This study examined the effects of platelet lysate (PL)-fortified post-thaw human amniotic membranes (HAMs) on the proliferation and maintenance of overlying cultivated human limbal stem cells (hLSCs).

PL was produced by isolating platelet-rich plasma from the peripheral blood of three volunteers and by adding thrombin solution at a 1:5 ratio. HAMs were frozen, thawed, and fortified with the PL. hLSCs derived from donated corneo-scleral rings were cultivated on the post-thaw fortified HAMs and were compared with hLSCs cultivated on post-thaw unfortified HAMs (control) with respect to cell viability and genotypic/phenotypic alterations.

The hLSCs cultivated on the post-thaw fortified HAMs showed a significant increase in viability on days 1 (P = 0.029) and 3 (P = 0.042) and a borderline increase in viability after 1 week (P = 0.069) compared with the hLSCs cultivated on the post-thaw unfortified HAMs. Gene expression assay showed a remarkable decrease in Tp63 expression on day 7 (P = 0.028) and a significant increase in NGFR expression on days 1, 3, and 7 (P = 0.028, P = 0.0001, and P = 0.0001, respectively). However, ΔNp63 protein expression was not significantly different between the hLSCs cultivated on the post-thaw fortified HAMs and control cells after 1 week (P = 0.18).

PL-fortified post-thaw HAMs promote the proliferation of cultured hLSCs and their differentiation from stem cell stage to epithelial precursor stage and can be advantageous over unfortified HAMs for inducing ocular surface wound healing and re-epithelialization.