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T. T. Truong, D. M. Vu, D. K. Ha, S. X. Deng; The Effects of Different Culture Media on the Long-Term Preservation of Limbal Epithelial Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3754. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To compare KSFM, SHEM, and serum levels in their capacity to maintain and expand human limbal epithelial progenitor cells in culture.
Human limbal epithelial cells (LECs) were isolated from cadaveric donor tissues and serially cultured on 3T3 feeder layer in six growth media: KSFM plus 2% fetal bovine serum (KSFM2), KSFM plus 5% FBS (KSFM5), KSFM plus 10% FBS (KSFM10), SHEM without serum (SHEM0), SHEM plus 2% FBS (SHEM2), and SHEM plus 5% FBS (SHEM5). The phenotypes of LECs following serial passages were compared by rate of proliferation, expression of putative epithelial stem cell markers, morphology, and differentiation markers.
In primary culture (P0), all the media recovered similar numbers of progenitor cells from freshly isolated limbal epithelial cells shown by a comparable colony forming effeciency between 3-4%. Expression levels of cytokeratin14 (K14), ΔNp63α, and Ki67, and keratin 12 (K12) were similar. However, SHEM-based media promoted more than 3-fold higher proliferation but resulted in a heterogeneous morphology than those in KSFM-based media.Lower serum level led to greater proliferation of LECs however, reduced colony forming capacity and life span. KSFM2 and SHEM0 could support growth to P1, and KSFM5 and SHEM2 to P4, while cells can be propagated beyond P8 in KSFM10 and SHEM5.Cells grown in SHEM5 developed spindle-shaped fibroblast like morphology starting at P2 while those in KSFM10 maintained cuboidal epithelial morphology. Expression of a mesenchymal marker α-SMA was absent in human limbal and corneal epithelial tissues and in primary culture of LECs, but started to be expressed in early passages of the LECs and its level increased with subsequent passages. At P2, α-SMA expression was higher in cells cultured in SHEM5 than those in KSFM10, while K14 expression decreased with subsequent passages in both media. K12 expression was low in early and late passages in both media.A 12-fold increase of cells dead was observed in cells cultured in SHEM5 than those in KSFM10.
The current culture systems for expansion and maintenance of limbal epithelial progenitor cells on 3T3 feeder cells could not maintain an epithelial phenotype and lead to epithelial-mesenchymal transition after only 2 passages. This phenomenon is more pronounced in the SHEM medium which is used for ex vivo expansion of LEC in the treatment of limbal stem cell deficiency. Higher serum level increases the survival of LECs in vitro. Improvement in the current culturing technique is necessary to achieve successful expansion of limbal stem cells in culture.
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