Methods of evaluating cells and cell cultures
View Patent ↗Methods of evaluating the composition of a cell culture (e.g., to distinguish chondrocytes from fibroblasts) and methods for evaluating the phenotype of an individual cell (e.g., as a chondrocyte) are disclosed. The methods may be used, for example, for assessing chondrocyte cultures used for treatment of cartilage defects. In some embodiments, the invention involves identifying cell culture composition or the identity of a cell based on expression level of a fibroblast marker. In other embodiments, the invention involves comparing expression levels of at least one chondrocyte marker and at least one fibroblast marker in a cell culture sample or in an individual cell. In illustrative embodiments, the chondrocyte marker is hyaluronan and proteoglycan link protein 1 (HAPLN1), and the fibroblast marker is microfibrillar associated protein 5 (MFAP5).
1. A method of evaluating the composition of a cell culture, said method comprising:
a) obtaining a culture of cells for expansion of chondrocytes;
b) selecting a plurality of cells from the cell culture;
c) determining the average expression level of microfibrillar associated protein 5 (MFAP5) in the plurality of cells; and
d) determining the composition of the culture based on the average expression level of MFAP5;
wherein the average expression level of MFAP5 below a predetermined threshold indicates that the cell culture comprises chondrocytes.
2. The method of claim 1 , further comprising determining the average expression level of a chondrocyte marker in the plurality of cells; and, in step d), determining the composition of the culture based on the average expression level of the chondrocyte marker and the average expression level of MFAP5.
3. The method of claim 2 , wherein MFAP5 and the chondrocyte marker are such that the ratio of their expression levels (chondrocyte marker to MFAP5) in chondrocytes is equal to or greater than 5 times the ratio in dermal fibroblasts or synoviocytes.
4. The method of claim 2 , wherein the chondrocyte marker is selected from the group consisting of hyaluronan and proteoglycan link protein 1 (HAPLN1), matrix Gla protein (MGP), GF-like repeats and discoidin I-like domains 3 (EDIL3), WNT1 inducible signaling pathway protein 3 (WISP3), aggrecan 1 (AGC1), cartilage oligomeric matrix protein (COMP), type II collagen, alpha 1 (COL2A1), type IX collagen, alpha 1 (COL9A1), type XI collagen, alpha 1 (COL11A1), leukocyte cell derived chemotaxin 1 protein (LECT1), S100 calcium binding protein beta (S100B), cartilage acidic protein 1 (CRTAC1), SRY-box9 protein (SOX9), and nebulette (NEBL).
5. The method of claim 2 , wherein the chondrocyte marker is HAPLN1.
6. The method of claim 2 , wherein the expression levels of MFAP5 and the chondrocyte marker are determined at the RNA level.
7. The method of claim 6 , wherein the expression levels are determined using PCR.
8. The method of claim 7 , wherein the expression levels are determined using comparative C T PCR method.
9. The method of claim 2 , wherein an expression level ratio of the chondrocyte marker to MFAP5 of greater than 0.25, as determined using comparative C T PCR method, indicates that the cell culture contains chondrocytes.
10. The method of claim 2 , wherein a molar ratio of the chondrocyte marker to MFAP5 of greater than 0.55 indicates that the cell culture contains chondrocytes.
11. The method of claim 1 , wherein the cell culture comprises cells obtained from a cartilage biopsy.
12. The method of claim 11 , wherein the cartilage biopsy is taken from a knee joint.
13. The method of claim 11 , wherein, following the evaluation of the cell culture, the cells from the cell culture are administered to a patient in need thereof.
14. The method of claim 13 , wherein the cells from the culture are used for autologous chondrocyte implantation.
15. A method of evaluating the composition of a cell culture, the method comprising:
a) obtaining a culture of cells for expansion of chondrocytes;
b) determining the expression level of MFAP5 in the cell culture; and
c) determining the composition of the cell culture based on the expression level of MFAP5.
16. The method of claim 15 , further comprising determining the expression level of a chondrocyte marker in the cell culture; and, in step c), determining the composition of the cell culture based on the expression levels of the chondrocyte marker and MFAP5.
17. The method of claim 16 , wherein the chondrocyte marker is selected from the group consisting of hyaluronan and proteoglycan link protein 1 (HAPLN1), matrix Gla protein (MGP), GF-like repeats and discoidin I-like domains 3 (EDIL3), WNT1 inducible signaling pathway protein 3 (WISP3), aggrecan 1 (AGC1), cartilage oligomeric matrix protein (COMP), type II collagen, alpha 1 (COL2A1), type IX collagen, alpha 1 (COL9A1), type XI collagen, alpha 1 (COL11A1), leukocyte cell derived chemotaxin 1 protein (LECT1), S100 calcium binding protein beta (S100B), cartilage acidic protein 1 (CRTAC1), SRY-box9 protein (SOX9), and nebulette (NEBL).
18. The method of claim 16 , wherein the chondrocyte marker is HAPLN1.
19. The method of claim 16 , wherein the expression levels of the chondrocyte marker and MFAP5 are determined at the RNA level.
