Composite living skin equivalents

Abstract

A composite living skin equivalent is described comprising an epidermal layer of cultured keratinocyte cells, a layer of non-porous collagen, and a dermal layer of cultured fibroblast cells in a porous cross-linked collagen sponge matrix. Preferably the non-porous collagen is Type 1, Type 3 or mixtures of Types 1 and 3 bovine collagen, which has been pepsin treated. A process for preparing the skin equivalent is described, as well as a test kit for in vitro testing of the skin equivalent. The skin equivalent has use for skin grafting as well as in vitro testing of the effects of various substances on skin. The questions raised in reexamination request No. 90/003,418, filed Apr. 25, 1994, have been considered and the results thereof are reflected in this reissue patent which constitutes the reexamination certificate required by 35 U.S.C. 307 as provided in 37 CFR 1.570(e).

We claim: 
     
       1. A composite living skin equivalent which comprises relative to a horizontal plane: a) a sponge first layer comprising a cross-linked collagen sponge, said first layer having upper and lower surfaces, said sponge containing cultured fibroblast cells therein,   b) a .Iadd.non-porous .Iaddend.second layer comprising a high purity .[.non-porous.]. collagen .Iadd.essentially free of exogenous glycosaminoglycans.Iaddend., said second layer having upper and lower surfaces, the lower surface thereof being in contact with the upper surface of said first layer, and   c) a layer comprising cultured keratinocyte cells in contact with the upper surface of said non-porous collagen second layer.   
     
     
       2. The composite living skin equivalent of claim 1 wherein the collagen of the non-porous second layer is polymerized. 
     
     
       3. The composite living skin equivlent of claim .[.2.]. .Iadd.14 .Iaddend.wherein the collagen of at least one of the layers is bovine source derived. 
     
     
       4. The composite living skin equivalent of claim 3, wherein the non-porous collagen is selected from the group consisting of Type 1 collagen, Type 3 collagen, and mixtures thereof. 
     
     
       5. The composite living skin equivalent of claim 4, wherein the non-porous collagen has been highly purified by pre-treatment with pepsin. 
     
     
       6. The composite living skin equivalent of claim 5 wherein the pores of the cross-linked first layer of collagen are of sufficient size to permit the growth of fibroblast cells therein. 
     
     
       7. The composite living skin equivalent of claim 6 wherein the non-porous collagen second layer is applied to said upper surface of said second layer as a thin film from a solution of collagen. 
     
     
       8. The composite living skin equivalent of claim 7 wherein the second layer of collagen contacts the first layer of collagen only on the upper surface of said first layer. 
     
     
       9. The composite living skin equivalent of claim 8 wherein the fibroblast and keratinocytes cells are human source derived. 
     
     
       10. The composite living skin equivalent of claim 9, wherein the fibroblast cells are obtained from treating a dermal sample with collagenase. 
     
     
       11. The composite living skin equivalent of claim 10, wherein the layer of keratinocyte cells is prepared by obtaining a single-cell suspension of keratinocytes, and evenly and discontinuously distributing drops of the suspension onto the layer of non-porous collagen, which is then incubated. .Iadd. 
     
     
       12.  A composite living skin equivalent which comprises relative to a horizontal plane: a) a sponge first layer comprising a cross-linked collagen sponge, said first layer having upper and lower surfaces, said sponge containing cultured fibroblast cells therein,   b) a non-porous gel second layer comprising a high purity collagen essentially free of exogenous glycosaminoglycans, said second layer having upper and lower surfaces, the lower surface thereof being in contact with the upper surface of said first layer, and   c) a layer comprising cultured keratinocyte cells in contact with the upper surface of said non-porous collagen second layer. .Iadd.13. The composite living skin equivalent of claim 12 wherein said sponge first layer comprising a cross-linked collagen sponge is essentially free of exogenous   
     
     
        glycosaminoglycans. .Iaddend..Iadd.14.  The composite living skin equivalent of claim 13 wherein said sponge first layer is derived from collagen which is essentially free of exogenous denatured collagen. .Iaddend.