Limits...
Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture--primary culture cells markedly differ from fourth-passage cells.

Zimmermann T, Kunisch E, Pfeiffer R, Hirth A, Stahl HD, Sack U, Laube A, Liesaus E, Roth A, Palombo-Kinne E, Emmrich F, Kinne RW - Arthritis Res. (2000)

Bottom Line: To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies.This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta.This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

View Article: PubMed Central - HTML - PubMed

Affiliation: Experimental Rheumatology Unit, Friedrich Schiller University Jena, Jena, Germany.

ABSTRACT
To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

Show MeSH

Related in: MedlinePlus

Phenotype comparison of isolated primary-culture (left column) and conventional fourth-passage RA-SFB (right column) from one representative patient. Expression of Thy-1 (A, B), MHC-II (C, D), prolyl-4-hydroxylase (E, F), procollagen III (G, H), c-Fos (I, J), and c-Jun (K, L). While the percentages of cells positive for MHC-II (C) and the MFI for c-Jun (K) were significantly higher in isolated primary-culture RA-SFB, the percentages of cells positive for prolyl-4-hydroxylase (F), procollagen III (H), and c-Fos (J) were significantly higher in conventional fourth passage (see Table 5 for details). PE, phycoerythrine; FITC, fluoresceine isothiocyanate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC17827&req=5

Figure 7: Phenotype comparison of isolated primary-culture (left column) and conventional fourth-passage RA-SFB (right column) from one representative patient. Expression of Thy-1 (A, B), MHC-II (C, D), prolyl-4-hydroxylase (E, F), procollagen III (G, H), c-Fos (I, J), and c-Jun (K, L). While the percentages of cells positive for MHC-II (C) and the MFI for c-Jun (K) were significantly higher in isolated primary-culture RA-SFB, the percentages of cells positive for prolyl-4-hydroxylase (F), procollagen III (H), and c-Fos (J) were significantly higher in conventional fourth passage (see Table 5 for details). PE, phycoerythrine; FITC, fluoresceine isothiocyanate.

Mentions: The percentages of RA-SFB positive for MHC-II, as well as the MFI for VCAM-1 and c-Jun, were significantly decreased in conventional fourth passage in comparison with isolated primary RA-SFB (Fig. 7C,D,K,L and Table 5). The percentages of cells positive for MHC-I, CD13, prolyl-4-hydroxylase, vimentin, procollagen I and III, c-Fos and Jun-D were, in contrast, significantly increased in conventional fourth passage (Fig. 7E,F,G,H,I,J and Table 5).


Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture--primary culture cells markedly differ from fourth-passage cells.

Zimmermann T, Kunisch E, Pfeiffer R, Hirth A, Stahl HD, Sack U, Laube A, Liesaus E, Roth A, Palombo-Kinne E, Emmrich F, Kinne RW - Arthritis Res. (2000)

Phenotype comparison of isolated primary-culture (left column) and conventional fourth-passage RA-SFB (right column) from one representative patient. Expression of Thy-1 (A, B), MHC-II (C, D), prolyl-4-hydroxylase (E, F), procollagen III (G, H), c-Fos (I, J), and c-Jun (K, L). While the percentages of cells positive for MHC-II (C) and the MFI for c-Jun (K) were significantly higher in isolated primary-culture RA-SFB, the percentages of cells positive for prolyl-4-hydroxylase (F), procollagen III (H), and c-Fos (J) were significantly higher in conventional fourth passage (see Table 5 for details). PE, phycoerythrine; FITC, fluoresceine isothiocyanate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC17827&req=5

Figure 7: Phenotype comparison of isolated primary-culture (left column) and conventional fourth-passage RA-SFB (right column) from one representative patient. Expression of Thy-1 (A, B), MHC-II (C, D), prolyl-4-hydroxylase (E, F), procollagen III (G, H), c-Fos (I, J), and c-Jun (K, L). While the percentages of cells positive for MHC-II (C) and the MFI for c-Jun (K) were significantly higher in isolated primary-culture RA-SFB, the percentages of cells positive for prolyl-4-hydroxylase (F), procollagen III (H), and c-Fos (J) were significantly higher in conventional fourth passage (see Table 5 for details). PE, phycoerythrine; FITC, fluoresceine isothiocyanate.
Mentions: The percentages of RA-SFB positive for MHC-II, as well as the MFI for VCAM-1 and c-Jun, were significantly decreased in conventional fourth passage in comparison with isolated primary RA-SFB (Fig. 7C,D,K,L and Table 5). The percentages of cells positive for MHC-I, CD13, prolyl-4-hydroxylase, vimentin, procollagen I and III, c-Fos and Jun-D were, in contrast, significantly increased in conventional fourth passage (Fig. 7E,F,G,H,I,J and Table 5).

Bottom Line: To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies.This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta.This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

View Article: PubMed Central - HTML - PubMed

Affiliation: Experimental Rheumatology Unit, Friedrich Schiller University Jena, Jena, Germany.

ABSTRACT
To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1beta. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

Show MeSH
Related in: MedlinePlus