1 g, 100 mg
VitaCyte’s defined enriched (DE) collagenase products set a new standard of consistency and enables assessment of a broader range of collagenase:protease ratios for cell isolation than found with current collagenase products. Each DE product contains a fixed amount of protease activity and increasing amounts of collagenase activity, simplifying collagenase selection and minimizing the need for lot qualification.
DE Collagenase 20 (100 mg pack size, Cat#011-1120) and DE Collagenase 200 (1 g pack size, Cat#011-1020) may be used in the following applications:
1 g, 100 mg
DE Collagenase is an aseptically filled, lyophilized mixture of enriched Clostridium histolyticum collagenase and neutral protease from Bacillus polymyxa. Collagenase is purified from culture supernatants of C. histolyticum that contain porcine gelatin and pancreatic enzymes sterilized prior to bacterial fermentation. No animal derived products are used in the culture of B. polymyxa or in any step of the purification process for either enzyme. DE Collagenase contains a fixed amount of neutral protease activity (NP) units and variable amounts of enriched collagenase. DE Collagenase is prepared for research use only.
VitaCyte relies on several biochemical tools to characterize and ensure the consistency of components used to prepare DE Collagenase. The Collagenase activity is characterized using both the traditional Wünsch (Pz-peptide)1 substrate and a more recently developed fluorescent microplate collagen degrading activity (CDA) assays. The CDA relies on fluorecein isothiocyanate (FITC) labeled calf skin collagen fibrils as substrate2. The B. polymyxa neutral protease activity (NPA) is measured using a FITC labeled BSA substrate3.
Wünsch E and Heidrich H-G. (1963) Zur quantitativen bestimmung der kollagenase. Hoppe-Seyler’s Zeitschrift Physiologische Chemie 333, 149-151.
Thermolysin and BP Protease are entirely animal origin free.
A porcine gelatin peptone is used during the fermentation step of natural collagenase production. While this animal sourced material is required to stimulate collagenase production from the biosynthesis from the organism Clostridium histolyticum during anaerobic fermentation, it represents a very low adventitious agent risk as the media is sterilized at 121°C for 30 minutes prior to inoculation, which has been shown to inactivate many viruses.
The gelatin is removed during the purification process and no animal sourced materials are used in the remainder of the process.
Additional manufacturing details are available in product insert literature for each product.
There are two components to inactivation of the digestive enzymes commonly used for tissue dissociation. To inactivate protease activity, a serum-containing media or buffer is most effective. Buffers or media containing human serum albumin (HSA) are ineffective in suppressing protease activity, even at very high concentrations of protein (>10%). However, serum-containing buffers do not inhibit collagenase activity. The most effective method for inactivating collagenase activity is reducing temperature. Collagenases will lose about 80% their maximal activity at 26°C. Thus, introduction of an ice-cold, serum- containing media should virtually eliminate all collagenolytic and proteolytic activity of an enzyme mixture.
VitaCyte’s technical service team has a significant amount of experience in transitioning from crude enzyme products to the more defined purified ones and is glad to provide you with assistance. If you can provide us details of the cell of interest; the enzyme product that is currently being used for the isolation; the concentrations (or activities) being targeted; volumes utilized; and, a certificate of analysis for the particular lot you would like to mimic; we can provide some reasonable guidance to which product(s) and what target activities best fit your application. Our defined enriched (DE) collagenase products are specifically designed to encompass the broad range of activities found in the crude enzyme products and to make the transition to a highly purified product seamless.
In developing an Application Specific Formulation, we generally rely on individuals considered “expert in the art” to ß-test potential formulations and guide us in the refinement of the final composition. These formulations are usually engineered from existing protocols and crude enzyme targets. Once a formulation has been identified and deemed acceptable by at least a couple independent labs, it may be included in our product portfolio. We are always eager to assist you with virtually any request for a custom blend so do not hesitate to contact us to discuss your application.