To describe a reproducible oxidative injury model in ex vivo porcine corneas and to investigate the effects of corneal mesenchymal stem cell (Co-MSC) secretome and specific factors on the preservation of corneal endothelium after oxidative injury.
Porcine corneas underwent vital staining with trypan blue and alizarin red with different concentration and time points. Ex vivo porcine corneas were exposed (endothelial side) to varied concentrations of hydrogen peroxide. After injury, 3 groups of 5 corneas underwent treatment with secretome from either a wild-type (WT) murine Co-MSC, a pigment epithelium derived factor (PEDF) knock out (K/O) murine Co-MSC, or basal media for 4 hours at 37°C. The viability of the endothelium was evaluated using the optimized vital staining protocol.
The optimal vital staining was achieved with 0.4% trypan blue for 60 seconds and 0.5% alizarin red for 90 seconds. The optimal oxidative injury (for consistency and level of damage) was obtained with 1% hydrogen peroxide for 15 seconds. Treatment with both WT Co-MSC and PEDF K/O Co-MSC secretome significantly reduced the endothelial damage compared with control (17.2% ± 10.0%, 33.5% ± 11.6%, and 68% ± 17%, respectively, P < 0.01). The WT Co-MSC secretome was significantly more effective compared with PEDF K/O Co-MSC secretome (P < 0.05).
A reproducible model of vital staining and oxidative injury is described for studying porcine corneal endothelial survival. Our results demonstrate a beneficial role of a corneal MSC secretome in reducing oxidative damage to the corneal endothelium. In addition, it suggests a potential role for PEDF in this process.