Co-cultivation of cells in a micropatterned configuration

Abstract

Disclosed are methods for producing co-cultures of cells in which at least two cell types are present in a micropattern configuration.

What is claimed is:  
     
         1 . A method for producing a micropatterned co-culture containing at least two cell types, the method comprising: 
 i) providing a protein-coated substrate, wherein a protein coating the substrate defines a micropattern on the substrate;    ii) contacting the protein-coated substrate with cells of a first cell type suspended in a first cell medium under conditions such that cells of the first cell type bind the protein of the protein-coated substrate, thereby producing a micropatterned cell-coated substrate; and    iii) contacting the micropatterned cell-coated substrate with cells of a second cell type suspended in a second cell medium under conditions such that cells of the second cell type bind the substrate, thereby producing the micropatterned co-culture, wherein one of the cell media is a selective medium and one of the cell media is an attachment medium.    
     
     
         2 . The method of    claim 1   , wherein the cells of one of the first and second cell types are hepatocytes.  
     
     
         3 . The method of    claim 1   , wherein the cells of one of the first and second cell types are selected from the group consisting of Kupffer cells, Ito cells, endothelial cells, and biliary ductal cells.  
     
     
         4 . The method of    claim 1   , wherein the cells of one of the first and second cell types are fibroblasts.  
     
     
         5 . The method of    claim 1   , wherein the cells of the first cell type are hepatocytes, and the cells of the second cell type are fibroblasts.  
     
     
         6 . The method of    claim 1   , wherein the cells one or both of the first and second cell types are selected from the group consisting of muscle cells, bone marrow cells, hematopoietic cells, stromal cells, skin cells, mesenchymal cells, and parenchymal tumor cells.  
     
     
         7 . The method of    claim 6   , wherein the muscle cells are smooth muscle cells.  
     
     
         8 . The method of    claim 6   , wherein the skin cells are keratinocytes.  
     
     
         9 . The method of    claim 1   , wherein the selective medium is a serum-free medium.  
     
     
         10 . The method of    claim 1   , wherein the attachment medium comprises serum.  
     
     
         11 . The method of    claim 1   , wherein the attachment medium comprises at least one attachment factor selected from the group consisting of fibronectin, selectin, RGD peptides, ICAMs, E-cadherin, integrins, and antibodies that specifically bind a cell surface protein.  
     
     
         12 . The method of    claim 11   , wherein the antibody specifically binds a cell surface protein selected from the group consisting of integrins, ICAMs, selecting, RGD peptides, and E-cadherins.  
     
     
         13 . The method of    claim 1   , wherein the protein-coated substrate comprises collagen.  
     
     
         14 . The method of    claim 1   , wherein the protein-coated substrate comprises fibronectin, laminin, or entactin, and combinations thereof.  
     
     
         15 . The method of    claim 1   , wherein the first and second cell types define a micropattern in which an island of cells of either the first or second cell type is surrounded by cells of either the second or first cell type, respectively.  
     
     
         16 . The method of    claim 15   , wherein the island of cells is 25-1,000 μm in diameter.  
     
     
         17 . The method of    claim 16   , wherein the island of cells is 30-500 μm in diameter.  
     
     
         18 . The method of    claim 17   , wherein the island of cells is 100-500 μm in diameter.  
     
     
         19 . The method of    claim 1   , wherein the first and second cell types define a micropattern in which an island of cells of either the first or second cell type is surrounded by cells of either the second or first cell type, respectively, and wherein at least 30% of the cells of the island of cells are within 100 μm of an interface between the island of cells and the surrounding cells.  
     
     
         20 . The method of    claim 1   , wherein the substrate is selected from the group consisting of glass, polymers, and silicon substrates.  
     
     
         21 . A co-culture of at least two cell types produced by the method of    claim 1   .  
     
     
         22 . The co-culture of    claim 21   , wherein the co-culture comprises hepatocytes and fibroblasts.  
     
     
         23 . The co-culture of    claim 21   , wherein the co-culture comprises a combination of cells selected from the group consisting of: 
 a) hepatocytes and at least one cell type selected from the group consisting of Kupffer cells, Ito cells, endothelial cells, and biliary ductal cells;    b) endothelial cells and smooth muscle cells;    c) mesenchymal cells and tumorigenic parenchymal cells;    d) bone marrow cells and fibroblasts; and    e) keratinocytes and fibroblasts.    
     
