Unprecedented cell-selection using ultra-quick freezing combined with aquaporin expression.
Unprecedented cell-selection using ultra-quick freezing combined with aquaporin expression.
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DOI：
10.1371/journal.pone.0087644
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Yasui M
中科院分区：
文献类型：
--
作者：
Kato Y;Miyauchi T;Abe Y;Kojić D;Tanaka M;Chikazawa N;Nakatake Y;Ko SB;Kobayashi D;Hazama A;Fujiwara S;Uchida T;Yasui M
冷冻通常用于生物样品的保存和储存;然而，该过程可能具有一些不利影响，例如细胞膜损伤。水通道蛋白（Aquaporin，AQP）是一种水通道蛋白，其分子机制尚不清楚，但已被认为在低温保存中发挥一定作用。在这里，我们表明，超快速冷冻引起的膜损伤是由AQP 4的表达拯救。我们接下来检查AQP的表达结合超快速冷冻是否可以用于在大多数细胞死亡的冷冻条件下有效地选择细胞。从混合细胞培养物中专门选择稳定表达AQP 4的CHO细胞。已经使用生物信息学鉴定了在ES细胞分化成神经外胚层期间AQP 4的表达增加，我们证实了具有AQP 4表达的分化的ES细胞的存活改善。最后，我们表明，CHO细胞瞬时转染内皮素受体A和Aqp 4也选择和浓缩的多个循环的冷冻/解冻，这是证实了钙成像响应内皮素。此外，我们发现AQP的表达能够减少冷冻保护剂的量，从而降低渗透压和细胞毒性。综上所述，我们提出，这种简单但有效和安全的方法可能适用于选择哺乳动物细胞的再生医学以及基于细胞的功能测定或药物筛选方案中的应用。
Freezing is usually used for preservation and storage of biological samples; however, this process may have some adverse effects such as cell membrane damage. Aquaporin (AQP), a water channel protein, has been suggested to play some roles for cryopreservation although its molecular mechanism remains unclear. Here we show that membrane damage caused by ultra-quick freezing is rescued by the expression of AQP4. We next examine if the expression of AQP combined with ultra-quick freezing can be used to select cells efficiently under freezing conditions where most cells are died. CHO cells stably expressing AQP4 were exclusively selected from mixed cell cultures. Having identified the increased expression of AQP4 during ES cell differentiation into neuro-ectoderm using bioinformatics, we confirmed the improved survival of differentiated ES cells with AQP4 expression. Finally we show that CHO cells transiently transfected with Endothelin receptor A and Aqp4 were also selected and concentrated by multiple cycles of freezing/thawing, which was confirmed with calcium imaging in response to endothelin. Furthermore, we found that the expression of AQP enables a reduction in the amount of cryoprotectants for freezing, thereby decreasing osmotic stress and cellular toxicity. Taken together, we propose that this simple but efficient and safe method may be applicable to the selection of mammalian cells for applications in regenerative medicine as well as cell-based functional assays or drug screening protocols.
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