Despite years of experimentation, the tissue dissociation enzyme composition required to generate a sufficient number of islets for islet transplantation remains mostly undefined and not well understood. A new report from the University of Minnesota shows that intact class I collagenase is important for successful human islet isolation. This conclusion reflects the importance of properly characterizing purified tissue dissociation enzymes prior to use in human islet isolation procedures.

In 2008, VitaCyte and Drs. Hering, Balamurugan, and coworkers at the University of Minnesota began a collaboration to address this problem. This led to characterization of collagenase enzymes from major suppliers by analytical anion exchange high performance liquid chromatography (HPLC) and assessment of collagen degradation activity (CDA) using a fluorometic microplate assay.¹ These results are summarized in the report recently published in Transplantation.²

The key findings from this report are:

• Although is has been known for many years that class I (C1) and class II (C2) collagenase in combination with neutral protease(s) is required for successful cell isolation³, this is the first report showing that the molecular form of C1 in these mixtures appears to be critical for successful human islet isolation.
• A significantly higher proportion of successful islet isolations could be used for transplantation when the enzyme mixture contained a high proportion of intact C1 (VitaCyte CIzyme™ Collagenase HA enzyme) when compared to the Serva product that contained predominantly degraded C1
• Intact C1 is best detected by using analytical anion exchange HPLC and characterized by using the CDA assay. This combination of analytical tools provides insight into the amount of intact C1 present in the enzyme mixture.
• Reversed phase HPLC was shown to be ineffective in detecting intact from degraded C1.

VitaCyte is committed to developing improved assays used to characterize the enzymes used in cell isolation methods and to correlate these results to the outcome of cell isolation procedures. Please do not hesitate to visit http://www.vitacyte.com to obtain additional product or technical information on the use of tissue dissociation enzymes in cell isolation. We can also be contacted by phone at 888-664-2687.

References

1. Development and Characterization of a Collagen Degradation Assay to Assess Purified Collagenase Used in Islet Isolation

R.C. McCarthy, B. Spurlin, M.J. Wright, A.G. Breite, L.K. Sturdevant, C.S. Dwulet and F.E. Dwulet

Transplantation Proceedings, 2008 March; 40(2): 339-342.

2. Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay.

Balamurugan AN, Breite AG, Anazawa T, Loganathan G, Wilhelm JJ, Papas KK, Dwulet FE, McCarthy RC, Hering BJ.

Transplantation. 2010 Apr 27; 89(8): 954-61.PMID: 20300051

3. An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation

G.H.J. Wolters, G.H.Vos-Scheperkeuter, J.H.M. van Deijnen and R. van Schilfgaarde.

Diabetologia, 1992, 35: 735-742.