In the process of cell culture, cell digestion is often required, so the trypsin used to digest cells will be indispensable for cell culture, and it is also very important, which will affect the growth state of cells.

Trypsin can be broadly divided into two types, one is recombinant trypsin and the other is non-recombinant trypsin.

• Non-recombinant trypsin is derived from animal extraction, with animal origin, and is also the traditional use of trypsin, which is commonly found in the animal digestive system and is directly extracted from the pancreatic tissue of cattle or pigs.
• Recombinant trypsin is produced using genetic recombinant technology and is not of animal origin.

Product introduction and application

Non-recombinant trypsin (animal derived)

Product features：

Non-recombinant trypsin (also known as trypsin) is a serine proteolytic enzyme extracted from the pancreas of cattle, sheep and pigs, which can specifically cleave the carboxyl terminus of positively charged lysine and arginine, purified by multiple crystallization, and further prepared by chromatography and ultrafiltration technology, with animal origin.

Because pancreatic enzymes can act on transmembrane proteins and proteins in the extracellular matrix, such as cadherin, integrin, etc., they are now commonly used to dissociate tissues and monolayer cells. Although trypsin can hydrolyze the proteolysis between cells to discrete cells, the reaction of different tissues or cells to trypsin is different, and the activity of dispersed cells is also related to the concentration of trypsin, reaction temperature and time, and the ability of trypsin to disperse cells is strongest under the conditions of pH of 7-9 and temperature of 37 °C. The addition of EDTA to trypsin solution as a chelating agent binds calcium and magnesium into a six-tooth structure, which can weaken cell-cell adhesion and enhance trypsin's acquisition of hydrolyzed targeted peptide bonds.

The main application of trypsin in the early days in the biopharmaceutical field was the production of insulin, and soon it was also used in other cell culture fields. Animal pancreatic enzymes are commonly used in serum culture, and the composition and concentration of trypsin added to different cells are different.

Applications

1. production of insulin and inactivated vaccines;
2. Isolation and culture of tissue cells;
3. Clinical and surgical use;
4. Processing aids in the food industry;

Recombinant trypsin (animal origin-free)

Product features

Recombinant trypsin is an enzyme produced using genetic engineering technology, which has the advantages of good stability, high purity, and no animal origin. In the production process of modern biopharmaceuticals, the use of raw materials containing animal origin may bring potential virus sources that cause zoonotic diseases into the product, such as BSE of mad cow disease, foot-and-mouth disease virus, etc., in order to reduce the source of pollution, trypsin is synthesized through genetic engineering technology, which solves the risk of virus contamination that may be brought by traditional porcine or bovine trypsin.

Recombinant trypsin is mostly recombinant expressed by recombinant E. coli or yeast system and purified by multi-step chromatography, without heterozyme contamination such as chymotrypsin. Recombinant trypsin has the same properties as naturally extracted trypsin, with an amino acid sequence homologous to porcine trypsin sequence, which specifically cleaves the carboxyl groups in lysine and arginine residues in the polypeptide chain. Because of its non-animal source pollution and high purity, it has a wide range of applications in pharmaceutical development, biochemical research, proteomics and other fields.

The mainstream recombinant trypsin in the market is a recombinant porcine trypsin expressed in Escherichia coli or yeast using DNA recombinant technology, and its amino acid sequence is completely consistent with porcine trypsin, with the same activity and specificity as natural trypsin. It is composed of 223 amino acids and contains 6 pairs of disulfide bonds. The enzyme contains two active forms of trypsin: α-trypsin and β-trypsin. Its activity is inhibited by serine protease inhibitors such as PMSF and TLCK, metal ion chelators such as EDTA, etc. Recombinant trypsin is used in serum-free culture, and like trypsin, different cells are added with different components and concentrations of trypsin.

Applications

1. vaccine and recombinant insulin production;
2. Preparation of tissue samples for cell analysis;
3. Immune cell therapy;
4. Biopharmaceutical production;
5. Protein sequence, structure and omics research

