Method for separating cell from biological tissue

Abstract

Provided is a method for efficiently and stably separating cells having a high biological activity from a biological tissue, by using a degrading-enzyme composition, which is prepared by adding an enzyme for degrading a major protein of the biological tissue in an amount determined depending on the composition of the major protein to a predetermined amount of a neutral protease and/or a protease derived from Clostridium sp. According to this method, the type and amount of protein-degrading enzyme to be used for isolating cells can be determined from the composition of a major protein of the biological tissue. Thus, cells having a high biological activity can be efficiently separated while reducing the amount of protein-degrading enzyme to be used.

1 . A method for separating cells from a biological tissue, comprising using a degrading-enzyme composition prepared by adding an enzyme for degrading a major protein of the biological tissue in an amount determined depending on the composition of the major protein to a predetermined amount of a neutral protease and/or a protease derived from  Clostridium  sp. 
     
     
         2 . The method according to  claim 1 , comprising the steps of determining a composition of the major protein of the biological tissue; preparing a degrading-enzyme composition by adding an enzyme for degrading the major protein in an amount determined depending on the composition determined to the predetermined amount of a neutral protease and/or a protease derived from  Clostridium  sp.; and treating the biological tissue with the degrading-enzyme composition prepared. 
     
     
         3 . The method according to  claim 1 , wherein the enzyme for degrading the major protein is collagenase H and/or collagenase G of  Clostridium  sp. 
     
     
         4 . The method according to  claim 3 , wherein the major protein is collagen I and/or collagen III. 
     
     
         5 . The method according to  claim 4 , wherein the amount ratio of collagenase H and/or collagenase G to be added to the degrading-enzyme composition is determined in accordance with the composition of collagen I and/or collagen III. 
     
     
         6 . The method according to any one of  claims 1  to  5 , wherein the neutral protease is thermolysin or a neutral protease derived from  Clostridium  sp. 
     
     
         7 . The method according to any one of  claims 1  to  5 , wherein the protease derived from  Clostridium  sp. is a protease having an α-N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) degradation activity. 
     
     
         8 . The method according to any one of  claims 1  to  5 , wherein the protein composition of the biological tissue is determined by an enzymatic immunoassay. 
     
     
         9 . A method for separating pancreatic islets from a pancreas tissue, using a degrading-enzyme composition, which is prepared by adding collagenase H and/or collagenase G derived from  Clostridium  sp. in an amount sufficient to degrade collagen I and/or collagen III and in accordance with a composition of collagen I and/or collagen III of the pancreas tissue, to a predetermined amount of a neutral protease and/or a protease derived from  Clostridium  sp. 
     
     
         10 . The method according to  claim 9 , wherein the degrading-enzyme composition contains a neutral protease derived from  Clostridium  sp., as the neutral protease, and a protease having an α-N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) degradation activity, as the protease derived from the  Clostridium  sp. 
     
     
         11 . A method for separating liver cells from a liver tissue, using a degrading-enzyme composition, which is prepared by adding collagenase H and/or collagenase G of  Clostridium  sp. in an amount sufficient to degrade collagen I and/or collagen III and in accordance with a composition of collagen I and/or collagen III of the liver tissue, to a predetermined amount of a neutral protease and/or a protease derived from  Clostridium  sp.