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The present invention describes novel methods for making a substrate for selective cell patterning, and the substrates themselves, wherein the method comprises contacting reactive hydroxyl groups on the surface of a substrate with a hydroxyl-reactive bifunctional molecule to form a monolayer, and us

The present invention describes novel methods for making a substrate for selective cell patterning, and the substrates themselves, wherein the method comprises contacting reactive hydroxyl groups on the surface of a substrate with a hydroxyl-reactive bifunctional molecule to form a monolayer, and using stencils to deposit cell repulsive or cell adhesive moieties in controlled locations on the cell culture substrate. Methods comprising selective differentiation of stem cells to create tissue specific and organ-specific cell substrates, as well as the cell substrates themselves are also provided.

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1. A method of making a substrate for selective cell patterning comprising: a) providing a substrate with a surface, wherein the surface contains hydroxyl groups; b) contacting the hydroxyl groups on the surface of the substrate with a bifunctional molecule comprising a hydroxy-reactive moiety a

1. A method of making a substrate for selective cell patterning comprising: a) providing a substrate with a surface, wherein the surface contains hydroxyl groups; b) contacting the hydroxyl groups on the surface of the substrate with a bifunctional molecule comprising a hydroxy-reactive moiety and a nucleophilic moiety to form a monolayer; c) applying a stencil to the substrate; and d) further modifying the substrate by a technique selected from the group consisting of i) applying an electrophilic cell repulsive moiety to exposed regions of the monolayer to form a covalent bond between the cell repulsive moiety and the nucleophilic moiety of the bifunctional molecule deposited in step b to produce cell repulsive locations, removing the stencil, and applying cell adhesive molecules to the substrate to produce cell binding locations, wherein the cell adhesion molecules bind the substrate only in positions that were contacted by the stencil; and ii) applying cell adhesive molecules to exposed regions on the monolayer to produce cell binding locations, removing the stencil, and applying an electrophilic cell repulsive moiety to the substrate to produce cell repulsive locations, wherein the cell repulsive moiety forms a covalent bond with the nucleophilic moiety of the bifunctional molecule deposited in step b only in positions that were contacted by the stencil. 2. The method of claim 1 wherein the substrate is treated to introduce the hydroxyl groups onto the surface of the substrate. 3. The method of claim 2 wherein the substrate is oxygen plasma treated to introduce the hydroxyl groups onto the surface of the substrate. 4. The method of claim 1 wherein the bifunctional molecule comprises an organosilane. 5. The method of claim 1 wherein the bifunctional molecule comprises an aminosilane. 6. The method of claim 1 wherein the reactive cell repulsive moiety comprises a tresyl-chloride activated-poly(ethylene) glycol compound. 7. The method of claim 1 wherein vapor phase deposition is used for the contacting of the hydroxyl groups on the surface of the substrate with a bifunctional molecule. 8. The method of claim 1 further comprising contacting the cell binding locations with a cell-binding protein or peptide under conditions to permit binding of the cell-binding protein or peptide to the cell binding location. 9. The method of claim 1 further comprising contacting the cell binding locations with cells under conditions to permit binding of the cells to the cell binding locations. 10. The method of claim 8 further comprising contacting the cell-binding protein or peptide on the cell binding locations with cells under conditions to permit binding of the cells to the cell-binding protein or peptide. 11. The method of claim 8 further comprising lyophilizing the substrate. 12. The method of claim 9 further comprising cryopreserving or desiccating the substrate. 13. The method of claim 10 further comprising cryopreserving or desiccating the substrate. 14. A cell patterning substrate, prepared by the method of claim 1.