Diamond-like carbon-coated cell culture substrates

• US 7,931,938 B2
• Filed: 10/08/2004
• Issued: 04/26/2011
• Est. Priority Date: 10/10/2003
• Status: Active Grant

• Current Assignee: Cellular Bioengineering, Inc.
• Original Assignee: Cellular Bioengineering, Inc.
• Inventors: Lui, Ge Ming
• Primary Examiner(s): Ford; Allison M
• Application Number:
  US10/575,247
• Publication Number:
  US 20070274961A1
• Time in Patent Office: 2,391 Days
• Field of Search: None
• US Class Current: 427/249.1
• CPC Class Codes: A61L 27/303 Carbon
• CPC Class Codes: A61L 27/3633 Extracellular matrix [ECM]
• CPC Class Codes: A61L 27/383 Nerve cells, e.g. dendritic cells, Schwann cells
• CPC Class Codes: A61L 27/3878 Nerve tissue, brain, spinal cord, nerves, dura mater
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 2533/10 Mineral substrates
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062

Abstract

This present invention describes a method of coating a polymer surface with diamond-like carbon (DLC) to render it useful as a carrier for cells derived from neural crest origin, preferable neuronal cells that form dendrites. The biopolymer to be coated with the DLC will include biodegradable polymers and other implantable biopolymers to act as a carrier system for cell transplantation into the various parts of the body, including the brain, the eye, the central and peripheral nervous system, the lung, the liver, the spleen, the kidney, and the bone and cartilage. The biopolymer can be in sheet form or microparticle form, and can be imbedded with, or incorporated into during its synthesis, attachment or growth promoting reagents to enhance and support neuraonal call attachment and growth. This coating method can also augment other coating agents such as extracellular matrix (ECM) secreted by cultured bovine corneal endothelial cells, as well as adhesive molecules such as fibronectin, laminin, and RGDS. The coating step can be a sequential process where the DLC layer will be added on top of an ECM coated surface or an attachment factor coated surface.

Claims

1. A cell culture substrate, comprising a biodegradable biopolymer coated with hydrogen free diamond-like carbon, wherein cells can attach to and grow on the surface of the hydrogen free diamond-like carbon.

2. The cell culture substrate of claim 1, wherein the biopolymer is in a sheet form.

3. The cell culture substrate of claim 1, wherein the biopolymer is in micro particle form.

4. The cell culture substrate of claim 1, wherein the biopolymer has embedded or incorporated into it during its synthesis one or more attachment reagents selected from the group consisting of:

laminin, fibronectin, RGDS (SEQ ID NO;

     1), basic fibroblast growth factor (bFGF) conjugated with polycarbophyll, epidermal growth factor (EGF) conjugated with polycarbophyll, and heparin sulfate.

5. A method of growing neurons in culture comprising:

providing the cell culture substrate of claim 1;

seeding neurons onto the hydrogen free diamond-like carbon; and

culturing the neurons.

6. The method of claim 5, wherein the biopolymer is in sheet form.

7. The method of claim 5, wherein the biopolymer is in micro particle form.

8. A method of growing neurons in culture comprising:

providing the cell culture substrate of claim 4;

seeding neurons onto the hydrogen free diamond-like carbon; and

culturing the neurons.

9. A three dimensional cell culture substrate comprising a semi-solid biopolymer coated with Diamond-Like Carbon, wherein the biopolymer is comprised of chitosan or sodium alginate, and wherein neural cells can attach to and grow on the surface of the Diamond-Like Carbon.

10. The cell culture substrate of claim 9, wherein the biopolymer has embedded or incorporated into it during its synthesis one or more attachment reagents selected from the group consisting of laminin, fibronectin, RGDS (SEQ ID NO:

1), basic fibroblast growth factor (bFGF) conjugated with polycarbophyll, epidermal growth factor (EGF) conjugated with polycarbophyll, heparin sulfate, and nerve growth factor (NGF), in an amount sufficient to allow neural or nerve cells transplanted onto the growth medium at low density to proliferate and send out neural processes.

11. The cell culture substrate of claim 10, wherein said biopolymer is shaped into beads, sheets or micro-particles.

12. A method of transplanting neurons to a recipient comprising:

providing the three dimensional cell culture substrate of claim 10;

seeding neurons of interest onto the diamond-like carbon;

allowing the neurons to grow to sufficient density; and

implanting the neurons on the cell culture substrate into said recipient.

13. A three dimensional cell culture substrate comprising a semi-solid biopolymer coated with bovine corneal epithelial cell-extracellular matrix (BCE-ECM) and the BCE-ECM is further coated with Diamond-Like Carbon, wherein neural cells can attach to and grow on the surface of the Diamond-Like Carbon.

14. The cell culture substrate of claim 13, wherein the biopolymer is comprised of chitosan or sodium alginate.

15. The cell culture substrate of claim 13, wherein the biopolymer has embedded or incorporated into it during its synthesis one or more attachment reagents selected from the group consisting of:

laminin, fibronectin, RGDS (SEQ ID NO;

     1), basic fibroblast growth factor (bFGF) conjugated with polycarbophyll, epidermal growth factor (EGF) conjugated with polycarbophyll, heparin sulfate, and nerve growth factor (NGF), in an amount sufficient to allow neural or nerve cells transplanted onto the substrate at low density to proliferate and send out neural processes.

16. The cell culture substrate of claim 15, wherein said biopolymer is shaped into beads, sheets or micro-particles.

17. A laboratory apparatus having a coating suitable for inducing the growth and attachment of cells, the apparatus having an inside and outside surface, wherein the inside surface is the surface in contact with cells and cellular media and the inside surface of said apparatus is coated with a film of Diamond-like-Carbon layered over a biopolymer coating.

18. The apparatus of claim 17, wherein the apparatus is selected from the group consisting of cell culture dishes, petri dishes, tissue culture flasks, plates, bottles, slides, filter chambers, slide chambers, roller bottles, harvesters and tubing.

19. A laboratory apparatus having a coating suitable for inducing the growth and attachment of cells, the apparatus having an inside surface and an outside surface, wherein the inside surface is the surface in contact with cells and cellular media and the inside surface of said apparatus is coated with a film of Diamond-like-Carbon, the Diamond-like-Carbon being layered over a biopolymer coating and at least one other coating.

20. The apparatus of claim 19, wherein the at least one other coating is an extracellular matrix.

21. The apparatus of claim 20, wherein the coating is BCE-ECM.

22. A method of making a laboratory apparatus suitable for inducing the growth and attachment of cells, the method comprising:

a) obtaining a laboratory apparatus having an inside surface and an outside surface, wherein the inside surface is to be in contact with cells and cellular media;

b) applying to the inside surface of the apparatus a biopolymer coating;

then

c) applying a film of Diamond-like-Carbon over the biopolymer coating.

23. The method of claim 22, further comprising between steps b) and c), step b′

) applying at least one other coating over the biopolymer coating.