Cryopreservation of a Two-Dimensional Monolayer Using a Slow Vitrification Method with Polyampholyte to Inhibit Ice Crystal Formation
Cryopreservation of a Two-Dimensional Monolayer Using a Slow Vitrification Method with Polyampholyte to Inhibit Ice Crystal Formation
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DOI：
10.1021/acsbiomaterials.6b00150
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发表时间：
2016-06-01
影响因子：
5.8
通讯作者：
Hyon, Suong-Hyu
中科院分区：
文献类型：
--
作者：
Matsumura, Kazuaki;Kawamoto, Keiko;Hyon, Suong-Hyu
玻璃化冷冻用于冷冻保存卵母细胞和胚胎。成功的玻璃化冷冻和保存通常需要非常迅速的冷却。我们报道了一种新的缓慢玻璃化保存二维细胞结构的方法，使用含有6.5M乙二醇、0.5M蔗糖和10%w/w羧化多L赖氨酸的PBS玻璃化溶液(VS)，COOHPLL是一种可能抑制冰结晶的新型聚合物冷冻保护剂和稳定剂。热分析证实了玻璃态的稳定和晶化的抑制。加入10%COOH-PLL的VS以4.9℃/min和10.8℃/min的速度缓慢玻璃化冷冻后，培养的人骨髓间充质干细胞单层的存活率显著提高，诱导的细胞凋亡率明显低于不加COOH-PLL的VS。此外，细胞仍保持分化能力。COOH-PLL通过抑制去玻璃化而促进玻璃化。这种新颖、简单的缓慢玻璃化冷冻方法有望广泛应用于组织工程结构的保存，并可能促进再生医学的工业化。
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 degrees 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.