Stability study of Amniotic-membrane-derived lumican at different storage temperatures, as a potential eye drops treatment

Abstract

Purpose : Lumican is a small leucine rich proteoglycan present in the human amniotic membrane (AM) that promotes corneal epithelialization. The aim of the present study was determining whether lumican can be extracted from AM and evaluating its stability as AM extract (AME) stored at different temperatures, which could help design epitheliazing-eye drops.

Methods : One-hundred mg of AM were incubated with dispase II at 37°C for 30 min and mechanically de-epithelialized. The de-epithelialized AM was washed and immersed in liquid nitrogen for 40 min, then crushed until a fine powder was obtained, which was solubilized with 2.5 ml of saline buffer with protease inhibitors and centrifuged. Supernatant was collected and stored at -20°C, 4°C and room temperature (RT) for 6, 12, 20 and 32 days until lumican quantitation. Lumican was calculated in each sample according with: [ng/ml lumican / mg of AM tissue].

Results : We obtained similar protein concentrations among all the AME at basal protein quantification. There was no significant difference between protein concentration and time of storage; however, we found significant differences (p<0.05) between the AME of 32- and 20-days with respect their basal protein concentration. After that, we analyzed protein concentration with storage temperature, and found significant differences (p<0.05) in the AME of 32 days at 4°C and -20°C respect the RT condition, similarly the AME of 20 days had significant results at -20°C respect other temperature conditions. The quantification of lumican in AME showed that the concentration was affected by time of storage and temperature conditions. Lumican in the AME of 12 days was significantly higher (p<0.05) in comparison with the AME of 32, 20 and 6 days. There was no differences between lumican in the AME of 32 and 20 days with respect the temperature conditions of storage. Interestingly, when we analyzed the AME of 12 days we significantly (p<0.05) observed a high proportion of lumican in temperature conditions of -20°C and 4°C with respect of RT and -70°C. Similarly, we found a significant difference of lumican in the AME of 6 days stored at -20°C, respect other temperatures.

Conclusions : Lumican can be extracted from AM and stored for 12 days as AME, at both temperature conditions of -20°C and 4°C. Further studies are needed to determine AME lumican’s function on corneal epithelial cells.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.