- Manufactured for superior lot-to-lot enzyme consistency for improved performance consistency
- Formulated to contain an optimal amount of Collagen Degrading Activity (CDA) and Neutral Protease Activity (NPA) for rodent islet isolation
- Low contaminating endotoxin levels (<25 EU/OD)
- Lot specific Certificate of Analysis provided with complete QC analysis results
CIzyme RI is an aseptically filled, lyophilized mixture of 60% purified class I (C1) and 40% purified class II (C2) collagenase from Clostridium histolyticum pre-mixed with neutral protease from Bacillus polymyxa.
CIzyme RI was developed using C57BL/6 mice and has been successfully used to isolate islets from a wide variety of strains of mice and rats.1,2
- White lyophilized cake under vacuum in amber bottle sealed with butyl rubber stopper
- Contains approximately 375,000 CDA units and 75,000 NPA units.
- Unopened product stable for at least 2 years when stored between -15 to -25°C; internal studies indicate comparable stability for reconstituted, frozen solution for at least 3 months at the same temperatures
- Lyophilized in a buffer containing calcium to ensure enzyme stability during initial reconstitution.
CIzyme RI is purified from culture supernatants of C. histolyticum that contain porcine gelatin and pancreatic enzymes derived from US and Canadian sources. No animal derived products are used in the culture of B. polymyxa or in any step of the purification process for either enzyme. Standard chromatography techniques are used to purify the collagenase.
After assessing enzyme potency, the collagenase is aseptically dispensed into depyrogenated vials and lyophilized. When dry, the Collagenase AS vials are sealed under vacuum. A representative sample from the batch is reconstituted and analyzed by a variety of quality control tests. Results are compared to established specifications before release into inventory.
VitaCyte relies on several biochemical tools to characterize and ensure the consistency of components used to prepare CIzyme RI. The Collagenase activity is characterized using both the traditional Wünsch (Pz-peptide) 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 substrate. The B. polymyxa neutral protease activity (NPA) is measured using a FITC labeled BSA.
Breite AG, Stull N, McCarthy RC, Mirmira R, Dwulet FE. Cell Transplantation Society-International Xenotransplantation Association 2011 Joint Congress – Miami, FL P050 “Purified tissue dissociating enzyme performance in isolating mouse islets”
CIzyme RI FAQ
This product is stable for at least two years from date of manufacture if stored unopened between -15 to – 25°C. Internal studies have shown the reconstituted enzyme is stable as a frozen solution between -15 to – 25°C for at least 3 months. Additional studies have shown the reconstituted enzyme was successfully frozen and thawed at least twice without apparent loss of potency as assessed by the CDA and NPA assays.
General Product FAQ
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.
General Collagenase FAQ
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.