Composition for tissue dissociation containing collagenase I and II from clostridium histolyticum and a neutral protease

• US 5,830,741 A
• Filed: 12/06/1996
• Issued: 11/03/1998
• Est. Priority Date: 12/06/1996
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Original Assignee: Boehringer Mannheim Corporation
• Inventors: Dwulet, Francis E., Smith, Marilyn E.
• Primary Examiner(s): Weber, Jon P.
• Application Number:
  US08/760,893
• Time in Patent Office: 697 Days
• Field of Search: 435/220, 435/183
• US Class Current: 435/220
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/069 Vascular Endothelial cells
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea

Abstract

An enzyme composition for dissociating tissues is disclosed. The composition comprises purified collagenase I and collagenase II from Clostridium histolyticum in a mass ratio of about 0.3 to 0.6 and a neutral protease such that the ratio of total FITC casein units of activity to Wunsch units of activity in the composition is about 250:1 to about 600:1. The preferred neutral protease is thermolysin.

Claims

1. An enzyme composition comprising collagenase I and collagenase II from Clostridium histolyticum and an endoprotease, said enzyme composition prepared by combining collagenase I having a Wunsch unit of activity of about 0.2 U/mg to about 0.6 U/mg and collagenase II having a Wunsch unit of activity of about 9.5 U/mg to about 13.5 U/mg wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of collagenase II in the enzyme composition is about 0.3 to about 0.6, and adding an amount of the endoprotease sufficient to increase the ratio of the total FITC casein units of activity of the enzyme composition to the total Wunsch units of activity of the combined masses of collagenase I and collagenase II to about 250:

1 to about 600;

1.

2. The enzyme composition of claim 1, wherein the endoprotease is a neutral protease.

3. The enzyme composition of claim 1, wherein the endoprotease is thermolysin and the composition is substantially free of endotoxins.

4. An enzyme composition comprising collagenase I and collagenase II from Clostridium histolyticum and thermolysin, said enzyme composition prepared by combining collagenase I having a Wunsch unit of activity of about 0.2 U/mg to about 0.6 U/mg and collagenase II having a Wunsch unit of activity of about 9.5 U/mg to about 13.5 U/mg wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of collagenase II in the enzyme composition is about 0.3 to about 0.6, and adding an amount of the thermolysin sufficient to increase the ratio of the total FITC casein units of activity of the enzyme composition to the total Wunsch units of activity of the combined masses of collagenase I and collagenase II to about 400:

1.

5. A method of preparing an enzyme composition adapted for isolating living cells from tissue, said method comprising the step of combining collagenase I from Clostridium histolyticum having a Wunsch unit of activity of about 0.2 U/mg to about 0.6 U/mg and collagenase II from Clostridium histolyticum having a Wunsch unit of activity of about 9.5 U/mg to about 13.5 U/mg and an amount of an endoprotease sufficient to raise the FITC casein activity of the enzyme composition to a level such that the ratio of the total FITC casein units of activity of the enzyme composition to the total Wunsch units of activity of the combined masses of collagenase I and collagenase II is about 250:

1 to about 600;

1.

6. The method of claim 5, wherein the endoprotease is thermolysin.