Isolation of pure population of limbal epithelial stem cells for use in tissue engineering

Abstract

Abstract: : Purpose: To develop isolation method of epithelial stem cells in limbus for use in tissue engineering Methods: Three extraction technique was compared to get larger number of viable epithelial cells in limbus. Limbal tissue in human or white rabbit was incubated at 37° C for 1hour or at 4° C for 16 hours with 1.2U/ml Dispase. Another tissue was treated with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid(EDTA) at 37° C and suspended cells was repeatedly collected every 20 minutes for 4 times. The collected cells were cultured with NIH/3T3–seeded plate. Colony forming efficiency(CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed on a Coulter EPICS 753 after cells were incubated with Hoechst 33342 and propidium iodide(PI). Side population fraction was obtained using gates exhibiting low Hoechst blue and red fluororescence. The gated cells of each fraction were re–cultured to assess capability of colony formation. Results:The mean numbers of viable cells was 3 x 10⁴ cell/ml at 37° C, 8.06 x 10⁵ cell/ml at 4° C with dispase, and increased to 1.21 x 10⁶ cell/ml with trypsin/EDTA (p<0.05). CFE was 9.67±2.13 % in rabbit and 6.63±2.35% in human. Side population fraction was 3.61±0.42% in rabbit and 5.21±4.91% in human. The sorted cells were cultured for passage 4. The small round colonies were well formed in side population fraction, on contrary, more differentiated cells were found in Hoechst positive fraction. Conclusions: The epithelial stem cells in limbus was efficiently isolated using with trypsin/EDTA extraction and FACS. It may be very useful technique in tissue engineering.

Keywords: cornea: epithelium • clinical research methodology • clinical laboratory testing