Journal Article Cryopreservation of a two-dimensional monolayer using a slow vitrification method with polyampholyte to inhibit ice crystal formation

Matsumura, Kazuaki  ,  Kawamoto, Keiko  ,  Takeuchi, Masahiro  ,  Yoshimura, Shigehiro  ,  Tanaka, Daisuke  ,  Hyon, Suong-Hyu

2 ( 6 )  , pp.1023 - 1029 , 2016-04-18 , American Chemical Society
Vitrification methods have been developed to improve the preservation of oocytes and embryos. However, successful vitrification and preservation typically requires very high cooling speeds. Here, we report a novel slow vitrification method for cryopreservation of two-dimensional (2D) cell constructs using a vitrification solution (VS) in 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 is likely to inhibit ice crystallization. Stabilization of the glassy state and inhibition of devitrification was confirmed by thermal analysis using differential scanning calorimetry. The viability of cultured human mesenchymal stem cell (MSC) monolayers after freezing by our novel slow vitrification method at a rate of 4.9°C/min in VS with 10% COOH-PLL was significantly higher than that of cells frozen using our slow vitrification method in VS without COOH-PLL. Moreover, cells maintained the capacity for differentiation. We further confirmed that COOH-PLL improved the vitrification properties of the current vitrification system through inhibition of recrystallization properties. This novel, simple method for slow vitrification can be widely applicable for the preservation of tissue-engineered constructs and may facilitate the industrialization of regenerative medicine.

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