Conditioned media and methods of making a conditioned media
View Patent ↗A stable and scalable process is provided to manufacture reduced serum umbilical cord tissue-derived (UTC)-conditioned media. The method includes the culture of a UTC under reduced serum conditions. Subsequently, the UTC is washed and grown in serum-free basal media. After approximately 24 hours, the conditioned media is collected, filtered and concentrated by use of an approximately 5 kDa or similar cut-off membrane.
1. A method of preparing a conditioned media, the method comprising:
providing an isolated human umbilical tissue-derived cell (UTC) in a serum-containing microcarrier bead culture media in a spinner flask;
reducing serum content of the culture media in one or more incremental steps;
transferring the UTC from the reduced serum content culture media to a serum-free basal media when the serum content reaches a predetermined level;
growing the UTC in the serum-free basal media for no more than 24 hours; and
isolating the UTC from the serum-free basal media leaving a conditioned media, wherein serum proteins are undetectable in the conditioned media using SDS-PAGE in a 4-20% tris-glycine gel.
2. The method of claim 1 , further comprising filtering the conditioned media.
3. The method of claim 1 , further comprising concentrating the conditioned media.
4. The method of claim 1 , wherein the human umbilical tissue-derived cell is positive for CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, HLA-A, B, C and negative for CD31, CD34, CD45, CD117, CD141 and HLA-DR, DP, DQ.
5. The method of claim 1 , wherein the microcarrier in the microcarrier bead culture media is polystyrene modified with cationic amine.
6. A method of preparing a conditioned media, the method comprising:
seeding a human umbilical cord tissue-derived cell (UTC) in a serum-containing microcarrier bead culture media in a spinner flask;
reducing serum content of the culture media in one or more incremental steps;
transferring the UTC from the reduced serum-content culture media to a serum-free basal media;
growing the UTC in the serum-free basal media for no more than 24 hours; and
isolating the UTC from the serum-free basal media leaving a conditioned media, wherein serum proteins are undetectable in the conditioned media using SDS-PAGE in a 4-20% tris-glycine gel;
wherein the protein content in the conditioned media is at least about two times more than from a static flask.
7. The method of claim 6 , wherein the conditioned media is filtered.
8. The method of claim 7 , wherein the conditioned media is filtered using an approximately 0.22 micron filter.
9. The method of claim 6 , wherein the conditioned media is concentrated.
10. The method of claim 9 , wherein the conditioned media is concentrated with a cut-off membrane.
11. The method of claim 10 , wherein the cut-off membrane is an approximately 5 kDa cut-off membrane.
12. The method of claim 6 , wherein the serum content is reduced in increments of about 5% to about 60%.
13. The method of claim 6 , wherein the serum content is reduced in increments of about 50%.
14. The method of claim 6 , wherein the UTC grows for about 1 to 3 passages in each increment.
15. The method of claim 14 , wherein the UTC grows for about 2 passages in each increment.
16. The method of claim 6 , further comprising preliminarily culturing the UTC in a standard culture media and isolating the UTC from the standard culture media before seeding.
17. The method of claim 6 , wherein the human umbilical tissue-derived cell is positive for CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, HLA-A, B, C and negative for CD31, CD34, CD45, CD117, CD141 and HLA-DR, DP, DQ.
18. The method of claim 6 , wherein the microcarrier in the microcarrier bead culture media is polystyrene modified with cationic amine.
19. A method of preparing a conditioned media, the method comprising:
seeding a human umbilical cord tissue-derived cell (UTC) in a serum-containing microcarrier bead culture media in a spinner flask;
reducing serum content of the culture media in one or more incremental steps;
transferring the UTC from the reduced serum-content culture media to a serum-free basal media;
growing the UTC in the serum-free basal media for up to about 24 hours; and
isolating the UTC from the serum-free basal media leaving a conditioned media, wherein serum proteins are undetectable in the conditioned media using Western blot analysis, wherein the Western Blot analysis comprises protein separation in a 4-20% tris-glycine gel and a mouse monoclonal anti-BSA antibody.
20. The method of claim 19 , wherein the conditioned media is filtered.
21. The method of claim 20 , wherein the conditioned media is filtered using an approximately 0.22 micron filter.
22. The method of claim 19 , wherein the conditioned media is concentrated.
23. The method of claim 22 , wherein the conditioned media is concentrated with a cut-off membrane.
24. The method of claim 23 , wherein the cut-off membrane is an approximately 5 kDa cut-off membrane.
25. The method of claim 19 , wherein the serum content is reduced in increments of about 5% to about 60%.
26. The method of claim 19 , wherein the serum content is reduced in increments of about 50%.
27. The method of claim 19 , wherein the UTC grows for about 1 to 3 passages in each increment.
28. The method of claim 27 , wherein the UTC grows for about 2 passages in each increment.
29. The method of claim 19 , further comprising preliminarily culturing the UTC in a standard culture media and isolating the UTC from the standard culture media before seeding.
30. The method of claim 19 , wherein the microcarrier in the microcarrier bead culture media is polystyrene modified with cationic amine.
31. A method of preparing a conditioned media, the method comprising:
providing an isolated human umbilical tissue-derived cell (UTC) in a serum-containing microcarrier bead culture media in a spinner flask;
reducing serum content of the culture media in one or more incremental steps;
transferring the UTC from the reduced serum content culture media to a serum-free basal media when the serum content reaches a predetermined level;
growing the UTC in the serum-free basal media for no more than 24 hours; and
isolating the UTC from the serum-free basal media leaving a conditioned media, wherein serum proteins are undetectable in the conditioned media using Western blot analysis, wherein the Western Blot analysis comprises protein separation in a 4-20% tris-glycine gel and a mouse monoclonal anti-BSA antibody.
32. The method of claim 31 , further comprising filtering the conditioned media.
33. The method of claim 31 , further comprising concentrating the conditioned media.
34. The method of claim 31 , wherein the microcarrier in the microcarrier bead culture media is polystyrene modified with cationic amine.