Our ContributionsA History of Innovative Solutions
We’re proud of our history because our purified, rigorously characterized Clostridium histolyticum collagenases enabled us and others to achieve many firsts in improving enzyme-mediated cell isolation methods. Here’s a brief look at some of those firsts, including the first patents for manufacture of defined and purified mixtures of protease and collagenase (now expired) initially assigned to Boehringer Mannheim Corporation, where the first author was Francis Dwulet, one of VitaCyte’s co-founders.
Enzyme mixtures of purified C. histolyticum collagenase and bacterial neutral protease to isolate mammalian cells from tissue.1,2
Significance
First cost-effective, commercial manufacture of high-quality purified collagenase-protease enzyme mixtures to isolate mammalian cells, validating earlier reports that indicated both enzymes required for releasing cells from tissue.
Development of a specific, functional collagenase assay where specific activity of different forms of purified enzymes correlate with physico-chemical analysis.3
Significance
First report of specific collagen degradation activity (CDA U/mg protein) correlating with the structure function of three different functional isoforms of collagenase enzymes: intact class I (C1116kDa), truncated class I (C1100kDa), intact class II (C2114kDa).
Indication that molecular form of class I collagenase is associated with higher human islet yields when used in enzyme mixtures that also contain class II collagenase and supplemental protease.4
Significance
First report showing that C1116kDa was more effective in human islet recovery than C1100kDa in C1-C2-protease enzyme mixtures used to isolate human islets using the Ricordi method.
Proposed mechanism of interaction between class I and class II C. histolyticum collagenase and neutral protease to release cells from tissue.5
Significance
First report describing a proposed mechanism of collagenase-mediated release of cells from tissue. The mechanism, illustrated in a 2-minute medical animation video, shows collagenases’ dual role in thinning the jungle of collagen fibrils and “loosening” the tissue matrix. The loosened matrix exposes protease-sensitive sites on the cell-anchoring proteins, that are cut by neutral proteases, releasing cells from the tissue.
5
First commercial-scale manufacture of purified recombinant C. histolyticum collagenases, where the optimal dose of these collagenases used for human islet isolation was determined prospectively using a design of experiment (DOE) approach.6
Significance
The animal-origin-free enzyme mixtures used in the DOE were recombinant intact C1 and C2 collagenases mixed with a fixed dose of BP Protease. The lowest dose of recombinant C1 and C2 collagenases among the 4 enzyme mixtures evaluated was approximately 40% of the dose of natural collagenase currently used for human islet isolation.
6
Development of a purified collagenase-protease enzyme mixture for human hepatocyte isolation.7
Significance
VitaCyte collaborated with Stephen Strom’s lab at the University of Pittsburgh to develop a purified collagenase-protease mixture, eliminating the need for collagenase lot pre-qualification when using traditional collagenase products for this application.
7
Development of a simple method to optimize C. histolyticum collagenases-protease mixtures for enzyme-mediated cell isolation.8
Significance
Insights into the mechanism of action of collagenase mediated tissue dissociation provided a new approach to optimize collagenase-protease mixtures for cell isolation. The new concept is that if purified collagenase is used in excess, then the selection and dose of neutral protease activity determine the recovery of viable, functional cells.
8
Manufacture of a GMP Grade, purified, animal-origin-free Dispase equivalent enzyme (BP Protease) is an effective enzyme for enzyme-mediated cell isolation. 6,7
Significance
BP Protease is a more effective enzyme for human islet and hepatocyte isolation than Thermolysin.
References
Dwulet FE, Ellis BB, Gill JF, Jacobsen LB, Smith ME, Waters DG, inventors; Purified mixture of collagenase I, collagenase II, and two other proteases patent US Patent # 5,753,485. 1998.
Dwulet FE, Smith ME, inventors; Composition for tissue dissociation containing collagenase I and II from Clostridium histolyticum and a neutral protease. United States patent 5,830,741. 1998.
Balamurugan AN, Breite AG, Anazawa T, et al. Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay. Transplant 2010; 89(8): 954-61.
- 7.
Balamurugan AN, Green ML, Breite AG, et al. Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation. Transplant Direct 2016; 2(1): e54.
Gramignoli R, Green ML, Tahan V, et al. Development and application of purified tissue dissociation enzyme mixtures for human hepatocyte isolation. Cell Transplant (2012) 21:1245-1260.