title: Yield and Viability of Human Limbal Stem Cells From Fresh and Stored Tissue.
abstract: OBJECTIVE
  We compared cell number, putative stem cell markers, and clonogenic ability in fresh uncultured human limbal epithelial cells to that obtained from stored organ-cultured tissue.
  
  
  METHODS
  Cell suspensions were formed from fresh and organ culture-stored human limbal epithelium. Expression of putative stem cell markers ΔNp63 and TrkA was performed using immunofluorescent staining before culture. Colony-forming efficiency (CFE) assays were performed at first passage. The effects of tissue storage, age, and postmortem/culture times were analyzed in a general linear model.
  
  
  RESULTS
  Limbal tissue from 94 donors (34 fresh and 60 stored) was compared. Three times more cells were obtained per eye from fresh (35.34 × 104; SD, 17.39) than stored (11.24 × 104; SD, 11.57; P < 0.01) tissue. A higher proportion of cells from fresh tissue were viable (91.9%; SD, 5.7 vs. 85%; SD, 10.8) P < 0.01. Higher total cell expression of ΔNp63 (20.19 × 104; SD, 15.5 vs. 3.28 104; SD, 4.33) and TrkA (59.24 × 104; SD, 13.21 vs. 7.65 × 104; SD, 1.05) was observed in fresh than stored tissue (P < 0.01). Colony-forming efficiency was higher for fresh (1.42; SD, 0.12) than stored (0.43; SD, 0.15; P < 0.01) cells. For stored tissue only, there was a significant inverse relationship between donor age and total number of cells isolated (R2 = 0.27, P < 0.001).
  
  
  CONCLUSIONS
  Storage of corneoscleral discs in organ culture medium leads to significant reduction in limbal epithelial cell number, expression of ΔNp63 and TrkA, and viability compared to fresh tissue. There is a smaller basal stem cell population in stored compared to fresh tissue.
authors.affiliation: Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Mason
authors.affiliation: Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom 2St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom.
authors.forename: Rosalind M K
authors.initials: RM
authors.lastname: Stewart
authors.affiliation: Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Carl M
authors.initials: CM
authors.lastname: Sheridan
authors.affiliation: Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Keshtkar
authors.affiliation: Tissue Services, National Health Service Blood and Transplant, Speke, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Rooney
authors.affiliation: Tissue Services, National Health Service Blood and Transplant, Speke, United Kingdom.
authors.forename: Eric
authors.initials: E
authors.lastname: Austin
authors.affiliation: Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Schlötzer-Schrehardt
authors.affiliation: Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: Friedrich E
authors.initials: FE
authors.lastname: Kruse
authors.affiliation: Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom 2St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom.
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Kaye
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002102:Cadaver; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D008875:Middle Aged; D013234:Stem Cells / Q000502:physiology*
chemical_list: D015415:Biomarkers