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E. A. Blazejewska, F. E. Kruse, J. Lampel, U. Schlötzer-Schrehardt; Growth Characteristics of Human Limbal Epithelial Cells in vitro: Effect of Isolation Methods and Culture Conditions. Invest. Ophthalmol. Vis. Sci. 2007;48(13):458.
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© ARVO (1962-2015); The Authors (2016-present)
To analyze the effect of different dissociation methods and culture conditions on growth capacity and differentation of human limbal epithelial cells in order to develop an optimized method for the in vitro expansion of limbal progenitor cells.
Epithelial cells were isolated from small human limbal biopsies (1x2 mm) obtained from 20 donor eyes (mean age 68 ± 7 years) by (1) dispase/trypsin-EDTA digestion, (2) thermolysin/trypsin-EDTA digestion, (3) trypsin-EDTA digestion and magnetic stirring, and (4) trypsin-EDTA digestion and agitation at 37 °C, seeded at 1000 cells/cm2 on a 3T3 feeder cell layer and cultured for 2 to 4 weeks. Clonal growth, measured by colony density and colony size, was compared using different culture media (MCDB151, DMEM/F12, Progenitor Cell Targeting (PCT), EpiLife), different serum concentrations (0-10% FCS), and different growth factor concentrations (0-20 ng/ml human EGF). The epithelial phenotype was verified by electron microscopy and immunohistochemistry using antibodies to markers of a differentiated (K3/K12) or rather undifferentiated state (ABCG-2, Nestin).
Limbal epithelial cell isolation using trypsin-EDTA with agitation at 37 °C significantly improved both cell yield and colony growth over the other dissociation methods. Limbal epithelial cells cultured in DMEM/F12 and PCT media showed a significantly higher colony density and size than those grown in MCDB151 and EpiLife after two weeks of culture; however, a prolonged culture time (4 weeks) promoted clonal growth in MCDB151 medium. Addition of 10% FCS and 5 ng/ml hEGF resulted in significantly greater colony density and size. Transmission electron microscopy and immunohistochemistry of colonies identified as holoclones showed monolayers of small rather undifferentiated, cuboid cells with central foci of stratification in MCDB151 medium, a largely multilayered and differentiated cell sheet in DMEM/F12 medium, and rather unstructured layers of cells with degenerative alterations in PCT and EpiLife media.
Tissue dissociation by trypsin-EDTA digestion with agitation at 37 °C is advantageous for cell isolation from small biopsies. Clonal growth of putative limbal progenitor cells appears to be most effective in MCDB151 medium supplemented with 10% FCS and 5 ng/ml hEGF after prolonged culture time. This work provides an important step toward the development of an improved protocol for efficient in vitro expansion of limbal progenitor cells for ocular surface reconstruction.
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