The Effects of Amniotic Membrane Extract on Primary Human Corneal Epithelial Cells

Abstract

 

Purpose

 

1) To assess the effects of amniotic membrane extract (AMX) on the cellular activity of primary human corneal epithelial (HCE) cells. 2) To assess the effects of AMX on HCE activity under mechanical and oxidative stress conditions.

 

Methods

 

A lyophilized form of AMX was obtained from Keera Inc; later reconstituted in balanced salt solution. Primary human corneal epithelial cells were cultured. MTT assay for cellular viability over 48 hours using various AMX concentrations was carried out with 10% fetal bovine serum and serum-free media controls. The second phase involved mechanical “scratch” stress of HCE culture simulating corneal injury. 0.1% AMX was incubated for 48 and 72 hours in culture and comparison of scratch healing was made versus serum-free control. The oxidative phase involved use of 0.5 mM tertiary-butylhydroperoxide (t-BOOH) addition to HCE culture for 1 hour, washout of cells, then addition of 0.1% AMX versus control for 48 hours. Further oxidative study was undertaken using 12 hour pre-treatment with 0.1% AMX.

 

Results

 

MTT assay cellular viability was 100% for control, 101% for 0.01% AMX, 95.5% for 0.05% AMX, 95.3% for 0.1% AMX, 78.5% for 0.5% AMX, and 91% for 10% fetal bovine serum. 0.1% AMX was further studied due to highest viability for highest concentration. Mechanical “scratch” test on the HCE cultures revealed a significant healing distance ratio at 48 and 72 hours in favor of 0.1% AMX treated cultures (p = 0.021, 0.035). Oxidative stress with 0.5 mM t-BOOH 1 hour administration did not reveal significant difference in MTT assay of AMX treated versus control cultures (71% vs. 69%). Pre-treatment with 0.1% AMX for 12 hours before repeat of t-BOOH protocol revealed significant difference at 24 hours (73% AMX vs. 66% control, p=0.04) but not at 48 hours (81% AMX vs. 71% control, p=0.38).

 

Conclusions

 

The concentration of AMX with the best cellular viability in relation to highest concentration was 0.1%. The 0.1% AMX treated cells healed faster with a mechanical insult compared to controls, suggesting benefit in an acute corneal injury with increased HCE proliferation. The oxidative impact on cellular viability of t-BOOH did not improve with 0.1% AMX administration vs. control unless the cells were pre-treated with AMX. In more chronic corneal stress, AMX pre-treatment may provide some benefit.

 

 

Photographic comparison of mechanical "scratch" healing of AMX-treated HCE cells vs. control

 

Keywords: 480 cornea: basic science • 482 cornea: epithelium