Decelluarlized Tissue Engineered Constructs and Tissues

Abstract

New methods for producing tissue engineered constructs and engineered native tissues are disclosed. The methods include producing a tissue engineered construct by growing cells in vitro on a substrate and then decellularizing the construct to produce a decellularized construct consisting largely of extracellular matrix components. The construct can be used immediately or stored until needed. The decellularized construct can be used for further tissue engineering, which may include seeding the construct with cells obtained from the intended recipient of the construct. During any of the growth phases required for production of the construct, the developing construct may be subjected to various tissue engineering steps such as application of mechanical stimuli including pulsatile forces. The methods also include producing an engineered native tissue by harvesting tissue from an animal or human, performing one or more tissue engineering steps on the tissue, and subjecting the tissue to decellularization. The decellularized, engineered native tissue may then be subjected to further tissue engineering steps.

1 . A method for preparing a construct for use as a tissue engineering scaffold or for implanting into a human comprising:
 culturing and maintaining human smooth muscle cells, endothelial cells, or fibroblast cells under conditions suitable for growth of the cells, whereby a proteinaceous extracellular matrix surrounding said cells is formed; and,   decellularizing the proteinaceous extracellular matrix,   
       thereby forming a tissue engineered construct comprising a 3-dimensional proteinaceous extracellular matrix. 
     
     
         2 . The method of  claim 1  further comprising:
 implanting the tissue engineered construct into a human.   
     
     
         3 . The method of  claim 2  further comprising:
 testing serum concentrations in the human of IgG and IgM antibodies against antigens present in extracts made from the proteinaceous extracellular matrix surrounding said cells which has not been decellularized.   
     
     
         4 . The method of  claim 1  further comprising:
 testing the tissue engineered construct comprising a 3-dimensional proteinaceous extracellular matrix for reduced immunogenicity by histopathologic analysis of tissue samples obtained from rabbits implanted with said tissue engineered construct compared to the proteinaceous extracellular matrix surrounding said cells that have not been decellularized.   
     
     
         5 . A method for preparing a construct for use as a tissue engineering scaffold or for implanting into a human comprising:
 maintaining human smooth muscle, endothelial or fibroblast cells under conditions suitable for growth of the cells, whereby a proteinaceous extracellular matrix surrounding said cells is formed, and   decellularizing said proteinaceous extracellular matrix.   
     
     
         6 . A method of treating a tissue-engineered construct comprising a 3-dimensional proteinaceous extracellular matrix, comprising:
 decellularizing a tissue-engineered construct comprising a 3-dimensional proteinaceous extracellular matrix, thereby providing a matrix with reduced immunogenicity.   
     
     
         7 . The method of  claim 6  wherein the matrix is at least 50 microns in its smallest dimension. 
     
     
         8 . The method of  claim 6  further comprising:
 testing the matrix for reduced immunogenicity by assaying IgG or IgM antibodies in serum of a human in whom the matrix has been implanted, wherein the antibodies are against antigens present in extracts made from non-decellularized constructs.   
     
     
         9 . The method of  claim 6  wherein the step of decellularizing employs a nuclease. 
     
     
         10 . The method of  claim 6  wherein the step of decellularizing employs a protease inhibitor. 
     
     
         11 . The method of  claim 6  wherein the step of decellularizing employs freezing and thawing. 
     
     
         12 . The method of  claim 6  wherein the step of decellularizing employs a phospholipase. 
     
     
         13 . The method of  claim 6  wherein the step of decellularizing employs a detergent.