Research Papers:

Long term storage in liquid nitrogen leads to only minor phenotypic and gene expression changes in the mammary carcinoma model cell line BT474

Judit Fazekas, Thomas W. Grunt, Erika Jensen-Jarolim and Josef Singer _

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Oncotarget. 2017; 8:35076-35087. https://doi.org/10.18632/oncotarget.16623

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Judit Fazekas1,2, Thomas W. Grunt3, Erika Jensen-Jarolim1,2 and Josef Singer2,4

1Comparative Medicine, The Interuniversity Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University of Vienna and University of Vienna, Vienna, Austria

2Institute of Pathophysiology and Allergy Research, Center of Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria

3Comprehensive Cancer Center, Department of Medicine I & Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria

4Department of Internal Medicine II, University Hospital Krems, Karl Landsteiner University of Health Sciences, Krems, Austria

Correspondence to:

Josef Singer, email: [email protected]

Keywords: long term storage in liquid nitrogen, cell line stability, BT474 cells, gene expression, EGFR

Received: October 04, 2016     Accepted: March 08, 2017     Published: March 28, 2017


Background/Aim: 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.

Conclusion: 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.

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