title: Preservation of the limbal stem cell phenotype by appropriate culture techniques.
abstract: OBJECTIVE
  To evaluate the effect of several culture variables on clonal growth and differentiation of limbal stem cells ex vivo and provide an improved culture technique that supports preferential expansion and preservation of stem cells for therapeutic applications.
  
  
  METHODS
  Corneal epithelial stem cells were isolated from human limbal specimens and clonally expanded on a 3T3 feeder layer, followed by subcultivation of holoclones on fibrin gels. The effect of different limbal regions, enzymatic dissociation methods, and culture media supplemented with different calcium, serum, and growth factor concentrations on colony-forming efficiency, colony size, and colony density was compared. A panel of putative stem cell and differentiation markers was used to analyze the epithelial phenotype by morphologic and immunohistochemical methods.
  
  
  RESULTS
  Limbal cells obtained from the superior limbus, isolated by a two-step enzymatic dissociation method (dispase II/trypsin-EDTA), and cultured in low to medium (0.03-0.4 mM) calcium concentrations with proper serum levels (10% FCS) and growth factor combinations (EGF, NGF) yielded the highest clonal growth capacity and an undifferentiated cellular phenotype. Subcultivation of holoclones supported the preservation of stem and progenitor cells in the basal layer of the fibrin-based epithelial sheets, as demonstrated by multiple molecular stem cell markers (p63alpha, Bmi-1, K15, and ABCG2), whereas increased calcium concentrations and air-lifting induced terminal differentiation and gradual loss of stem cells.
  
  
  CONCLUSIONS
  The proposed culture system supports enrichment and survival of limbal stem and progenitor cells during the entire cultivation process and may be essential for long-term restoration of the damaged ocular surface.
authors.affiliation: Department of Ophthalmology, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany. ewa.meyer-blazejewska@uk-erlangen.de
authors.forename: Ewa Anna
authors.initials: EA
authors.lastname: Meyer-Blazejewska
authors.forename: Friedrich E
authors.initials: FE
authors.lastname: Kruse
authors.forename: Katrin
authors.initials: K
authors.lastname: Bitterer
authors.forename: Christian
authors.initials: C
authors.lastname: Meyer
authors.forename: Carmen
authors.initials: C
authors.lastname: Hofmann-Rummelt
authors.forename: Peter H
authors.initials: PH
authors.lastname: Wünsch
authors.forename: Ursula
authors.initials: U
authors.lastname: Schlötzer-Schrehardt
mesh_terms: D016475:3T3 Cells; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000502:physiology; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000502:physiology; D018920:Coculture Techniques; D003470:Culture Media; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008875:Middle Aged; D010641:Phenotype; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D003470:Culture Media