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Flow cytometric characterization of freshly isolated and culture expanded human synovial cell populations in patients with chronic arthritis.

Van Landuyt KB, Jones EA, McGonagle D, Luyten FP, Lories RJ - Arthritis Res. Ther. (2010)

Bottom Line: Within the hematopoietic CD45-positive populations, we found no differences in relative amounts of macrophages, T-lymphocytes and B-lymphocytes between patient groups.Culture expanded cells were found to fulfill the multipotent mesenchymal stromal cell marker profile, except for CD34 negativity.Detection of peripheral blood macrophage and B-cell markers was decreased after enzymatic exposure and mechanical forces, respectively, but stromal markers were not affected.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Skeletal Development and Joint Disorders, Division of Rheumatology, Katholieke Universiteit Leuven, Herestraat 49, Leuven, B3000, Belgium. Kristel.Vanlanduyt@med.kuleuven.be

ABSTRACT

Introduction: The synovium is a major target tissue in chronic arthritis and is intensively studied at the cellular and molecular level. The aim of this study was to develop flow cytometry for the quantitative analysis of synovial cell populations pre and post culture and to characterize mesenchymal cell populations residing in the inflammatory synovium.

Methods: Knee synovium biopsies from 39 patients with chronic arthritis and from 15 controls were treated in a short, standardized tissue digestion procedure. Stored, thawed digests were routinely analyzed with flow cytometry including live-dead staining and use of the markers CD45, CD3, CD14, CD20, CD34, CD73, CD105, CD90, CD146, CD163 and HLA-DR to distinguish inflammatory and stromal cells. The influence of the digestion method on the detection of the different surface markers was studied separately. In addition, we studied the presence of a specific cell population hypothesized to be mesenchymal stem cells (MSC) based on the CD271 marker. Cell expansion cultures were set up and a MSC-related surface marker profile in passages 3 and 6 was obtained. Immunohistochemistry for CD34 and von Willebrand factor (vWF) was done to obtain additional data on synovium vascularity.

Results: The cell yield and viability normalized to tissue weight were significantly higher in inflammatory arthritis than in controls. Within the hematopoietic CD45-positive populations, we found no differences in relative amounts of macrophages, T-lymphocytes and B-lymphocytes between patient groups. Within the CD45-negative cells, more CD34-positive cells were seen in controls than in arthritis patients. In arthritis samples, a small CD271 positive population was detected. Culture expanded cells were found to fulfill the multipotent mesenchymal stromal cell marker profile, except for CD34 negativity. Detection of peripheral blood macrophage and B-cell markers was decreased after enzymatic exposure and mechanical forces, respectively, but stromal markers were not affected.

Conclusions: Flow cytometry can distinguish synovial cell populations in tissue digests. The preparation method can influence the detection levels of macrophage and B-cell populations. However, stromal cell markers seem not affected and quantification is possible, supporting flow cytometry tissue analysis as a tool to study these cell populations in arthritis.

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Microscopic evaluation of synovial cell yield and viability directly after isolation. Viability was counted as cells positive for trypan blue.
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Figure 1: Microscopic evaluation of synovial cell yield and viability directly after isolation. Viability was counted as cells positive for trypan blue.

Mentions: Control patients (seven males/eight females) had a median age of 44 years. The reason for orthopedic arthroscopy was knee pain due to cartilage lesions (five patients), meniscal lesions (three patients), combined cartilage-meniscal injury (five patients), cruciate ligament repair (one patient), and removal of a non-resorbed intra-articular screw (one patient). Characteristics of the patients from the arthritis group with established diagnosis are shown in Table 1. The median cell yield after digestion was 2286 cells/mg in the control group vs. 20,222 cells/mg in the arthritis group (P < 0.001; Figure 1). The viability of the cells by trypan blue exclusion was statistically different between both groups (median 52% vs. 72%; P < 0.001). There was no correlation between cell yield and viability for both groups.


