Methods, surface modified plates and compositions for cell attachment, cultivation and detachment

• US 10,066,203 B2
• Filed: 02/19/2009
• Issued: 09/04/2018
• Est. Priority Date: 02/21/2008
• Status: Active Grant

• Current Assignee: Nunc A/S (Fisher Bioimage APS)
• Original Assignee: Nunc A/S (Fisher Bioimage APS), Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Fryer, Benjamin, Nelson, Shelley, Nielsen, Villy, Marwood, Tina Kristensen, Brevig, Thomas
• Primary Examiner(s): Singh, Anoop K
• Assistant Examiner(s): Sgagias, Magdalene K
• Application Number:
  US12/388,930
• Publication Number:
  US 20090215177A1
• Time in Patent Office: 3,484 Days
• 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 human pluripotent stem cells to a surface lacking a feeder cell layer and lacking an adlayer, comprising the steps of:

a. obtaining a suspension of human pluripotent stem cells;

b. treating the suspension of cells of step (a) 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;

c. adding the suspension of cells of step (b) to a surface having a static sessile contact angle of at least about 14.3 degrees, and wherein the surface comprises polystyrene and contains from 1.6% N to 1.8% N, a sum of O and N of greater than or equal to 29.6%;

d. allowing the cells of step (c) to attach to the surface;

wherein attachment is enhanced compared to cells not treated with the at least one compound, and wherein the pluripotency of the attached cells is maintained; and

e. culturing the attached cells without differentiating the cells.

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

3. The method of claim 2, wherein the at least one compound is removed after the cells attach to the surface.

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

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

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

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

9. The method of claim 1, wherein the method comprises treating the suspension of cells with a compound capable of inhibiting Rho kinase activity.

10. The method of claim 1, wherein the method comprises treating the suspension of cells with a compound capable of inhibiting Rho activity.

17. The method of claim 1, wherein the surface is corona plasma treated.

20. The method of claim 1, wherein the surface has a static sessile contact angle from about 14.3 degrees to about 18.8 degrees.

8. A method to enhance the attachment of human pluripotent stem cells to a surface lacking a feeder cell layer and lacking an adlayer, and comprising the steps of:

a. obtaining a suspension of human pluripotent stem cells;

b. treating the suspension of cells of step (a) 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;

c. adding the suspension of cells of step (b) to a surface having a static sessile contact angle of at least about 14.3 degrees, and wherein the surface comprises polystyrene and contains from 1.6% N to 1.8% N, a sum of O and N of greater than or equal to 29.6%;

d. allowing the cells of step (c) to attach to the surface; and

wherein attachment is enhanced compared to cells not treated with the at least one compound;

e. maintaining the cells of step (d) in culture after the cells attach to the surface without differentiating the cells, and wherein the pluripotency of the attached cells is maintained; and

f. removing the at least one compound from the culture after the cells attach to the surface without differentiating the cells and cells remain attached.

11. The method of claim 8, wherein the suspension of cells is a suspension of clusters of cells.

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

13. The method of claim 8, wherein the surface is part of a vessel or matrix.

14. The method of claim 8, wherein the method comprises treating the suspension of cells with a compound capable of inhibiting Rho kinase activity.

15. The method of claim 8, wherein the method comprises treating the suspension of cells with a compound capable of inhibiting Rho activity.

18. The method of claim 8, wherein the surface is corona plasma treated.

21. The method of claim 8, wherein the surface has a static sessile contact angle from about 14.3 degrees to about 18.8 degrees.

16. A method to enhance the attachment of human pluripotent stem cells to a surface lacking a feeder cell layer and comprising:

a. adding a suspension of human pluripotent cells treated with a compound capable of inhibiting Rho kinase activity or compound capable of inhibiting Rho activity to a surface having a static sessile contact angle of at least about 14.3 degrees, wherein the surface comprises polystyrene and contains from 1.6% N to 1.8% N, a sum of O and N of greater than or equal to 29.6%, and wherein attachment is enhanced compared to cells not treated with the at least one compound; and

allowing the cells to attach; and

b. culturing the attached cells of step (a) without differentiating the cells.

19. The method of claim 16, wherein the surface is corona plasma treated.

22. The method of claim 16, wherein the surface has a static sessile contact angle from about 14.3 degrees to about 18.8 degrees.