Mechanism and Optimization Strategy of Cryopreservation Damage in Microcarrier-Hepatocyte Complex

The aim of this study is to optimize the cryopreservation scheme for microcarrier hepatocyte complexes used in artificial liver support systems to improve cell survival and adhesion rates. The effects of cryoprotectant concentration, loading temperature, and method on cell viability were evaluated experimentally. It was found that cell toxicity and osmotic damage were reduced significantly, and higher cell survival and adhesion rates were maintained by using two-step loading of 5% volume fraction dimethyl sulfoxide (Me₂SO) at 4 ℃. In addition, we investigated the effects of intracellular ice formation and cooling rate on cell viability and attachment. By performing ice seeding at -6 ℃, the intracellular ice damage was effectively reduced, and the adhesion rate of cells after recovery was improved. The experimental results show that a 10% volume fraction of Me₂SO and a cooling rate of 1 ℃/min, despite having high toxicity and osmotic damage, have the best freezing effect due to the smallest difference in thermal expansion. This study provides important techniques for the cryopreservation of microcarrier hepatocyte complexes for artificial liver support systems.