Introduction & Objective: One of the important issues in long term storage of cells is removal of animal serum from cell culture environments. The aim of this study was to evaluate amniotic fluid (AF), which is full of growth factors, as substitute for fetal bovine serum (FBS) in the cryopreservation protocol.

Materials & Methods: In this experimental study human amniotic epithelial cells were isolated from placentas which were seronegative for microbial infections. The cells were preserved in 24 different patterns for 12 months in -196 °C (liquid nitrogen) and viability of cells were determined before and after cryopreservation by trypan blue and MTT assay. Moreover, Oct-4 expression was studied to determine pluripotency before and after cryopreservation with immunocytochemistry. Results were compared between groups with ANOVA (Tukey Post-Test). P.value under 0.01 and 0.05 was considered statistically significant.

Results: The presence of DMEM, FBS or AF is necessary for amniotic cell cryopreservation. Trypan-blue, MTT and immunocytochemistry showed that there isn’t significant difference between using AF and FBS in viability and pluripotency of cells. Moreover, results showed that DMSO is a better cryoprotectant compared to glycerol.

Conclusion: Results showed that amniotic fluid can be a proper substitute for FBS in amniotic epithelial cells cryopreservation.