Defined enzyme mixtures for obtaining cells and treating wounds

Details Defined enzyme mixtures for obtaining cells and treating wounds Defined enzyme mixtures for obtaining cells and treating wounds

1997-09-16

2000-11-14

Lilling, Herbert J.

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Hydrolases

424 941, 424 9466, 435183, 435212, 435218, 435219, A61K 3848, A61K 3846, C12N 948

Patent

active

061466263

DESCRIPTION:

BRIEF SUMMARY
Methods for isolating cells from tissues ought to be reproducible and guarantee minimum damage to the cells obtained. In general, collagenase-containing preparations with an indefinite composition from Clostridium histolyticum are used for this purpose, but these aims are not reliably achieved therewith. The use of other enzyme preparations or nonenzymatic methods is not customary or provides only poor isolation results (1), (9).
The collagenase-containing preparations (2) normally used and recommended for use are obtained from filtrates of cultures of the bacterial strain Clostridium histolyticum and, besides various collagenases and proteases (3a), (3b), also contain cleavage products of these enzymes formed by proteolysis, and other constituents, some of which have injurious effects and are unknown.
According to the present state of knowledge, it is not possible with a single enzyme from Clostridium histolyticum to obtain viable cells in good yield. On the contrary, it is necessary for various enzymes from this bacterium to act together in order to achieve efficient tissue breakdown. However, the ratio of the amounts of enzymes necessary for this has not hitherto been disclosed. Nor have defined mixtures of enzymes from Clostridium histolyticum been available for the user.
Based on the problem described, various research groups have attempted to employ purified enzymes for isolating cells from tissues. However, this has never entailed use of pure enzymes and thus any clearly defined mixtures either.
Suggs et al. (4) isolated human vein endothelial cells using a mixture composed of a purified collagenase fraction and purified trypsin from beef pancreas. The enriched collagenase fraction employed did not, however, have a defined content of the various enzymes and still contained, for example, small amounts of clostripain. The isolation result was not significantly different from that obtained on use of collagenase-containing preparations with an indefinite composition. It was not possible to achieve satisfactory tissue disintegration using single components of the mixture. However, the use of trypsin for cell isolation is not without problems because this proteolytic enzyme attacks membrane proteins. Thus, for example, there is an adverse affect on insulin binding to liver membranes and to adipocytes (5).
Hefley (6), (7) employed mixtures of purified collagenase fractions (6) to isolate bone cells from the cranium of mice. Earlier (7), the author reported that it is possible to employ a mixture of a purified collagenase fraction together with a neutral protease for successful isolation of these cells. However, in both studies there was use of eluates from column fractionations whose content of various collagenases differing in their substrate specificity, and of other components, was unknown.
Wolters et al. (8) used mixtures of neutral protease and "collagenase type VII" supplied by Sigma, although it is unknown which types and amounts of various collagenases this contained, to isolate islet cells from the rat pancreas. In addition, it had a small content of clostripain und "nonspecific protease". The neutral protease was employed in a highly purified form. Mixtures of the two said components afforded good yields of viable islet cells.
The present invention relates to the individual enzymes collagenase HP, collagenase AZ and elastase in high purity, and to mixtures thereof.
Collagenase HP has a specific activity of at least 20 U/mg, preferably at least 50 U/mg, in the assay of Gra.beta.mann and Nordwig (11) with the synthetic hexapeptide Z-Gly-Pro-Gly-Gly-Pro-Ala as substrate. For pharmaceutical purposes it preferably has a specific activity of 100 U/mg or more.
Collagenase AZ has a specific activity of at least 10 U/mg, preferably at least 30 U/mg, in the assay of Mandl et al. (12) using azocoll as substrate. For pharmaceutical purposes it preferably has a specific activity of 50 U/mg or more.
Elastase has a specific activity of at least 2 U/mg, preferably at least 5 U/mg, in the assay with elastin from

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