     
         24 . A method for modulating a metabolic or synthetic function of a cell of a first cell type, the method comprising: 
 i) providing a protein-coated substrate, wherein a protein coating the substrate defines a micropattern on the substrate;    ii) contacting the protein-coated substrate with cells of a first cell type suspended in a first cell medium under conditions such that cells of the first cell type bind the protein of the protein-coated substrate, thereby producing a micropatterned cell-coated substrate; and    iii) contacting the micropatterned cell-coated substrate with cells of a second cell type suspended in a second cell medium under conditions such that cells of the second cell type bind the substrate, thereby producing the micropatterned co-culture, wherein:    a) one of the cell media is a selective medium and one of the cell media is an attachment medium; and    b) the cells of the first and second cell types define a micropattern wherein cells of the second cell type surround cells of the first cell type, and at least 30% of the cells of the first cell type are within 100 μm of an interface between the cells of the first cell type and the cells of the second cell type,    thereby producing a micropatterned co-culture, wherein a metabolic or synthetic function of a cell of the first cell type is modulated relative to cells of the first cell type in an unpatterned co-culture that comprises cells of the first and second cell types.    
     
     
         25 . The method of    claim 24   , wherein modulation is detected as an increase in protein production of a cell of the first cell type.  
     
     
         26 . The method of    claim 25   , wherein the cells of the first cell type are hepatocytes and modulation is detected as a change in intracellular or secreted albumin of a hepatocyte.  
     
     
         27 . The method of    claim 25   , wherein the cells of the first cell type are hepatocytes and modulation is detected as an change in urea synthesis in a hepatocyte.  
     
     
         28 . The method of    claim 25   , wherein modulation is detected as an change in DNA synthesis in a cell of the first cell type.  
     
     
         29 . The method of    claim 24   , wherein the co-culture comprises a combination of cells selected from the group consisting of: 
 a) hepatocytes and at least one cell type selected from the group consisting of Kupffer cells, Ito cells, endothelial cells, and biliary ductal cells;    b) endothelial cells and smooth muscle cells;    c) mesenchymal cells and tumorigenic parenchymal cells;    d) bone marrow cells and fibroblasts; and    e) keratinocytes and fibroblasts.    
     
     
         30 . The method of    claim 24   , wherein the co-culture comprises hepatocytes and fibroblasts.  
     
     
         31 . A co-culture of cells produced according to the method of    claim 24   .  
     
     
         32 . The method of    claim 24   , wherein the selective medium is a serum-free medium.  
     
     
         33 . The method of    claim 24   , wherein the attachment medium comprises serum.  
     
     
         34 . The method of    claim 24   , wherein the protein-coated substrate comprises a protein selected from the group consisting of collagen, fibronectin, laminin, and entactin, or combinations thereof.  
     
     
         35 . The method of    claim 24   , wherein the micropattern defined by cells of the first and second cell types comprises an island of cells of the first cell type surrounded by cells of the second cell type.  
     
     
         36 . The method of    claim 35   , wherein the island of cells is 25-1,000 μm in diameter.  
     
     
         37 . The method of    claim 36   , wherein the island of cells is 30-500 μm in diameter.  
     
     
         38 . The method of    claim 37   , wherein the island of cells is 100-500 μm in diameter.  
     
     
         39 . The method of    claim 24   , wherein the rate at which a metabolic or synthetic function is modulated in the micropatterned co-culture is increased relative to the rate at which a metabolic or synthetic function is modulated in an unpatterned co-culture.  
     
     
         40 . The method of    claim 24   , wherein a metabolic or synthetic function of cells of the first cell type is modulated at least 1.5-fold in a micropatterned co-culture, relative to a metabolic or synthetic function of cells of the first cell type in an unpatterned co-culture.  
     
     
         41 . The method of    claim 40   , wherein a metabolic or synthetic function of cells of the first cell type is modulated at least 5-fold in a micropatterned co-culture, relative to a metabolic or synthetic function of cells of the first cell type in an unpatterned co-culture.  
     
