Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts

INTRODUCTION Rheumatoid arthritis synovial fibroblasts (RASF) are key players in synovial pathophysiology and are therefore examined extensively in various experimental approaches. We evaluated, whether passaging during culture and freezing has effects on gene expression and cell proliferation. METHODS RASF were passaged for up to 8 passages. RNA was isolated after each passage and cDNA arrays were performed to evaluate the RNA expression pattern during passaging. In addition, doubling time of the cells was also measured. RESULTS From passages 2-4, mRNA expression did not change significantly. Gene expression in RASF started to change in passages 5-6 with 7-10% differentially expressed genes. After passages 7-8, more than 10% of the genes were differentially expressed. The doubling rate was constant for up to 5 passages and decreased after passages 6-8. After freezing, gene expression of the second passage is comparable to gene expression prior to freezing. CONCLUSIONS The results of this study show, that experiments, which examine gene expression of RASF and shall reflect or imitate an in vivo situation, should be limited to early culture passages to avoid cell culture effects. It is not necessary to stop culturing SF after a few passages, but to keep the problems of cell culture in mind to avoid false positive results. Especially, when large-scale screening methods on mRNA level are used. Of note, freezing does not affect gene expression substantially.