Repair and/or regeneration of cells with a composition produced by culturing fibroblast cells under hypoxic conditions
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 tissue regeneration patch comprising a composition made by culturing fibroblast cells under hypoxic conditions for at least 2 weeks on a microcarrier bead or three-dimensional surface in a suitable growth medium containing basic FGF (bFGF), under 1-5% oxygen thereby producing multipotent stem cells, wherein the multipotent stem cells produce and secrete a soluble composition, collecting the composition after said 2 weeks and preparing a tissue regeneration patch comprising the soluble composition.
2. A tissue regeneration patch comprising a composition made by culturing fibroblast cells under hypoxic conditions for at least 2 weeks on a microcarrier bead or three-dimensional surface in a suitable growth medium containing basic FGF (bFGF), under 1-5% oxygen thereby producing multipotent stem cells, wherein the multipotent stem cells produce and secrete into the medium and onto the bead, cell, culture or three-dimensional surfaces an insoluble composition, collecting the composition after said 2 weeks and preparing the tissue regeneration patch comprising the insoluble composition.
3. A tissue regeneration patch comprising a composition made by culturing fibroblast cells under hypoxic conditions for at least 2 weeks on a microcarrier bead or three-dimensional surface in a suitable growth medium containing basic FGF (bFGF), under 1-5% oxygen thereby producing multipotent stem cells, wherein the multipotent stem cells produce and secrete into the medium a soluble composition and onto the bead, cell, culture or three-dimensional surface an insoluble composition, collecting a composition after said 2 weeks and preparing the tissue regeneration patch comprising the soluble and insoluble composition.
4. A tissue regeneration patch of any of claim 1 , 2 , or 3 , wherein the composition comprises extracellular matrix proteins.
5. A method of repair and/or regeneration of cells comprising contacting cells to be repaired or regenerated with a composition made by culturing fibroblast cells under hypoxic conditions for at least 2 weeks on a microcarrier bead or three-dimensional surface in a suitable growth medium containing basic FGF (bFGF), under 1-5% oxygen thereby producing multipotent stem cells, wherein the multipotent stem cells produce and secrete a soluble composition, collecting the composition after said 2 weeks producing a soluble composition and administering the composition to the cells to be repaired or regenerated, wherein repair or regeneration is effected.
6. A method of repair and/or regeneration of cells comprising contacting cells to be repaired or regenerated with a composition made by culturing fibroblast cells under hypoxic conditions for at least 2 weeks on a microcarrier bead or three-dimensional surface in a suitable growth medium containing basic FGF (bFGF), under 1-5% oxygen thereby producing multipotent stem cells, wherein the multipotent stem cells produce and secrete into the medium and onto the bead, cell, culture or three-dimensional surfaces an insoluble composition, collecting the composition after said 2 weeks and administering the composition to the cells to be repaired or regenerated, wherein repair or regeneration is effected.
7. A method of repair and/or regeneration of cells comprising contacting cells to be repaired or regenerated with a composition made by culturing fibroblast cells under hypoxic conditions for at least 2 weeks on a microcarrier bead or three-dimensional surface in a suitable growth medium containing basic FGF (bFGF), under 1-5% oxygen thereby producing multipotent stem cells, wherein the multipotent stem cells produce and secrete into the medium a soluble composition and onto the bead, cell, culture or three-dimensional surface an insoluble composition, collecting a composition after said 2 weeks and administering the composition to the cells to be repaired or regenerated, wherein repair or regeneration is effected.
8. A method of repair and/or regeneration of cells comprising contacting cells to be repaired or regenerated with a composition made by culturing fibroblast cells under hypoxic conditions for at least 2 weeks on a microcarrier bead or three-dimensional surface in a suitable growth medium containing basic FGF (bFGF), under 1-5% oxygen thereby producing multipotent stem cells, wherein the multipotent stem cells produce and secrete into the medium and onto surfaces, a composition, collecting the composition after said 2 weeks and administering the composition to the cells to be repaired or regenerated, wherein repair or regeneration is effected.
9. The method of any of claim 5 , 6 , 7 , or 8 , wherein the cells to which the composition is administered are osteochondral cells.
10. The method of any of claim 5 , 6 , 7 or 8 , wherein the composition comprises extracellular matrix proteins.
11. The method of any of claim 5 , 6 , 7 or 8 , wherein the fibroblasts are neonatal fibroblasts.
12. The method of any of claim 5 , 6 , 7 or 8 , wherein the three-dimensional surface comprises mesh.
13. The method of any of claim 5 , 6 , 7 or 8 , wherein the growth medium comprises serum.
14. The method of any of claim 5 , 6 , 7 or 8 , wherein the growth medium is serum-free.
15. The method of any of claim 5 , 6 , 7 or 8 , wherein collagen species are upregulated as compared with media produced in oxygen conditions of about 15-20% oxygen.
16. The method of claim 15 , 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.
17. The method of any of claim 5 , 6 , 7 or 8 , wherein Wnt species are upregulated as compared with media produced in oxygen conditions of about 15-20% oxygen.
18. The method of claim 17 , wherein the Wnt species are Wnt7a and Wnt11.
19. The method of any of claim 5 , 6 , 7 or 8 , wherein laminin species are upregulated as compared with media produced in oxygen conditions of about 15-20% oxygen.
20. The method of claim 19 , wherein the laminin species is laminin 8.