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 of isolated primary-culture RA-SFB (lower panels) in comparison with primary-culture normal skin-FB (upper panel) and isolated primary-culture OA-SFB. The double-staining experiments were performed with the anti-Thy-1 mAb AS02. (A)-(C) The expression of Thy-1 (A), MHC-II/Thy-1 (B) (double-staining), and vimentin/Thy-1 (C) (double-staining) in normal skin-FB; (D)-(F) the expression of Thy-1 (D), MHC-II/Thy-1 (E) (double-staining), and vimentin/Thy-1 (F) (double-staining) in OA-SFB; (G)-(I) the expression of the antigens Thy-1 (G), MHC-II/Thy-1 (H) (double-staining), vimentin/Thy-1 (I) (double-staining) on RA-SFB; (J)-(L) the expression of the cytoplasmic antigens procollagen I (J) and procollagen III/Thy-1 (K) (single-staining for procollagen III) and (L) (double-staining) in RA-SFB. Expression of prolyl-4-hydroxylase (M) and the proto-oncogenes c-Fos (N), and c-Jun (O) in RA-SFB is also shown. See Results and Table 4 for mean values and statistical comparison among the different FB types. PE, phycoerythrine; FITC, fluoresceine isothiocyanate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC17827&req=5

Figure 6: Phenotype of isolated primary-culture RA-SFB (lower panels) in comparison with primary-culture normal skin-FB (upper panel) and isolated primary-culture OA-SFB. The double-staining experiments were performed with the anti-Thy-1 mAb AS02. (A)-(C) The expression of Thy-1 (A), MHC-II/Thy-1 (B) (double-staining), and vimentin/Thy-1 (C) (double-staining) in normal skin-FB; (D)-(F) the expression of Thy-1 (D), MHC-II/Thy-1 (E) (double-staining), and vimentin/Thy-1 (F) (double-staining) in OA-SFB; (G)-(I) the expression of the antigens Thy-1 (G), MHC-II/Thy-1 (H) (double-staining), vimentin/Thy-1 (I) (double-staining) on RA-SFB; (J)-(L) the expression of the cytoplasmic antigens procollagen I (J) and procollagen III/Thy-1 (K) (single-staining for procollagen III) and (L) (double-staining) in RA-SFB. Expression of prolyl-4-hydroxylase (M) and the proto-oncogenes c-Fos (N), and c-Jun (O) in RA-SFB is also shown. See Results and Table 4 for mean values and statistical comparison among the different FB types. PE, phycoerythrine; FITC, fluoresceine isothiocyanate.

Mentions: To verify whether isolated RA-SFB displayed the features observed in situ in the RASM [1,2,12,13,14,15, 21,22,23,24,25,28], the expression of several surface or intracellular/nuclear molecules was investigated by FACS analysis (Fig. 6 and Table 4). The phenotype was compared with that of primary-culture normal skin-FB and isolated primary-culture OA-SFB (Fig. 6 and Table 4).


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 of isolated primary-culture RA-SFB (lower panels) in comparison with primary-culture normal skin-FB (upper panel) and isolated primary-culture OA-SFB. The double-staining experiments were performed with the anti-Thy-1 mAb AS02. (A)-(C) The expression of Thy-1 (A), MHC-II/Thy-1 (B) (double-staining), and vimentin/Thy-1 (C) (double-staining) in normal skin-FB; (D)-(F) the expression of Thy-1 (D), MHC-II/Thy-1 (E) (double-staining), and vimentin/Thy-1 (F) (double-staining) in OA-SFB; (G)-(I) the expression of the antigens Thy-1 (G), MHC-II/Thy-1 (H) (double-staining), vimentin/Thy-1 (I) (double-staining) on RA-SFB; (J)-(L) the expression of the cytoplasmic antigens procollagen I (J) and procollagen III/Thy-1 (K) (single-staining for procollagen III) and (L) (double-staining) in RA-SFB. Expression of prolyl-4-hydroxylase (M) and the proto-oncogenes c-Fos (N), and c-Jun (O) in RA-SFB is also shown. See Results and Table 4 for mean values and statistical comparison among the different FB types. PE, phycoerythrine; FITC, fluoresceine isothiocyanate.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Phenotype of isolated primary-culture RA-SFB (lower panels) in comparison with primary-culture normal skin-FB (upper panel) and isolated primary-culture OA-SFB. The double-staining experiments were performed with the anti-Thy-1 mAb AS02. (A)-(C) The expression of Thy-1 (A), MHC-II/Thy-1 (B) (double-staining), and vimentin/Thy-1 (C) (double-staining) in normal skin-FB; (D)-(F) the expression of Thy-1 (D), MHC-II/Thy-1 (E) (double-staining), and vimentin/Thy-1 (F) (double-staining) in OA-SFB; (G)-(I) the expression of the antigens Thy-1 (G), MHC-II/Thy-1 (H) (double-staining), vimentin/Thy-1 (I) (double-staining) on RA-SFB; (J)-(L) the expression of the cytoplasmic antigens procollagen I (J) and procollagen III/Thy-1 (K) (single-staining for procollagen III) and (L) (double-staining) in RA-SFB. Expression of prolyl-4-hydroxylase (M) and the proto-oncogenes c-Fos (N), and c-Jun (O) in RA-SFB is also shown. See Results and Table 4 for mean values and statistical comparison among the different FB types. PE, phycoerythrine; FITC, fluoresceine isothiocyanate.
Mentions: To verify whether isolated RA-SFB displayed the features observed in situ in the RASM [1,2,12,13,14,15, 21,22,23,24,25,28], the expression of several surface or intracellular/nuclear molecules was investigated by FACS analysis (Fig. 6 and Table 4). The phenotype was compared with that of primary-culture normal skin-FB and isolated primary-culture OA-SFB (Fig. 6 and Table 4).

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