulations of the TrypLE enzyme, a gentle, trypsin-like enzyme. To avoid unplanned protein degradation, organic trypsin must be inactivated with trypsin inhibitors. In contrast, synthetic trypsin, such as Gibco TrypLE products, are deactivated via dilution, eliminating the need for additional solutions.
NOTE: If you have fetal bovine serum (FBS) in your cell culture media, the culture will need to be washed with a balanced salt solution before trypsinization to ensure trypsin functionality.
Trypsin EDTA
EDTA, a chelating agent, improves trypsin’s ability to detach adherent cells. Binding calcium and magnesium into a hexadentate, EDTA weakens cell-cell adhesion and enhances trypsin’s access to hydrolysis-targeted peptide bonds.
Gibco Trypsin-EDTA is made from irradiated porcine-derived protease with e-beam verification as well as tested for PPV, mycoplasma, and PCV 1/2 contamination.
Help enable gentle cell dissociation with TrypLE reagents
Gibco TrypLE reagents are highly purified, recombinant cell-dissociation enzymes that replace porcine trypsin. These reagents are ideal for dissociating attachment-dependent cell lines in both serum-containing and serum-free conditions, and can be directly substituted for trypsin without protocol changes. TrypLE reagents are gentle on cells, stable at room temperature, and animal origin–free.
| Trypsin | Trypsin-EDTA | TrypLE Express | TrypLE Select | |
|---|---|---|---|---|
| Characteristics | Traditional standard | Trypsin with enhanced cleavage | Gentler, stabler, and cost comparable | Gentler, stabler, animal and human-component free |
| Use case | General purpose | General purpose | General purpose | Bioproduction/industrial application |
| Origin source | Porcine pancreas | Porcine pancreas | Fungus (animal origin-free) | Fungus (animal origin-free) |
| Manufacturing standards | cGMP manufacturing | cGMP manufacturing | cGMP manufacturing | cGMP, manufactured on dedicated animal origin-free machinery |
| Storage temperature | –5 to –20° C | –5 to –20° C | Room temperature | Room temperature |
| Higher cell viability | ✓ | ✓ | ||
| Inactivation method | Trypsin inhibitors | Trypsin inhibitors | Inhibition by dilution | Inhibition by dilution |
Trypsin inhibitors
Trypsin inhibitors destructively alter trypsin, nullifying the proteolytic enzyme’s ability to bind with, and so digest, proteins. Used post dissociation, trypsin inhibitors protect your cells against further protein degradation by trypsin.
NOTE: Soybean trypsin inhibitors also bind chymotrypsin, albeit to a lesser degree.
Trypsin inhibitors comparison chart
| Soybean trypsin inhibitor (powder) | Defined trypsin inhibitor | Trypsin neutralizer solution | |
|---|---|---|---|
| Animal origin-free | ✓ | ||
| Calcium and magnesium free | ✓ | ||
| Sterile | ✓ | ✓ | |
| With 0.5% newborn bovine serum | ✓ |
Collagenase
Relatively gentle, collagenase works by digesting connective tissue’s native triple-helical collagen fibrils. These tissues are often found in skin, tendon, blood vessels, and bone—making collagenase disaggregation suitable for human tumor, mouse kidney, human brain, and epithelium cultures.
Choose the right collagenase for your cell type
| Collagenase | Tissue/Cell type |
|---|---|
| Type I | Epithelial, adrenal, lung, fat |
| Type II | Heart, thyroid, salivary, liver, bone, cartilage |
| Type IV | Islet (insulin receptor sites) |
Dispase
Dispase, or neutral protease, is a Bacillus polymyxa–produced metalloenzyme. Rapid and gentle, dispase can be used to harvest and transfer normal diploid cells and cell lines. Dispase effectively separates cell clumps and splits cells from intact tissue with little impact to membrane integrity or viability.
Non-enzymatic cell dissociation
Versene solution
Versene solution, an EDTA solution in PBS, dissociates cells gently and non-enzymatically. This chelating agent detaches some cell types (such as epithelial cells) from vessel surfaces for suspension. It can also be used as a wash prior to trypsinization.
For further guidance on Versene solution applications, please see the cell dissociation selection guide by culture type.
Non-enzymatic cell dissociation buffers
Recommended for weakly adherent cells, such as epithelial cells, Gibco cell dissociation buffers dissociate gently and maintain surface protein integrity. Applications include cells involved in ligand binding, flow cytometry, and immunohistochemistry research.
Please consult the enzyme-free cell dissociation buffer protocol for more information.
Try the Gibco MediaSelect Tool
Easily find a media solution suited to your specific application. Use this interactive tool to select classical basal, protein expression, or viral vector media.
Your selections will allow you to choose from a catalog menu of media – or customize your own formulation to fit your experimental setup and budget. Where available, you can compare media formulations side-by-side.
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Gibco cell culture products are manufactured in facilities compliant with current good manufacturing practices (GMP) and adhere to a robust quality management system, helping to ensure the consistency, reliability, and high quality you can rely on.
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