Cryopreservation in a Standard Freezer: −28 °C as Alternative Storage Temperature for Amniotic Membrane Transplantation

Abstract

Human amniotic membrane (hAM) is usually stored at −80 °C. However, in many regions, cryopreservation at −80 °C is not feasible, making hAM unavailable. Therefore, the possibility of cryopreservation at −28 °C (household freezer) was investigated. hAMs (n = 8) were stored at −80 °C or −28 °C for a mean time of 8.2 months. hAM thickness, epithelial integrity and basement membrane were assessed histologically. The collagen content, concentration of hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) were determined. Elastic modulus and tensile strength were measured. The mean thickness of hAM stored at −28 °C was 33.1 ± 21.6 µm (range 9.7–74.9); thickness at −80 °C was 30.8 ± 14.7 µm (range 13.1–50.7; p = 0.72). Mean collagen content, epithelial cell number and integrity score showed no significant difference between samples stored at −28 °C or −80 °C. Basement membrane proteins were well preserved in both groups. Mean tensile strength and elastic modulus were not significantly different. Concentration of bFGF at −28 °C was 1063.2 ± 680.3 pg/g (range 369.2–2534.2), and 1312.1 ± 778.2 pg/g (range 496.2–2442.7) at −80 °C (p = 0.11). HGF was 5322.0 ± 2729.3 pg/g (range 603.3–9149.8) at −28 °C, and 11338.5 ± 6121.8 pg/g (range 4143.5 to 19806.7) at −80 °C (p = 0.02). No microbiological contamination was detected in any sample. The cryopreservation of hAM at −28 °C has no overt disadvantages compared to −80 °C; the essential characteristics of hAM are preserved. This temperature could be used in an alternative storage method whenever storage at −80 °C is unavailable.