Bioengineered Tissue Constructs and Methods for Producing and Using Thereof

Abstract

Cultured tissue constructs comprising cultured cells and endogenously produced extracellular matrix components without the requirement of exogenous matrix components or network support or scaffold members. Some tissue constructs of the invention are comprised of multiple cell layers or more than one cell type. The tissue constructs of the invention have morphological features and functions similar to tissues their cells are derived and their strength makes them easily handleable. Preferred cultured tissue constructs of the invention are prepared in defined media, that is, without the addition of chemically undefined components.

1 - 30 . (canceled)  
     
     
         31 . A cultured tissue construct comprising fibroblasts cells grown under conditions to produce a layer of extracellular matrix which is synthesized and assembled by the fibroblast cells, with the fibroblast cells surrounded by the synthesized extracellular matrix layer, wherein the extracellular matrix is produced by the fibroblast cells in the absence of exogenous matrix components or synthetic members during the culturing conditions, and wherein the conditions comprise a chemically defined medium.  
     
     
         32 . The cultured tissue construct of  claim 31 , wherein fibroblasts are derived from tissue selected from the group consisting of neonate male foreskin, dermis, tendon, lung, urethra, umbilical cord, corneal stroma, oral mucosa, intestine.  
     
     
         33 . The cultured tissue construct of  claim 31 , wherein said cultured cells are dermal fibroblasts.  
     
     
         34 . The cultured tissue construct of  claim 31 , wherein said cultured cells are from dermal papilla of hair follicles.  
     
     
         35 . The cultured tissue construct of  claim 31 , wherein said layer has cultured cells from dermal papilla of hair follicles are localized on said layer.  
     
     
         36 . The cultured tissue construct of  claim 31 , wherein said cultured cells are derived from human tissue and are cultured in medium containing no animal-derived components.  
     
     
         37 . The cultured tissue construct of  claim 31 , wherein the cultured cells are recombinant cells or genetically-engineered cells.  
     
     
         38 . (canceled)  
     
     
         39 . A method for producing a cultured tissue construct, comprising, 
 (a) seeding fibroblast cells capable of synthesizing an extracellular matrix on a porous membrane in a culture vessel in a first cell culture medium;    (b) culturing the fibroblast cells of step (a) in the first cell culture medium to between about 80% to about 100% confluence on the porous membrane;    (c) stimulating the fibroblast cells to synthesize, secrete and organize extracellular matrix components under culturing conditions in a second culture medium; and,    (d) continued culturing of the fibroblast cells until the cells form a layer of synthesized extracellular matrix of at least about 30 microns thick, with the cultured fibroblast cells surrounded by the synthesized extracellular matrix layer, wherein said extracellular matrix is produced by the cultured fibroblast cells in the absence of exogenous matrix components or synthetic members during the culturing conditions.    
     
     
         40 . The method of  claim 39 , wherein either the first culture medium, or the second culture medium, or both the first and second culture mediums are chemically defined.  
     
     
         41 . The method of  claim 39 , wherein the first and second culture mediums contain no animal components.  
     
     
         42 . The method of  claim 39 , wherein the second culture medium comprises an ascorbate derivative.  
     
     
         43 . The method of  claim 42 , wherein the ascorbate derivative is sodium ascorbate, ascorbic acid, or L-ascorbic acid phosphate magnesium salt n-hydrate.  
     
     
         44 . The method of  claim 39 , wherein the second culture medium comprises proline, glycine, or hydroxylproline.  
     
     
         45 . The method of  claim 42 , wherein the second culture medium further comprises proline, glycine, or hydroxylproline.  
     
     
         46 . The method of  claim 39 , wherein the layer synthesized extracellular matrix of at least about 60 microns thick.  
     
     
         47 . The method of  claim 46 , wherein the layer synthesized extracellular matrix of at least about 120 microns thick.  
     
     
         48 . The method of  claim 39 , wherein the second culture medium comprises a neutral polymer.  
     
     
         49 . The method of  claim 48 , wherein the neutral polymer is polyethylene glycol (PEG), dextran, polyvinylpyrrolidone (PVP).  
     
     
         50 . The method of  claim 39 , wherein in step (a) the fibroblast cells are seeded at a density between about 1×10 5  cells/cm 2  to about 6.6×10 5  cells/cm 2 .  
     
     
         51 . The method of  claim 39 , wherein the fibroblast cells are derived from tissue selected from the group consisting of neonate male foreskin, umbilical cord, dermis, tendon, lung, urethra, corneal stroma, oral mucosa, and intestine.  
     
     
         52 . A method for producing a bilayered cultured tissue construct, comprising: 
 (a) seeding fibroblast cells capable of synthesizing an extracellular matrix on a porous membrane in a culture vessel in a first cell culture medium;    (b) culturing the fibroblast cells of step (a) in the first cell culture medium to between about 80% to about 100% confluence on the porous membrane;    (c) stimulating the fibroblast cells of step (a) to synthesize, secrete and organize extracellular matrix components under culturing conditions in a second culture medium;    (d) continued culturing of the fibroblast cells until the cells form a layer of synthesized extracellular matrix of at least 30 microns thick, with the cultured fibroblast cells surrounded by the synthesized extracellular matrix layer, wherein said extracellular matrix is produced by the cultured fibroblast cells in the absence of exogenous matrix components or synthetic members during the culturing conditions;    (e) seeding epithelial cells to the top surface the synthesized extracellular matrix of step (d), and,    (f) stimulating the epithelial cells of step (e) under culturing conditions to form a bilayered tissue construct of an extracellular matrix, with the cultured fibroblast cells surrounded by the synthesized extracellular matrix layer, and a second layer of epithelial cells.    
     
     
         53 . The method of  claim 52 , wherein either the first culture medium, or the second culture medium, or both the first and second culture mediums are chemically defined.  
     
     
         54 . The method of  claim 52 , wherein the first and second culture mediums contain no animal components.  
     
     
         55 . The method of  claim 52 , wherein the second culture medium comprises an ascorbate derivative.  
     
     
         56 . The method of  claim 55 , wherein the ascorbate derivative is sodium ascorbate, ascorbic acid, or L-ascorbic acid phosphate magnesium salt n-hydrate.  
     
     
         57 . The method of  claim 52 , wherein the second culture medium comprises proline, glycine, or hydroxylproline.  
     
     
         58 . The method of  claim 55 , wherein the second culture medium further comprises proline, glycine, or hydroxylproline.  
     
     
         59 . The method of  claim 52 , wherein the layer synthesized extracellular matrix of at least about 60 microns thick.  
     
     
         60 . The method of  claim 59 , wherein the layer synthesized extracellular matrix of at least about 120 microns thick.  
     
     
         61 . The method of  claim 52 , wherein the second culture medium comprises a neutral polymer.  
     
     
         62 . The method of  claim 61 , wherein the neutral polymer is polyethylene glycol (PEG), dextran, polyvinylpyrrolidone (PVP).  
     
     
         63 . The method of  claim 52 , wherein in step (a) the fibroblast cells are seeded at a density between about 1×10 5  cells/cm 2  to about 6.6×10 5  cells/cm 2 .  
     
     
         64 . The method of  claim 52 , wherein the fibroblast cells are derived from tissue selected from the group consisting of neonate male foreskin, umbilical cord, dermis, tendon, lung, urethra, corneal stroma, oral mucosa, and intestine.  
     
     
         65 . The method of  claim 52 , wherein the epithelial cells are selected from the group consisting of keratinocytes, corneal epithelial cells, epithelial cells from oral mucosa, esophageal epithelial cells, and uroepithelial cells.  
     
     
         66 - 105 . (canceled)