Potency assay
The present invention relates to a method for determining the biological activity or therapeutic efficacy of cultured mesenchymal lineage precursor cells or stem cells based on their released TGF-9 levels in culture. The present invention also relates to isolated populations of mesenchymal lineage precursor cells or stem cells selected based on the level of TGF-9 levels released by such cells in culture. The present invention further relates to treatment of a subject suffering from a degenerative disc disease by administering such selected cell populations.
1. A method for determining if human mesenchymal lineage precursor or stem cells in a population of cells are sufficiently potent for use in treating a degenerative disc disease, the method comprising:
(i) culturing a population of cells comprising human mesenchymal lineage precursor cells or stem cells in a culture medium for a suitable culture time period;
(ii) determining at the end of the culture period the amount of TGFβ1 released by the cultured population of cells into the culture medium; and
(iii) determining that the human mesenchymal lineage precursor or stem cells in the population are sufficiently potent for such use if the [determined] amount of TGFβ1 determined in step (ii) is at least 2800 pg/10 6 cells present at the end of the culture period.
2. The method of claim 1 , wherein the population of step (i) is a population enriched for mesenchymal lineage precursor or stem cells.
3. The method of claim 1 , wherein the method comprises seeding the cells in a culture vessel at a density of about 50,000 viable cells/cm 2 .
4. The method of claim 1 , wherein the method comprises culturing the cells in chondrogenic basal medium supplemented with 0.5% bovine serum albumin.
5. The method of claim 1 , wherein the culture period is at least 68 [to 76] hours.
6. The method of claim 1 , wherein the method comprises collecting a sample of the culture medium in which the cells were cultured.
7. The method of claim 6 , wherein the method comprises activating latent TGFβ1 in the culture medium prior to determining the amount of TGFβ1 in the culture medium.
8. The method of claim 7 , wherein activating latent TGFβ1 comprises adding an acid to the culture medium sample to lower the pH of the culture medium.
9. The method of claim 8 , wherein the method comprises concentrating the culture medium sample prior to lowering the pH.
10. The method of claim 8 , wherein, following addition of the acid, the culture medium is neutralised to a pH of 7.2 to 7.6.
11. The method of claim 1 , wherein the method comprises determining the amount of TGFβ1 in the culture medium by enzyme-linked immunosorbent assay (ELISA) .