Flow cytometric characterization of freshly isolated and culture expanded human synovial cell populations in patients with chronic arthritis.

Van Landuyt KB, Jones EA, McGonagle D, Luyten FP, Lories RJ - Arthritis Res. Ther. (2010)

Microscopic evaluation of synovial cell yield and viability directly after isolation. Viability was counted as cells positive for trypan blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875643&req=5

Figure 1: Microscopic evaluation of synovial cell yield and viability directly after isolation. Viability was counted as cells positive for trypan blue.
Mentions: Control patients (seven males/eight females) had a median age of 44 years. The reason for orthopedic arthroscopy was knee pain due to cartilage lesions (five patients), meniscal lesions (three patients), combined cartilage-meniscal injury (five patients), cruciate ligament repair (one patient), and removal of a non-resorbed intra-articular screw (one patient). Characteristics of the patients from the arthritis group with established diagnosis are shown in Table 1. The median cell yield after digestion was 2286 cells/mg in the control group vs. 20,222 cells/mg in the arthritis group (P < 0.001; Figure 1). The viability of the cells by trypan blue exclusion was statistically different between both groups (median 52% vs. 72%; P < 0.001). There was no correlation between cell yield and viability for both groups.

Bottom Line: Within the hematopoietic CD45-positive populations, we found no differences in relative amounts of macrophages, T-lymphocytes and B-lymphocytes between patient groups.Culture expanded cells were found to fulfill the multipotent mesenchymal stromal cell marker profile, except for CD34 negativity.Detection of peripheral blood macrophage and B-cell markers was decreased after enzymatic exposure and mechanical forces, respectively, but stromal markers were not affected.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Skeletal Development and Joint Disorders, Division of Rheumatology, Katholieke Universiteit Leuven, Herestraat 49, Leuven, B3000, Belgium. Kristel.Vanlanduyt@med.kuleuven.be

ABSTRACT

Introduction: The synovium is a major target tissue in chronic arthritis and is intensively studied at the cellular and molecular level. The aim of this study was to develop flow cytometry for the quantitative analysis of synovial cell populations pre and post culture and to characterize mesenchymal cell populations residing in the inflammatory synovium.

Methods: Knee synovium biopsies from 39 patients with chronic arthritis and from 15 controls were treated in a short, standardized tissue digestion procedure. Stored, thawed digests were routinely analyzed with flow cytometry including live-dead staining and use of the markers CD45, CD3, CD14, CD20, CD34, CD73, CD105, CD90, CD146, CD163 and HLA-DR to distinguish inflammatory and stromal cells. The influence of the digestion method on the detection of the different surface markers was studied separately. In addition, we studied the presence of a specific cell population hypothesized to be mesenchymal stem cells (MSC) based on the CD271 marker. Cell expansion cultures were set up and a MSC-related surface marker profile in passages 3 and 6 was obtained. Immunohistochemistry for CD34 and von Willebrand factor (vWF) was done to obtain additional data on synovium vascularity.

Results: The cell yield and viability normalized to tissue weight were significantly higher in inflammatory arthritis than in controls. Within the hematopoietic CD45-positive populations, we found no differences in relative amounts of macrophages, T-lymphocytes and B-lymphocytes between patient groups. Within the CD45-negative cells, more CD34-positive cells were seen in controls than in arthritis patients. In arthritis samples, a small CD271 positive population was detected. Culture expanded cells were found to fulfill the multipotent mesenchymal stromal cell marker profile, except for CD34 negativity. Detection of peripheral blood macrophage and B-cell markers was decreased after enzymatic exposure and mechanical forces, respectively, but stromal markers were not affected.

Conclusions: Flow cytometry can distinguish synovial cell populations in tissue digests. The preparation method can influence the detection levels of macrophage and B-cell populations. However, stromal cell markers seem not affected and quantification is possible, supporting flow cytometry tissue analysis as a tool to study these cell populations in arthritis.

Show MeSH
Related in: MedlinePlus