     
         42 . A method for modulating a metabolic or synthetic function of a cell of a second cell type, the method comprising: 
 i) providing a protein-coated substrate, wherein a protein coating the substrate defines a micropattern on the substrate;    ii) contacting the protein-coated substrate with cells of a first cell type suspended in a first cell medium under conditions such that cells of the first cell type bind the protein of the protein-coated substrate, thereby producing a micropatterned cell-coated substrate; and    iii) contacting the micropatterned cell-coated substrate with cells of a second cell type suspended in a second cell medium under conditions such that cells of the second cell type bind the substrate, thereby producing the micropatterned co-culture, wherein:    a) one of the cell media is a selective medium and one of the cell media is an attachment medium; and    b) the cells of the first and second cell types define a micropattern wherein cells of the first cell type surround cells of the second cell type, and at least 30% of the cells of the second cell type are within 100 μm of an interface between the cells of the second cell type and the cells of the first cell type,    thereby producing a micropatterned co-culture, wherein a metabolic or synthetic function of a cell of the second cell type is modulated relative to cells of the second cell type in an unpatterned co-culture that comprises cells of the second and first cell types.    
     
     
         43 . The method of    claim 42   , wherein the co-culture comprises a combination of cells selected from the group consisting of: 
 a) hepatocytes and at least one cell type selected from the group consisting of Kupffer cells, Ito cells, endothelial cells, and biliary ductal cells;    b) endothelial cells and smooth muscle cells;    c) mesenchymal cells and tumorigenic parenchymal cells;    d) bone marrow cells and fibroblasts;    e) keratinocytes and fibroblasts and    f) hepatocytes and fibroblasts.    
     
     
         44 . A co-culture produced according to the method of    claim 42   .  
     
     
         45 . The method of    claim 24   , wherein modulation comprises upregulation of a metabolic or synthetic function of a cell.  
     
     
         46 . A method for producing a micropatterned co-culture containing at least two cell types, the method comprising: 
 i) providing a protein coated substrate wherein a protein coated substrate defines a micropattern on the substrate;    ii) contacting the protein-coated substrate with cells of a first cell type suspended in a first cell medium under conditions such that the cells of the first cell type bind the protein of the protein-coated substrate, thereby producing a micropatterned cell-coated substrate; and    iii) contacting the micropatterned cell-coated substrate with cells of a second cell type suspended in a second cell medium under conditions such that the cells of the second cell type bind to the substrate, thereby producing the micropatterned co-culture, wherein the first cell type is in non-attachment medium and second cell type has natural attachment capabilities to attach it to the substrate.    
     
     
         47 . The method of    claim 46   , wherein the cells are fibroblasts.  
     
     
         48 . The method of    claim 46   , wherein the substrate is charged.  
     
     
         49 . The method of    claim 1   , wherein said first cell type has been genetically engineered to produce a desired product, and said second cell type produces a protein, and said first cell type enables said second cell type to reproduce and grow.  
     
     
         50 . The method of    claim 46   , wherein the cells of one of the first and second cell types are fungi or bacteria.  
     
     
         51 . The method of    claim 1   , wherein the cells of one of the first and second cell types are fungi or bacteria.  
     
     
         52 . A method for producing a micropatterned co-culture containing at least two cell types, the method comprising: 
 i) providing a repellent-coated substrate, wherein a cell repellent coating the substrate defines a micropattern on the substrate;    ii) contacting the repellent-coated substrate with cells of a first cell type suspended in a first cell medium under conditions such that cells of the first cell type bind the substrate, thereby producing a micropatterned cell/repellent-coated substrate; and    iii) contacting the micropatterned cell/repellent-coated substrate with cells of a second cell type suspended in a second cell medium under conditions such that cells of the second cell type bind the repellent, thereby producing the micropatterned co-culture.    
     
     
         53 . The method of    claim 52   , wherein the cells one of the first and second cell types are hepatocytes.  
     
     
         54 . The method of    claim 52   , wherein the cells of one of the first and second cell types are selected form the group consisting of Kupffer cells, Ito cells, endothelial cells, and biliary ductal cells.  
     
     
         55 . The method of    claim 52   , wherein the cells of one of the first and second cell types are fibroblasts.  
     
     
         56 . The method of    claim 52   , wherein the cells of one of the first and second cell types are fungi or bacteria.