title: Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes.
abstract: OBJECTIVE
  Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed.
  
  
  METHODS
  Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR.
  
  
  RESULTS
  Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C.
  
  
  CONCLUSIONS
  HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.|Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.|Department of Ophthalmology, Vestre Viken HF Trust, Drammen, Norway.|Faculty of Health Sciences, National Centre for Optics, Vision and Eye Care, Buskerud and Vestfold University College, Kongsberg, Norway.
authors.forename: Tor Paaske
authors.initials: TP
authors.lastname: Utheim
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.|Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
authors.forename: Rakibul
authors.initials: R
authors.lastname: Islam
authors.affiliation: Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
authors.forename: Ida G
authors.initials: IG
authors.lastname: Fostad
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.|Department of Ophthalmology, Oslo University Hospital, Oslo, Norway.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Eidet
authors.affiliation: Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
authors.forename: Amer
authors.initials: A
authors.lastname: Sehic
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.
authors.forename: Ole K
authors.initials: OK
authors.lastname: Olstad
authors.affiliation: Schepens Eye Research Institute, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
authors.affiliation: Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
authors.forename: Edward B
authors.initials: EB
authors.lastname: Messelt
authors.affiliation: Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
authors.forename: May
authors.initials: M
authors.lastname: Griffith
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.|Faculty of Medicine, University of Oslo, Oslo, Norway.
authors.forename: Lara
authors.initials: L
authors.lastname: Pasovic
mesh_terms: D017209:Apoptosis / Q000235:genetics; D015415:Biomarkers / Q000378:metabolism; D002450:Cell Communication / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics*; D049109:Cell Proliferation; D002478:Cells, Cultured; D016000:Cluster Analysis; D015536:Down-Regulation / Q000235:genetics; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism*; D016850:Limbus Corneae / Q000166:cytology; D009055:Mouth / Q000166:cytology*; D020411:Oligonucleotide Array Sequence Analysis; D011309:Preservation, Biological; D025341:Principal Component Analysis; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000235:genetics; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D013312:Stress, Physiological / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics
chemical_list: D015415:Biomarkers