Contributions of supercritical fluid technology for advancing decellularization and postprocessing of viable biological materials.

The demand for tissue and organ transplantation worldwide has led to an increased interest in the development of new therapies to restore normal tissue function through transplantation of injured tissue with biomedically engineered matrices. Among these developments is decellularization, a process that focuses on the removal of immunogenic cellular material from a tissue or organ. However, decellularization is a complex and often harsh process that frequently employs techniques that can negatively impact the properties of the materials subjected to it. The need for a more benign alternative has driven research on supercritical carbon dioxide (scCO2) assisted decellularization. scCO2 can achieve its critical point at relatively low temperature and pressure conditions, and for its high transfer rate and permeability. These properties make scCO2 an appealing methodology that can replace or diminish the exposure of harsh chemicals to sensitive materials, which in turn could lead to better preservation of their biochemical and mechanical properties. The presented review covers relevant literature over the last years where scCO2-assisted decellularization is employed, as well as discussing major topics such as the mechanism of action behind scCO2-assisted decellularization, CO2 and cosolvents' solvent properties, effect of the operational parameters on decellularization efficacy and on the material's properties.

Sterilization of epidermal growth factor with supercritical carbon dioxide and peracetic acid; analysis of changes at the amino acid and protein level.

Aseptic processing and terminal sterilization become increasingly challenging as medical devices become more complex and include active biologics. Terminal sterilization is preferred for patient safety and production costs. We aimed to determine how sterilization using supercritical CO2 (scCO2) with low levels of peracetic acid (PAA) affects amino acids and human epidermal growth factor (EGF) as a model protein. In a benchtop reactivity test, the amino acids methionine, tryptophan, arginine and lysine reacted with low levels of PAA in solution. At PAA levels used for scCO2 sterilization, however, mass spectrometry only identified oxidative adducts on methionine and tryptophan. Mass spectrometry analysis of EGF exposed to scCO2/PAA identified oxidative adducts on residues Met21, Trp49 and Trp50, as well as a low level of truncations after residues Trp49 and Trp50. Importantly, processing of EGF in solution with scCO2 did not affect its native conformation, and sterilized EGF maintained its activity in cell proliferation assays. When processing samples in lyophilized form with scCO2/PAA, amino acids did not react with PAA and the presence of adducts was strongly reduced on methionine and tryptophan, both as single amino acids and in EGF. Truncation after tryptophan residues did not occur. EGF sterilized in the lyophilized form retained its activity when processing occurred with added moisture. These results have significant implications for the maintenance of biological function in sterilized decellularized scaffolds and the ability to manufacture terminally sterilized combination devices containing therapeutic peptides or proteins.