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 method of preparing a stable, ready-to-use cell culturing system comprising:
applying an extracellular matrix coating solution comprising laminin and at least one other extracellular matrix component to a surface of a substrate, 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 surface;
removing the incubated coating solution from the substrate;
washing the substrate after the removing step; and
drying the washed substrate in a drying oven at a relative humidity of 40-60%.
2. The method of claim 1 , further comprising sterilizing the dried substrate.
3. The method of claim 2 , wherein the sterilizing comprises UV sterilization.
4. The method of claim 2 , further comprising packaging the sterilized dried substrate in moisture-resistant packaging.
5. The method of claim 4 , further comprising storing the packaged dried substrate at −20° C. until use.
6. The method of claim 1 , further comprising blocking the surface of the substrate after the removing step and prior to or during the washing step with a blocking composition including at least one blocking agent and an aqueous solvent.
7. The method of claim 1 , wherein the blocking agent is selected from the group consisting of gelatin, sucrose and trehalose.
8. The method of claim 1 , wherein the washing comprises washing the substrate with the blocking composition.
9. The method of claim 6 , wherein the blocking composition comprises sucrose.
10. The method of claim 9 , further comprising sterilizing the dried substrate and storing the sterilized dried substrate at room temperature for at least 2 weeks.
11. The method of claim 1 , wherein the washing comprises washing the substrate with distilled water or a buffer.
12. The method of claim 1 , wherein the washed substrate is dried in a drying oven at room temperature.
13. The method of claim 1 , wherein the washed substrate is dried in a drying oven at a temperature in the range of 22° C. to 30° C.
14. The method of claim 1 , wherein at least one other extracellular matrix component is entactin.
15. The method of claim 14 , wherein the ratio of laminin/entactin is about 5 to about 9.
16. The method of claim 1 , wherein at least one other extracellular matrix component is collagen IV.
17. The method of claim 16 , wherein the ratio of laminin/collagen IV is about 1.5 to about 2.5.
18. The method of claim 1 , wherein at least one other extracellular matrix component comprises entactin and collagen IV.
19. The method of claim 1 , wherein the total protein concentration in the coating solution is about 30 μg/ml to about 300 μg/ml.
20. The method of claim 1 , wherein the total protein concentration in the coating solution is about 30 μg/ml to about 200 μg/ml.