Tissue Engineering of the Corneoscleral Stem Cell Niche: Limbal Cell-Containing Fibrin Constructs (LCFC)

Abstract

Purpose: : Stem cell-based therapy of the ocular surface is currently focused on the ex vivo expansion of corneal epithelial stem cells. The niche in which this population resides, the corneoescleral limbal area, is essential for the maintenance of their stemness. The aim of this study was to construct a novel autologous limbal tissue equivalent, the limbal cell-containing fibrin construct (LCFC), which contains the two main limbal cell types: epithelial and fibroblastic cells.

Methods: : LCFCs were prepared from plasma-derived fibrin gels combined with confluent epithelial primary cultures (P0) and fibroblastic first passaged (P1) cells obtained from porcine limbal tissue samples. A fibroblast-containing fibrin gel (FFG) was prepared mixing fibroblastic cells with fresh frozen plasma. P0 limbal-derived epithelial cells were then seeded onto the FFG to construct the final LCFC, which were maintained under in vitro culture conditions during 14 or 21 days. Fibroblastic cell proliferation into the fibrin gels and cell-mediated shrinkage were determined on the FFGs. Epithelial cell growth and structural properties of the LCFCs were examined by light microscopy and scanning electron microscopy (SEM).

Results: : Fibroblastic limbal-derived cells showed a high proliferative activity on the FFGs, reaching a final cell number of 1.19 ± 0.35x10⁶ (n=20) cells/FFG after 14 days. SEM images showed clear adhesion of fibroblasts to the fibrin fibrils. FFGs underwent a slight cell-mediated contraction after 14 days. Epithelial cells growing on top of the FFG became confluent after 14 culture days and exhibited typical cobblestone morphology. Histological sections of the LCFCs showed the growth of an epithelial layer on the FFG in which the fibroblastic population was proliferating, and showed an incipient stratification.

Conclusions: : We have developed a new bioengineered autologous limbal stem cell niche equivalent (LCFC), which support both limbal epithelial and stromal cells growth. These results warrant animal studies to test whether this LCFC can efficiently reconstruct a damaged limbal niche.

Keywords: cornea: epithelium • cornea: basic science • anterior segment