Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation
Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation
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DOI：
10.1016/j.cryobiol.2020.02.008
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发表时间：
2020-04-01
期刊：
影响因子：
2.7
通讯作者：
Murray, Benjamin J.
中科院分区：
文献类型：
--
作者：
Daily, Martin I.;Whale, Thomas F.;Murray, Benjamin J.
迄今为止，哺乳动物细胞的冷冻保存通常在冷冻管中进行，但是存在将原代细胞冷冻保存在多孔板中将是有利的应用。然而，多孔板的每个孔中的小体积液体中的过度过冷在没有干预的情况下是不可避免的，并且倾向于导致高且可变的细胞死亡率。在这里，我们描述了一种技术，在96孔板中的粘附的原代牛颗粒细胞的冷冻保存控制速率冷冻使用控制冰成核。在低温保存期间，使用手动接种技术在温暖的过冷温度（低于熔点5 ℃）下诱导冰成核，显著改善了解冻后的回收率，从成核不受控制的29.6%（SD = 8.3%）提高到8个重复培养物的平均值时的57.7%（9.3%）（p < 0.001）。定性观察到解冻细胞的脱离在没有冰成核控制的威尔斯孔中更普遍，这表明冻存细胞单层脱离可能是深度过冷的结果。使用红外热成像技术，我们表明，许多等份的冷冻保护剂溶液在96孔板可以过冷到-20 ° C以下的温度时，成核不受控制，而且观察到的冷冻温度是高度可变的，尽管严格的尝试，以消除作为成核位点的污染物。我们的结论是，在96孔板，或任何小体积格式的细胞成功冷冻保存，需要控制冰成核。
Cryopreservation of mammalian cells has to date typically been conducted in cryovials, but there are applications where cryopreservation of primary cells in multiwell plates would be advantageous. However excessive supercooling in the small volumes of liquid in each well of the multiwell plates is inevitable without intervention and tends to result in high and variable cell mortality. Here, we describe a technique for cryopreservation of adhered primary bovine granulosa cells in 96-well plates by controlled rate freezing using controlled ice nucleation. Inducing ice nucleation at warm supercooled temperatures (less than 5 degrees C below the melting point) during cryopreservation using a manual seeding technique significantly improved post-thaw recovery from 29.6% (SD = 8.3%) where nucleation was left uncontrolled to 57.7% (9.3%) when averaged over 8 replicate cultures (p < 0.001). Detachment of thawed cells was qualitatively observed to be more prevalent in wells which did not have ice nucleation control which suggests cryopreserved cell monolayer detachment may be a consequence of deep supercooling. Using an infra-red thermography technique we showed that many aliquots of cryoprotectant solution in 96-well plates can supercool to temperatures below -20 degrees C when nucleation is not controlled, and also that the freezing temperatures observed are highly variable despite stringent attempts to remove contaminants acting as nucleation sites. We conclude that successful cryopreservation of cells in 96-well plates, or any small volume format, requires control of ice nucleation.