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 liver cells from a liver tissue, using a degrading-enzyme composition, comprising:
 measuring an abundance of collagen I and collagen III in the liver tissue,   determining an amount ratio of collagenase H to collagenase G derived from  Clostridium  sp. based on the measured abundance of collagen I and collagen III, wherein a higher amount ratio is used when the measured abundance is higher and a lower amount ratio is used when the measured abundance is lower,   preparing the degrading-enzyme composition by adding collagenase H and collagenase G derived from  Clostridium  sp. with the determined ratio, to a predetermined amount of one or more of a neutral protease or a protease derived from  Clostridium  sp.,   treating the liver tissue with the degrading-enzyme composition, and   separating liver cells from the liver tissue treated with the degrading-enzyme composition,   wherein collagenase H degrades collagen I and collagen III, and   wherein the amount ratio (H/G) of collagenase H to collagenase G in the degrading-enzyme composition is 0.25 or less.   
     
     
         2 . The method according to  claim 1 , wherein the abundance of collagen I and collagen III in the liver tissue is measured by enzymatic immunoassay using specific antibodies against collagen I and collagen III. 
     
     
         3 . The method according to  claim 1 , wherein the neutral protease is thermolysin or a neutral protease derived from  Clostridium  sp. 
     
     
         4 . The method according to  claim 3 , wherein the degrading-enzyme composition contains thermolysin 0.5 mg/10 ml, collagenase G 14.4 mg/10 ml and collagenase H 3.6 mg/10 ml.