Pressurized carbon dioxide as a potential tool for decellularization of pulmonary arteries for transplant purposes

Vascular bio-scaffolds produced from decellularized tissue offer a promising material for treatment of several types of cardiovascular diseases. These materials have the potential to maintain the functional properties of the extracellular matrix (ECM), and allow for growth and remodeling in vivo. The most commonly used methods for decellularization are based on chemicals and enzymes combinations, which often damage the ECM and cause cytotoxic effects in vivo. Mild methods involving pressurized CO₂-ethanol (EtOH)-based fluids, in a supercritical or near supercritical state, have been studied for decellularization of cardiovascular tissue, but results are controversial. Moreover, data are lacking on the amount and type of lipids remaining in the tissue. Here we show that pressurized CO₂-EtOH-H₂O fluids (average molar composition, Χ_CO2 0.91) yielded close to complete removal of lipids from porcine pulmonary arteries, including a notably decrease of pro-inflammatory fatty acids. Pressurized CO₂-limonene fluids (Χ_CO2 0.88) and neat supercritical CO₂ (scCO₂) achieved the removal of 90% of triacylglycerides. Moreover, treatment of tissue with pressurized CO₂-limonene followed by enzyme treatment, resulted in efficient DNA removal. The structure of elastic fibers was preserved after pressurized treatment, regardless solvent composition. In conclusion, pressurized CO₂-ethanol fluids offer an efficient tool for delipidation in bio-scaffold production, while pressurized CO₂-limonene fluids facilitate subsequent enzymatic removal of DNA.