Abstract

SO.04.12

Preservation of the limbal stem cell phenotype by appropriate culture techniques

Schlötzer-Schrehardt U., Blazejewska E., Kruse F. E.
Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen

Objective: Successful transplantation of ex vivo-expanded corneal epithelial stem cells relies on the preservation of stem cell characteristics in culture, which can, however, not be satisfactorily accomplished by the conventional explant culture technique. In this study, we took advantage of the clonogenic, self-renewing capacity of stem cells and analyzed the effect of different culture conditions on clonal growth capacity and differentiation state of human limbal progenitor cells.
Methods: Epithelial cells were isolated from small human limbal specimens obtained from 20 donor eyes by enzymatic digestion, seeded on a 3T3 feeder 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, PCT, KSFM), and different serum and growth factor concentrations. The epithelial phenotype was verified by electron microscopy and immunohistochemistry using antibodies to markers of a differentiated (K3/K12) or undifferentiated state (ABCG2, Nestin, p63).
Results: About 0.3-0.4% of seeded cells grew to stable large colonies forming holoclones on the feeder layer. Limbal epithelial cells cultured in DMEM/F12, PCT, and KSFM media showed a significantly higher colony density and size than those grown in MCDB151 after 2 weeks of culture; however, a prolonged culture time of 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. Electron microscopy and immunohistochemistry of holoclones showed monolayers of small, rather undifferentiated cuboid cells with foci of stratification in MCDB151 medium, and largely multilayered and differentiated cell sheets in DMEM/F12, PCT, and KSFM media. Stem cell clones could be isolated from the feeder layer and subcultured without serum on transplantable substrates such as fibrin gels or amniotic membrane as confluent monolayers.
Conclusions: Optimized culture conditions support the enrichment of limbal progenitor cells and may improve the long-term outcome of ocular surface reconstruction in patients with limbal stem cell deficiency.

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