Methods for culturing human keratinocytes

Abstract

The present disclosure relates to methods for culturing human epidermal keratinocytes. When keratinocytes are cultured on plates coated with a laminin containing an alpha-4 chain or an alpha-5 chain, in a xeno-free, chemically defined cell culture medium, they expand efficiently in vitro. Useful cell culture kits for culturing keratinocytes are also described herein, as are methods of using such cells for treatment of burns or chronic wounds.

1 . A method for expanding human keratinocytes, comprising:
 plating human keratinocytes on a substrate having a coating thereon, the coating comprising laminin-421; and   culturing the keratinocytes in a xeno-free cell culture medium.   
     
     
         2 . The method of  claim 1 , wherein the keratinocytes plated on the substrate are freshly isolated. 
     
     
         3 . The method of  claim 1 , wherein the keratinocytes are plated on the substrate at a density of about 0.7×10 5 /cm 2  to about 1.2×10 5 /cm 2 . 
     
     
         4 . The method of  claim 1 , wherein the keratinocytes are cultured on the substrate at a temperature from about 30° C. to about 40° C. 
     
     
         5 . The method of  claim 1 , wherein the keratinocytes are cultured in an atmosphere containing from about 5% CO 2  to about 15% CO 2 . 
     
     
         6 . The method of  claim 1 , further comprising periodically dissociating and passaging the keratinocytes. 
     
     
         7 . The method of  claim 6 , wherein the passaging of the keratinocytes occurs upon reaching a density of 1 cell to about 1.5×10 4  cells/cm 2 . 
     
     
         8 . The method of  claim 1 , wherein the cell culture medium is also chemically defined. 
     
     
         9 . A system for expanding keratinocytes, comprising:
 a substrate containing laminin-421; and   a xeno-free cell culture medium.   
     
     
         10 . The system of  claim 9 , wherein the cell culture medium does not contain any animal serum. 
     
     
         11 . The system of  claim 9 , wherein the system does not contain any feeder cells. 
     
     
         12 . The system of  claim 9 , wherein the cell culture medium is also chemically defined. 
     
     
         13 . A method for treating any injuries or conditions of a patient, comprising:
 plating keratinocyte cells taken from a donor site of the patient on a substrate having a coating that comprises laminin-421;   expanding the keratinocyte cells on the substrate in a xeno-free cell culture medium; and   covering the injury or condition with the keratinocyte cells from the substrate.   
     
     
         14 . The method of  claim 13 , wherein the cell culture medium is also chemically defined. 
     
     
         15 . The method of  claim 13 , wherein the injury or condition is a burn wound, or a chronic wound, or a case in which only the epithelium is compromised, or in which an epithelium defect is a component of the injury. 
     
     
         16 . The method of  claim 13 , wherein the injuries or conditions are in an eye, the bladder, the oral cavity, or the intestine.