In vitro synthesis of a layered cell sorted tissue

Abstract

The present invention relates to a layered cell sorted tissue that is formed in vitro. The tissue is generated by the spontaneous sorting of cells from a homogenous cell mixture into discrete layers by cell type. Connective tissue components, such as fibronectin, may be used to manipulate orientation of the layers during the cell sorting process. The layered cell sorted tissue may be used as a skin graft for burns, wounds, and ulcers. The tissue may also be used in assays to determine effects of chemicals or drugs on human tissue in vitro as well as provide an in vitro assay for tumor cell metastasis.

1 . A layered cell sorted tissue comprising a discrete first cell layer comprising epithelial cells and a discrete second cell layer comprising mesenchymal cells, wherein the cells spontaneously sorted from a homogenous cell mixture to form said discrete first and second layers upon contact with a sufficiently high concentration of one or more components present in serum to facilitate cell sorting, and wherein said tissue is formed in vitro.  
     
     
         2 . (canceled)  
     
     
         3 . The tissue  claim 1  wherein said first cell layer comprises keratinocytes.  
     
     
         4 . The tissue of  claim 1  wherein said first cell layer comprises colon epithelial cells.  
     
     
         5 . The tissue of  claim 4  wherein said first cell layer comprises CaCo2 cells.  
     
     
         6 . The tissue of  claim 1  wherein said first cell layer comprises vaginal epithelial cells.  
     
     
         7 . The tissue of  claim 6  wherein said first cell layer comprises A431 cells.  
     
     
         8 . The tissue of  claim 1  wherein said first cell layer comprises urogenital epithelial cells.  
     
     
         9 . The tissue of  claim 8  wherein said first cell layer comprises HUC18 cells.  
     
     
         10 . (canceled)  
     
     
         11 . The tissue of  claim 1  wherein said second cell layer comprises fibroblasts.  
     
     
         12 - 13 . (canceled)  
     
     
         14 . The tissue of  claim 1  wherein said first cell layer is an epidermal layer comprising keratinocytes and said second cell layer is a dermal layer comprising fibroblasts.  
     
     
         15 . The tissue of  claim 14  further comprising a plurality of integral proteins that connect said epidermal and dermal layers to form a basal lamina.  
     
     
         16 . The tissue of  claim 14  wherein the epidermal layer is the top layer of said tissue.  
     
     
         17 . The tissue of  claim 14  wherein the epidermal layer is the bottom layer of said tissue.  
     
     
         18 - 22 . (canceled)  
     
     
         23 . An assay for testing a response to a chemical or a drug comprising: 
 providing the layered cell sorted tissue of  claim 1;     contacting said tissue with said chemical or said drug;    determining whether said chemical or said drug has an effect on said tissue by comparing a difference in phenotype, genotype, or both, between said tissue before and after contact with said chemical or said drug.    
     
     
         24 . An in vitro assay for studying tumor cell metastasis comprising: 
 providing the layered cell sorted tissue of  claim 1  on a light blocking plate;    contacting said tissue with an anti-metastatic compound;    contacting said tissue with at least one fluorescently labeled tumor cell;    determining whether said anti-metastatic compound has an anti-metastatic effect on said tissue by measuring fluorescence at the bottom of said plate before and after contact of said tissue with said anti-metastatic compound.    
     
     
         25 . The method of  claim 24  wherein said fluorescently labeled tumor cell is a HT-1080 human fibrosarcoma cell.  
     
     
         26 . A skin graft comprising the layered cell sorted tissue of  claim 14 .  
     
     
         27 . A method for grafting skin comprising the steps of: 
 providing a homogenous mixture of keratinocytes and fibroblasts;    applying the mixture to a graft site; and    allowing the mixture to spontaneously sort into a discrete layer comprising keratinocytes and a discrete layer comprising fibroblasts.    
     
     
         28 . The method of  claim 27 , wherein said graft site is a wound.  
     
     
         29 . The method of  claim 27 , wherein said graft site is a burn.  
     
     
         30 . A method for grafting skin comprising the steps of: 
 providing the tissue of  claim 17;  and    flipping the tissue to orient the epidermal layer as the top layer of said graft during placement onto a graft site.    
     
     
         31 . The method of  claim 30 , wherein said graft site is a wound.  
     
     
         32 . The method of  claim 30 , wherein said graft site is a burn.  
     
     
         33 . A composition of cells for making a layered cell sorted tissue in vitro comprising a homogenous cell mixture comprising fibroblasts and epithelial cells, and a sufficiently high concentration of at least one component in serum to facilitate cell sorting.  
     
     
         34 . The composition of  claim 33  wherein the mixture comprises at least about 4×10 6  fibroblasts.  
     
     
         35 . The composition of  claim 33  wherein the mixture comprises at least about 4×10 6  of said epithelial cells.  
     
     
         36 . The composition of  claim 42  wherein the mixture comprises at least about 4×10 6  fibroblasts and at least about 4×10 6  of said epithelial cells.  
     
     
         37 . The composition of  claim 33  wherein said epithelial cells are selected from the group consisting of keratinocytes, CaCo2 cells, A431 cells, and HUC18 cells.  
     
     
         38 . The layered cell sorted tissue of  claim 1 , wherein the orientation of the sorted layers is manipulated by contact with a sufficient amount of a connective tissue component.  
     
     
         39 . The layered cell sorted tissue of  claim 1 , wherein said homogeneous cell mixture comprises about an equal number of mesenchymal cells and epithelial cells.  
     
     
         40 . The layered cell sorted tissue of  claim 39 , wherein said homogeneous cell mixture comprises at least about 4×10 6  mesenchymal cells and at least about 4×10 6  epithelial cells.  
     
     
         41 . The composition of cells of  claim 33 , wherein the orientation of the layered cell sorted tissue is manipulated by contact with a sufficient amount of a connective tissue component.  
     
     
         42 . The composition of cells of  claim 33 , wherein said homogeneous cell mixture comprises about an, equal number of mesenchymal cells and epithelial cells.