Extracellular matrix coated surface for culturing cells
View Patent ↗A cell culture product is provided for propagating embryonic stem cells, and maintaining their self-renewal and pluripotency characteristics for extended periods of time in culture. The cell culturing product includes a substrate; and a coating thereon deposited from a coating solution. The coating solution includes a mixture of extracellular matrix proteins and an aqueous solvent, wherein the total protein concentration in the coating solution is about 10 μg/ml to about 1 mg/ml.
1. A cell culture product comprising:
a substrate; and
a coating thereon;
wherein the coating is produced by a method comprising:
applying a coating solution to the substrate, wherein the coating solution comprises a mixture of extracellular matrix proteins including laminin and at least one other extracellular matrix component and an aqueous solvent, wherein the total protein concentration in the coating solution is about 10 μg/ml to about 1 mg/ml;
incubating the coating solution on the substrate; and then
washing the substrate to remove unbound extracellular matrix proteins.
2. The cell culture product of claim 1 , wherein the coating is present on the substrate in a dried state.
3. The cell culture product of claim 1 , wherein the total protein concentration in the coating solution is about 30 μg/ml to about 300 μg/ml.
4. The cell culture product of claim 1 , wherein the total protein concentration in the coating solution is about 30 μg/ml to about 200 μg/ml.
5. The cell culture product of claim 1 , wherein the at least one other extracellular matrix component is entactin.
6. The cell culture product of claim 5 , wherein the coating further comprises a component selected from the group consisting of type IV collagen, heparan sulfate proteoglycans (HSPG), growth factors and combinations thereof.
7. The cell culture product of claim 1 , wherein the aqueous solvent is a buffer or cell culture media.
8. The cell culture product of claim 1 , wherein the coating on the substrate further comprises a blocking composition comprising at least one blocking agent and an aqueous solvent.
9. The cell culture product of claim 8 , wherein the blocking agent is selected from the group consisting of gelatin, sucrose and trehalose.
10. The cell culture product of claim 8 , wherein the blocking agent is present in the blocking composition in an amount of about 0.1 to about 5.0% w/v.
11. A cell culture product comprising:
a substrate; and
an extracellular matrix coating thereon, wherein the coating is provided to at least one surface of the substrate in a solution at a minimal concentration sufficient to saturate the substrate surface with extracellular matrix proteins; and then the substrate is washed to remove unbound extracellular matrix proteins.
12. The cell culture product of claim 11 , wherein the total amount of extracellular matrix protein adsorbed by or bound the substrate surface is from about 200 ng/cm 2 to about 650 ng/cm 2 .
13. An apparatus for propagating pluripotent embryonic stem cells comprising:
a) a container suitable for culturing cells; and
b) an extracellular matrix in a fixed concentration on at least one surface of the container;
wherein said extracellular matrix comprises a mixture of laminin and at least one other extracellular matrix component, and
wherein the extracellular matrix is provided to the at least one surface by coating at least one surface of the container with the extracellular matrix, washing the surface coated with extracellular matrix to remove unbound extracellular matrix proteins, and then drying the container such that the extracellular matrix is in a dried state on at least one surface of said container.
14. The apparatus of claim 13 , wherein the extracellular matrix comprises a mixture of laminin and entactin.