A Three-Dimensional Culture Method to Expand Limbal Stem/Progenitor Cells

Abstract

Purpose: To reduce the feeder cell contamination and to separate the feeder cells from the cultured human limbal stem/progenitor cells (LSCs), a novel three-dimensional (3-D) method was employed to culture LSCs in vitro.

Methods: Limbal epithelial cells in the form of single-cell suspensions, cell clusters, and tissue explants were subjected to the standard culture or to a 3-D sandwich culture method. The standard culture method was to culture LSCs directly on a layer of murine 3T3 feeder cells. The 3-D culture method was to culture LSCs and feeder cells separately on the opposite sides of a porous membrane. Cell morphology, proliferate rate, and stem cell phenotypes were examined.

Results: LSC clusters and explants, but not single cells could be expanded using the 3-D method. The expanded LSCs exhibited a small, compact, cuboidal stem-cell morphology and expressed a comparable levels of putative stem cell markers including ABCG2, ΔNp63a and keratin (K) 14 and the maturation marker K12 to those of LSCs derived from the standard culture method. LSC clusters cultured with the 3-D method had a significantly higher proliferation rate than did those cultured with the standard method. Electron microscopy revealed 2 to 3 layers of limbal epithelial cells on the porous membrane. The basal cells were small and cuboidal, and the upper layers of cells were larger and flattened. Tight conjunctions were present between the neighboring cells in the upper layers. No cell-cell contact was observed between cultured LSCs and feeder cells in the 3-D method.

Conclusions: The 3-D method permits complete separation between cultured cells and feeder cells, while providing an even and close proximity between them. This alternative method permits culturing of LSCs without the risk of feeder cell contamination.

Keywords: 482 cornea: epithelium • 721 stem cells