Amniotic Membrane Extract Preparation: What is the Best Method?

PURPOSE: To compare different preparation methods for a suitable amniotic membrane (AM) extract containing a given amount of growth factors. METHODS: In this interventional case series, we dissected the AM from eight placentas within 24 hours after delivery, under clean conditions. After washing and mixing, AM extracts (AMEs) were prepared using pulverization and homogenization methods, and different processing and storing conditions. Main outcome measures were the amount of added protease inhibitor (PI), the relative centrifugal force (g), in-process temperature, repeated extraction times, drying percentage, repeated pulverization times, and the effect of filtering with 0.2 μm filters. Extract samples were preserved at different temperature and time parameters, and analyzed for hepatic growth factor (HGF) and total protein using ELISA and calorimetric methods, respectively. RESULTS: The extracted HGF was 20% higher with pulverization as compared to homogenization, and increased by increasing the PI to 5.0 μl/g of dried AM. Repeating centrifugation up to 3 times almost doubled the extracted HGF and protein. Storing the AME at −170° for 6 months caused a 50% drop in the level of HGF and protein. Other studied parameters showed no significant effect on the extracted amount of HGF or total protein. CONCLUSION: Appropriate extraction methods with an adequate amount of PI increases the level of extractable components from harvested AMs. To achieve the maximal therapeutic effects of AMEs, it is necessary to consider the half-life of its bioactive components.