Low-Concentration Vitrification Agent Based on Polyglutamic Acid and Nanowarming for Porcine Blood Vessel Cryopreservation

Long-term cryopreservation of blood vessels is crucial for cardiovascular disease treatments. To avoid ice crystal-induced injuries and achieve higher recovery, viability, function, and scalability of blood vessels, ice-free vitrification technology is required. However, traditional ice-free vitrification often necessitates high concentrations of toxic cryopreservation agents (CPAs), such as VS55 at concentrations up to 8.4 M. In this study, we successfully achieved low-concentration vitrification using a novel CPA formulation (BTP solution) containing zwitterionic betaine and polyglutamic acid at only 3.6 M. The incorporation of polyglutamic acid significantly enhanced hydration properties, enabling a substantial reduction in the betaine concentration, while enhancing effective vitrification. To achieve uniform rewarming in a large system, gum-acid-coated magnetic nanoparticles were synthesized to facilitate rapid and even rewarming, thereby reducing tissue damage. With this low-concentration BTP, we successfully preserved porcine blood vessels for at least 2 months. After cryopreservation, the vessels retained 92.1% structural integrity, 92.6% biomechanical properties, and normal fiber content and maintained contraction-relaxation capabilities. This low-concentration vitrification technique holds considerable promise for further development and broader application in the cryopreservation of biological tissues and organs.