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 - 14 . (canceled) 
     
     
         15 . A live cell construct, comprising:
 (a) a neutralized collagen hydrogel support comprising:
 i) a first surface; 
 ii) a second surface; and 
 iii) an intermediate surface between the first surface and second surface and comprising a gradient of cross-linked neutralized collagen hydrogel, wherein the gradient of cross-linked neutralized collagen hydrogel has a higher density near the second surface than near the first surface; and 
   (b) a self-renewing monolayer of live primary epithelial cells comprising undifferentiated and/or differentiated live primary epithelial cells formed on the first surface of the cross-linked neutralized collagen hydrogel support, wherein the self-renewing monolayer is maintained or increased for at least 1 day.   
     
     
         16 . The construct of claim  14 , wherein the gradient of cross-linked neutralized collagen comprises a stiffness between about 100 Pa to about 1,000 Pa. 
     
     
         17 . The construct of  15 , wherein the epithelial cells are selected from the group consisting of mammalian, avian, reptilian, amphibian, and insect cells. 
     
     
         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 . The construct of  claim 15 , wherein the live undifferentiated epithelial cells are human cells. 
     
     
         20 . The construct of  claim 15 , wherein the epithelial cells are not malignant cells. 
     
     
         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, ducts of the reproductive tract, endometrium, thyroid gland, adrenal gland, parathyroid gland, ventricular ependyma and/or brain. 
     
     
         22 . The construct of  claim 15 , further comprising:
 a culture medium contacting said self-renewing monolayer of live primary epithelial cells, which culture medium sustains said monolayer of live cells.   
     
     
         23 . The construct of  claim 22 , wherein said culture medium comprises a short-chain fatty acid. 
     
     
         24 . The construct of  claim 23 , wherein:
 (i) said culture medium contains not more than 10 milliMolar of monosaccharides plus disaccharides; and   (ii) said culture medium contains at least 2, 20, 50, or 100 milliMolar of said short chain fatty acids.   
     
     
         25 . (canceled) 
     
     
         26 . The construct of  claim 15 , wherein said support is porous. 
     
     
         27 . The construct of  claim 15 , wherein said support second surface is on a porous carrier, a mesh, an inorganic grid, a hydrogel, or a combination thereof. 
     
     
         28 . The construct of  claim 15 , said first 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 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 first surface. 
     
     
         30 . The construct of  claim 28 , wherein:
 said live cells in said monolayer comprise both differentiated cells and undifferentiated cells in combination;   said differentiated cells and said undifferentiated cells are positioned in said monolayer in a 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 first surface; and   (c) contacting a second culture medium to said second 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 ,   (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.   
     
     
         33 . The method of  claim 32 , wherein said test compound is selected from the group consisting of aromatic organic compounds, aliphatic organic compounds, and mixed aromatic and aliphatic organic compounds. 
     
     
         34 . The method of  claim 32 , wherein said test microbe is selected from the group consisting of gram negative bacteria, gram positive bacteria, yeast, and molds. 
     
     
         35 . A method of making a live cell construct, comprising:
 (a) providing a neutralized collagen hydrogel support comprising:
 i) a first surface; 
 ii) a second surface; and 
 iii) an intermediate surface between the first surface and second surface and comprising a gradient of cross-linked neutralized collagen hydrogel, wherein the gradient of cross-linked neutralized collagen hydrogel has a higher density near the second surface than near the first surface; 
   (b) contacting live undifferentiated epithelial cells to the first surface of the cross-linked neutralized collagen hydrogel support, and   (c) propagating a self-renewing monolayer of live primary epithelial cells comprising undifferentiated and/or differentiated live primary epithelial cells on said first surface of the cross-linked neutralized collagen hydrogel support, wherein the self-renewing monolayer is maintained or increased for at least 1 day.