Skin equivalent culture

Abstract

Disclosed is a method of preparing a collagenous construct comprising (i) casting viable collagen-producing human dermal fibroblasts in a support matrix comprising fibrin onto a support material, (ii) incubating in situ said support matrix with said viable collagen-producing human dermal fibroblasts in a collagen-inducing medium thereby (a) inducing or enhancing collagen production by said viable collagen-producing human dermal fibroblasts to form a collagenous construct, and (b) degrading said fibrin, (iii) rendering said collagenous construct free of said viable collagen-producing human dermal fibroblasts, and (iv) cross-linking said collagenous construct with a chemical or exposure to ultraviolet light.

1 . A method of preparing a collagenous construct comprising:
 (i) casting viable collagen-producing human dermal fibroblasts in a support matrix comprising fibrin onto a support material;   (ii) incubating in situ said support matrix with said viable collagen-producing human dermal fibroblasts in a collagen-inducing medium thereby:
 (a) inducing or enhancing collagen production by said viable collagen-producing human dermal fibroblasts to form a collagenous construct, and 
 (b) degrading said fibrin; 
   (iii) rendering said collagenous construct free of said viable collagen-producing human dermal fibroblasts; and   (iv) cross-linking said collagenous construct with a chemical or exposure to ultraviolet light.   
     
     
         2 . The method of  claim 1 , wherein said support material is a glass coverslip, a polycarbonate membrane, a tissue culture plastic, a plastic backing material, or petrolated/paraffin gauze. 
     
     
         3 . The method of  claim 1 , wherein said collagenous construct has a size range of 4 cm 2 -100 cm 2 . 
     
     
         4 . The method of  claim 1 , wherein said collagenous construct is less than 2 mm thick. 
     
     
         5 . The method of  claim 1 , wherein said collagenous construct has a convex shape. 
     
     
         6 . The method of  claim 1 , wherein said collagenous construct has an ultimate tensile strength of at least 1 N/cm 2 . 
     
     
         7 . The method of  claim 1 , wherein said collagenous construct is fibrin-free. 
     
     
         8 . The method of  claim 1 , wherein said support matrix is incubated in said collagen-inducing medium for 21 to 63 days. 
     
     
         9 . The method of  claim 8 , wherein said collagen-inducing medium is replaced daily or at least 3 times per week. 
     
     
         10 . The method of  claim 1 , wherein said human dermal fibroblasts are allogeneic human dermal fibroblasts. 
     
     
         11 . The method of  claim 1 , wherein said human dermal fibroblasts are human neonatal dermal fibroblasts. 
     
     
         12 . The method of  claim 1 , wherein said fibrin in said support matrix is formed by thrombin-mediated polymerization of fibrinogen. 
     
     
         13 . The method of  claim 1 , wherein said collagen-inducing medium comprises one or more of phenytoin, ascorbic acid, vaiproic acid, cyclosporin A, nifedipine, diltiazem, verapamil HCl, amolldipine, Dulbecco's Modified Eagle's Medium (DMEM), Hams F-12 medium, newborn calf serum, fetal calf serum, L-glutamine, epidermal growth factor (EGF), hydrocortisone, ethanolamine, o-phosphoryl-ethanolamine, transferrin, triiodothyronine, selenium, L-proline, glycine insulin, TGF-β, polyethylene glycol (PEG), platelet-derived growth factor (PDGF), or plasmin. 
     
     
         14 . The method of  claim 1 , further comprising culturing said human dermal fibroblasts prior to incubating said support matrix. 
     
     
         15 . The method of  claim 14 , wherein said human dermal fibroblasts are cultured for a period of up to 21 days in a medium comprising one or more of Dulbecco's Modified Eagle's Medium (DMEM), newborn calf serum, fetal calf serum, or L-glutamine. 
     
     
         16 . The method of  claim 14 , wherein said human dermal fibroblasts are cultured for a period of up to 7 days in a serum free medium. 
     
     
         17 . The method of  claim 1 , further comprising culturing said human dermal fibroblasts during incubation of said support matrix. 
     
     
         18 . The method of  claim 1 , wherein collagen production is induced or enhanced by one or more of electrical stimulation, tension, movement, ultrasound, or infrared light. 
     
     
         19 . The method of  claim 1 , wherein said collagenous construct comprises 10%-99% collagen. 
     
     
         20 . The method of  claim 1 , wherein said collagenous construct comprises components of the extracellular matrix (ECM) in skin. 
     
     
         21 . The method of  claim 1 , wherein said collagenous construct is a single-layered construct or a multi-layered construct. 
     
     
         22 . The method of  claim 21 , wherein the layers of said multi-layered construct are separated by an interlaying substance. 
     
     
         23 . The method of  claim 22 , wherein said interlaying substance comprises fibrin. 
     
     
         24 . The method of  claim 1 , wherein said viable collagen-producing cells are rendered non-viable by sterilizing or by freeze drying said collagenous construct. 
     
     
         25 . The method of  claim 1 , wherein said collagenous construct is sterile. 
     
     
         26 . The method of  claim 1 , wherein said collagenous construct is freeze-dried. 
     
     
         27 . The method of  claim 26 , wherein said freeze dried collagenous construct is reconstituted and then applied to a skin lesion. 
     
     
         28 . The method of  claim 1 , wherein said collagenous construct is applied to a skin lesion.