DEFINED CELL CULTURING SURFACES AND METHODS OF USE

• US 20100021998A1
• Filed: 07/24/2009
• Published: 01/28/2010
• Est. Priority Date: 07/25/2008
• Status: Active Grant

• Current Assignee: Corning Incorporated
• Original Assignee: Becton, Dickinson & Co
• Inventors: Sanyal, Suparna, Abraham, Elizabeth, Saxena, Deepa, Qian, Susan Xiuqi
• Granted Patent: US 8,288,513 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2533/30 Synthetic polymers
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0602 Vertebrate cells
• 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
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: C12N 5/0662 Stem cells
• CPC Class Codes: C12N 5/0663 Bone marrow mesenchymal stem cells (BM-MSC)
• CPC Class Codes: C12N 5/0664 Dental pulp stem cells, Dental follicle stem cells
• CPC Class Codes: C12N 5/0665 Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
• CPC Class Codes: C12N 5/0666 Mesenchymal stem cells from hair follicles
• CPC Class Codes: C12N 5/0667 Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
• CPC Class Codes: C12N 5/0668 Mesenchymal stem cells from other natural sources

Abstract

In one aspect, there is provided a cell culturing substrate including: a cell culture surface having a film attached thereto, wherein the film includes one or more plasma polymerized monomers; and a coating on the film-coated surface, the coating deposited from a coating solution comprising one or more extracellular matrix proteins and an aqueous solvent, where the total extracellular matrix protein concentration in the coating solution is about 1 ng/mL to about 1 mg/mL.

Claims

1. A cell culturing substrate comprising:

a cell culture surface having a film attached thereto, wherein said film comprises one or more plasma polymerized monomers; and

a coating on said film-coated surface, said coating deposited from a coating solution comprising one or more extracellular matrix proteins and an aqueous solvent, wherein the total extracellular matrix protein concentration in the coating solution is about 1 ng/mL to about 1 mg/mL.

2. The cell culturing substrate of claim 1, wherein said one or more monomers is selected from the group consisting of acrylic acid, methacrylic acid, acetic acid, vinylacetic acid and combinations thereof.

3. The cell culturing substrate of claim 1, wherein said one or more monomers is selected from the group consisting of allylamine, methylamine, propylamine, heptylamine and diaminopropane.

4. The cell culturing substrate of claim 1, wherein said monomer is selected from the group consisting of alkanes, alkenes, dienes, styrenes and combinations thereof.

5. The cell culturing substrate of claim 1, wherein said one or more monomers is selected from the group consisting of amines, hydrocarbons and combinations thereof, wherein the ratio of amine to hydrocarbon is between about 100:

0 and about 20;

80.

6. The cell culturing substrate of claim 1, wherein the total protein concentration in the coating composition is about 1 μ

g/mL to about 300 μ

g/mL.

7. The cell culturing substrate of claim 1, wherein the total protein concentration in the coating composition is about 5 μ

g/mL to about 200 μ

g/mL.

8. The cell culturing substrate of claim 1, wherein the coating composition comprises extracellular matrix proteins selected from the group consisting of natural, recombinant, synthetic extracellular matrix proteins and combinations thereof.

9. The cell culturing substrate of claim 1, wherein the coating composition comprises a whole extra cellular matrix protein or a fragment of the extracellular matrix protein

10. The cell culturing substrate of claim 1, wherein the coating composition further comprises a component selected from the group consisting of entactin, heparan sulfate proteoglycans (HSPG), growth factors and combinations thereof.

11. The cell culturing substrate of claim 1, wherein the aqueous solvent is selected from the group consisting of a buffer, a cell culture media and combinations thereof.

15. A method of culturing stem cells comprising:

providing a cell culturing substrate according to claim 1;

applying a suspension of stem cells to said cell culturing substrate;

incubating said suspension of stem cells on said cell culturing substrate at 5% CO₂ in humidified air at 37°

C.; and

permitting said stem cells to attach to said cell culturing substrate of the cells, wherein the attached cells remain in a predominately undifferentiated state.

16. The method of claim 15, further comprising introducing a cell culture medium to said cell culturing substrate prior to application of said suspension of eukaryotic stem cells.

17. The method of claim 15, wherein said cell culture medium comprises growth media.

18. The cell culturing substrate of claim 15, wherein said stem cells are eukaryotic stem cells.

19. The cell culturing substrate of claim 15, wherein said stem cells are embryonic stem cells.

20. The cell culturing substrate of claim 15, wherein said stem cells are human embryonic stem cells.

21. The cell culturing substrate of claim 15, wherein said stem cells are human mesenchymal stem cells.

22. The cell culturing substrate of claim 15, wherein said stem cells are human neuronal stem cells.

12. A method of preparing a cell culturing substrate comprising:

providing a cell culture surface;

plasma polymerizing a film onto said surface to form a film-coated surface, wherein said plasma polymerizing utilizes one or more monomers; and

introducing a coating solution to said film-coated surface to form a cell culture substrate,

said coating solution comprising one or more extracellular matrix proteins and an aqueous solution, wherein the total extracellular matrix protein concentration in the coating solution is about 1 ng/mL to about 1 mg/mL.

13. The method of claim 12, further comprising:

incubating said cell culture substrate, wherein said one or more extracellular matrix proteins is immobilized on said film-coated surface.

14. The method of claim 12, further comprising:

freezing said cell culture substrate for a period of time, then incubating said cell culture substrate, wherein said one or more extracellular matrix proteins is immobilized on said film-coated surface.