Cell cryopreservation & resuscitation trivia

AMNGENT CONSUMABLES 

In the science fiction three-body novel, a low-temperature dormancy technology has been described, by freezing the human body in an extremely low temperature environment, causing the human metabolism to stagnate or slow down, waiting for centuries, and then using high-tech technology to wake up people in hibernation.

In reality, although the current science and technology is still difficult to reach the level of freezing complete individuals in science fiction, it is completely feasible for the cryopreservation and recovery of biological samples such as embryos, stem cells, sperm, and tissue sections.

Since samples such as cells are sensitive to temperature, the process of freezing is relatively simple, but it is not an easy task to freeze well and maintain high resuscitation activity when used. The difficulty is that when the cells are cold to below zero, the organelles are dehydrated, the concentration of soluble substances in the cells increases, and ice crystals are formed in the cells, and the production of ice crystals will greatly reduce the activity of the cells.

So, what methods can be used in the freezing process to reduce cell damage?

The formation of ice crystals is closely related to the cooling rate, and the freezing rate is too fast or too slow, which can easily lead to the formation of large ice crystals by water in the cells, which is not conducive to cell survival.

Therefore, it is necessary to reduce intracellular water as evenly as possible during cell cryopreservation, and reducing the formation of intracellular ice crystals is the key to reducing cell damage. If frozen slowly, the cells can be gradually dehydrated and large ice crystals will not be produced in the cells. On the contrary, in the process of quickly putting cells into the liquid nitrogen tank, the cooling rate is too fast, and the water in the cell does not have time to penetrate outside the cell membrane, forming a large number of ice crystals, and large crystals will cause damage and rupture of cell membranes and organelles, resulting in a decrease in cell recovery rate.

Slow freezing and fast thawing is a principle that everyone has always followed when cryopreservation and resuscitation of cells.

Slow freezing can gradually dehydrate the cells, and a large number of ice crystals will not be produced in the cells, fast thawing is to make the ice crystals during extracellular cryopreservation quickly thaw, to avoid the ice crystals slowly thawing into the cells to form recrystallization, and at the same time to prevent the Xiaoice crystals from forming large ice crystals, resulting in the recrystallization of ice crystals, thereby causing damage to cells.

For the vast majority of mammalian cells, the cooling rate of 1 °C/min is the most suitable cryopreservation rate, which is suitable for the cryopreservation of immune cells such as T/NK and stem cells such as MSCs in cell therapy.

It was found that the structure of ice crystals is large and balanced when slow cooling (1 °C/min or even lower), no matter how much the rate of resuscitation, the structure of ice crystals does not change significantly, but when rapidly cooling (10 °C/min), the ice structure shows a high degree of amorphous shape, showing a more subtle dendritic structure, which is easier to form recrystallization during the recovery process, especially when it is revived at a lower heating rate (6.2 °C/min or lower), it is easy to recrystallize the phenomenon, thereby causing damage to cells. Affects cell biological activity. However, with a higher rate of resuscitation of rapidly cooled (10°C/min) cell samples, the ice crystals did not have enough time to form recrystallization, which is why fast melting can avoid damage to cells caused by recrystallization.

The program cooling box often used in scientific research and exploration adopts the gradient cooling method. Like Amngent gradient cooling box, in the absence of alcohol and any liquid, it can ensure that the cooling rate of samples in cryogenic freezers is -1°C/min, and can be used in various cell types, including stem cells, primary cell box cell lines. It is easy to use, just insert the cryovial, and then put it in an environment of -80°C. It not only greatly improves the user's work efficiency and the survival rate of preserved cells, but also makes it safer for researchers and samples.

The Amngent gradient cooling box adopts new material design and core cooling technology, which can be used directly without pre-cooling, which is energy-saving and environmentally friendly. Suitable for 1.5ml and 2.0ml cryopreservation tubes, the cooling rate is consistent, the cell resuscitation performance is stable and the survival rate is high.

Gradient cooling box

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Image source: Amngent

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