Cryopreservation of a Two-Dimensional Monolayer Using a Slow Vitrification Method with Polyampholyte to Inhibit Ice Crystal Formation.

Vitrification is used for cryopreserving oocytes and embryos. Successful vitrification and preservation typically require very rapid cooling. We report a novel slow vitrification method for the cryopreservation of two-dimensional cell constructs using a vitrification solution (VS) of PBS containing 6.5 M ethylene glycol, 0.5 M sucrose, and 10% w/w carboxylated poly-l-lysine (COOH-PLL), a novel polymeric cryoprotectant and stabilizing agent that likely inhibits ice crystallization. Stabilization of the glassy state and inhibition of devitrification were confirmed by thermal analysis. Slow vitrification at rates of 4.9 and 10.8 °C/min using VS with 10% COOH-PLL significantly improved the viability of cultured human mesenchymal stem cell monolayers after freezing and induced less apoptosis than when VS was used without COOH-PLL. Moreover, the cells maintained differentiation capacity. COOH-PLL improved vitrification through the inhibition of devitrification. This novel, simple method for slow vitrification is expected to be widely applicable for the preservation of tissue-engineered constructs and may facilitate the industrialization of regenerative medicine.