Amniotic membrane (AM) transplantation is increasingly used in ophthalmological and dermatological surgeries to promote re-epithelialization and wound healing. Biologically active cells in the epithelial and stromal layers deliver growth factors and cytokines with anti-inflammatory, anti-bacterial, anti-immunogenic and anti-fibrotic properties. In this work, confocal microscopy was used to show that our cryopreservation protocol for AM yielded viable cells in both the stromal and epithelial layers with favorable post-transplant outcome. AM was obtained from Caesarean-section placenta, processed into allograft pieces of different sizes (3 cm × 3 cm, 5 cm × 5 cm, and 10 cm × 10 cm) and cryopreserved in 10 % dimethyl sulfoxide using non-linear controlled rate freezing. Post-thaw cell viability in the entire piece of AM and in the stromal and epithelial cell layers was assessed using a dual fluorescent nuclear dye and compared to hypothermically stored AM, while surveys from surgical end-users provided information on post-transplant patient outcomes. There was no significant statistical difference in the cell viability in the entire piece, epithelial and stromal layers regardless of the size of allograft piece (p = 0.092, 0.188 and 0.581, respectively), and in the entire piece and stromal layer of hypothermically stored versus cryopreserved AM (p = 0.054 and 0.646, respectively). Surgical end-user feedback (n = 49) indicated that 16.3 % of AM allografts were excellent and 61.2 % were satisfactory. These results support the expanded clinical use of different sizes of cryopreserved AM allografts and address the issue of orientation of the AM during transplant for the treatment of dermatological defects and ocular surface disorders.

No abstract