Purified mixture of collagenase I, collagenase II and two other proteases

• US 5,753,485 A
• Filed: 01/24/1996
• Issued: 05/19/1998
• Est. Priority Date: 06/24/1994
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Original Assignee: Boehringer Mannheim Corporation
• Inventors: Dwulet, Francis E., Gill, John F., Ellis, Bernice B., Waters, David G., Jacobsen, Linda B., Smith, Marilyn E.
• Primary Examiner(s): Saucier, Sandra E.
• Application Number:
  US08/590,957
• Time in Patent Office: 846 Days
• Field of Search: 435/220, 435/183, 435/219
• US Class Current: 435/220
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea

Abstract

A purified enzyme mixture useful for isolating cells or cell clusters from tissue is disclosed. The mixture includes at least two collagenase enzymes, at least two other proteases, and additional non-protease components. The mixture is purified by removing at least some of the non-protease components. The purified mixture may then be used to isolate cells or cell clusters from tissue. Also disclosed are the essential components of the purified enzyme mixture as well as preferred ranges and ratios of these essential components for isolating cells or cell clusters from tissue. Finally, a dissociation system is disclosed that can be used with the purified enzyme mixture to dissociate tissue and recover cells or cell clusters.

Claims

1. An enzyme composition prepared by combining defined amounts of proteases said composition comprising collagenase I and collagenase II from C. histolyticum, having an A₂₈₀ /A₃₆₀ ratio of greater than 30 and two other proteases, wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of the collagenase II in the composition is about 0.3 to about 0.6.

2. The composition of claim 4 wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of collagenase II is about 0.4 to about 0.45.

3. The composition of claim 1 wherein the collagenase I and collagenase II have an A₂₈₀ /A₃₆₀ ratio greater than 100.

4. The enzyme composition of claim 1 wherein at least one of the two other proteases is a neutral protease.

5. The enzyme position of claim 4 wherein the neutral protease is selected from the group consisting of thermolysin, dispase and C. histolyticun neutral protease.

6. The composition of claim 5 wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of collagenase II is about 0.4 to about 0.45.

7. The enzyme composition of claim 4 wherein the neutral protease is thermolysin.

8. The enzyme composition of claim 7 wherein the ratio of the total FITC casein activity of the enzyme composition to the total Wunsch units of activity of the masses of collagenase I and collagenase II in the composition is about 19:

1 to about 70;

1.

9. The enzyme composition of claim 4 wherein the neutral protease is dispase.

10. The enzyme composition of claim 9 wherein the ratio of the total FITC casein activity of the enzyme composition to the total Wunsch units of activity of the masses of collagenase I and collagenase II in the composition is about 19:

1 to about 62;

1.

11. The enzyme composition of claim 4 wherein the neutral protease is dispase or thermolysin and the ratio of total FITC casein activity of the enzyme composition to the total Wunsch units of the masses of collagenase I and collagenase II in the composition is about 41:

1.

12. The enzyme composition of claim 4 wherein the two other proteases are a neutral protease and clostripain.

13. The enzyme composition of claim 12 wherein the neutral protease is selected from the group consisting of thermolysin, dispase and C. histolyticum neutral protease.

14. The enzyme composition of claim 12 wherein the neutral protease is thermolysin.

15. The enzyme composition of claim 14 wherein the ratio of the total FITC casein activity of the enzyme composition to the total Wunsch units of activity of the masses of collagenase I and collagenase II in the composition is about 19:

1 to about 70;

1.

16. The enzyme composition of claim 12 wherein the neutral protease is dispase.

17. The enzyme composition of claim 16 wherein the ratio of the total FITC casein activity of the enzyme composition to the total Wunsch units of activity of the masses of collagenase I and collagenase II in the composition is about 19:

1 to about 62;

1.

18. The enzyme composition of claim 12 wherein the neutral protease is dispase or thermolysin and the ratio of total FITC casein activity of the enzyme composition to the total Wunsch units of the masses collagenase I and collagenase II in the composition is about 41:

1.

19. The composition of claim 12 wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of collagenase II is about 0.4 to about 0.45.

20. A method of preparing an enzyme composition adapted for isolating living cells from tissue, said method comprising the steps of combining collagenase I, collagenase II, and clostripain from C. histolyticum wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of collagenase II is about 0.3 to about 0.6;

and

adding an amount of a neutral protease sufficient to raise the total FITC casein activity of the enzyme composition to a level such that the ratio of the total FITC casein activity of the enzyme composition to the total Wunsch unit activity of the masses of collagenase I and collagenase II in the enzyme composition is about 19;

1 to about 70;

1.

21. The method of claim 20 wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of collagenase II is about 0.4 to about 0.45.