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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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Immunohistochemical staining of primary-culture RA synovial cells in chamber slides: (A), (B), (D)-(H) phase contrast, and (C) lightfield. While (A) control isotype-matched mAbs (peroxidase) or (B) rabbit serum (APAAP) showed no positive reaction, (C) the anti-Thy-1 mAb AS02 stained large, flat, spindle-shaped or stellate-shaped cells (ie cells with FB morphology; alkaline phosphatase, purple staining) and (D) the anti-CD14 mAb RMO52 identified small, round cells, conceivably macrophages (peroxidase, brown staining). The RA-SFB clearly expressed (E) procollagen I and (H) procollagen III (APAAP, red staining). The primary culture contained no endothelial cells, as documented by the lack of staining for (F) von Willebrand factor (APAAP) and (G) CD144 (APAAP). Original magnification: (A), (B), (F), and (G), 184 ×; (C)-(E) and (H) 368 ×.
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Figure 1: Immunohistochemical staining of primary-culture RA synovial cells in chamber slides: (A), (B), (D)-(H) phase contrast, and (C) lightfield. While (A) control isotype-matched mAbs (peroxidase) or (B) rabbit serum (APAAP) showed no positive reaction, (C) the anti-Thy-1 mAb AS02 stained large, flat, spindle-shaped or stellate-shaped cells (ie cells with FB morphology; alkaline phosphatase, purple staining) and (D) the anti-CD14 mAb RMO52 identified small, round cells, conceivably macrophages (peroxidase, brown staining). The RA-SFB clearly expressed (E) procollagen I and (H) procollagen III (APAAP, red staining). The primary culture contained no endothelial cells, as documented by the lack of staining for (F) von Willebrand factor (APAAP) and (G) CD144 (APAAP). Original magnification: (A), (B), (F), and (G), 184 ×; (C)-(E) and (H) 368 ×.

Mentions: The primary culture of RA synovial cells resulting from trypsin/collagenase digestion of the RASM contained large, spindle-shaped Thy-1+ SFB (CD90+; Fig. 1C) (monoclonal antibody [mAb] AS02; Dianova, Hamburg, Germany) and small, round CD14+ cells, most probably macrophages (Fig. 1D) (mAb Tyk4; Dako, Hamburg, Germany), 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; Immunotech, Hamburg, Germany) and CD144 (Fig. 1G) (mAb Cadherin 5; Immunotech), which clearly identified human umbilical vein endothelial cells (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; Prof. Schuppan, Berlin, Germany). An average of 62% of the cells stained with the anti-Thy-1 mAb AS02 (n = 4 RA patients; Table 1a) in flow cytometry (FACS) [10]; the average percentage of CD14+ cells was approximately 15% (n = 4; Table 1a). There were <1% T cells (CD3+) (mAb UCHT-1; ATCC, Manassas, VA, USA), B cells (CD19+/20+) (mAbs HD 37 and B-Ly 1; Dako), plasma cells (CD38+) (mAb AT 13/5; Dako), natural killer (NK) cells (CD56+) (mAb NKH/1; Immunotech), dendritic cells (CD83+) (mAb HB 15a; Immunotech), endothelial cells (CD144+), or PMN (CD15+) (mAb80H5; Immunotech), indicating that non-adherent cells had been efficiently removed during primary culture. The total yield of cells following 7 days of primary culture averaged (5.2 ± 1.1) × 107 cells (mean ± SEM; n = 7).


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)

Immunohistochemical staining of primary-culture RA synovial cells in chamber slides: (A), (B), (D)-(H) phase contrast, and (C) lightfield. While (A) control isotype-matched mAbs (peroxidase) or (B) rabbit serum (APAAP) showed no positive reaction, (C) the anti-Thy-1 mAb AS02 stained large, flat, spindle-shaped or stellate-shaped cells (ie cells with FB morphology; alkaline phosphatase, purple staining) and (D) the anti-CD14 mAb RMO52 identified small, round cells, conceivably macrophages (peroxidase, brown staining). The RA-SFB clearly expressed (E) procollagen I and (H) procollagen III (APAAP, red staining). The primary culture contained no endothelial cells, as documented by the lack of staining for (F) von Willebrand factor (APAAP) and (G) CD144 (APAAP). Original magnification: (A), (B), (F), and (G), 184 ×; (C)-(E) and (H) 368 ×.
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Figure 1: Immunohistochemical staining of primary-culture RA synovial cells in chamber slides: (A), (B), (D)-(H) phase contrast, and (C) lightfield. While (A) control isotype-matched mAbs (peroxidase) or (B) rabbit serum (APAAP) showed no positive reaction, (C) the anti-Thy-1 mAb AS02 stained large, flat, spindle-shaped or stellate-shaped cells (ie cells with FB morphology; alkaline phosphatase, purple staining) and (D) the anti-CD14 mAb RMO52 identified small, round cells, conceivably macrophages (peroxidase, brown staining). The RA-SFB clearly expressed (E) procollagen I and (H) procollagen III (APAAP, red staining). The primary culture contained no endothelial cells, as documented by the lack of staining for (F) von Willebrand factor (APAAP) and (G) CD144 (APAAP). Original magnification: (A), (B), (F), and (G), 184 ×; (C)-(E) and (H) 368 ×.
Mentions: The primary culture of RA synovial cells resulting from trypsin/collagenase digestion of the RASM contained large, spindle-shaped Thy-1+ SFB (CD90+; Fig. 1C) (monoclonal antibody [mAb] AS02; Dianova, Hamburg, Germany) and small, round CD14+ cells, most probably macrophages (Fig. 1D) (mAb Tyk4; Dako, Hamburg, Germany), 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; Immunotech, Hamburg, Germany) and CD144 (Fig. 1G) (mAb Cadherin 5; Immunotech), which clearly identified human umbilical vein endothelial cells (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; Prof. Schuppan, Berlin, Germany). An average of 62% of the cells stained with the anti-Thy-1 mAb AS02 (n = 4 RA patients; Table 1a) in flow cytometry (FACS) [10]; the average percentage of CD14+ cells was approximately 15% (n = 4; Table 1a). There were <1% T cells (CD3+) (mAb UCHT-1; ATCC, Manassas, VA, USA), B cells (CD19+/20+) (mAbs HD 37 and B-Ly 1; Dako), plasma cells (CD38+) (mAb AT 13/5; Dako), natural killer (NK) cells (CD56+) (mAb NKH/1; Immunotech), dendritic cells (CD83+) (mAb HB 15a; Immunotech), endothelial cells (CD144+), or PMN (CD15+) (mAb80H5; Immunotech), indicating that non-adherent cells had been efficiently removed during primary culture. The total yield of cells following 7 days of primary culture averaged (5.2 ± 1.1) × 107 cells (mean ± SEM; n = 7).

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