Tissue Dissociation Guide: Collagenase, Dispase, and Liberase Enzyme Types
General Information about our Tissue Dissociation Enzymes
- Description of our Collagenase Enzymes
- Collagenase Enzyme Products
- Collagenase and Liberase® Enzymes from Roche
- Collagenase Assays
- Collagenase Tissue Digestion/Dissocation Troubleshooting and References
Collagenases are, enzymes that break down the native collagen that holds animal tissues together and, are made by a variety of microorganisms and by many different animal cells1. The most potent collagenase is the "crude" collagenase secreted by the anaerobic bacteria Clostridium histolyticum. We originally adopted the 1953 fermentation and purification process described by MacLennan, Mandl and Howes2, but eventually improved upon for higher activity products. “Crude” collagenase refers to the fact that the material is a mixture of several different enzymes besides collagenase that act together to break down tissue. It is now known that two forms of the collagenase enzyme are present3,4. With a few exceptions different commercial collagenase are all made from C. histolyticum, or are recombinant versions where Escherichia coli expresses a gene cloned from C. histolyticum.
Description of our Collagenase Enzymes
The different Collagenase products in the tables below were developed by because they each digest some types of tissue (muscle, pancreas, heart, adipose) better than others. In addition to meeting enzyme activity specifications, every lot of our collagenase products must pass digestion tests with various tissues from rats. Products that are also described as “cell culture tested” have undergone additional testing with mammalian cell lines to verify that they are not cytotoxic.
Sterile-filtered (0.2 mm) versions prepared from some of the more popular collagenase products are also listed below.
Our purified collagenase products have only trace amounts of caseinase (proteolytic) or clostripain activities. The purified Type VII Collagenase is also offered in Cell Culture Tested and sterile-filtered versions.
Crude Collagenase, Use-Tested
Chromatographically Purified Collagenase
Crude Collagenase with Proteolytic Activity Inhibitor
Collagenase Blends™
These products were developed to allow more lot-to-lot reproducibility in collagenase digestions. The proportion of purified collagenase (Blend F) to purified clostridial neutral protease is varied in Blends H and L to provide researchers with a range of digestion options.
Collagenase and Liberase® Enzymes from Roche
Collagenase enzymes from Roche are intuitively designed and trusted to deliver consistent performance and reproducible results for your routine and critical applications. Collagenase is suitable for preparation of cells from many types of tissue, such as hepatocytes, adipocytes, pancreatic islets, epithelial cells, muscle cells, and endothelial cells. However, suitability of each lot of the enzyme for disruption of a tissue should be determined empirically. Additionally, our Liberase® enzyme technology combines highly purified collagenase I and II enzymes with Dispase® or Thermolysin to facilitate the dissociation of a broad range of tissue types.
Liberase Research Grade Application Guide
Dispase Enzymes
Along with enzymes available from Roche, we are committed to providing tissue dissociation reagents that are highly purified and dependable for your specific application needs. A gentle enzyme that does not damage cell membranes, dispase is suitable for the separation of diverse tissues and cells that are grown in vitro and to prevent cell clumping for suspension cultures.
Collagenase Assays
The Type I and Type II forms of the purified collagenase enzymes differ in their specificities and relative activities on native collagen and synthetic substrates. These two collagenases can be mostly distinguished by their preference for one of the two different substrates used in our assays. The Collagenase Digestive Unit (CDU) assay10,11 measures predominantly the Collagenase I activity, which cleaves two of the three helical chains in the long, undenatured collagen protein. Collagenase II activity is measured by this enzyme’s ability to cut a short synthetic peptide, N-[3-(2-Furyl)acryloyl)]-Leu-Gly-Pro-Ala (FALGPA, see Product No. F5135), in a second collagenase digestive assay12,13. Purified preparations of either Collagenase I or II have been shown to be less effective at digesting various types of collagen or mammalian tissue when compared to a mixture of both forms of this enzyme. A purified collagenase containing only the Collagenase I and II forms of this enzyme is less effective at digesting tissue than the whole crude collagenase or combinations of the purified collagenase and various proteases. Obviously, the combination of true collagenase and the different native proteases, Clostripain and aminopeptidases that have evolved in nature assist each other in digesting the collagen in different animal tissues. For tissue digestions the crude collagenase products have always been the most effective. Some researchers have tried mixtures of chromatographically purified collagenase with a protease such as trypsin or subtilisin to digest tissue.
In addition to the CDU and FALGPA assays for Collagenase activities, we test each product lot for Caseinase14,15, Clostripain and Tryptic activities to look at the proteolytic enzymatic activities in the collagenase products. The Caseinase assay is the most important of the three for measuring the proteolytic activity that assists the digestion of animal tissue. Because the Clostripain present in crude collagenase must be reduced (e.g. by treatment with Dithiothreitol) in order to be active this enzyme probably contributes little to the tissue dissociation process in the laboratory. It is monitored because some researchers have reported that Clostripain may be damaging or toxic.
Many collagenase products that meet enzymatic specifications are also use-tested with various tissues obtained from rats. Type II (C6885, C1764) and Type VIII (C2139) collagenase lots are tested for the ability to release adipose (fat) cells from rat epididymal fat pads5. Fat cells are then screened for metabolic activity by measuring glucose oxidation rates with and without insulin addition. Type IV (C5138, C1889) and Type VIII (C2139) lots have been tested for the ability to release viable cells from rat liver7. Type V (C9263, C2014), Type XI (C7657, C4785, C9407, and C9697) and Type S (C6079) Collagenase lots must release intact islets of Langerhans from rat pancreas to pass their product test8.
Collagenase Tissue Digestion/Dissocation Troubleshooting and References
Based on our own R&D and from discussions with customers it is clear that the way a particular tissue is dissected and prepared has a significant effect on the speed and efficiency of any tissue digestion-dissociation with collagenase. Differences in the ages of the tissue donors can also be a major source of variation over time. Make sure that calcium ions are present in the digestion buffers at 5 mM. Chelating agents EGTA and EDTA can severely inhibit Collagenase activity by removing Calcium ions required for enzyme stability and activity. β-mercaptoethanol16, cysteine16 and 8-hydroxyquinoline-5-sulfonate16 ;are other inhibiting substances. A new lot of Collagenase with higher specific activity could cause excessive cell death at an established concentration. In that case use less collagenase and/or add BSA or serum (up to 0.5% and 5-10% respectively) to stabilize the cells to further digestion.
* The separately prepared Collagenase and protease enzymes in the “Blend” products (No. C7926, C8051 or C8176) give reproducible control of how much of each is used.
** DNAse will be inactivated by the shear of excessive stirring, and added enzymes may be digested by the neutral protease present in the Collagenase.
*** Use EGTA (or EDTA) to remove Ca++ and flush away microorganisms, then wash tissue with buffer to remove the chelating agent. Do not add EGTA or EDTA to the enzyme solutions!
References
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