title: Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts.
abstract: BACKGROUND
  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.
authors.affiliation: Department of Internal Medicine and Rheumatology, University of Giessben, Kerckhoff-Klinik, D-61231 Bad Nauheim, Benekestr, 2-8, Germany. e.neumann@kerckhoff-klinik.de
authors.forename: Elena
authors.initials: E
authors.lastname: Neumann
authors.forename: Birgit
authors.initials: B
authors.lastname: Riepl
authors.forename: Anette
authors.initials: A
authors.lastname: Knedla
authors.forename: Stephanie
authors.initials: S
authors.lastname: Lefèvre
authors.forename: Ingo H
authors.initials: IH
authors.lastname: Tarner
authors.forename: Joachim
authors.initials: J
authors.lastname: Grifka
authors.forename: Jurgen
authors.initials: J
authors.lastname: Steinmeyer
authors.forename: Jurgen
authors.initials: J
authors.lastname: Schölmerich
authors.forename: Steffen
authors.initials: S
authors.lastname: Gay
authors.forename: Ulf
authors.initials: U
authors.lastname: Müller-Ladner
mesh_terms: D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D009584:Nitrogen; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D013583:Synovial Membrane / Q000378:metabolism / Q000473:pathology*
chemical_list: D009584:Nitrogen
references.citation: Arthritis Res. 1999;1(1):71-80
references.pmid: 11056662
references.citation: Nucleic Acids Res. 1996 Apr 15;24(8):1504-7
references.pmid: 8628684
references.citation: Ann Rheum Dis. 2004 May;63(5):483-8
references.pmid: 15082476
references.citation: Arthritis Res. 2000;2(5):361-7
references.pmid: 11094449
references.citation: Arthritis Rheum. 2004 May;50(5):1448-56
references.pmid: 15146414
references.citation: Arthritis Rheum. 1999 May;42(5):954-62
references.pmid: 10323451
references.citation: Exp Cell Res. 1994 May;212(1):132-40
references.pmid: 8174635
references.citation: Rheum Dis Clin North Am. 1995 Aug;21(3):675-90
references.pmid: 8619094
references.citation: Am J Pathol. 1996 Nov;149(5):1607-15
references.pmid: 8909250
references.citation: Biotechniques. 1999 Sep;27(3):554-60, 562, 564
references.pmid: 10489615
references.citation: Gene Ther. 2002 Nov;9(22):1508-19
references.pmid: 12407423
references.citation: Ann Rheum Dis. 2004 Jul;63(7):884-6
references.pmid: 15194591
references.citation: Gene Ther. 2004 Jul;11(13):1040-7
references.pmid: 15164093
references.citation: Arthritis Rheum. 1996 Nov;39(11):1781-90
references.pmid: 8912499
references.citation: Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10895-900
references.pmid: 9380731
references.citation: Cancer Res. 1997 Aug 15;57(16):3554-61
references.pmid: 9270028
references.citation: Biochem Biophys Res Commun. 1997 May 8;234(1):153-6
references.pmid: 9168980
references.citation: Clin Exp Rheumatol. 2004 Mar-Apr;22(2):158-64
references.pmid: 15083882
references.citation: Arthritis Rheum. 1995 Apr;38(4):477-84
references.pmid: 7536415
references.citation: Arthritis Rheum. 1988 Mar;31(3):315-24
references.pmid: 3358796
references.citation: J Autoimmun. 2003 Sep;21(2):161-6
references.pmid: 12935785
references.citation: Arthritis Rheum. 1999 Aug;42(8):1594-600
references.pmid: 10446856
references.citation: Arthritis Res. 2000;2(1):59-64
references.pmid: 11219390
references.citation: Rheumatol Int. 1989;9(3-5):105-13
references.pmid: 2692124
references.citation: Med Klin (Munich). 1999 Apr 15;94(4):228-32
references.pmid: 10373760
references.citation: J Rheumatol. 1997 Oct;24(10):1873-80
references.pmid: 9330926
references.citation: Arthritis Rheum. 2002 Jan;46(1):52-63
references.pmid: 11817609
references.citation: Arthritis Res. 2002;4(5):R8
references.pmid: 12223111
references.citation: Curr Opin Rheumatol. 1998 May;10(3):212-9
references.pmid: 9608324
references.citation: Arthritis Rheum. 1998 Aug;41(8):1356-64
references.pmid: 9704633
references.citation: J Immunol. 1994 Jun 15;152(12):5940-8
references.pmid: 8207219
references.citation: Ann Rheum Dis. 1997 Jan;56(1):45-51
references.pmid: 9059141
references.citation: Ann Rheum Dis. 1993 Mar;52 Suppl 1:S39-47
references.pmid: 8481058