Epidermal and dermal equivalents

Abstract

The present invention relates to the treatment of skin defects by organotypically-cultured autologous keratinocytes isolated from the outer root sheath of anagen or growing hair. Methods for primary, as well as subsequent organotypic cultures (i.e., epidermal equivalents) in fully-defined media supplemented by autologous human serum and substances isolated form blood components, with minimal allogeneic biological supplements, are disclosed herein. Techniques to prepare epidermal equivalents for transplantation by use of a biocompatible glue are also disclosed herein.

1 - 71 . (canceled)  
   
   
       72 . A method for preparing an epidermal or complex skin equivalent suitable for subsequent treatment of a skin defect, said method comprising: 
 a) culturing an anagenic hair follicle to obtain outer root sheath cells;    b) culturing said outer root sheath cells to obtain keratinocyte precursor cells; and    c) preparing an epidermal or complex skin equivalent comprising said keratinocyte precursor cells,    wherein said hair follicle is intact.    
   
   
       73 . The method of  claim 72 , wherein said culturing steps (a) and (b) are performed in a medium containing human serum at a concentration less than 5%.  
   
   
       74 . The method of  claim 72 , wherein said keratinocyte precursor cells are seeded at a density of between 3×10 4  cells/cm 2  and 1×10 5  cells/cm 2 .  
   
   
       75 . The method of  claim 72 , wherein said keratinocyte precursor cells are selected by: 
 d) primary-culturing said outer root sheath-derived keratinocyte precursor cells by adhering said intact anagenic hair to a microporous membrane, said membrane possessing growth-arrested/limited feeder cells on its undersurface so as to select for keratinocyte precursor cells from the outer root sheath of hair;    e) organotypically-culturing the outer root sheath cells harvested from said primary cultures by modulating a microporous membrane which also possesses growth-arrested limited feeder cells on its undersurface; and    f) generating said epidermal or complex skin equivalent for subsequent use as a graft insert by placing a carrier membrane on top of said organotypic-culture from step d) and detaching said complex skin or epidermal equivalent;    whereby said graft comprises the keratinocyte precursor cells and carrier membrane as a single laminar unit, said keratinocyte precursor cells being seeded on said carrier membrane at a density of between 3×10 4  cells/cm 2  and 1×10 5  cells/cm 2 .    
   
   
       76 . The method of  claim 75 , wherein the culture density of said growth-arrested/limited feeder cells on said microporous membrane is between about 1×10 4  cells/cm 2  and about 5×10 4  cells/cm 2 .  
   
   
       77 . The method of  claim 75 , wherein said growth-arrested/limited feeder cells are banked or immortalized cells.  
   
   
       78 . The method of  claim 75 , wherein said epidermal or complex skin equivalent comprises outer root sheath cells cultured in a medium containing only homologous or autologous releasates from blood components.  
   
   
       79 . The method of  claim 78 , wherein said epidermal or complex skin equivalent comprises outer root sheath cells cultured in a medium containing only homologous or autologous releasates from blood components at a concentration of about 0.1% to about 20%.  
   
   
       80 . The method of  claim 75 , wherein said microporous membrane is coated by one or more extracellular matrix substances selected from the group consisting off fibrin, fibronectin, collagens, laminins and hyaluronan.  
   
   
       81 . The method of  claim 75 , wherein said microposous membrane possesses a growth-arrested/limited feeder cells system on its undersurface with said feeder cells of at least one type of cell selected from the group consisting of human dermal fibroblasts, epidermal cells, mesenchymal cells, neuronal cells and endothelial cells.  
   
   
       82 . The method of  claim 75 , wherein said carrier membrane is made from one or more types of materials selected from the group consisting of polyester, PTFE, polyurethane, hyaluronic acid, polyactic acid, collagen and a silicone or Vasiline gauze dressing.  
   
   
       83 . The method of  claim 75 , wherein the size of said epidermal or complex skin equivalent is selected from the group consisting of 1.0 cm, 1.5 cm, 2.0 cm and 2.5 cm in diameter.  
   
   
       84 . The method of  claim 72 , wherein said epidermal or complex skin equivalent is coated on its top or cornified side with a fibrin glue.  
   
   
       85 . The method of  claim 84 , wherein said fibrin glue comprises one or more antimicrobial, anti-fungal or anti-viral agents emulsified therein.  
   
   
       86 . The method of  claim 72 , wherein said outer root sheath cells are homologous cells.  
   
   
       87 . The method of  claim 72 , wherein said epidermal or complex skin equivalent comprises outer root sheath cells cultured in a medium containing only homologous or autologous biological supplements.  
   
   
       88 . The method of  claim 72 , wherein said epidermal equivalent is coated on its top or cornified side with a carrier membrane.  
   
   
       89 . The method of  claim 72 , wherein said outer root sheath cells are autologous cells obtained from an individual who will subsequently undergo treatment for a skin defect.  
   
   
       90 . The method of  claim 72 , further comprising shipping or transporting said epidermal or complex skin equivalent by: 
 detaching said epidermal or complex skin equivalent from said culture medium;    transferring said equivalent onto a carrier; and    contacting said epidermal equivalent and carrier with a solidified or gelled medium.    
   
   
       91 . The method of  claim 90 , wherein said equivalent is coated on its top or cornified side with a carrier membrane.  
   
   
       92 . The method of  claim 91 , wherein said equivalent is further sealed and shipped for future use in grafting.  
   
   
       93 . The method of  claim 90 , wherein said solidified or gelled medium is selected from the group consisting of agarose, methyl cellulose and another gelifying substance.