Multi-well micropatterning by ablation

Abstract

The present invention is drawn to the generation of micropatterns of biomolecules and cells on standard laboratory materials through selective ablation of a physisorbed biomolecule with oxygen plasma. In certain embodiments, oxygen plasma is able to ablate selectively physisorbed layers of biomolecules (e.g., type-I collagen, fibronectin, laminin, and Matrigel) along complex non-linear paths which are difficult or impossible to pattern using alternative methods. In addition, certain embodiments of the present invention relate to the micropatterning of multiple cell types on curved surfaces, multiwell plates, and flat bottom flasks. The invention also features kits for use with the subject methods.

1 . A method of forming a micropatterned substrate, comprising the steps of:
 adsorbing molecules onto a surface of a substrate, thereby forming a coated surface of the substrate;   comprising a micropatterned etch mask onto the coated surface of said substrate; and   exposing the compressed micropatterned etch mask and coated surface of the substrate to a gas plasma for a period of time, thereby ablating the exposed surfaces of the substrate.   
     
     
         2 . The method of  claim 1 , further comprising rinsing and drying said coated surface after the adsorbing step. 
     
     
         3 . The method of  claim 1 , wherein said exposing step is carried out in a plasma asher. 
     
     
         4 . The method of  claim 1 , wherein the micropatterned etch mask is one solid elastomericpiece. 
     
     
         5 . The method of  claim 1 , wherein the micropatterned etch mask comprises a plurality of pillars. 
     
     
         6 . The method of  claim 1 , wherein the micropatterned etch mask comprises chrome or elastomeric poly(dimethylsiloxane) or rubber or plastic. 
     
     
         7 . The method of  claim 1 , wherein the adsorbed molecules are different. 
     
     
         8 . The method of  claim 7 , wherein the different molecules each have a different pattern. 
     
     
         9 . The method of  claim 1 , wherein the micropatterned etch mask comprises plastic. 
     
     
         10 . The method of  claim 1 , wherein the micropatterned etch mask comprises an about 50 μm to about 1 mm thick piece of plastic. 
     
     
         11 . The method of  claim 1 , wherein said substrate surface is ceramic, metal, glass, or plastic. 
     
     
         12 . The method of  claim 1 , wherein said substrate comprises fluoropolymers, fluorinatedethylene propylene, polyvinylidene, polydimethylsiloxane, polystyrene, polycarbonate, and polyvinyl chloride, fused silica, polysilicon, or single silicon crystals. 
     
     
         13 . The method of  claim 1 , wherein said substrate is a tissue culture flask, a tissue culture bottle, or a cell culture multiwall plate. 
     
     
         14 . The method of  claim 1 , wherein said substrate is a 24-well or a 96-well or a 384-well cell culture plate. 
     
     
         15 . The method of  claim 1 , wherein said molecules are biomolecules. 
     
     
         16 . The method of  claim 1 , wherein said molecules are biomolecules; and said biomolecules are selected from the group consisting of peptides, polypeptides, nucleic acids, nucleic acid binding partners, proteins, receptors, antibodies, enzymes, carbohydrates, oligosaccharides, polysaccharides, cells, cell aggregates, cell components, lipids, arrays of ligands, non-protein ligands, liposomes, and microorganisms. 
     
     
         17 . The method of  claim 1 , wherein said molecules are hyaluronic acid, collagen, fibronectin, lamanin, or matrigel. 
     
     
         18 . The method of  claim 1 , further comprising the steps of:
 removing the micropatterned etch mask; and   contacting said micropatterned substrate with cells.   
     
     
         19 . The method of  claim 3 , wherein said cells are hepatocytes, endothelial cells, kidney, muscle, pancreas, epithelium cells, tissue/skin cells, intestinal cells or stem-cell derived cells. 
     
     
         20 . The method of  claim 19 , wherein said cells are rat cells, human cells, mouse cells, monkey cells, or guinea pig cells. 
     
     
         21 . A multi-well cell culture plate wherein each cell is micropatterned with a material, wherein said plate is prepared by a process comprising the steps of:
 adsorbing molecules onto a surface of a substrate, thereby forming a coated surface of the substrate;   compressing a micropatterned etch mask onto the coated surface of said substrate; and   exposing the compressed micropatterned etch mask and coated surface of the substrate to a gas plasma for a period of time, thereby ablating the exposed surfaces of the substrate.   
     
     
         22 . The multi-well plate of  claim 21 , wherein said multi-well cell culture plate is a 24-well or a 96-well or a 384-well cell culture plate. 
     
     
         23 . The multi-well plate of  claim 21 , wherein said material is a biomolecule; and said biomolecule is selected from the group consisting of peptides, polypeptides, nucleic acids, nucleic acid binding partners, proteins, receptors, antibodies, enzymes, carbohydrates, oligosaccharides, polysaccharides, cells, cell aggregates, cell components, lipids, arrays of ligands, non-protein ligands, liposomes, and microorganisms. 
     
     
         24 . The multi-well plate of  claim 23 , wherein said material is not a biomolecules. 
     
     
         25 . The multi-well plate of  claim 24 , wherein said cells are human or rat hepatocytes. 
     
     
         26 . A microscale human liver tissue comprising primary human hepatocytes cultures in micropatterned colonies surrounded by supportive stromal cells. 
     
     
         27 . An assay for genes involved in stimulation or maintenance of hepatocyte differentiation comprising comparing a gene expression profile from the human liver tissue of  claim 26  with a second gene expression profile from a control hepatocyte culture and identifying one or more genes differentially expressed in said profiles. 
     
     
         28 . The assay of  claim 27 , wherein the gene is a drug metabolism gene. 
     
     
         29 . An assay for agents involved in stimulation or maintenance of hepatocyte differentiation comprising contacting the human liver tissue of  claim 26  with a test agent and assaying for effect on expression of a gene identified in  claim 27 . 
     
     
         30 . A method for testing toxicity of agents comprising contacting the human liver tissue of  claim 26  with a test agent and assaying for hepatocyte viability within said human liver tissue.