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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.

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Morphology of negatively isolated RA-SFB and macrophages upon reculture. When recultured, the RA-SFB obtained by negative isolation with Dynabeads® M-450 CD14 showed almost exclusively spindle-shaped, flat or stellate morphology (A). Recultured CD14+ cells (B) exhibited small, round, macrophage-like morphology with attached magnetobeads, and contained only very few cells with FB morphology. Original maginification: 368 ×.
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Figure 5: Morphology of negatively isolated RA-SFB and macrophages upon reculture. When recultured, the RA-SFB obtained by negative isolation with Dynabeads® M-450 CD14 showed almost exclusively spindle-shaped, flat or stellate morphology (A). Recultured CD14+ cells (B) exhibited small, round, macrophage-like morphology with attached magnetobeads, and contained only very few cells with FB morphology. Original maginification: 368 ×.

Mentions: Negatively isolated RA-SFB showed almost exclusively spindle-shaped or stellate, flat morphology when recultured (Fig. 5A). Recultured CD14+ cells (Fig. 5B) exhibited small, round morphology with attached Dynabeads® and contained only very few cells with FB morphology.


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)

Morphology of negatively isolated RA-SFB and macrophages upon reculture. When recultured, the RA-SFB obtained by negative isolation with Dynabeads® M-450 CD14 showed almost exclusively spindle-shaped, flat or stellate morphology (A). Recultured CD14+ cells (B) exhibited small, round, macrophage-like morphology with attached magnetobeads, and contained only very few cells with FB morphology. Original maginification: 368 ×.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Morphology of negatively isolated RA-SFB and macrophages upon reculture. When recultured, the RA-SFB obtained by negative isolation with Dynabeads® M-450 CD14 showed almost exclusively spindle-shaped, flat or stellate morphology (A). Recultured CD14+ cells (B) exhibited small, round, macrophage-like morphology with attached magnetobeads, and contained only very few cells with FB morphology. Original maginification: 368 ×.
Mentions: Negatively isolated RA-SFB showed almost exclusively spindle-shaped or stellate, flat morphology when recultured (Fig. 5A). Recultured CD14+ cells (Fig. 5B) exhibited small, round morphology with attached Dynabeads® and contained only very few cells with FB morphology.

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