Methods to generate polymer scaffolds having a gradient of crosslinking density

Abstract

The present invention is directed to a method of making a live cell construct or a support, comprising: (a) providing a non-cellular organic polymer support having a top surface, a bottom surface, and an intermediate portion there between, and (b) contacting a cross-linking agent to one surface of said support for a time sufficient to generate a gradient of cross-linking of said polymer in said intermediate portion. Also provided are live cell constructs, supports, and methods of use of the supports and live cell constructs.

1 . A method of making a live cell construct or a support, comprising:
 (a) providing a non-cellular organic polymer support having a top surface, a bottom surface, and an intermediate portion there between,   (b) contacting a cross-linking agent to one surface of said support (e.g., under aqueous conditions) for a time sufficient to generate a gradient of cross-linking of said polymer in said intermediate portion;   (c) optionally, Wherein said gradient of cross-linking in said intermediate portion produces a corresponding gradient of free amino and/or carboxy groups in said intermediate portion, coupling a compound of interest to said free amino and/or carboxy groups to produce a gradient of said compound of interest in said intermediate portion;   (d) optionally contacting live undifferentiated epithelial cells to said non-cellular support, and   (e) optionally propagating an undifferentiated and/or differentiated epithelial cell monolayer on said top surface.   
     
     
         2 . The method of  claim 1 , wherein the cells in the monolayer comprise:
 (i) undifferentiated cells (e.g., stem or progenitor cells); and   (ii) optionally, differentiated cells in combination with the undifferentiated cells.   
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein the live undifferentiated epithelial cells are gastrointestinal epithelial cells, urinary epithelial cells, respiratory epithelial cells, reproductive epithelial cells, endocrine and endocrine gland epithelial cells, lymph vessel epithelial cells, blood vessel epithelial cells, or ventricular ependyma epithelial cells. 
     
     
         5 - 6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the live undifferentiated epithelial cells are from the colon, small intestine, stomach, esophagus, tongue, nasopharynx, oropharynx, laryngeopharynx, pancreas, kidney, bladder, trachea, lungs, testes, ovaries, ducts of the reproductive tract, endometrium thyroid gland, adrenal gland, parathToid gland, ventricular ependyma and/or brain. 
     
     
         8 . The method of  claim 1 , further comprising:
 (d) contacting a culture media to said monolayer of live cells (e.g., which culture media is in or on said support), which culture media sustains said monolayer of live cells.   
     
     
         9 - 10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein said support is a porous support. 
     
     
         12 . The method of  claim 1 , wherein said support comprises collagen, gelatin, laminin, elastin, fibronectin, heparin sulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid, gelatinous protein mixture secreted by EnRelbreth-Holm-Swarm mouse sarcoma cells (e.g. Matrigel®, Geltrex®, MaxGel™, etc.), and combinations thereof; and/or said support comprises a hydrogel formed from a natural or synthetic polymers chitosan, agarose, alginate, polyvinyl alcohol, sodium polyacrylate, acrylate polymers, polyethylene glycol, synthetic peptides, etc., optionally surface modified by the compounds described above). 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , said top surface having a plurality of wells formed therein; each of said wells having a top opening, side walls and a floor; said epithelial cell monolayer extending onto said well side walls and floors, with the well top openings remaining open, to form open lumens (or “crypts”) lined with cells in said wells. 
     
     
         15 . A live cell construct or a support useful for producing a live cell construct, comprising:
 (a) a non-cellular organic polymer support having a top surface, a bottom surface, and an intermediate portion there between, said intermediate portion having a gradient of cross-linking of said polymer formed therein;   (b) optionally, a monolayer of live undifferentiated and/or differentiated epithelial cells formed on said top surface; and   (c) optionally, a gradient of (i) free reactive amino acid and/or carboxylic acid groups in said intermediate portion, or (ii) a gradient of a compound of interest covalently coupled to amino acid and/or carboxylic acid groups in said intermediate portion.   
     
