Potency assay of secretomes
The present invention relates to a method for determining the potency of a supernatant of a mammalian cell culture to be used in the treatment of an inflammatory condition.
1. A method for determining potential of a supernatant of a mammalian cell culture to be used in treatment of an inflammatory condition comprising steps of
a) incubating SH-SY5Y cells in a culture medium comprising or consisting of said supernatant,
b) measuring a promoter activity of an activator protein 1 (AP-1) promoter, wherein the supernatant of the mammalian cell culture has the potential to be used in the treatment of the inflammatory condition, if the promoter activity of the AP-1 promoter is at least 50% higher compared to the AP-1 promoter activity measured when the SH-SY5Y cells are cultivated in a culture medium lacking said supernatant.
2. The method according to claim 1 , wherein the supernatant of a mammalian cell culture is a supernatant of a peripheral blood mononuclear cell (PB MC) culture.
3. The method according to claim 1 , wherein the inflammatory condition is a condition associated with ischemia, a skin condition, or an internal inflammatory condition.
4. The method according to claim 1 , wherein the SH-SY5Y cells of step a) are incubated in the cell culture medium for at least 2 hours.
5. The method according to claim 4 , wherein the SH-SY5Y cells of step a) are incubated in the cell culture medium for at least 4 hours, or at least 6 hours, or at least 12 hours, or at least 18 hours, or at least 24 hours.
6. The method according to claim 1 , wherein the culture medium comprises at least 20% of said supernatant.
7. The method according to claim 6 , wherein the culture medium comprises at least 50%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, of said supernatant.
8. The method according to claim 1 , wherein the culture medium of step a) is a Dulbecco's Modified Eagle Medium (DMEM), a Ham's F12 Medium (F12), a Minimum Essential Medium or a combination of one or more of these media.
9. The method according to claim 1 , wherein the culture medium of step a) comprises 2 to 20% fetal bovine serum (FBS) and/or L-alanyl-L-glutamine.
10. The method according to claim 9 , wherein the culture medium of step a) comprises 5 to 15% fetal bovine serum (FBS).
11. The method according to claim 1 , wherein the SH-SY5Y cells comprise at least one expression cassette comprising an AP-1 promoter operably linked to at least one nucleic acid molecule encoding for a reporter protein.
12. The method according to claim 11 , wherein the reporter protein is selected from the group consisting of a luciferase and a fluorescent protein.
13. The method according to claim 12 , wherein the luciferase is a firefly luciferase and the fluorescent protein is a green fluorescent protein.
14. The method according to claim 1 , wherein the promoter activity of the AP-1 promoter is at least 80% higher compared to the promoter activity measured when the SH-SY5Y cells are cultivated in a culture medium lacking said supernatant.
15. The method according to claim 14 , wherein the promoter activity of the AP-1 promoter is at least 100% higher compared to the promoter activity measured when the SH-SY5Y cells are cultivated in a culture medium lacking said supernatant.
16. The method according to claim 14 , wherein the promoter activity of the AP-1 promoter is at least 150% higher compared to the promoter activity measured when the SH-SY5Y cells are cultivated in a culture medium lacking said supernatant.
17. The method according to claim 14 , wherein the promoter activity of the AP-1 promoter is at least 200%, higher compared to the promoter activity measured when the SH-SY5Y cells are cultivated in a culture medium lacking said supernatant.
18. A method for determining potential of a supernatant of a mammalian cell culture to be used in treatment of an inflammatory condition comprising steps of
a) incubating SH-SY5Y cells in a culture medium comprising or consisting of said supernatant,
b) measuring a promoter activity of an activator protein 1 (AP-1) promoter, wherein the supernatant of the mammalian cell culture has the potential to be used in the treatment of the inflammatory condition, if the promoter activity of the AP-1 promoter is up to 10% higher or lower compared to the promoter activity measured when the SH-SY5Y cells are cultivated in a culture medium comprising a reference supernatant of mammalian cells known to be effective for the treatment of the inflammatory condition.
19. The method according to claim 18 , wherein the supernatant of a mammalian cell culture is a supernatant of a peripheral blood mononuclear cell (PBMC) culture.
20. The method according to claim 18 , wherein the inflammatory condition is a condition associated with ischemia, a skin condition, or an internal inflammatory condition.