학술논문
Isolation of Putative Corneal Epithelial Stem Cells from Cultured Limbal Tissue
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- 영문명
- 발행기관
- 대한안과학회
- 저자명
- Mee Kum Kim Jae Lim Lee Kyeong Seon Shin Gyung-Ah Jung Won Ryang Wee Jin Hak Lee Ki Sook Park Young Sook Son
- 간행물 정보
- 『The Korean Journal of Ophthalmology』Vol.20 No.1, 55~61쪽, 전체 7쪽
- 주제분류
- 의약학 > 기타의약학
- 파일형태
- 발행일자
- 2006.03.30
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국문 초록
영문 초록
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℃ or 4℃ 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℃ 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 3×104 cell/ml and 8.06×105 cell/ml at 37℃and 4℃ incubations; the number increased to 1.21×106 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. Korean Journal of Ophthalmology 20(1):55-61, 2006
목차
Materials and Methods
Results
Discussion
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- Isolation of Putative Corneal Epithelial Stem Cells from Cultured Limbal Tissue
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