title: The effects of storage temperature on PBMC gene expression.
abstract: BACKGROUND
  Cryopreservation of peripheral blood mononuclear cells (PBMCs) is a common and essential practice in conducting research. There are different reports in the literature as to whether cryopreserved PBMCs need to only be stored ≤ -150 °C or can be stored for a specified time at -80 °C. Therefore, we performed gene expression analysis on cryopreserved PBMCs stored at both temperatures for 14 months and PBMCs that underwent temperature cycling 104 times between these 2 storage temperatures. Real-time RT-PCR was performed to confirm the involvement of specific genes associated with identified cellular pathways. All cryopreserved/stored samples were compared to freshly isolated PBMCs and between storage conditions.
  
  
  RESULTS
  We identified a total of 1,367 genes whose expression after 14 months of storage was affected >3 fold in PBMCs following isolation, cryopreservation and thawing as compared to freshly isolated PBMC aliquots that did not undergo cryopreservation. Sixty-six of these genes were shared among two or more major stress-related cellular pathways (stress responses, immune activation and cell death). Thirteen genes involved in these pathways were tested by real-time RT-PCR and the results agreed with the corresponding microarray data. There was no significant change on the gene expression if the PBMCs experienced brief but repetitive temperature cycling as compared to those that were constantly kept ≤ -150 °C. However, there were 18 genes identified to be different when PBMCs were stored at -80 °C but did not change when stored < -150 °C. A correlation was also found between the expressions of 2'-5'- oligoadenylate synthetase (OAS2), a known interferon stimulated gene (IFSG), and poor PBMC recovery post-thaw. PBMC recovery and viability were better when the cells were stored ≤ -150 °C as compared to -80 °C.
  
  
  CONCLUSIONS
  Not only is the viability and recovery of PBMCs affected during cryopreservation but also their gene expression pattern, as compared to freshly isolated PBMCs. Different storage temperature of PBMCs can activate or suppress different genes, but the cycling between -80 °C and -150 °C did not produce significant alterations in gene expression when compared to PBMCs stored ≤ -150 °C. Further analysis by gene expression of various PBMC processing and cryopreservation procedures is currently underway, as is identifying possible molecular mechanisms.
authors.affiliation: Leidos Biomedical Research, Inc., Frederick, MD, 21702, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
authors.affiliation: Leidos Biomedical Research, Inc., Frederick, MD, 21702, USA.
authors.forename: Norma
authors.initials: N
authors.lastname: Diaz
authors.affiliation: Leidos Biomedical Research, Inc., Frederick, MD, 21702, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Adelsberger
authors.affiliation: Leidos Biomedical Research, Inc., Frederick, MD, 21702, USA.
authors.forename: Xueyuan
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Leidos Biomedical Research, Inc., Frederick, MD, 21702, USA.
authors.forename: Randy
authors.initials: R
authors.lastname: Stevens
authors.affiliation: Leidos Biomedical Research, Inc., Frederick, MD, 21702, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Rupert
authors.affiliation: Division of Clinical Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Rockville, MD, 20852, USA.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Metcalf
authors.affiliation: Leidos Biomedical Research, Inc., Frederick, MD, 21702, USA.
authors.forename: Mike
authors.initials: M
authors.lastname: Baseler
authors.affiliation: Biogen, 125 Broadway, Cambridge, MA, 02142, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Barbon
authors.affiliation: Leidos Biomedical Research, Inc., Frederick, MD, 21702, USA.
authors.forename: Tomozumi
authors.initials: T
authors.lastname: Imamichi
authors.affiliation: Leidos Biomedical Research, Inc., Frederick, MD, 21702, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Lempicki
authors.affiliation: Biogen, 125 Broadway, Cambridge, MA, 02142, USA. louis.cosentino@biogen.com.
authors.forename: Louis M
authors.identifier: http://orcid.org/0000-0001-8017-2571
authors.initials: LM
authors.lastname: Cosentino
mesh_terms: D016923:Cell Death / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D015925:Cryopreservation; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007372:Interferons / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics; D013312:Stress, Physiological / Q000235:genetics; D013696:Temperature; D059467:Transcriptome / Q000235:genetics
chemical_list: D007372:Interferons
keywords: Cryopreservation; Gene expression; Peripheral blood mononuclear cells