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Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation
Cryobiology ( IF 2.3 ) Pub Date : 2020-04-01 , DOI: 10.1016/j.cryobiol.2020.02.008
Martin I Daily 1 , Thomas F Whale 1 , Riitta Partanen 2 , Alexander D Harrison 1 , Peter Kilbride 3 , Stephen Lamb 3 , G John Morris 3 , Helen M Picton 2 , Benjamin J Murray 1
Affiliation  

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 °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 °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.

中文翻译:

在 96 孔板中冷冻保存哺乳动物体细胞原代培养物得益于冰核控制

迄今为止,哺乳动物细胞的冷冻保存通常在冷冻管中进行,但在某些应用中,在多孔板中冷冻保存原代细胞将是有利的。然而,在没有干预的情况下,多孔板每个孔中小体积液体的过度过冷是不可避免的,并且往往会导致高和可变的细胞死亡率。在这里,我们描述了一种在 96 孔板中通过控制速率冷冻使用受控冰核对粘附的原代牛颗粒细胞进行冷冻保存的技术。在低温保存过程中使用手动接种技术在温暖的过冷温度(低于熔点低于 5°C)诱导冰成核显着提高了解冻后的恢复率,从 29.6% (SD = 8.3%) 在成核不受控制的情况下提高到 57.7% (9 . 3%) 当平均超过 8 次重复培养时 (p < 0.001)。定性地观察到解冻细胞的脱离在没有冰成核控制的孔中更为普遍,这表明冷冻保存的细胞单层脱离可能是深度过冷的结果。我们使用红外热成像技术表明,当不控制成核时,96 孔板中的许多冷冻保护剂溶液可以过冷至低于 -20 °C 的温度,并且尽管严格尝试去除,但观察到的冷冻温度变化很大。污染物充当成核位点。我们得出结论,在 96 孔板或任何小体积格式中成功冷冻保存细胞需要控制冰核。定性地观察到解冻细胞的脱离在没有冰成核控制的孔中更为普遍,这表明冷冻保存的细胞单层脱离可能是深度过冷的结果。我们使用红外热成像技术表明,当不控制成核时,96 孔板中的许多冷冻保护剂溶液可以过冷至低于 -20 °C 的温度,并且尽管严格尝试去除,但观察到的冷冻温度变化很大。污染物充当成核位点。我们得出结论,在 96 孔板或任何小体积格式中成功冷冻保存细胞需要控制冰核。定性地观察到解冻细胞的脱离在没有冰成核控制的孔中更为普遍,这表明冷冻保存的细胞单层脱离可能是深度过冷的结果。我们使用红外热成像技术表明,当不控制成核时,96 孔板中的许多冷冻保护剂溶液可以过冷至低于 -20 °C 的温度,并且尽管严格尝试去除,但观察到的冷冻温度变化很大。污染物充当成核位点。我们得出结论,在 96 孔板或任何小体积格式中成功冷冻保存细胞需要控制冰核。我们使用红外热成像技术表明,当不控制成核时,96 孔板中的许多冷冻保护剂溶液可以过冷至低于 -20 °C 的温度,并且尽管严格尝试去除,但观察到的冷冻温度变化很大。污染物充当成核位点。我们得出结论,在 96 孔板或任何小体积格式中成功冷冻保存细胞需要控制冰核。我们使用红外热成像技术表明,当不控制成核时,96 孔板中的许多冷冻保护剂溶液可以过冷至低于 -20 °C 的温度,而且尽管严格尝试去除,但观察到的冷冻温度变化很大。污染物充当成核位点。我们得出结论,在 96 孔板或任何小体积格式中成功冷冻保存细胞需要控制冰核。
更新日期:2020-04-01
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