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

Proliferation rates, as assessed by incorporation of [3H]-thymidine (counts per minute [cpm]) in: first-passage (white bars) and conventional (conv.) fourth-passage (dark gray bars) normal skin-FB (n = 3 each) (A) isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) OA-SFB (n = 3, 3, and 4, respectively) (B) and isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) RA-SFB (n = 4, 3, and 5, respectively) (C), at rest (control) or following stimulation with IL-1β (50, 100, or 150 U/ml) or PDGF-BB (2.5, 5, or 10 U/ml). See Results for details. @ P ≤ 0.05 for the comparison between cytokine-stimulated FB and non-stimulated control FB within the same FB preparation. *P ≤ 0.05 for the comparison between OA-SFB and RA-SFB.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC17827&req=5

Figure 8: Proliferation rates, as assessed by incorporation of [3H]-thymidine (counts per minute [cpm]) in: first-passage (white bars) and conventional (conv.) fourth-passage (dark gray bars) normal skin-FB (n = 3 each) (A) isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) OA-SFB (n = 3, 3, and 4, respectively) (B) and isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) RA-SFB (n = 4, 3, and 5, respectively) (C), at rest (control) or following stimulation with IL-1β (50, 100, or 150 U/ml) or PDGF-BB (2.5, 5, or 10 U/ml). See Results for details. @ P ≤ 0.05 for the comparison between cytokine-stimulated FB and non-stimulated control FB within the same FB preparation. *P ≤ 0.05 for the comparison between OA-SFB and RA-SFB.

Mentions: At rest, the proliferation rates of conventional fourth-passage normal skin-FB did not significantly differ from those of the corresponding cells in first passage (Fig. 8A). The same was true for conventional fourth passage OA-SFB and RA-SFB (Fig. 8B,C). Upon stimulation with PDGF (2.5 and 5 U/ml for all cells, 10 U/ml only for OA-SFB), however, the proliferation rates of skin, RA, and OA conventional fourth-passage FB were significantly higher (maximum mean increase, 5.4-fold) than those observed in first-passage cells (Fig. 8A,B,C). Following stimulation with IL-1β, in contrast, only conventional fourth-passage RA-SFB (150 U/ml IL-1β) showed significantly higher proliferation rates than first-passage cells (Fig. 8C).


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)

Proliferation rates, as assessed by incorporation of [3H]-thymidine (counts per minute [cpm]) in: first-passage (white bars) and conventional (conv.) fourth-passage (dark gray bars) normal skin-FB (n = 3 each) (A) isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) OA-SFB (n = 3, 3, and 4, respectively) (B) and isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) RA-SFB (n = 4, 3, and 5, respectively) (C), at rest (control) or following stimulation with IL-1β (50, 100, or 150 U/ml) or PDGF-BB (2.5, 5, or 10 U/ml). See Results for details. @ P ≤ 0.05 for the comparison between cytokine-stimulated FB and non-stimulated control FB within the same FB preparation. *P ≤ 0.05 for the comparison between OA-SFB and RA-SFB.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Proliferation rates, as assessed by incorporation of [3H]-thymidine (counts per minute [cpm]) in: first-passage (white bars) and conventional (conv.) fourth-passage (dark gray bars) normal skin-FB (n = 3 each) (A) isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) OA-SFB (n = 3, 3, and 4, respectively) (B) and isolated first-passage (white bars), isolated (isolat.) fourth-passage (light gray bars), and conventional fourth-passage (dark gray bars) RA-SFB (n = 4, 3, and 5, respectively) (C), at rest (control) or following stimulation with IL-1β (50, 100, or 150 U/ml) or PDGF-BB (2.5, 5, or 10 U/ml). See Results for details. @ P ≤ 0.05 for the comparison between cytokine-stimulated FB and non-stimulated control FB within the same FB preparation. *P ≤ 0.05 for the comparison between OA-SFB and RA-SFB.
Mentions: At rest, the proliferation rates of conventional fourth-passage normal skin-FB did not significantly differ from those of the corresponding cells in first passage (Fig. 8A). The same was true for conventional fourth passage OA-SFB and RA-SFB (Fig. 8B,C). Upon stimulation with PDGF (2.5 and 5 U/ml for all cells, 10 U/ml only for OA-SFB), however, the proliferation rates of skin, RA, and OA conventional fourth-passage FB were significantly higher (maximum mean increase, 5.4-fold) than those observed in first-passage cells (Fig. 8A,B,C). Following stimulation with IL-1β, in contrast, only conventional fourth-passage RA-SFB (150 U/ml IL-1β) showed significantly higher proliferation rates than first-passage cells (Fig. 8C).

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