     
         16 . The construct of  claim 14 , wherein said live cells in said monolayer comprise:
 (i) undifferentiated cells (e.g., stem or progenitor cells); and   (ii) optionally, differentiated cells in combination with said undifferentiated cells.   
     
     
         17 . (canceled) 
     
     
         18 . The construct of  claim 15 , wherein the epithelial cells are gastrointestinal epithelial cells, urinary epithelial cells, respiratory epithelial cells, reproductive epithelial cells, endocrine and endocrine gland epithelial cells, lymph vessel epithelial cells, blood vessel epithelial cells, or ventricular ependyma epithelial cells. 
     
     
         19 - 20 . (canceled) 
     
     
         21 . The construct of  claim 15 , wherein the epithelial cells are from the colon, small intestine, stomach, esophagus, tongue, nasopharynx, oropharynx, laryngeopharynx, pancreas, kidney, bladder, trachea, lungs, testes, ovaries, duets of the reproductive tract, endometrium, thyroid gland, adrenal gland, parathyroid gland, ventricular ependyma and/or brain. 
     
     
         22 . The construct of  claim 15 , further comprising:
 (c) a culture medium contacting said monolayer of live cells: (e.g., which culture medium is in or on said support), which -culture medium sustains said monolayer of live cells.   
     
     
         23 - 24 . (canceled) 
     
     
         25 . The construct of  claim 15 , wherein said support comprises collagen, gelatin, laminin, elastin, fibronectin, heparan sulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid, gelatinous protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (a g Matrigel®, Geltrex®, MaxGel™, etc.), and combinations thereof; and/or said support comprises a hydrogel formed from a natural or synthetic polymers (e.g.,chitosan, agarose, alginate, polyvinyl alcohol, sodium polyacrylate, acrylate polymers, polyethylene glycol, synthetic peptides, etc., optionally surface modified by the compounds described above). 
     
     
         26 . The construct of  claim 15 , wherein said support is porous. 
     
     
         27 . (canceled) 
     
     
         28 . The construct of  claim 15 , said top surface having a plurality of wells formed therein, each of said wells having a top opening, side walls and a floor;
 said epithelial cell monolayer extending onto said well side walls and floors, with said well top openings remaining uncovered, to form open cell lumens (or “crypts”) in said wells.   
     
     
         29 . The construct of  claim 28 , wherein said wells are from 100, 200 or 300 microns deep, up to 800 or 1000 microns deep or more, and/or said wells are from 10 or 50 microns wide, up to 100 or 200 microns wide or more; and/or at least 10, 50, or 100 of said wells are formed in said top surface. 
     
     
         30 . The construct of  claim 28 , wherein:
 said live cells in said monolayer comprise both differentiated cells and undifferentiated cells (e.g, stern or progenitor cells) in combination;   said differentiated cells and said undifferentiated cells are positioned in said monolayer a gradient (e.g., with a greater concentration of differentiated cells on one end of the gradient; and a greater concentration of undifferentiated cells on the other end of said gradient);   said gradient oriented with or along said well side walls.   
     
     
         31 . A method of sustaining a live cell construct, comprising;
 (a) providing a construct according to  claim 15 ;   (b) contacting a first culture medium to said top surface; and   (c) contacting a second culture medium to said bottom surface,   wherein one of said culture media induces the differentiation of propagating stem and progenitor cells and the other of said culture media induces the propagation of undifferentiated cells.   
     
     
         32 . A method of screening a test compound or microbe for a toxicological, physiological, or carcinogenic effect, comprising:
 (a) providing a construct according to  claim 15 , or a construct sustained according to the method of  claim 31 ,   (b) contacting a test compound or microbe to said construct; and then   (c) detecting a toxicological, physiological, or carcinogenic effect of said test compound or microbe on the cells of said construct (e.g., by comparing the construct after said contacting to a like construct to which said test compound or microbe has not been contacted, and/or by comparing the construct after said contacting step to said construct before said contacting step).   
     
     
         33 - 34 . (canceled)