T-cell independent pathways, such as upregulation of proto-oncogenes, production of growth factors and the release of matrix-degrading enzymes lead to progressive destruction of the affected joints [2
]. Transformed-appearing, activated SF are key players in this synovial activation [1
]. To identify the underlying mechanisms of the destructive behavior of RASF, in vitro
cultured RASF are used in various experimental settings [10
]. The analysis of the pathways that may help to understand the progressive growth at the invasion zone and the active cartilage and bone degradation without interfering with physiologic matrix remodeling is therefore mandatory to identify novel therapeutic targets to inhibit the progressive joint destruction by RASF.
Several cytogenetic and molecular biology techniques are currently used to identify differentially expressed genes under different biological conditions, for example differential display, subtractive hybridization, and cDNA arrays [9
]. They are currently used to analyze molecular changes and mechanisms involved in the pathogenesis of RA [9
]. The advantages of the current techniques include analysis of gene subsets, comparison of more than two biological conditions in combination with a high sensitivity. In additon, to control the effects of potential new drug targets such as proliferation inhibitors, antiinflammatory and destruction inhibiting molecules, in vitro
analysis using RASF or animal models including the use of human RASF such as the SCID mouse model for RA are helpful tools [9
]. Unfortunately, high amounts of cellular material (mRNA or protein) are required in most cases for the different gene or gene product analysis.
To evaluate the critical effects of this passaging procedure in cell culture on gene expression, RASF were therefore cultured over several passages followed by cDNA array analysis for up to eight passages and comparison of early passages to higher culture passages was performed. In addition, the effects of the storage of RASF in liquid nitrogen on gene expression were examined.
As shown in Figure , the gene expression pattern of the RASF populations were constant at passages 2 to 4. Passage 0 was different when compared with passages 1 to 8 (>10%), which is most likely due to the presence of macrophages on the culture plate (detectable by immunohistochemistry [1
]), which are not present at the later passages 1 to 8. In addition, changes in gene expression of RASF populations could be detected after passages 5 to 6, showing changes of 7 to 10% of the analyzed genes (Figure ). After passages 7 to 8, more than 10% of the analyzed genes were differentially expressed combined with an increasingly inconstant expression pattern at higher passages.
Thawing of the cells after storage in liquid nitrogen affected mainly the gene expression of the first passage of the cells, possibly due to the stress of thawing and the remaining DMSO until the DMSO was completely removed. Thereafter, the cells showed a similar pattern of gene expression when compared with the freshly cultured RASF as the non-frozen cells (Figures and ). Moreover, the proliferation rates of the fibroblast cultures decreased in later passages, showing a decreased doubling rate after five to eight passages (Table ).
Taken together, the data of the study show that experiments, which involve analysis of gene expression and the phenotype of RASF, should be limited to early cell culture passages, that is passages 2 to 5, to avoid cell culture effects, diverging gene expression at higher passages, and decreased proliferation of the analyzed RASF populations. In case of a need for larger cell numbers, for example for transduction or animal experiments, an internal long-term cultivation control should be performed, which also includes the comparison of early passaged cells to later passaged cells. In addition, storage of cells in liquid nitrogen affect mainly gene expression of the first culture passage after thawing of the cells and therefore, the second passage should be used for experiments.
Therefore, this paper addresses researchers who perform experimental approaches with cultured SF on the RNA expression level. We want to highlight that culturing of the cells for a too high number of passages will produce differences in gene expression in comparison to the cells used at low passages. Many researchers address the ability to proliferate, the induction of apoptosis and the cytokine expression by these experiments. The intention of the paper is not to recommend excluding culturing RASF after four or five passages, but to keep the problems of culturing in mind to avoid false-positive results and additional, rather labor-consuming, work when verification of the obtained expression data with fresh cell cultures is performed. Culture conditions should be kept constant during the experiments. In addition, we want to emphasize, that the functional ability of the cells or the regulation on protein level are not necessarily changed.