Conditioned media and methods of making a conditioned media

Patent metadata

Field	Value
Publication number	US-10179900-B2
Application number	US-33987208-A
Country	US
Kind code	B2
Filing date	Dec 19, 2008
Priority date	Dec 19, 2008
Publication date	Jan 15, 2019
Grant date	Jan 15, 2019

Abstract

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.

First claim

What is claimed:

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.

Assignees

• Harmon Alexander M
• Ang Abel
• Depuy Synthes Products Inc

Inventors

• Harmon Alexander M
• Ang Abel

Classifications

• A61K35/51

  Umbilical cord; Umbilical cord blood; Umbilical stem cells · CPC title

• C12N5/0668

  Mesenchymal stem cells from other natural sources · CPC title

• C12N2500/95

  Protein-free medium and culture conditions · CPC title

• C12N2500/90

  Serum-free medium, which may still contain naturally-sourced components · CPC title

• C12N5/0605 Primary

  Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly · CPC title