Yes and No. The most effective inhibitor of protease activity is to use serum-containing media. Adult serum is more effective than fetal serum. The protease activity is likely inhibited by alpha-2-macroglobulin. Buffers or media containing human serum albumin (HSA) are ineffective in suppressing protease activity, even at very high concentrations of protein (>10%). Collagenase activity
The initial fermentation procedures to generate collagenase or other bacterial enzymes required the use of mammalian meat peptones that contained collagen peptides. As collagenase fermentation methods were refined, gelatin peptones were used in place of meat peptones. You can request a certificate of origin from the manufacturer to determine if animal proteins were used in
Yes. The supplemental protease used for cell isolation is determined by the precedent set by the enzyme suppliers. However, it is likely that other proteases will also work but it needs to be determined by experimentation. Three different proteases have been used to isolate human islets: thermolysin, BP Protease (a Dispase equivalent enzyme), and C.
Yes. Lot qualification is required because of the difference in enzymatic/biochemical compositions between different lots of poorly defined, crude or enriched collagenase products. Replacing poorly defined collagenase with defined collagenase usually requires a single set of lot qualification experiments. Once the enzyme composition and dose is defined, then the same product and dose can be
C. histolyticum collagenase and many other bacterial neutral proteases are metallo-proteases. They require excess calcium to maintain their enzyme activity. One mM calcium ion is sufficient to protect this activity. Cation chelating agents such as EDTA or EGTA should not be used in the presence of these enzymes. As many of you know, these chelating
No and yes. For poorly defined collagenase products, many isolators use these values as a guideline in selecting a specific lot of collagenase. These analyses are an unreliable predictor of successful cell isolation because the collagenase is often degraded by proteases during the manufacturing or manufacturing procedure. For defined collagenase products, if the manufacturer claims
Each lot of poorly defined crude or enriched collagenase is unique because it represents the enzymatic activities present in the C. histolyticum culture supernatant. The enzyme activities listed on the Certificate of Analysis can be used as a guide but they are not sufficient to predict the effectiveness of a specific lot to recover cells.
Initially, crude collagenase manufactured and sold by Worthington Biochemicals in the early 1960’s and later by Sigma-Aldrich. These products are minimally processed bacterial culture supernatants. Later, Sigma manufactured Collagenase Type XI, an enriched collagenase product prepared by further processing of crude collagenase. Enriched collagenase has higher enzyme activities than found in crude collagenase. All these
Nearly all collagenases used for cell isolation are derived from culture supernatants recovered after anaerobic culture of Clostridium histolyticum. A rich broth containing digested animal meat is frequently used to ensure growth of C. histolyticum. C. histolyticum is a saprophyte that survives by secreting a diverse number of enzymes that breaks down decaying organic matter.