Co-cultivation of cells in a micropatterned configuration

• US 6,133,030 A
• Filed: 10/03/1997
• Issued: 10/17/2000
• Est. Priority Date: 05/14/1997
• Status: Expired due to Term

• Current Assignee: Massachusetts Institute of Technology, The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: Massachusetts Institute of Technology, The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Bhatia, Sangeeta, Toner, Mehmet, Yarmush, Martin
• Primary Examiner(s): Lankford, Jr., Leon B.
• Assistant Examiner(s): TATE, CHRISTOPHER ROBIN
• Application Number:
  US08/943,143
• Time in Patent Office: 1,110 Days
• Field of Search: 435/373, 435/395, 435/402, 435/289.1, 435/305.1, 435/309.1, 435/177
• 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 producing a micropatterned co-culture containing at least two cell types, the method comprising:

i) providing a substrate coated with a cell-binding protein which defines a micropattern on the substrate;

ii) contacting the cell-binding protein 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 cell-binding protein, 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 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.

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 of 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 1, wherein the cells of one or both of the first and second cell types are smooth muscle cells.

8. The method of claim 1, wherein the cells of one or both of the first and second cell types 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 1, wherein the cell-binding protein comprises collagen.

13. The method of claim 1, wherein the cell-binding protein comprises fibronectin, laminin, entactin, or combinations thereof.

14. 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 the other cell type.

15. The method of claim 14, wherein the island of cells is 25-1,000 μ

m in diameter.

16. The method of claim 15, wherein the island of cells is 30-500 μ

m in diameter.

17. The method of claim 16, wherein the island of cells is 100-500 μ

m in diameter.

18. 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 the other cell type, and wherein at least 30% of the cells of the island of cells are within 100 μ

m of an interface between the island of cells and the surrounding cells.

19. The method of claim 1, wherein the substrate is selected from the group consisting of glass, polymers, and silicon substrates.

20. A micropatterned co-culture of at least two cell types produced by the method of claim 1.

21. The micropatterned co-culture of claim 20, wherein the co-culture comprises hepatocytes and fibroblasts.

22. The micropatterned co-culture 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; and

e) keratinocytes and fibroblasts.

23. A method for producing a micropatterned co-culture containing at least two cell types, the method comprising:

i) providing a substrate coated with a cell-binding protein which defines a micropattern on the substrate;

ii) contacting the cell-binding protein with cells of a first cell type suspended in a selective cell medium that lacks serum and attachment factors and/or includes a non-adhesive factor to inhibit attachment under conditions such that the cells of the first cell type bind the cell-binding protein, 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 second cell type has natural attachment capabilities to attach it to the substrate.

24. The method of claim 23, wherein the cells of either the first or second cell type are fibroblasts.

25. The method of claim 23, wherein the substrate is electrically charged.