Methods and tools for detecting collagen degradation

Abstract

Fluorophore-labeled collagen covalently bound to a cell culture vessel can be used to assay collagen degradation by a variety of cell types, including osteoclasts and tumor cells. Such assays provide a high throughput platform for rapid screening of large numbers of potential modulators of, for example, tumor metastasis or osteoclast differentiation and/or function.

1 . A cell culture vessel comprising fluorophore-labeled collagen covalently bound to a culture surface of the cell culture vessel. 
   
   
       2 . The cell culture vessel of  claim 1  wherein the fluorophore is a lanthanide chelate. 
   
   
       3 . The cell culture vessel of  claim 2  wherein the lanthanide chelate is a europium chelate. 
   
   
       4 . The cell culture vessel of  claim 3  wherein the europium chelate is Eu 3+ —N 1 -(p-isothiocyanatobenzyl)diethylenetriamine-N 1 ,N 2 ,N 3 -tetraacetic acid. 
   
   
       5 . The cell culture vessel of  claim 1  wherein the collagen is type I. 
   
   
       6 . The cell culture vessel of  claim 1  wherein the collagen is type IV. 
   
   
       7 . The cell culture vessel of  claim 1  wherein the collagen is mammalian. 
   
   
       8 . The cell culture vessel of  claim 1  wherein the collagen is human. 
   
   
       9 . The cell culture vessel of  claim 1  wherein fluorescence of the covalently bound collagen is quenched. 
   
   
       10 . A cell culture vessel comprising Eu 3+ —N 1 -(p-isothiocyanatobenzyl)diethylenetriamine-N 1 ,N 2 ,N 3 -tetraacetic acid-labeled human collagen type I covalently bound to a culture surface of the cell culture vessel. 
   
   
       11 . A kit for detecting collagen degradation, comprising:
 (a) a cell culture vessel comprising fluorophore-labeled collagen covalently bound to a culture surface of the cell culture vessel; and   (b) instructions for a method comprising steps of:
 (1) culturing cells in culture medium on the fluorophore-labeled collagen, wherein the cells can degrade the collagen or can differentiate into cells which can degrade the collagen; and 
 (2) detecting the presence or absence of a fluorescence signal in a sample of the culture medium, wherein fluorescence signal intensity reflects the concentration of fluorophore-labeled collagen fragments in the sample of the culture medium. 
   
   
   
       12 . The kit of  claim 11  wherein the fluorophore is a lanthanide chelate. 
   
   
       13 . The kit of  claim 12  wherein the lanthanide chelate is a europium chelate. 
   
   
       14 . A kit for detecting collagen degradation, comprising:
 (a) a cell culture vessel comprising Eu 3+ -N 1 -(p-isothiocyanatobenzyl)diethylenetriamine-N 1 ,N 2 ,N 3 -tetraacetic acid-labeled human collagen type I covalently bound to a culture surface of the cell culture vessel;   (b) an enhancing solution; and   (c) instructions for a method comprising:
 (1) culturing cells in culture medium on the Eu 3+ —N 1 -(p-isothiocyanatobenzyl)diethylenetriamine-N 1 , N 2 ,N 3 -tetraacetic acid-labeled collagen, wherein the cells can degrade the collagen or can differentiate into cells which can degrade the collagen; 
 (2) transferring the sample of the culture medium from the cell culture vessel to an assay vessel containing an enhancing solution; and 
 (3) detecting the presence or absence of a fluorescence signal in a sample of the culture medium, wherein fluorescence signal intensity reflects the concentration of Eu 3+ —N 1 -(p-isothiocyanatobenzyl)diethylenetriamine-N 1 ,N 2 ,N 3 -tetraacetic acid-labeled collagen fragments in the sample of the culture medium. 
   
   
   
       15 . A kit for detecting collagen degradation, comprising:
 (a) a cell culture vessel comprising fluorophore-labeled collagen covalently bound to a culture surface of the cell culture vessel, wherein fluorescence of the covalently bound fluorophore-labeled collagen is quenched; and   (b) instructions for a method comprising:
 (1) culturing cells in culture medium on the fluorophore-labeled collagen, wherein the cells can degrade the collagen or can differentiate into cells which can degrade the collagen; 
 (2) transferring the sample of the culture medium from the cell culture vessel to an assay vessel containing an enhancing solution; and 
 (3) detecting in the cell culture vessel the presence or absence of a fluorescence signal in a sample of the culture medium, wherein fluorescence signal intensity reflects the concentration of fluorophore-labeled collagen fragments in the sample of the culture medium.