CONDITIONED MEDIA AND METHODS OF MAKING A CONDITIONED MEDIA

• US 20100159588A1
• Filed: 12/19/2008
• Published: 06/24/2010
• Est. Priority Date: 12/19/2008
• Status: Active Grant

• Current Assignee: Depuy Synthes Products Incorporated (Johnson & Johnson)
• Original Assignee: Ethicon Incorporated (Johnson & Johnson)
• Inventors: Ang, Abel, Harmon, Alexander M.
• Granted Patent: US 10,179,900 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61K 35/51 Umbilical cord; Umbilical cord blood; Umbilical stem cells
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/95 Protein-free medium and culture conditions
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• CPC Class Codes: C12N 5/0668 Mesenchymal stem cells from other natural sources

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.

Claims

1. A method of preparing a conditioned media, the method comprising:

seeding a mammalian umbilical cord tissue-derived cell (UTC) in a culture media;

reducing serum content of the culture media;

transferring the UTC from the culture media to a serum-free basal media;

growing the UTC in the serum-free basal media; and

isolating the UTC from the serum-free basal media leaving a conditioned media.

2. The method of claim 1, wherein the UTC is grown in the serum-free basal media for up to 24 hours.

3. The method of claim 1, wherein the conditioned media is filtered.

4. The method of claim 3, wherein the conditioned media is filtered using an approximately 22 micron filter.

5. The method of claim 1, wherein the conditioned media is concentrated.

6. The method of claim 5, wherein the conditioned media is concentrated with a cut-off membrane.

7. The method of claim 6, wherein the cut-off membrane is an approximately 5 kDa cut-off membrane.

8. The method of claim 1, wherein the reducing serum content comprises weaning the UTC from the culture media.

9. The method of claim 8, wherein the weaning comprises reducing serum content of the culture media in increments.

10. The method of claim 9, wherein the serum content is reduced in increments of about 5% to about 60%.

11. The method of claim 9, wherein the serum content is reduced in increments of about 50%.

12. The method of claim 9, wherein the UTC grows for about 1 to 3 passages in each increment.

13. The method of claim 12, wherein the UTC grows for about 2 passages in each increment.

14. The method of claim 1, wherein the culture media is a static culture.

15. The method of claim 1, wherein the culture media is a microcarrier bead culture.

16. The method of claim 1, 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 1, wherein the UTC is a human umbilical cord tissue-derived cell.

18. A method of preparing a conditioned media, the method comprising:

providing an isolated mammalian umbilical tissue-derived cell (UTC) in a culture media;

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.

19. The method of claim 18, wherein the transferring comprises weaning the UTC from the culture media.

20. The method of claim 18, wherein the transferring comprises replacing the reduced serum culture media with the serum-free basal media.

21. The method of claim 18, further comprising filtering the conditioned media.

22. The method of claim 18, further comprising concentrating the conditioned media.

23. The method of claim 18, wherein the UTC is a human umbilical cord tissue-derived cell.

24. A conditioned media generated by seeding a human umbilical tissue-derived cell (UTC) in a culture media, wherein serum content of the culture media is reduced prior to transfer of the UTC to a serum-free basal media, and wherein the UTC is then isolated from the serum-free basal media leaving a conditioned media.

25. The conditioned media of claim 24, wherein the UTC is grown in the serum-free basal media for up to 24 hours.

26. The conditioned media of claim 24, wherein the conditioned media is filtered.

27. The conditioned media of claim 26, wherein the conditioned media is filtered using an approximately 22 micron filter.

28. The conditioned media of claim 24, wherein the conditioned media is concentrated.

29. The conditioned media of claim 28, wherein the conditioned media is concentrated with a cut-off membrane.

30. The conditioned media of claim 29, wherein the cut-off membrane is an approximately 5 kDa cut-off membrane.

31. The conditioned media of claim 24, wherein reducing the serum content comprises weaning the UTC from the culture media.

32. The conditioned media of claim 31, wherein the weaning comprises reducing serum content of the culture media in increments.

33. The conditioned media of claim 32, wherein the serum content is reduced in increments of about 5% to about 60%.

34. The conditioned media of claim 33, wherein the serum content is reduced in increments of about 50%.

35. The conditioned media of claim 32, wherein the UTC grows for about 1 to 3 passages in each increment.

36. The conditioned media of claim 35, wherein the UTC grows for about 2 passages in each increment.

37. The conditioned media of claim 24, wherein the culture media is a static culture.

38. The conditioned media of claim 24, wherein the culture media is a microcarrier bead culture.

39. The conditioned media of claim 24, further comprising preliminarily culturing the UTC in a standard culture media and isolating the UTC from the standard culture media before seeding.

40. The conditioned media of claim 24, wherein the UTC is a human umbilical cord tissue-derived cell.