Detailed comparison of products

products	Non-recombinant trypsin (animal derived)	Recombinant trypsin (animal origin-free)
Item	40123ES（phenol red(-),EDTA(+)） 40124ES（phenol red(-),EDTA(-)） 40126ES（phenol red(+),EDTA(-)） 40127ES（phenol red(+),EDTA(+)）	40512ES 20416ES（MS-SAFE）
Source	Taken from the pancreas of cattle, pigs, and sheep	colibacillus（40512ES） Pichia pastoris（20416ES）
Security	Contamination with potential pathogens	No exogenous viral contamination
Purity	More than 90% of the 1:250 trypsin solution is impurity	High purity (β-trypsin≥ 70%, α-trypsin≤20%）
Digestion cell type	Most contiguous strongly adherent cell lines	Most contiguous strongly adherent cell lines
Chymotrypsin limit	No more than 50 units of chymotrypsin per 2500 units of trypsin.	No myrosinase and other impurity enzyme contamination
Specific activity	≥2500 USP units/mg protein	≥3800 USP units/mg protein
Digestive effect	instability	Strong digestion and low amount of enzymes
Self-cutting rate	It is easy to self-cut and produce self-cutting fragments, which affects the results of mass spectrometry analysis	It is easy to self-cut and produce self-cutting fragments, which affects the results of mass spectrometry analysis
Dissolved liquid	HBSS solution with or without Ca2+, Mg2+	HBSS solution with or without Ca2+ and Mg2+ (40512ES); 1 mM HCL（20416ES）
Cell survival	Normal	Normal
Whether freeze-thaw is required	Yes	Yes

In addition to the most common trypsin used in cell digestion, there are many other types of digestive enzyme products that may also be used，such as follows.

About the application field of collagenase

Collagenase is a peptidase endonuclease that can specifically recognize the Pro-X-Gly-Pro sequence and cleave the peptide bond between the neutral amino acid (X) and glycine (Gly) in the sequence, collagenase is the only protease that can degrade the natural collagen fibrils with a three-strand supercoiled structure, and can effectively hydrolyze other proteins, polysaccharides and lipids in the extracellular matrix of connective tissue and epithelial tissue, making this product very suitable for tissue digestion.

1. Collagenase I., commonly used as a preparation of epithelial cells, liver, lung, fat, and adrenal tissue cells;
2. Collagenase II, which is used for the preparation of tissue-derived cells such as heart, bone, muscle, liver, thyroid and cartilage;
3. Collagenase III, commonly used in the preparation of mammary cells;
4. Collagenase IV, which is commonly used for the preparation of pancreatic islet cells, or for cell preparation experiments where receptor integrity needs to be maintained.
5. Collagenase V., suitable for the isolation of pancreatic islet cells and connective tissue cells.

About the application field of compound trypsin

Accutase is a cell digest that contains proteolytic and collagenase activity, and is a perfect replacement for trypsin-EDTA digests for digesting cells from conventional tissue culture vessels and adherent culture vessels. Accutase is suitable for tissue dissociation and cell isolation, as the isolated cells retain intact surface antigens and do not contain any animal- or bacterial-derived components, which can meet the requirements of subsequent experiments such as cell surface labeling, viral growth analysis, flow cytometry, and bioreactor-related assays.

Application areas for pancreatic enzyme substitutes

PerfCell Tryzyme™ Express enzyme products are highly pure, have little cell damage, are mild in digestion, and can be stably stored at room temperature, making them an ideal digestive enzyme for different cell line cultures. This type of product does not require the use of trypsin inhibitors for inactivation, and does not contain animal origin, which is more friendly to some serum-free growth adherent cell experiments.

About the application area of dispase

Dispase II dispase II, a non-specific metalloproteinase, is commonly used in cell biology to isolate single cells from different tissues or organs, and is used in subsequent cell cultures, such as isolation of primary cells, cell passaging, etc., and can also be used to eliminate cell aggregation that occurs during suspension cell culture. Compared with other proteases commonly used in cell biology (e.g., trypsin, collagenase, pronase, etc.), this enzyme has the following advantages:

1. Fast, effective and gentle cell digestion enzymes, with little damage to cells, and can maintain the integrity of cell membranes;
2. Derived from bacteria, no mycoplasma or other animal virus contamination;
3. Strong stability, not affected by temperature, pH and serum components;
4. It can be used for the isolation of various types of tissues and cells.

Related product recommendations

Catalog number	Product name	Size
40123ES60	PerfCel™l Trypsin-EDTA (0.25%), Phenol red	100 mL
40124ES60	PerfCell™ Trypsin-no EDTA (0.25%), no phenol red	100 mL
40126ES60	PerfCell™ Trypsin (0.25%), phenol red	100 mL
40127ES60	PerfCell™ Trypsin-EDTA (0.25%), Phenol red	100 mL
40101ES25	Trypsin 1:250(powder)	25 g
40512ES10/60	PerfCell™ Recombinant Trypsin（powder）	10 mg/100 mg
20416ES60	Recombinant Trypsin（MS-SAFE）	100 µg
40134ES60	PerfCell Tryzyme™Express	100 mL
40135ES60	PerfCell Tryzyme™Express	100 mL
40506ES60	ACCUTASE	100 mL
40104ES60/80	Dispase II	100 mg/1 g