20. A method of evaluating the composition of a cell culture, said method comprising:
a) obtaining a cartilage biopsy from a mammal;
b) isolating cells from the biopsy;
c) culturing the isolated cells in a cell culture to produce chondrocytes;
d) determining the expression levels of MFAP5 and HAPLN1 in a plurality of cells obtained from the cell culture; and
e) determining the composition of the culture based on the expression levels of MFAP5 and HAPLN1.
21. The method of claim 20 , wherein the expression levels of MFAP5 and HAPLN1 are determined at the RNA level.
22. The method of claim 21 , wherein the expression levels are determined using PCR.
23. The method of claim 22 , wherein the expression levels are determined using comparative C T PCR method.
24. The method of claim 20 , wherein an expression level ratio of HAPLN1 to MFAP5 greater than 0.25, as determined using comparative C T PCR method, indicates that the cell culture contains chondrocytes.
25. The method of claim 20 , wherein a molar ratio of HAPLN1 to MFAP5 greater than 0.55 indicates that the cell culture contains chondrocytes.
26. A method of evaluating the composition of a cell culture comprising:
a) obtaining a cartilage biopsy from a mammal;
b) isolating cells from the biopsy;
c) culturing the isolated cells in a cell culture to produce chondrocytes;
d) determining the expression levels of MFAP5 and a chondrocyte marker in a plurality of cells obtained from the cell culture; and
e) determining the presence of chondrocytes in the cell culture based on the expression levels of MFAP5 and the chondrocyte marker.
27. The method of claim 26 , wherein the chondrocyte marker is selected from the group consisting of hyaluronan and proteoglycan link protein 1 (HAPLN1), matrix Gla protein (MGP), GF-like repeats and discoidin I-like domains 3 (EDIL3), WNT1 inducible signaling pathway protein 3 (WISP3), aggrecan 1 (AGC1), cartilage oligomeric matrix protein (COMP), type II collagen, alpha 1 (COL2A1), type IX collagen, alpha 1 (COL9A1), type XI collagen, alpha 1 (COL11A1), leukocyte cell derived chemotaxin 1 protein (LECT1), S100 calcium binding protein beta (S100B), cartilage acidic protein 1 (CRTAC1), SRY-box9 protein (SOX9), and nebulette (NEBL).
28. The method of claim 26 , wherein the chondrocyte marker is HAPLN1.
29. A method of evaluating the composition of a cell culture to confirm the presence of chondrocytes comprising:
a) obtaining a plurality of cells from a cell culture comprising cells derived from cartilage or synovium; and
b) determining the average expression level of microfibrillar associated protein 5 (MFAP5) in the plurality of cells;
wherein the average expression level of MFAP5 below a predetermined threshold indicates that the cell culture comprises chondrocytes.
30. A method of evaluating the composition of a chondrocyte cell culture to identify the presence of fibroblasts and/or synoviocytes comprising:
a) obtaining a plurality of cells from a chondrocyte cell culture; and
b) determining the average expression level of microfibrillar associated protein 5 (MFAP5) in the plurality of cells;
wherein an average expression level of MFAP5 above a predetermined threshold indicates that the cell culture comprises fibroblasts and/or synoviocytes.
31. A method of evaluating the composition of a cell culture to determine the ratio of chondrocytes to fibroblasts and/or synoviocytes comprising:
a) performing a cell count of a chondrocyte cell culture
b) obtaining a plurality of cells from the cell culture; and
c) determining the average expression level of microfibrillar associated protein 5 (MFAP5) in the plurality of cells;
wherein an average expression level of MFAP5 above a predetermined threshold indicates that the cell culture comprises more fibroblasts and/or synoviocytes than chondrocytes and an average expression level of MFAP5 below a predetermined threshold indicates that the cell culture comprises more chondrocytes than fibroblasts and/or synoviocytes.
32. The method of claim 31 , further comprising determining the expression level of a chondrocyte marker in the plurality of cells; and determining the ratio of chondrocytes to fibroblasts and/or synoviocytes based on the expression levels of the chondrocyte marker and MFAP5.
33. The method of claim 32 , wherein the chondrocyte marker is selected from the group consisting of hyaluronan and proteoglycan link protein 1 (HAPLN1), matrix Gla protein (MGP), GF-like repeats and discoidin I-like domains 3 (EDIL3), WNT1 inducible signaling pathway protein 3 (WISP3), aggrecan 1 (AGC1), cartilage oliqomeric matrix protein (COMP), type II collagen, alpha 1 (COL2A1), type IX collagen, alpha 1 (COL9A1), type XI collagen, alpha 1 (COL11A1), leukocyte cell derived chemotaxin 1 protein (LECT1), S100 calcium binding protein beta (S100B), cartilage acidic protein 1 (CRTAC1), SRY-box9 protein (SOX9), and nebulette (NEBL).
34. The method of claim 32 , wherein the chondrocyte marker is HAPLN1.