Bissoyi Akalabya, Pramanik K
Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, India.
Cryo Letters. 2013 Sep-Oct;34(5):453-65.
The present work investigates the effects of a variety of natural cryoprotectants in combination on post-thaw viability and apoptosis of cryopreserved mononuclear cells (MNCs) derived from umbilical cord blood. The extracellular cryoprotectants (10 mM) namely trehalose, hydroxyl ethyl starch, polyvinyl pyrrolidine and intracellular CPAs (5 mM) like erythritol, taurine and ectoine were used to prepare different combinations of freezing medium following L9 (3(4)) Taguchi orthogonal array. Catalase, coenzyme Q10 and n-acetyl cystine (100 microg/m) were added as antioxidants. Among various combinations, freezing medium consisting of hydroxyl ethyl starch, ectoin and co-enzyme Q10 with 10% FBS is found to be most effective combination achieving maximum cell viability of 93%, 5.6% early apoptotic, 0.7% late apoptotic and 0.1% necrotic cells. SEM and phase contrast microscopy confirmed the normal cell morphology of the post-thaw cultured cells with retaining their membrane integrity. The survival rate of MNCs is higher than the rate achieved using conventional Me2SO.
本研究探讨了多种天然冷冻保护剂联合使用对脐带血来源的冷冻保存单个核细胞（MNCs）解冻后活力及凋亡的影响。使用细胞外冷冻保护剂（10 mM）海藻糖、羟乙基淀粉、聚乙烯吡咯烷酮以及细胞内冷冻保护剂（5 mM）赤藓糖醇、牛磺酸和四氢嘧啶，按照L9（3⁴）田口正交表制备不同组合的冷冻培养基。添加过氧化氢酶、辅酶Q10和N-乙酰半胱氨酸（100 μg/mL）作为抗氧化剂。在各种组合中，由羟乙基淀粉、四氢嘧啶和辅酶Q10与10%胎牛血清组成的冷冻培养基被发现是最有效的组合，可实现93%的最大细胞活力，早期凋亡细胞为5.6%，晚期凋亡细胞为0.7%，坏死细胞为0.1%。扫描电子显微镜和相差显微镜证实了解冻后培养细胞的正常细胞形态，且细胞膜完整性得以保留。MNCs的存活率高于使用传统二甲基亚砜（Me2SO）所达到的存活率。
