Development of purified enzyme blends for optimized tissue dissociation

F. E. Dwulet, T. J. Fetterhoff, T. J. Cavanagh, L. Jacobsen, M. Smith, M. J. Wright, Timothy E Albertson, S. Calvin

Research output: Contribution to journalArticlepeer-review

Abstract

Many applications in biomedical research require the dissociation of tissue into its constituent cells. Collagenase is widely used for tissue dissociation. Commercial collagenase comprises concentrated culture supernatant from the fermentation of Clostridium histolyticum. This reagent is heterogeneous, containing up to 30 different enzymes, cellular debris, pigments and endotoxin. The conditions and age of the fermentation greatly affects levels of specific enzymes and contaminants from one preparation to the next. This lot to lot variation has limited the usefulness of collagenase to biomedical research. The enzymes required for tissue dissociation were identified in crude collagenase. Collagenases I & II, clostripain, and neutral protease were each purified to greater than 95% homogeneity. Formulations of enzyme mixtures (Liberase) for optimal tissue dissociation were predicted by response surface modeling. Where indicated, purified non-Clostridial proteases were used. Liberase was compared to crude collagenase in the isolation of hepatocytes and pancreatic islets. Hepatocyte yield and viability were increased by 70 and 30%, respectively. Hepatocyte function was increased 30% (cytochromeP450 assay). Canine islet yields increased 160% to over 4000 islets per gram. Islet function (insulin secretion in response to glucose challenge) increased 3 fold to a stimulation index of over 11. Liberase purified enzyme blends enable reproducible tissue dissociation with significantly increased cell yield, viability, and a higher level of maintained native cell functionality.

Original languageEnglish (US)
JournalFASEB Journal
Volume12
Issue number5
StatePublished - Mar 20 1998
Externally publishedYes

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

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