Forming cell structure with transient linker in cage

• US 8,389,277 B2
• Filed: 10/13/2008
• Issued: 03/05/2013
• Est. Priority Date: 10/11/2007
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Agency For Science Technology and Research
• Inventors: Hanry, Yu, Siew Min, Ong
• Primary Examiner(s): Ketter, Jim
• Application Number:
  US12/682,751
• Publication Number:
  US 20100216241A1
• Time in Patent Office: 1,604 Days
• Field of Search: None
• US Class Current: 435/370
• 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 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 23/34 Internal compartments or partitions
• CPC Class Codes: C12M 25/06 Plates; Walls; Drawers; Multilayer plates

Abstract

In a method of forming a cellular structure, cells and a transient linker are supplied to a volume partially enclosed by a cage. The linker facilitates initial attachment of adjacent cells to form a cell aggregate. The cage defines distributed openings that are sized to retain the cell aggregate. A fluid comprising a cell culture medium is supplied to the volume. The fluid is withdrawn from the volume through the openings. Aggregated cells retained in the volume are cultured to form a cell structure. A cell culturing device is provided which comprises a conduit and a cage in the conduit. A fluid flows in the conduit. The fluid comprises the cells, the transient linker and the cell culture medium. The cage retains aggregated cells formed in the fluid, and defines distributed openings that allow the fluid to flow through.

Claims

1. A method of forming a cellular structure, comprising:

supplying cells and a transient linker to a volume partially enclosed by a cage, said linker facilitating initial attachment of adjacent cells to form a cell aggregate, said cage defining distributed openings that are sized to retain said cell aggregate;

supplying a fluid comprising a cell culture medium to said volume;

withdrawing said fluid from said volume through said openings; and

culturing aggregated cells retained in said volume to form a cell structure.

2. The method of claim 1, wherein said cells are suspended in said fluid and said linker is dissolved in said fluid before said fluid is supplied to said volume.

3. The method of claim 1, comprising maintaining a flow of said fluid through said volume.

4. The method of claim 3, wherein said cells in said fluid have a density of about 5 to about 6 million cells/ml and said transient linker in said fluid has a concentration of about 6 to about 8 μ

M.

5. The method of claim 1, wherein said openings are distributed to facilitate perfusion of said cell culture medium through said cell structure.

6. The method of claim 1, wherein said cage is disposed in a conduit, and said fluid flows through said conduit.

7. The method of claim 6, wherein said conduit comprises a bottom and opposing side walls extending from said bottom.

8. The method of claim 7, wherein said cage comprises a plurality of projections extending from said bottom and between said side walls.

9. The method of claim 8, wherein said projections comprise micro-pillars.

10. The method of claim 9, wherein said micro-pillars are arranged in a substantially U-shaped pattern.

11. The method of claim 10, wherein a gap between two adjacent ones of said micro-pillars is about 10 to about 50 micrometers.

12. The method of claim 1, wherein said linker comprises a polyethyleneimine backbone and hydrazide groups bonded to said backbone.

13. The method of claim 12, wherein said linker has a molecular weight of about 2000 to about 20000 Dalton.

14. The method of claim 1, wherein said cells comprise an aldehyde group.

15. The method of claim 14, wherein said cells comprise cells that have been modified to form aldehyde groups on surfaces of said modified cells.

16. The method of claim 1, wherein said cells comprise HepG2 cells or rat bone marrow stem cells.

17. The method of claim 3, wherein said flow of said fluid is actuated by applying a withdrawal force downstream of said distributed openings.

18. A cell culturing device, comprising:

a conduit;

a fluid flowing in said conduit, said fluid comprising cells, a transient linker and a cell culture medium, said linker facilitating initial attachment of adjacent cells to form a cell aggregate; and

a cage in said conduit for retaining aggregated cells formed in said fluid, said cage defining distributed openings that are sized to retain said aggregated cells and allow said fluid to flow through.

19. The device of claim 18, wherein said cage comprises a plurality of projections, arranged in a substantially U-shaped pattern.

20. The device of claim 19, wherein said projections comprise micro-pillars.

21. The device of claim 19, wherein said conduit has a bottom and opposing side walls extending from said bottom, said projections extending from said bottom.

22. The device of claim 18, wherein said cells in said fluid have a density of about 5 to about 6 million cells/ml and said transient linker in said fluid has a concentration of about 6 to about 8 μ

M.

23. The device of claim 18, wherein said cells are suspended in said fluid.

24. The device of claim 18, wherein said linker is dissolved in said fluid.

25. The device of claim 18, wherein said linker comprises a polyethyleneimine backbone and hydrazide groups bonded to said backbone.

26. The device of claim 25, wherein said linker has a molecular weight of about 2000 to about 20000 Dalton.

27. The device of claim 18, wherein said cells comprise an aldehyde group.

28. The device of claim 27, wherein said cells comprise cells that have been modified to form aldehyde groups on surfaces of said modified cells.

29. The device of claim 18, wherein said cells comprise HepG2 cells or rat bone marrow stem cells.

30. The device of claim 18, wherein said fluid is actuated by a withdrawal force applied downstream of said distributed openings.