Comparison of Limbal Epithelial Cell Growth on a Synthetic Polymer Scaffold and Human Amniotic Membrane

Abstract

Purpose: : To evaluate the suitability of a synthetic biodegradable electrospun membrane, a co-polymer of poly-lactic and -glycolic acid (PLGA), for culturing limbal epithelial cells as an alternative to the use of human amniotic membrane (hAM).

Methods: : After obtaining IRB approval and informed consent, limbal biopsies of 1x2mm were harvested from normal individuals. Suspension and explant cultures of human and rabbit limbal epithelial cells were established on hAM and PLGA scaffolds. The electrospun scaffolds of 150μm thickness with fibers of 3-5μm degraded predictably over several weeks. The influence of extracellular matrix proteins (collagen and laminin) and fibrin on cell outgrowth from the explants on PLGA was determined. Cultured cells were characterized by immunolabeling using markers to identify differentiated (Cytokeratin 3/12) and stem cell populations (p63, ABCG2 and p63α). An ex vivo corneal organ culture model was employed to study the transfer of cultured cells from scaffolds onto the denuded corneas.

Results: : Limbal cultures could be established by suspension and explant culture techniques on hAM and PLGA in 2 weeks. The addition of fibrin to the PLGA scaffold greatly improved cell outgrowth from the limbal explants. Immunostaining revealed the presence of both stem cell and differentiated cell populations similar to that reported in hAM. Results show that cells cultured on PLGA can be successfully transferred onto the denuded ex vivo cornea in 2 weeks.

Conclusions: : Data show that PLGA provides sufficient support for the proliferation and migration of limbal epithelial cells with the maintenance of stem cell population. The PLGA scaffold will provide a xenobiotic free biodegradable carrier membrane for transplanting cultured cells to the human cornea as an alternative to using donor human amnion.

Keywords: cornea: epithelium • regeneration • transplantation