Numerous techniques have been reported for preparing and sterilizing amniotic membrane (AM) for use in clinical applications. However, these preparations either do not produce completely sterile tissue or are detrimental to molecules unique to the tissue matrix, thus compromising beneficial wound-healing properties of the AM graft. The objective of this work was to produce a sterile human AM tissue graft using a novel preparation technique involving supercritical carbon dioxide (SCCO2). AM tissue was subjected to various sterilization treatment groups that optimized the duration of exposure to SCCO2 and the amount of peracetic acid (PAA) required to achieve a sterility assurance level of 10(-6) log reduction in bacterial load. Effects of sterilization treatment on the histological, biophysical, and biochemical properties of the sterile AM were evaluated and compared with those of native AM tissue. Exposure of the AM tissue to combined SCCO2 and PAA sterilization treatment did not significantly alter tissue architecture, the amounts of pertinent extracellular matrix proteins (type IV collagen, glycosaminoglycans, elastin) present in the tissue, or the biophysical properties of the tissue. AMs treated with SCCO2 were also found to be excellent substrates for adipose-derived stem cell (ASC) attachment and proliferation in vitro. Human ASCs, attached to all treatment groups after 24 h of culture and continued to proliferate over the next few days. The current study's results indicate that SCCO2 can be used to sterilize AM tissue grafts while simultaneously preserving their biological attributes. The preservation of these features make AM appealing for use in numerous clinical and tissue engineering applications.