Co-cultivation of cells in a micropatterned configuration

• US 6,221,663 B1
• Filed: 01/13/2000
• Issued: 04/24/2001
• Est. Priority Date: 05/14/1997
• Status: Expired due to Term

• Current Assignee: General Hospital Corporation
• Original Assignee: General Hospital Corporation
• Inventors: Bhatia, Sangeeta, Yarmush, Martin, Toner, Mehmet
• Primary Examiner(s): TATE, CHRISTOPHER ROBIN
• Application Number:
  US09/482,017
• Time in Patent Office: 467 Days
• Field of Search: 435/373, 435/375, 435/395, 435/370-372, 435/402, 435/392, 435/177, 435/70.1, 435/70.3, 435/69.1
• US Class Current: 435/373
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12M 21/08 for producing artificial tissue or for ex-vivo cultivation of tissue prostheses A61F2/00, grafts A61L27/00
• CPC Class Codes: C12M 33/00 Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00
• CPC Class Codes: C12M 35/08 Chemical, biochemical or biological means, e.g. plasma jet, co-culture
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/14 hepatocytes
• CPC Class Codes: C12N 2535/10 Patterned coating
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
• CPC Class Codes: C12N 5/0697 Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691

Abstract

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

Claims

1. 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 that lacks serum and attachment factors and/or includes a non-adhesive factor to inhibit attachment and one of the cell media is an attachment medium that contains an effective amount of serum and/or at least one attachment factor; and

b) the cells of the first and second cell types define a micropattern wherein at least 30% of the cells of the first cell type are within 100 μ

m 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.

2. The method of claim 1, wherein modulation is detected as an increase in protein production of a cell of the first cell type.

3. The method of claim 2, 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.

4. The method of claim 2, wherein the cells of the first cell type are hepatocytes and modulation is detected as an change in urea synthesis in a hepatocyte.

5. The method of claim 2, wherein modulation is detected as an change in DNA synthesis in a cell of the first cell type.

6. The method of claim 1, 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.

7. The method of claim 1, wherein the co-culture comprises hepatocytes and fibroblasts.

8. A co-culture of cells produced according to the method of claim 1.

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 protein-coated substrate comprises a protein selected from the group consisting of collagen, fibronectin, laminin, and entactin, or combinations thereof.

12. The method of claim 1, 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.

13. The method of claim 12, wherein the island of cells is 25-1,000 μ

m in diameter.

14. The method of claim 13, wherein the island of cells is 30-500 μ

m in diameter.

15. The method of claim 14, wherein the island of cells is 100-500 μ

m in diameter.

16. The method of claim 1, 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.

17. The method of claim 1, 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.

18. The method of claim 17, 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.

19. The method of claim 1, wherein modulation comprises upregulation of a metabolic or synthetic function of a cell.

20. 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 that lacks serum and attachment factors and/or includes a non-adhesive factor to inhibit attachment, and one of the cell media is an attachment medium that contains an effective amount of serum and/or at least one attachment factor; and

b) the cells of the first and second cell types define a micropattern wherein at least 30% of the cells of the second cell type are within 100 μ

m 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.

21. The method of claim 20, 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.

22. A co-culture produced according to the method of claim 20.