Abstract
IntroductionThe 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.MethodsKnee 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.ResultsThe 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.ConclusionsFlow 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.
Highlights
The synovium is a major target tissue in chronic arthritis and is intensively studied at the cellular and molecular level
This study reports on the use of enzymatic and cytometric techniques for the characterization of synovial cell populations
These findings will help open up the way for direct and functional studies of synovial cell populations ex vivo in humans
Summary
The synovium is a major target tissue in chronic arthritis and is intensively studied at the cellular and molecular level. The synovium has been a preferred tissue for the investigation of cellular and molecular mechanisms in arthritic. Immunohistochemistry has become a standard method of analysis for the detection and quantification of synovial cell populations [2]. With this method, both semi-quantitative and quantitative digital image analyses have been proposed as tools to compare samples [1,2]. Even if under perfect circumstances specific molecular signals can be detected and localized reliably, this information represents the endpoint of investigation because the cells cannot be studied further with respect to their function
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