Skin equivalent culture

Abstract

Disclosed is a method of preparing a collagenous construct comprising casting a support matrix comprising fibrin and viable collagen-producing cells onto a support material, wherein said cells include human dermal fibroblasts, incubating in situ said support matrix in a collagen-inducing medium thereby, inducing or enhancing collagen production by said cells to form a collagenous construct, and degrading said fibrin, and rendering said collagenous construct free of said viable collagen-producing cells.

1 . A method of preparing a collagenous construct comprising:
 (i) casting a support matrix comprising fibrin and viable collagen-producing cells onto a support material, wherein said cells include human dermal fibroblasts;   (ii) incubating in situ said support matrix in a collagen-inducing medium thereby:
 (a) inducing or enhancing collagen production by said cells to form a collagenous construct, and 
 (b) degrading said fibrin; and 
   (iii) rendering said collagenous construct free of said viable collagen-producing cells.   
     
     
         2 . The method of  claim 1 , wherein said support material is a glass coverslip, a polycarbonate membrane, or a tissue culture plastic. 
     
     
         3 . The method of  claim 1 , wherein said support material is a plastic backing material or petrolated/paraffin gauze. 
     
     
         4 . The method of  claim 1 , wherein said collagenous construct has a size range of 4 cm 2 -100 cm 2 . 
     
     
         5 . The method of  claim 1 , wherein said collagenous construct is less than 2 mm thick. 
     
     
         6 . The method of  claim 1 , wherein said collagenous construct has a size range of 4 cm 2 -100 cm 2  and is less than 2 mm thick. 
     
     
         7 . The method of  claim 1 , wherein said collagenous construct has a convex shape. 
     
     
         8 . The method of  claim 1 , wherein said collagenous construct has an ultimate tensile strength of at least 1 N/cm 2 . 
     
     
         9 . The method of  claim 1 , wherein said collagenous construct is fibrin-free. 
     
     
         10 . The method of  claim 1 , wherein said collagenous construct is storage stable. 
     
     
         11 . The method of  claim 1 , wherein said support matrix is incubated in said collagen-inducing medium for 21 to 49 days. 
     
     
         12 . The method of  claim 1 , wherein said support matrix is incubated in said collagen-inducing medium for 21 to 63 days. 
     
     
         13 . The method of  claim 12 , wherein said collagen-inducing medium is replaced daily or at least 3 times per week. 
     
     
         14 . The method of  claim 1 , wherein said collagenous construct is capable of being suturable, stapable, or meshable. 
     
     
         15 . The method of  claim 1 , wherein said viable collagen-producing cells comprise 90%-100% human dermal fibroblasts. 
     
     
         16 . The method of  claim 1 , wherein said human dermal fibroblasts are allogeneic human dermal fibroblasts. 
     
     
         17 . The method of  claim 1 , wherein said human dermal fibroblasts are human neonatal dermal fibroblasts. 
     
     
         18 . The method of  claim 1 , wherein said support matrix is formed by thrombin-mediated polymerization of fibrinogen. 
     
     
         19 . 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, or glycine. 
     
     
         20 . The method of  claim 19 , wherein said collagen-inducing medium further comprises one or more of insulin, TGF-β, polyethylene glycol (PEG), platelet-derived growth factor (PDGF), or plasmin. 
     
     
         21 . The method of  claim 1 , further comprising culturing said human dermal fibroblasts prior to incubating said support matrix. 
     
     
         22 . The method of  claim 21 , 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. 
     
     
         23 . The method of  claim 21 , wherein said human dermal fibroblasts are cultured for a period of up to 7 days in a serum free medium. 
     
     
         24 . The method of  claim 1 , further comprising culturing said human dermal fibroblasts during incubation of said support matrix. 
     
     
         25 . 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. 
     
     
         26 . The method of  claim 1 , wherein said collagenous construct comprises 10%-99% collagen. 
     
     
         27 . The method of  claim 26 , wherein said collagen comprises collagen I. 
     
     
         28 . The method of  claim 27 , wherein said collagen further comprises collagen III. 
     
     
         29 . The method of  claim 1 , wherein said collagenous construct comprises elastin. 
     
     
         30 . The method of  claim 1 , wherein said collagenous construct comprises components of the extracellular matrix (ECM) in skin. 
     
     
         31 . The method of  claim 1 , wherein said collagenous construct is a single-layered construct. 
     
     
         32 . The method of  claim 31 , wherein said collagenous construct is a multi-layered construct 
     
     
         33 . The method of  claim 32 , wherein the layers of said multi-layered construct are separated by an interlaying substance. 
     
     
         34 . The method of  claim 33 , wherein the interlaying substance comprises fibrin. 
     
     
         35 . The method of  claim 1 , wherein said collagenous construct is packaged for shipment. 
     
     
         36 . The method of  claim 35 , wherein said package include a transport medium. 
     
     
         37 . The method of  claim 1 , wherein said viable collagen-producing cells are rendered non-viable by fixing said collagenous construct. 
     
     
         38 . The method of  claim 1 , wherein said viable collagen-producing cells are rendered non-viable by sterilizing said collagenous construct. 
     
     
         39 . The method of  claim 1 , wherein said viable collagen-producing cells are rendered non-viable by freeze drying said collagenous construct. 
     
     
         40 . The method of  claim 1 , wherein said collagenous construct is sterile. 
     
     
         41 . The method of  claim 1 , wherein said collagenous construct is freeze-dried. 
     
     
         42 . The method of  claim 41 , wherein said freeze dried collagenous construct is reconstituted and then applied to a skin lesion. 
     
     
         43 . The method of  claim 1 , wherein said collagenous construct is applied to a skin lesion. 
     
     
         44 . The method of  claim 43 , wherein said viable human dermal fibroblasts are allogeneic human dermal fibroblasts. 
     
     
         45 . A method of preparing a collagenous construct comprising:
 (i) casting a support matrix comprising fibrin and viable collagen-producing cells onto a support material, wherein said cells include human dermal fibroblasts;   (ii) incubating in situ said support matrix in a collagen-inducing medium for 21 to 63 days thereby:
 (a) inducing or enhancing collagen production by said cells to form a collagenous construct, and 
 (b) degrading said fibrin; 
 wherein said medium is replaced at least 3 time per week; and 
   (iii) rendering said collagenous construct free of said viable collagen-producing cells,   wherein said collagenous construct has a size range of 4 cm 2 -100 cm 2  and is less than 2 mm thick,   wherein said collagenous construct has an ultimate tensile strength of at least 1 N/cm 2 ,   wherein said collagenous construct comprises 10%-99% collagen, and   wherein said collagenous construct is sterile and comprised within a package.