Controlled Release of Cryoprotectants by Near-Infrared Irradiation for Improved Cell Cryopreservation

Cryopreservation is essential to store living cells and tissues for future use while maintaining the proper levels of cell functions. The use of cryoprotective agents (CPAs) to inhibit intracellular ice formation during cryopreservation is vital for cell survival, but the addition and removal of CPAs and ice recrystallization during rewarming will cause fatal injury to cells. The conventional CPA loading and unloading methods generate osmotic shocks and cause mechanical injury to biological samples, and the conventional method of rewarming using a water bath also leads to ice recrystallization and devitrification. A new CPA-loaded microparticle-based method for loading and photothermal rewarming under near-infrared (NIR) laser irradiation was proposed to overcome these difficulties. We have successfully achieved the controlled release of CPAs (2 M EG, 2 M PG, and 0.5 M trehalose) with a graphene oxide (GO, 0.04% w/v) core from a 1.5% (w/v) sodium alginate shell to the human umbilical vein endothelial cells (HUVECs) within 60 s using NIR laser irradiation (808 nm Lasever at 5000 mW/cm²) and successfully recovered the CPA-loaded cells with 0.04% (w/v) GO in 8-10 s using the same NIR irradiation. The results show that this method achieved 25% higher viability of HUVECs compared to the conventional method. In short, this study proposes a new approach for achieving controlled CPA loading to cells with a photothermal-induced strategy for cell cryopreservation.