Determination of the Minimal Size of a Biopsy for the Reconstruction of an Autologous Corneal Endothelium by Tissue Engineering

Abstract

Purpose: : To determine the minimal size of the feline corneal autologous biopsy to harvest in order to produce an autologous reconstructed endothelium.

Methods: : Different size of biopsy punches (1.77, 4.9, 12.6 mm²) were used on freshly dissected corneas to obtain small pieces of Descemet’s membrane. The feline corneal endothelial cells (FCEC) were isolated, seeded on gelatin-coated plates and cultured with 10% serum supplemented with epidermal growth factor and bovine pituitary extract. Cell number, size and morphology were assessed on different cell populations for up to three passages. Cell cultures were also maintained at confluence to observe the post-confluent changes in cell morphology at every passage.

Results: : The FCEC isolated from the 1.77 mm² Descemet’s membrane were sufficient to initiate primary cultures. However, cells quickly lost their small cell size and did not preserve their characteristic morphology at passage two. In contrast, punches of 4.9 and 12.6 mm² permitted the growth of respectively 150,000 and 300,000 cells in primary cultures and 500,000 and 1,500,000 cells at passage 1. Cells isolated from the 4.9 and 12.6 mm² punches presented a normal endothelial morphology and small cell size at passage two.

Conclusions: : These results show that the minimal size of a biopsy punch to isolate enough cells for the reconstruction of an autologous corneal endothelium is 4.9 mm². Reconstruction of an autologous endothelium is a promising approach for the treatment of diseases affecting the corneal endothelium.

Keywords: cornea: endothelium • cornea: basic science • growth factors/growth factor receptors