Methods and compositions for cell attachment and cultivation on planar substrates

• US 9,969,973 B2
• Filed: 11/19/2009
• Issued: 05/15/2018
• Est. Priority Date: 11/20/2008
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Chen, Ya Xiong, Fryer, Benjamin
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US12/621,702
• Publication Number:
  US 20100124783A1
• Time in Patent Office: 3,099 Days
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• 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 2533/78 Cellulose
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• 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 is directed to methods for the growth, expansion and differentiation of pluripotent stem cells on planar substrates lacking an adlayer and a feeder cell layer.

Claims

1. A method to attach human pluripotent stem cells to a surface of a porous planar substrate containing from at least 8% N to about 12% N, from at least 12% O to at least 55% O, a contact angle from 18 degrees to 32 degrees, and lacking an adlayer and a feeder cell layer, comprising the steps of:

a) adding Y-27632 to a suspension of pluripotent stem cells;

b) adding the cell suspension of step a) to the porous planar substrate;

c) allowing the cells to attach to the surface of the porous planar substrate; and

d) removing the Y-27632 after the cells attach to the surface of the porous planar substrate,

wherein removal of the Y-27632 does not result in detachment of the cells,

wherein the porous planar substrate comprises mixed cellulose esters, nylon or cellulose acetate, and

wherein the attaching comprises culturing the cells and the porous planar substrate under conditions that allow for growth without differentiation.

2. The method of claim 1, wherein the cells are maintained in culture after they have attached to the surface of the porous planar substrate.

3. The method of claim 1, wherein the cells are further differentiated after they have attached to the surface of the porous planar substrate.

4. The method of claim 1, wherein the method comprises from about 0.1 μ

M to about 100 μ

M of Y-27632.

5. The method of claim 1, wherein at least 95% of the cells maintain expression of markers associated with pluripotency.

6. The method of claim 1, wherein the Y-27632 is required for the attachment of the cell suspension to the porous planar substrate.

13. The method of claim 1, wherein the porous planar substrate comprises mixed cellulose esters.

14. The method of claim 1, wherein the porous planar substrate comprises nylon.

15. The method of claim 1, wherein the porous planar substrate comprises cellulose acetate.

16. The method of claim 1, wherein the Y-27632 enhances attachment of the cell suspension to the porous planar substrate.

17. The method of claim 1, wherein the Y-27632 is required for the attachment of the cell suspension to the porous planar substrate.

7. A method of enhancing the attachment of human pluripotent stem cells to a surface of a porous planar substrate containing from at least 8% N to about 12% N, from at least 12% O to at least 55% O, a contact angle from 18 degrees to 32 degrees, and lacking an adlayer and a feeder cell layer, comprising the steps of:

a) treating a suspension of pluripotent stem cells with Y-27632;

b) contacting the porous planar substrate with the treated suspension of pluripotent human stem cells;

c) allowing the cells to attach to the surface of the porous planar substrate; and

d) removing the Y-27632 after the cells attach to the surface of the porous planar substrate,

wherein at least 95% of the cells maintain expression of markers associated with pluripotency,

wherein the porous planar substrate comprises mixed cellulose esters, nylon or cellulose acetate, and

wherein the Y-27632 enhances attachment of the cell suspension to the porous planar substrate.

8. The method of claim 7, wherein the cells are maintained in culture after they have attached to the surface of the porous planar substrate.

9. The method of claim 7, wherein the cells are further differentiated after they have attached to the surface of the porous planar substrate.

10. The method of claim 7, wherein the Y-27632 is removed after the pluripotent stem cells attach to the surface of the porous planar substrate.

11. The method of claim 7, wherein the method comprises from about 0.1 μ

M to about 100 μ

M of Y-27632.

12. The method of claim 7, wherein removal of the Y-27632 does not result in detachment of the cells.

18. The method of claim 7, wherein the porous planar substrate comprises mixed cellulose esters.

19. The method of claim 7, wherein the porous planar substrate comprises nylon.

20. The method of claim 7, wherein the porous planar substrate comprises cellulose acetate.