METHODS, SURFACE MODIFIED PLATES AND COMPOSITIONS FOR CELL ATTACHMENT, CULTIVATION AND DETACHMENT

• US 20090215177A1
• Filed: 02/19/2009
• Published: 08/27/2009
• Est. Priority Date: 02/21/2008
• Status: Active Grant

• Current Assignee: Nunc A/S (Fisher Bioimage APS)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Nielsen, Villy, Marwood, Tina Kristensen, Brevig, Thomas, Fryer, Benjamin, Nelson, Shelley
• Granted Patent: US 10,066,203 B2
• Time in Patent Office: Days
• US Class Current: 435/395
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 2533/00 Supports or coatings for cell culture, characterised by material
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

The present invention relates to the field of mammalian cell culture, and provides methods and compositions for cell attachment to, cultivation on and detachment from a solid substrate surface containing from at least about 0.5% N, a sum of O and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacking an adlayer. In one embodiment of the present invention, the cells are treated with a compound capable of inhibiting Rho kinase activity. In another embodiment, the cells are treated with a compound capable of inhibiting Rho activity.

Claims

1. A method to enhance the attachment of cells to a surface containing from at least about 0.5% N, a sum of O and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacking an adlayer, comprising the steps of:

a. Obtaining a suspension of the cells,

b. Treating the suspension of cells with at least one compound selected from the group consisting of;

a compound capable of inhibiting Rho kinase activity, and a compound capable of inhibiting Rho activity, and

c. Adding the suspension of cells to the surface and allowing the cells to attach.

2. The method of claim 1, wherein the sum of O and N is greater than or equal to 19.5%.

3. The method of claim 1, wherein the surface has an adlayer.

4. The method of claim 1, wherein the cells are maintained in culture after the cells attach to the surface.

5. The method of claim 4, wherein at least one compound is removed after the cells attach to the surface.

6. The method of claim 1, wherein the cells are detached from the surface by removing at least one compound.

7. The method of claim 1, wherein the suspension of cells is a suspension of clusters of cells.

8. The method of claim 1, wherein the suspension of cells is a suspension of single cells.

9. The method of claim 1, wherein the surface is part of a vessel or matrix.

10. A method to enhance the attachment of cells to a surface containing from at least about 0.9% N, a sum of O and N of greater than or equal to 22.3% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacking an adlayer, comprising the steps of:

a. Obtaining a suspension of the cells, and

b. Adding the suspension of cells to the surface and allowing the cells to attach.

11. The method of claim 10, wherein the surface has an adlayer.

12. The method of claim 10, wherein the cells are maintained in culture after the cells attach to the surface.

13. The method of claim 10, wherein the suspension of cells is a suspension of clusters of cells.

14. The method of claim 10, wherein the suspension of cells is a suspension of single cells.

15. The method of claim 10, wherein the surface is part of a vessel or matrix.

16. A surface that is part of a vessel or matrix intended for use in cell culture or analysis, containing from at least about 0.9% N, a sum of O and N of greater than or equal to 22.3% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacking an adlayer, wherein the surface allows the attachment and cultivation of cells.

17. The method of claim 16, wherein the surface has an adlayer.

18. The surface of claim 16, wherein the cells are maintained in culture after the cells attach to the surface.

19. A composition comprising:

a. A surface that is part of a vessel or matrix intended for use in cell culture or analysis, containing from at least about 0.5% N, a sum of O and N of greater than or equal to 17.2% and a contact angle of at least about 13.9 degrees, lacking a feeder cell layer and lacking an adlayer, and

b. At least one compound selected from the group consisting of;

a compound capable of inhibiting Rho kinase activity, and a compound capable of inhibiting Rho activity.

20. The composition of claim 19, wherein the sum of O and N is greater than or equal to 19.5%.

21. The method of claim 19, wherein the surface has an adlayer.