title: Long term storage in liquid nitrogen leads to only minor phenotypic and gene expression changes in the mammary carcinoma model cell line BT474.
abstract: OBJECTIVE
  Cancer cell lines are indispensible surrogate models in cancer research, as they can be used off-the-shelf, expanded to the desired extent, easily modified and exchanged between research groups for affirmation, reproduction or follow-up experiments.As malignant cells are prone to genomic instability, phenotypical changes may occur after certain passages in culture. Thus, cell lines have to be regularly authenticated to ensure data quality. In between experiments these cell lines are often stored in liquid nitrogen for extended time periods.Although freezing of cells is a necessary evil, little research is performed on how long-term storage affects cancer cell lines. Therefore, this study investigated the effects of a 28-year long liquid nitrogen storage period on BT474 cells with regard to phenotypical changes, differences in cell-surface receptor expression as well as cytokine and gene expressional variations.
  
  
  METHODS
  Two batches of BT474 cells, one frozen in 1986, the other directly purchased from ATCC were investigated by light microscopy, cell growth analysis, flow cytometry and cytokine as well as whole-transcriptome expression profiling.
  
  
  RESULTS
  The cell lines were morphologically indifferent and showed similar growth rates and similar cell-surface receptor expression. Transcriptome analysis revealed significant differences in only 26 of 40,716 investigated RefSeq transcripts with 4 of them being up-regulated and 22 down-regulated.
  
  
  CONCLUSIONS
  This study demonstrates that even after very long periods of storage in liquid nitrogen, cancer cell lines display only minimal changes in their gene expression profiles. However, also such minor changes should be carefully assessed before continuation of experiments, especially if phenotypic alterations can be additionally observed.
authors.affiliation: Comparative Medicine, The Interuniversity Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University of Vienna and University of Vienna, Vienna, Austria.|Institute of Pathophysiology and Allergy Research, Center of Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Judit
authors.initials: J
authors.lastname: Fazekas
authors.affiliation: Comprehensive Cancer Center, Department of Medicine I & Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Grunt
authors.affiliation: Comparative Medicine, The Interuniversity Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University of Vienna and University of Vienna, Vienna, Austria.|Institute of Pathophysiology and Allergy Research, Center of Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Erika
authors.initials: E
authors.lastname: Jensen-Jarolim
authors.affiliation: Institute of Pathophysiology and Allergy Research, Center of Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria.|Department of Internal Medicine II, University Hospital Krems, Karl Landsteiner University of Health Sciences, Krems, Austria.
authors.forename: Josef
authors.initials: J
authors.lastname: Singer
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000276:immunology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003451:Cryoprotective Agents / Q000009:adverse effects*; D016207:Cytokines / Q000235:genetics; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D061986:MCF-7 Cells; D009368:Neoplasm Transplantation; D009584:Nitrogen / Q000009:adverse effects*; D011309:Preservation, Biological / Q000379:methods*
chemical_list: D003451:Cryoprotective Agents; D016207:Cytokines; D009584:Nitrogen
keywords: BT474 cells; EGFR; cell line stability; gene expression; long term storage in liquid nitrogen