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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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FACS analysis of primary-culture RA synovial cells (following 7 days of culture). The primary culture contained virtually only cells staining either with the FB-directed anti-Thy-1 mAb AS02 (A) or with the monocyte/macrophage-specific anti-CD14 mAb Tyk4 (B). FITC, fluoresceine isothiocyanate.
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Figure 4: FACS analysis of primary-culture RA synovial cells (following 7 days of culture). The primary culture contained virtually only cells staining either with the FB-directed anti-Thy-1 mAb AS02 (A) or with the monocyte/macrophage-specific anti-CD14 mAb Tyk4 (B). FITC, fluoresceine isothiocyanate.

Mentions: RA synovial cells were subjected to extensive analysis to carefully characterize the starting population before negative isolation (ie the 7-day primary culture of synovial cells resulting from trypsin/collagenase digestion of the RASM). The primary culture contained large, spindle-shaped Thy-1+ SFB (Fig. 1C) and small, round CD14+ cells, most probably macrophages (Fig. 1D; mAb Tyk4), as detected by immunohistochemical staining [6,9]. Endothelial cells were absent, as confirmed by lack of staining for von Willebrand factor (Fig. 1F; mAb 4F9) and CD144 (Fig. 1G; mAb Cadherin 5), which clearly identified HUVEC (data not shown). The FB nature of the spindle-shaped cells was confirmed by intracellular staining for procollagen I and III (Fig. 1E,H; rabbit antibodies MP I and MP III). An average of approximately 62% of the cells stained with the anti-Thy-1 mAb AS02 (n = 4 RA patients; Table 1a and Fig. 4A) in FACS analysis [10]. The average percentage of CD14+ cells was approximately 15% (n = 4; Table 1a and Fig. 4B). There were <1% T cells (CD3+; mAb UCHT-1), B cells (CD19+/20+; mAbs HD 37 and B-Ly 1), plasma cells (CD38+; mAb AT 13/5), NK cells (CD56+; mAb NKH/1), dendritic cells (CD83+; mAb HB 15a), endothelial cells (CD144+), or PMN (CD15+; mAb 80H5), indicating that non-adherent cells had been efficiently removed during primary culture. Endothelial cells presumably did not adhere to the culture dish due to the absence of gelatin coating and unfavorable medium composition (see Materials and Methods).


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)

FACS analysis of primary-culture RA synovial cells (following 7 days of culture). The primary culture contained virtually only cells staining either with the FB-directed anti-Thy-1 mAb AS02 (A) or with the monocyte/macrophage-specific anti-CD14 mAb Tyk4 (B). FITC, fluoresceine isothiocyanate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC17827&req=5

Figure 4: FACS analysis of primary-culture RA synovial cells (following 7 days of culture). The primary culture contained virtually only cells staining either with the FB-directed anti-Thy-1 mAb AS02 (A) or with the monocyte/macrophage-specific anti-CD14 mAb Tyk4 (B). FITC, fluoresceine isothiocyanate.
Mentions: RA synovial cells were subjected to extensive analysis to carefully characterize the starting population before negative isolation (ie the 7-day primary culture of synovial cells resulting from trypsin/collagenase digestion of the RASM). The primary culture contained large, spindle-shaped Thy-1+ SFB (Fig. 1C) and small, round CD14+ cells, most probably macrophages (Fig. 1D; mAb Tyk4), as detected by immunohistochemical staining [6,9]. Endothelial cells were absent, as confirmed by lack of staining for von Willebrand factor (Fig. 1F; mAb 4F9) and CD144 (Fig. 1G; mAb Cadherin 5), which clearly identified HUVEC (data not shown). The FB nature of the spindle-shaped cells was confirmed by intracellular staining for procollagen I and III (Fig. 1E,H; rabbit antibodies MP I and MP III). An average of approximately 62% of the cells stained with the anti-Thy-1 mAb AS02 (n = 4 RA patients; Table 1a and Fig. 4A) in FACS analysis [10]. The average percentage of CD14+ cells was approximately 15% (n = 4; Table 1a and Fig. 4B). There were <1% T cells (CD3+; mAb UCHT-1), B cells (CD19+/20+; mAbs HD 37 and B-Ly 1), plasma cells (CD38+; mAb AT 13/5), NK cells (CD56+; mAb NKH/1), dendritic cells (CD83+; mAb HB 15a), endothelial cells (CD144+), or PMN (CD15+; mAb 80H5), indicating that non-adherent cells had been efficiently removed during primary culture. Endothelial cells presumably did not adhere to the culture dish due to the absence of gelatin coating and unfavorable medium composition (see Materials and Methods).

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