Isolation of Putative Corneal Epithelial Stem Cells from Cultured Limbal Tissue.
- Author:
Mee Kum KIM
1
;
Jae Lim LEE
;
Kyeong Seon SHIN
;
Gyung Ah JUNG
;
Won Ryang WEE
;
Jin Hak LEE
;
Ki Sook PARK
;
Young Sook SON
Author Information
- Publication Type:Original Article ; Comparative Study ; In Vitro ; Research Support, Non-U.S. Gov't
- Keywords: Corneal epithelial stem cell; EDTA; FACS; Hoechst 33342; Trypsin
- MeSH: Trypsin/pharmacology; Stem Cells/*cytology/drug effects; Rabbits; Limbus Corneae/*cytology/drug effects; Humans; Epithelium, Corneal/*cytology/drug effects; Edetic Acid/pharmacology; Cells, Cultured; Cell Culture Techniques; Cell Count; Animals
- From:Korean Journal of Ophthalmology 2006;20(1):55-61
- CountryRepublic of Korea
- Language:English
- Abstract: PURPOSE: 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 1.2U/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 3x10(4) cell/ml and 8.06x10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21x10(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.
