Extracellular matrix compositions produced under hypoxic culture conditions
View Patent ↗The present invention is directed to a method of producing compositions including embryonic proteins. The method includes culturing cells under hypoxic conditions on a biocompatible three-dimensional surface in vitro. The culturing method produces both soluble and non-soluble fractions, which may be used separately or in combination to obtain physiologically acceptable compositions useful in a variety of medical and therapeutic applications.
1. A method of making a composition comprising one or more embryonic proteins comprising:
culturing cells under hypoxic conditions of about 1-5% oxygen for at least 2 weeks on a surface of beads in a suitable growth medium, thereby producing a soluble and a non-soluble composition comprising one or more embryonic proteins.
2. The method of claim 1 , wherein the growth medium comprises serum.
3. The method of claim 1 , wherein the growth medium is serum-free.
4. The method of claim 1 , wherein collagen species are upregulated as compared with media produced in oxygen conditions of about 15-20% oxygen.
5. The method of claim 4 , wherein the collagen is selected from type V alpha 1; IX alpha 1; IX alpha 2; VI alpha 2; VIII alpha 1; IV, alpha 5; VII alpha 1; XVIII alpha 1; or XII alpha 1.
6. The method of claim 1 , wherein Wnt species are upregulated as compared with media-produced in oxygen conditions of about 15-20% oxygen.
7. The method of claim 6 , wherein the Wnt species are wnt 7a and wnt 11.
8. The method of claim 1 , wherein laminin species are upregulated as compared with media produced in oxygen conditions of about 15-20% oxygen.
9. The method of claim 8 , wherein the laminin species is laminin 8.
10. The method of claim 1 , wherein the cell-free supernatant is dialyzed, lyophilized and reconstituted in a buffer.
11. The method of claim 1 , wherein the cell-free supernatant is dialyzed, desiccated and reconstituted in a buffer.
12. The method of claim 1 , wherein the cells are fibroblasts.
13. The method of claim 12 , wherein the fibroblasts are neonatal fibroblasts.
14. The method of claim 1 , wherein the surface is three-dimensional.
15. The method of claim 1 , wherein the cells are species specific.
16. A hypoxic tissue culture system including a composition as in claim 1 .
17. The tissue culture system of claim 16 , wherein the system is used to support the growth of stem cells.
18. The tissue culture system of claim 17 , wherein the stem cells are embryonic stem cells, mesenchymal stem cells or neuronal stem cells.
19. A method of producing a Wnt protein and a vascular endothelial growth factor (VEGF) comprising:
culturing cells under hypoxic conditions of about 1-5% oxygen for at least 2 weeks on a surface of beads in a suitable growth medium, thereby producing the Wnt protein and the VEGF.
20. The method of claim 19 , wherein the growth medium is serum-free.
21. The method of claim 19 , wherein Wnt species are upregulated as compared with media produced in oxygen conditions of about 15-20% oxygen.
22. The method of claim 21 , wherein the Wnt species are wnt 7a and wnt 11.
23. The method of claim 19 , wherein VEGF species are upregulated as compared with media produced in oxygen conditions of about 15-20% oxygen.
24. The method of claim 23 , wherein the VEGF species is VEGF-A or isoform thereof.
25. The method of claim 19 , wherein the cells are fibroblasts.
26. The method of claim 19 , wherein the surface is three-dimensional.