Method of isolating cells from umbilical cord

Abstract

The present invention relates a method for isolating four cell types from a single umbilical cord as pure cultures. These cell lines (epithelial cells, fibroblasts, smooth muscle cells and endothelial cells) can be characterised and utilised in experimental models and for therapeutic purposes. Particularly, the umbilical cells isolated herein are used to form a tissue replacement or engineered living composition. Also, the isolated umbilical cells of the invention may have the potential of progenitor cells.

1 . A method for the simultaneous isolation of four cell types from one umbilical cord sample comprising the steps of: 
 a) isolating endothelial cells by perfusing the vein of an umbilical cord biopsy with a solution comprising thermolysin for a period of time to allow detachment of said endothelial cells from said vein;    b) treating said umbilical cord biopsy of step a) with a solution of trypsin-EDTA for a time sufficient to allow detachment of epithelial cells;    c) mechanically removing smooth muscle cells and fibroblast-like cells from said umbilical cord biopsy followed by a culture period of sufficient time to allow separation and isolation of smooth muscle and fibroblast-like cells from laminae; and    d) harvesting the isolated cells of steps a) to c).    
     
     
         2 . The method of  claim 1 , wherein said cell types are progenitor cells.  
     
     
         3 . A biological composition comprising the four cell types obtained with the method of  claim 1 .  
     
     
         4 . A method for the simultaneous isolation of four cell types from one umbilical cord sample comprising the steps of: 
 a) isolating endothelial cells by perfusing the vein of an umbilical cord biopsy with a solution comprising at least one of thermolysin or collagenase for a period of time to allow detachment of said endothelial cells from said vein;    b) allowing the digestion of media of said umbilical cord vein of step a) through a second perfusion with a solution comprising collagenase for detachment and isolation of smooth muscle cells from said vein;    c) extracting epithelial cells by treating the umbilical cord biopsy in a solution comprising trypsin-EDTA for a time allowing detachment of said epithelial cells;    d) culturing a sample of the Warton's jelly layer of said umbilical cord section in a solution comprising collagenase for a period of time sufficient to allow separation and isolation of fibroblast-like cells from said layer; and    e) harvesting isolated cells of steps a) to d).    
     
     
         5 . The method of  claim 4 , wherein said cell types are progenitor cells.  
     
     
         6 . A biological composition comprising the four cell types obtained with the method of  claim 4 .  
     
     
         7 . A method for the simultaneous isolation of four cell types from one umbilical cord sample comprising the steps of: 
 a) extracting epithelial cells by treating an umbilical cord biopsy in a solution comprising trypsin-EDTA for a time allowing detachment of said epithelial cells;    b) mechanically removing endothelial cells from surface of an umbilical cord vein;    c) mechanically removing smooth muscle cells and fibroblast-like cells from said umbilical cord biopsy followed by a culture period of sufficient time to allow separation and isolation of smooth muscle and fibroblast-like cells from laminae;    d) harvesting the isolated cells of steps a) to c).    
     
     
         8 . The method of  claim 7 , wherein said removing of step b) is performed by scraping said umbilical cord vein.  
     
     
         9 . The method of  claim 7 , wherein said cell types are progenitor cells.  
     
     
         10 . A biological composition comprising the four cell types obtained with the method of  claim 7.