title: Isolation of putative corneal epithelial stem cells from cultured limbal tissue.
abstract: OBJECTIVE
  To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue.
  
  
  METHODS
  Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2 U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation.
  
  
  RESULTS
  The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3 x 10(4) cell/ml and 8.06 x 10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21 x 10(6) cell/ml with a trypsin/EDTA treatment (p < 0.05). CFE was 9.67 +/- 2.13% and 6.63 +/- 2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61 +/- 0.42% and 5.21 +/- 4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67 +/- 2.24% and 1.17 +/- 6.13%, respectively (p < 0.05).
  
  
  CONCLUSIONS
  Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.
authors.affiliation: Department of Ophthalmology, Seoul National University Hospital, Seoul, Korea.
authors.forename: Mee Kum
authors.initials: MK
authors.lastname: Kim
authors.forename: Jae Lim
authors.initials: JL
authors.lastname: Lee
authors.forename: Kyeong Seon
authors.initials: KS
authors.lastname: Shin
authors.forename: Gyung-Ah
authors.initials: GA
authors.lastname: Jung
authors.forename: Won Ryang
authors.initials: WR
authors.lastname: Wee
authors.forename: Jin Hak
authors.initials: JH
authors.lastname: Lee
authors.forename: Ki Sook
authors.initials: KS
authors.lastname: Park
authors.forename: Young Sook
authors.initials: YS
authors.lastname: Son
mesh_terms: D000818:Animals; D002452:Cell Count; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D004492:Edetic Acid / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology* / Q000187:drug effects; D011817:Rabbits; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects; D014357:Trypsin / Q000494:pharmacology
chemical_list: D004492:Edetic Acid; D014357:Trypsin