Isolation of putative corneal epithelial stem cells from cultured limbal tissue.
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.
Kim
Mee Kum
MK
Department of Ophthalmology, Seoul National University Hospital, Seoul, Korea.
Lee
Jae Lim
JL
Shin
Kyeong Seon
KS
Jung
Gyung-Ah
GA
Wee
Won Ryang
WR
Lee
Jin Hak
JH
Park
Ki Sook
KS
Son
Young Sook
YS
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
D004492:Edetic Acid; D014357:Trypsin