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Tissue dissociation tips (2)
Tissue Enzymes Considerations

While many enzyme systems have been applied for a particular type of cell isolation, the enzymes discussed here have traditionally been used for a wide variety of tissues from many different species of various ages.

Collagenase: The most common collagenase which is commercially available is derived from Clostridium histolyticum.  Clostridial collagenase degrades the helical regions in native collagen preferentially at the X-Gly bond in the sequence Pro-X-Gly-Pro where X is most frequently a neutral amino acid.  Purified clostridiopeptidase alone is usually inefficient in dissociating tissues due to incomplete hydrolysis of all collagenous polypeptides and its limited activity against the high concentrations of non-collagen proteins and other macromolecules found in the extracellular matrix.  The collagenase most commonly used for tissue dissociation is a crude preparation containing clostridiopeptidase A in addition to a number of other proteases, polysaccharidases and lipases.  Crude collagenase is well suited for tissue dissociation since it contains the enzyme required to attack native collagen and reticular fibers in addition to the enzymes that hydrolyze the other proteins, polysaccharides and lipids in the extracelluar matrix of connective and epithelial tissues.  There are four basic subtypes of collagenases with different enzymatic activity have been identified and commercially available on the market (Table 1):

Table 1                                Collagenases Subtypes

Type 1

Containing average amounts of assayed activities (collagenase, caseinase, clostripain, and tryptic activities). It is generally recommended for epithelial, liver, lung, fat, and adrenal tissue cell preparations.

Type 2

Containing greater clostripain activity. It is generally used for heart, bone, muscle, thyroid and cartilage.

Type 3

Selected because of low proteolytic activity. It is usually used for mammary cells.

Type 4

Selected because of low tryptic activity.. It is commonly used for islets and other applications where receptor integrity is crucial.

Trypsin:  Trypsin is a pancreatic serine protease with specificity for peptide bonds involving the carboxyl group of the basic amino acids, arginine and lysine. Trypsin is one of the most highly specific proteases known, although it also exhibits some esterase and amidase activity.  Purified trypsin alone is usually ineffective for tissue dissociation since it shows little selectivity for extracellular proteins. Combinations of purified trypsin and other enzymes such as elastase and/or collagenase have proven effective for dissociation.

Elastase: Pancreatic elastase is a serine protease with specificity for peptide bonds adjacent to neutral amino acids. It also exhibits esterase and amidase activity. While elastase will hydrolyze a wide variety of protein substrates, it is unique among proteases in its ability to hydrolyze native elastin, a substrate not attacked by trypsin, chymotrypsin or pepsin. It is produced in the pancreas as an inactive zymogen, proelastase, and activated in the duodenum by trypsin. Elastin mostly appears in the elastic fibers of connective tissues, thus elastase is frequently used with other enzymes like trypsin or collagenase to dissociate tissues which contain extensive intercellular fiber networks.

Hyaluronidase: Hyaluronidase is a polysaccharidase specifically cleavage of endo-N-acetylhexosaminic bonds between 2-acetoamido-2-deoxy-beta-D-glucose and D-glucuronate. These bonds are common in hyaluronic acid and chondroitin sulfate A and C. Because these substances are found in high concentrations in the ground substance of virtually all connective tissues, hyaluronidase is often used in combination with a crude protease such as collagenase, for the dissociation of connective tissues.

Papain: Papain is a sulfhydryl protease from Carica papaya latex. Papain has wide specificity and degrades most protein substrates more extensively than the pancreatic proteases.  It also exhibits esterase activity.  With some tissues papain has proved less damaging and more effective than other proteases.  Papain has been widely used for obtaining high yields of viable, morphologically intact cortical neurons.

Chymotrypsin: Chymotrypsin is a protease that preferentially catalyzes the hydrolysis of peptide bonds involving the aromatic amino acids tyrosine, phenylalanine, and tryptophan.  In addition it acts upon the peptide bonds of leucyl, methionyl, asparagenyl and glutamyl residues, and the amides and esters of susceptible amino acids.  Chymotrypsin is used to a limited extent in tissue dissociation, usually in combination with trypsin and elastase.

Deoxyribonuclease I: Often as a result of cell damage, deoxyribonucleic acid leaks into the dissociation medium increasing viscosity and causing handling problems. Purified deoxyribonuclease is sometimes included in cell isolation procedures to digest the nucleic acids without damaging the intact cells.

Neutral Protease (Dispase): Neutral Protease (Dispase) is a bacterial enzyme produced by Bacillus polymyxa that hydrolyses N-terminal peptide bonds of non-polar amino acid residues and is classified as an amino-endopeptidase. The unique feature of this enzyme is that it possesses a mild proteolytic dissociation activity while preserving the integrity of the cell membrane.   It makes the enzyme especially useful for the isolation of primary cells and the subsequent sub-culture. Neutral Protease (Dispase) is also frequently used as a secondary enzyme in conjunction with collagenase and/or other proteases in many primary cell isolation and tissue dissociation applications. Neutral Protease (Dispase) dissociates fibroblast-like cells more efficiently than epithelial-like cells. Thus it has been used for differential isolation and culture applications.

Trypsin Inhibitor (soybean): The trypsin inhibitor from soybean inactivates trypsin in  equimolar ratios, however, it exhibits no effects on the esterolytic, proteolytic or elastolytic activities of porcine elastase. It inhibits trypsin enzymatic activity during tissue dissociation procedure to increase yield of a particular cell type.
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