Biomolecular evaluation of cryopreserved amniotic membranes for ophthalmological use by ELISA and RT-PCR at one and eighteen months

To study the presence of certain proteins - EGF (epidermal growth factor), KGF (keratinocyte growth factor), IL-10 (interleukin 10), HGF (hepatocyte growth factor), Alpha2-macroglobulin and IL-1RA (interleukin 1 receptor antagonist) in cryopreserved amniotic membranes at 1 and 18 months and, as a secondary objective, to detect mRNA corresponding to KGF, IL-1Ra, Alpha2-macroglobulin, Fas Ligand, TGF beta (transforming growth factor beta) and Lumican by RT-PCR in membranes preserved at 1 and 18 months.Four samples of amniotic membrane were divided into 2 groups: the first group (N=2) cryopreserved for 1 month and the second group (N=2) cryopreserved for 18 months, in order to be studied by RT-PCR and ELISA.RT-PCR detected KGF, IL-1Ra, Alpha2-macroglobulin, Fas Ligand, and Lumican. Of these, FAS Ligand mRNA was found in samples preserved for 1and 18 months. KGF, Lumican, and alpha2-microglobulin mRNA were found only at 1 month, and IL-1Ra mRNA was absent in both sample groups. RT-PCR for TGF-beta was inconclusive. ELISA was performed for detection and quantification of 6 proteins (EGF, KGF, IL-10, HGF, Alpha2-macroglobulin and IL-1Ra) in both amniotic membrane groups. All 6 proteins were found in all samples, with a lower concentration at 18 months compared to 1 month of preservation.This study shows that membranes cryopreserved in 50% glycerol for 18 months do retain the proteins necessary for regeneration of the corneal surface, giving these membranes their biochemical properties.