Human Corneal Endothelial Cell Seeding on a Stroma Reconstructed Using the Self-Assembly Approach of Tissue Engineering

Abstract

Purpose: : To optimize initial adherence time and cell density of human corneal endothelial cells (HCEC) seeded on a stroma reconstructed using the self-assembly approach.

Methods: : Human corneal fibroblasts were cultured in the presence of ascorbic acid in order to promote extracellular matrix secretion, forming sheets that were assembled to form a reconstructed corneal stroma. HCEC were then seeded on top, allowed to adhere, immersed in medium and cultured for up to 28 days. Initial cell adherence time was evaluated by calculating the number of non-adhered cells after 1, 2 and 4h. Different initial endothelial cell density (ECD) seedings were used (1000, 2000, 3000 cells/mm²) and final ECD and morphology were assessed using alizarin red staining and electron microscopy. Coverage was evaluated using histology and immunofluorescent (IF) staining of Na⁺K⁺-ATPase.

Results: : HCEC adhered quickly on the reconstructed stroma (91%, 98.5%, 99.5% adhesion after 1, 2, and 4h, respectively). On day 1, ECD was similar to the initial cell density seeding. However, regardless of initial cell density used, ECD on day 28 decreased to reach 1100 ±288 cells/mm². For the 2 higher initial cell density seedings, histology and electron microscopy showed a rearrangement of the cell monolayer over time. Initially the endothelium had some overlaying and elongated cells, which disappeared to produce a uniform monolayer of normal polygonal morphology on day 28, which was confirmed using Na⁺K⁺-ATPase IF staining.

Conclusions: : A 4h adhesion time and an initial ECD of 1000 cells/mm² were selected for the seeding of HCEC on a reconstructed corneal stroma. This study shows the feasibility of reconstructing a posterior cornea using the self-assembly approach of tissue engineering.

Keywords: cornea: endothelium • cornea: stroma and keratocytes