CIzyme Thermolysin



CIzyme Thermolysin protease  is an aseptically dispensed protein, in lyophilized form (12 mg) or as a frozen liquid (6 mg), containing purified thermolysin from Bacillus thermoproteolyticus.

Intended Use

CIzyme Thermolysin is a thermostable neutral protease that is commonly used with purified collagenase to isolate many different types of cells. It is commonly used with collagenase to isolate islets from rodent, cynomolgus monkey¹, and human pancreata.

Cat. # N/A
  • Manufactured for superior lot-to-lot enzyme consistency for improved performance consistency
  • When combined CIzyme Collagenase HA, 12 mg of thermolysin contained in this vial is suitable to isolate islets from an average human pancreas.
  • Low contaminating endotoxin levels (<50 EU/OD)
  • Lot specific Certificate of Analysis provided with complete QC analysis results
  • White lyophilized cake under vacuum in amber bottle sealed with butyl rubber stopper
  • Contains >2.3 Million Neutral Protease Activity (NPA) units.
  • Unopened product stable for at least 2 years when stored between -15 to – 25°C; internal studies indicate the reconstituted enzyme is stable at the same temperatures for at least 1 year, as long as no other protease enzyme has been added to the solution.
  • Product is shipped on dry ice to provide the most stable conditions during shipment.

Manufacturing Details

CIzyme Thermolysin is purified from B. thermoproteolyticus culture supernatants that contain bovine milk casein and is purified by re-crystallization and further purified by column chromatography.

After assessing enzyme potency, the collagenase is aseptically dispensed into depyrogenated vials and lyophilized. When dry, the CIzyme Thermolysin 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.

Suggested Assays

The working concentration of purified thermolysin is determined by the total enzyme activity contained in the product. Typically, this is expressed as the total neutral protease units as measured by using a FITC-BSA substrate. Several reports have shown the importance of using an optimal amount of neutral protease in an islet isolation procedure.


Product Documentation

Pack Inserts

Safety Data Sheet


Certificate of Origin

General Product FAQ

Is this product intended for use in diagnostic or therapeutic procedures?

No. This product is for research use only. Guidance for use of reagents in clinical cell transplantation procedures is governed by local Institutional Review Boards and regional Health Authorities. This product is manufactured in accordance with the principles for clinical trial material outlined in ICH Q7a. The document control system in place is in alignment with FDA guidance for Phase I material. Document controls are in place to minimize the chances of cross-contamination.

What are application-specific formulations?

Application-specific formulations are pre-blended mixtures of purified collagenase and protease in optimal ratios for isolation of cells from a particular tissue and/or species.

Currently, our CIzyme™ Collagenase AS and CIzyme™ Collagenase RI are our only two application-specific formulations.

What is the best way to inactivate the enzymes when a digestion is complete?

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 Protease FAQ

Can I substitute the type of protease I use for my application?

Yes.  Selection of a protease in many cases is simply due to the historical precedence of the given cell isolation protocol being followed; however, that is not to say a different one would not work.  The isolation of human islets is a perfect example in which thermolysin, BP Protease (a Dispase equivalent), and C. histolyticum neutral protease have all been successfully used.  Generally, bacterial or fungal sources of protease are selected due to their broader specificity. Substitution of one protease for another may not be a seamless transition, however, as the kinetics of digestion can be dramatically different among the various general proteases.   For guidance in selecting a protease, contact VitaCyte’s technical support team.

Thermolysin FAQ

How should I store my CIzyme Thermolysin?

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 1 year. Additional studies have shown the enzyme can go through a freeze-thaw cycle at least once without loss of potency by neutral protease (NP) assay. The product is shipped on dry ice to provide the most stable conditions during shipment.