title: The impact of storage temperature on the morphology, viability, cell number and metabolism of cultured human conjunctival epithelium.
abstract: OBJECTIVE
  To evaluate the effect of storage temperature on the morphology, viability, cell number and metabolism of cultured human conjunctival epithelial cells (HCjEs).
  
  
  METHODS
  Three-day cultured HCjEs were stored at nine different temperatures between 4 °C and 37 °C for four and seven days. Phenotype was assessed by immunofluorescence microscopy, morphology by scanning electron microscopy, viability and cell number by a microplate fluorometer and glucose metabolism by a blood gas analyzer.
  
  
  RESULTS
  Cultured cells not subjected to storage expressed the conjunctival cytokeratins 7 and 19 and the proliferation marker proliferating cell nuclear antigen. Cell morphology was best maintained following four-day storage between 12 °C and 28 °C and following 12 °C storage after seven days. Assessed by propidium iodide uptake, the percentage of viable cells after four-day storage was maintained only between 12 °C and 28 °C, whereas it had decreased in all other groups (p < 0.05; n = 4). After seven days this percentage was maintained in the 12 °C group, but it had decreased in all other groups, compared to the control (p < 0.05; n = 4). The total number of cells remaining in the cultures after four-day storage, compared to the control, had declined in all groups (p < 0.05; n = 4), except 12 °C and 20 °C groups. Following seven days this number had decreased in all groups (p < 0.01; n = 4), except 12 °C storage. Four-day storage at 12 °C demonstrated superior preservation of the number of calcein-stained viable cells (p < 0.05) and the least accumulation of ethidium homodimer 1-stained dead cells (p < 0.001), compared to storage at 4 °C and 24 °C (n = 6). The total metabolism of glucose to lactate after four-day storage was higher in the 24 °C group compared to 4 °C and 12 °C groups, as well as the control (p < 0.001; n = 3).
  
  
  CONCLUSIONS
  Storage at 12 °C appears optimal for preserving the morphology, viability and total cell number in stored HCjE cultures. The superior cell preservation at 12 °C may be related to temperature-associated effects on cell metabolism.
authors.affiliation: Unit of Regenerative Medicine, Department of Medical Biochemistry, Oslo University Hospital , Oslo , Norway .
authors.forename: Jon R
authors.initials: JR
authors.lastname: Eidet
authors.forename: Øygunn A
authors.initials: ØA
authors.lastname: Utheim
authors.forename: Rakibul
authors.initials: R
authors.lastname: Islam
authors.forename: Torstein
authors.initials: T
authors.lastname: Lyberg
authors.forename: Edvard B
authors.initials: EB
authors.lastname: Messelt
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
authors.forename: Tor P
authors.initials: TP
authors.lastname: Utheim
mesh_terms: D001784:Blood Gas Analysis; D002452:Cell Count; D002470:Cell Survival / Q000502:physiology*; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism; D015925:Cryopreservation; D004848:Epithelium / Q000378:metabolism; D015590:Fluorophotometry; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D009926:Organ Preservation
chemical_list: D005947:Glucose
keywords: Cell culture; conjunctival epithelium; limbal stem cell deficiency; storage temperature; transplantation
references.citation: Cornea. 2003 Jul;22(5):478-81
references.pmid: 12827056
references.citation: Exp Eye Res. 2012 Jan;94(1):109-16
references.pmid: 22154551
references.citation: Bioessays. 1997 Aug;19(8):711-20
references.pmid: 9264254
references.citation: J Cell Sci. 1993 Oct;106 ( Pt 2):685-91
references.pmid: 8282773
references.citation: Invest Ophthalmol Vis Sci. 2007 Dec;48(12):5484-93
references.pmid: 18055796
references.citation: Biol Chem. 2002 Mar-Apr;383(3-4):477-88
references.pmid: 12033437
references.citation: Regen Med. 2011 Nov;6(6):767-82
references.pmid: 22050528
references.citation: Cornea. 2010 Apr;29(4):439-45
references.pmid: 20164752
references.citation: Regen Med. 2012 May;7(3):295-307
references.pmid: 22594324
references.citation: Invest Ophthalmol Vis Sci. 2011 Jul 29;52(8):5892-8
references.pmid: 21693612
references.citation: Methods Enzymol. 1979;63:234-57
references.pmid: 502860
references.citation: Free Radic Biol Med. 2001 Oct 15;31(8):954-61
references.pmid: 11595380
references.citation: Arch Ophthalmol. 2005 Mar;123(3):334-40
references.pmid: 15767475
references.citation: Graefes Arch Clin Exp Ophthalmol. 1988;226(4):353-6
references.pmid: 3169587
references.citation: Am J Ophthalmol. 2005 Jul;140(1):136-8
references.pmid: 16038659
references.citation: Br J Ophthalmol. 2007 Jun;91(6):797-800
references.pmid: 17124242
references.citation: Mol Vis. 2007 Jul 13;13:1138-43
references.pmid: 17653059
references.citation: Acta Ophthalmol Scand. 2007 Jun;85(4):377-86
references.pmid: 17263847
references.citation: Invest Ophthalmol Vis Sci. 2001 Jul;42(8):1757-61
references.pmid: 11431439
references.citation: Invest Ophthalmol Vis Sci. 2010 Feb;51(2):758-64
references.pmid: 19643956
references.citation: J Cell Biol. 1994 Apr;125(2):403-15
references.pmid: 8163556
references.citation: Indian J Ophthalmol. 2006 Mar;54(1):29-34
references.pmid: 16531667
references.citation: Stem Cells. 2011 Dec;29(12):1923-32
references.pmid: 21997829
references.citation: Br J Ophthalmol. 2009 Jul;93(7):980-3
references.pmid: 19211610
references.citation: Am J Ophthalmol. 2005 Apr;139(4):611-9
references.pmid: 15808155
references.citation: Biotechnol Bioeng. 2005 Jan 20;89(2):157-63
references.pmid: 15584025
references.citation: Curr Eye Res. 2009 Nov;34(11):913-24
references.pmid: 19958107
references.citation: Invest Ophthalmol Vis Sci. 2006 Sep;47(9):3820-7
references.pmid: 16936093
references.citation: J Ophthalmol. 2013;2013:216359
references.pmid: 24251032
references.citation: Am J Ophthalmol. 2011 Dec;152(6):917-924.e1
references.pmid: 21851920
references.citation: Cornea. 1995 May;14(3):300-10
references.pmid: 7600815
references.citation: Cornea. 2008 Apr;27(3):327-33
references.pmid: 18362662
references.citation: J Cell Physiol. 2010 Oct;225(1):15-9
references.pmid: 20506173