Methods of measuring cell viability in tissue engineered products

Abstract

This invention provides methods of measuring the viability of cultured cells by detecting one or more cell death-stable proteins or enzyme activities. Methods provided by the invention correlate viability to relative levels of enzyme activity in cell-containing and non-cell-containing fractions of a cell culture.

1 .- 7 . (canceled) 
     
     
         8 . A method of measuring the cytotoxicity of a treatment to cells in a tissue-engineered product maintained in a cell culture medium comprising:
 (a) applying the treatment to the tissue engineered product;   (b) detecting a cell death-stable enzyme activity in a portion of a cell culture conditioned medium not containing cells of the tissue-engineered product;   (c) detecting a cell death-stable enzyme activity in the cells and conditioned medium of a portion of the cell culture medium containing cells of the tissue-engineered product; and   (d) comparing the level of cell death-stable enzyme activity in the portion of the cell culture medium containing cells of the tissue-engineered product to the level of cell death-stable enzyme activity in the portion of the cell culture conditioned medium not containing cells of the tissue-engineered product,   
       wherein the cytotoxicity of the treatment is proportional to the difference in the fraction of viable cells in the treated tissue engineered product to an untreated tissue engineered product, and wherein the fraction of viable cells in the tissue-engineered product is directly proportional to the difference between the level of cell death-stable enzyme activity in the portion of the cell culture medium containing cells of the tissue-engineered product and the level of cell death-stable enzyme activity in the portion of the cell culture conditioned medium not containing cells of the tissue-engineered product. 
     
     
         9 . The method of  claim 8 , wherein the cells are mammalian. 
     
     
         10 . The method of  claim 9 , wherein the cells are human. 
     
     
         11 . The method of  claim 10 , wherein the cells are chondrocytes. 
     
     
         12 . The method of  claim 9 , wherein the cells are grown on a matrix. 
     
     
         13 . The method of  claim 9 , wherein the cells are present at a density of between 1.5×10 4  and 6×10 6  cells/cm 2 . 
     
     
         14 . The method of  claim 8 , wherein the cells are present at a high density. 
     
     
         15 . The method of  claim 14 , wherein the cells are present at between 2×10 6  and 6×10 6  cells/cm 2 . 
     
     
         16 . The method of  claim 8 , wherein the cell death-stable enzyme activity is measured by the steps comprising:
 (a) contacting a sample with a substrate of the cell death-stable enzyme activity wherein the substrate in conjugated to a detectable leaving group; and   (b) detection of the leaving group,   
       wherein the amount of leaving group detected is proportional to the level of cell death-stable enzyme activity. 
     
     
         17 . The method of  claim 16 , wherein the leaving group is chromogenic, luminogenic, or fluorescent. 
     
     
         18 . The method of  claim 17 , wherein the leaving group is fluorescent. 
     
     
         19 . The method of  claim 18 , wherein the leaving group is Rhodamine-110. 
     
     
         20 . The method of  claim 16  wherein the substrate is bis-(Ala-Ala-Phe)-Rhodamine-110. 
     
     
         21 . The method of  claim 18 , wherein the leaving group is a coumarin derivative. 
     
     
         22 . The method of  claim 21 , wherein the leaving group is AMC. 
     
     
         23 . The method of  claim 22 , wherein the substrate is Ala-Ala-Phe-AMC. 
     
     
         24 . The method of  claim 16 , further comprising the step of adding an agent which modulates the signal of the detectable leaving group. 
     
     
         25 . The method of  claim 24 , wherein the agent which modulates the signal of the leaving group, attenuates the signal of the leaving group. 
     
     
         26 . The method of  claim 25 , wherein the agent which attenuates the signal of the leaving group is phenol red. 
     
     
         27 . The method of  claim 26 , wherein the phenol red is present at a concentration of up to 500 mg/L. 
     
     
         28 . The method of  claim 8 , wherein the cell death-stable enzyme activity is proteolytic. 
     
     
         29 . The method of  claim 28 , wherein the cell death-stable enzyme activity comprises one or more tripeptidyl peptidases. 
     
     
         30 . The method of  claim 8 , wherein the cell death-stable enzyme activity is both a necrotically and programmed cell death-stable enzyme activity. 
     
     
         31 . The method of  claim 30 , wherein the cell death-stable enzyme activity is a programmed cell death-stable enzyme activity. 
     
     
         32 . The method of  claim 30 , wherein the cell death-stable enzyme activity is a necrotically stable enzyme activity.