Investigating in vitro effects of platelet lysate on restoration of frozen amniotic membrane and overlying cultivated human limbal stem cells

Purpose: The aim of this study was to evaluate the effects of platelet lysate on restoration of frozen amniotic membrane epithelial cells and overlying human limbal stem cells when cultivated on the corresponding amniotic membranes.Methods: In the experimental study, 5 amniotic membranes were prepared and after freezing process and treatment with platelet lysate, the viability of the cells was evaluated. In addition, the human limbal stem cells, after characterization by immunocytochemistry for P63 and Vimentin were cultured on amniotic membrane surface after freezing process, and after treatment with platelet lysate, were investigated in terms of cell viability by MTT and expression of EGF, PDGF, VEGF, TP63, NGFR genes by Real time PCR.Results: Post-freeze amniotic membranes revealed a significant increase of viability in 24h, 72h, and 1 week after treatment with platelet lysate as compared to the controls; although their viability rates were lower than those of fresh amniotic membranes. The nature of cultivated human limbal stem cells was confirmed through immune reactivity of the cells for p63 and p75. Viability of human limbal stem cells cultured on amniotic membranes treated with platelet lysate revealed a significant increase at 24 hours and 72 hours and a borderline increase at 1 week as compared to the controls. Human limbal stem cells cultivated on amniotic membrane and treated with platelet lysate showed increased expression of EGF, VEGF, and P75 genes and reduced expression of PDGF and P63 genes.Conclusion: The results of this study showed that platelet lysate was able to restore amniotic membrane epithelial cells after freezing and increase the viability of human limbal stem cells when cultivated on amniotic membranes treated with platelet lysate. Furthemore, platelet lysate, by increasing the expression of VEGF, EGF, and P75 genes in these cells can cause growth, proliferation, and differentiation of limbal stem cells and can improve the angiogenesis and healing process in persistant corneal epithelial defects. Thus, the use of cultured limbal stem cells on amniotic membrane and subsequent treatment with platelet lysate can be helpful in the autologous and allogeneic transplantation of limbal stem cells in cases with limbal stem cell deficiencies.