Cell frozen is a common and very important task in cell culture. Just like farmers keep seeds when they farm, we need to retain cells in the best condition to ensure that there is a continuous stream of seeds for cultivation in the future. For example, when we are relatively young or when we have better immunity, we can store good immune cells and good seeds, and when we are sick, aging, or have low immunity, we can take out these young and healthy seeds.
What is the "best cell"? Cells with vibrant and positive appearance are the best-in-class cells (Figure 1). We can judge the cell status by observing the cells by microscopy and counting them so that the cells can be frozen at the right time.
Figure 1 A549 human non- small cell lung cancer cell
(the left image has a low density and is not suitable for frozen storage. The right image has good cell morphology and density, which is suitable for frozen storage)
cells must also prevent frostbite
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Water will freeze under conditions below zero. When the cells are suspended in the solution, as the temperature drops, the water outside the cells will first freeze, and the ice crystals formed will cause the destruction of the cell membrane and organelles , causing cell death; the electrolyte concentration in the unfreezed solution increases. If the cells are exposed to such a high-solubil solution for too long, the lipid molecules on the cell membrane will be damaged and the cells will leak. Therefore, a large amount of water will enter the cells during re-heating, causing cell death.
If a cryoprotectant is added to the solution, the cells can be protected from solute damage and ice crystal damage. As shown in Figure 2, there is no frozen protective agent in the left picture, and there is more ice crystals in the cell. There is a frozen protective agent in the right picture, and there is obviously less ice crystals in the cell. The reason is that the cryoprotectant is easy to bind to the water molecules in the solution, thereby reducing the freezing point and reducing the formation of ice crystals. It also reduces the osmolarity of in the unicated solution by changing the concentration of electrolytes in the unicated solution, so as to prevent the cells from solute damage. In 1950, Smith AU successfully used glycerol to freeze red blood cell , and found that frozen storage protective agents are a key factor affecting the frozen storage effect.
Figure 2 The production of ice crystals after cell freezing is available when there is a freezing protective agent
1 velocity reduction is risky
freezing rate control is also very important. The freezing rate refers to the speed of cooling, which is directly related to the freezing effect. The following changes occur during the freezing process:
When the cells are cold to -5℃, the freezing point of the solution is reduced due to the addition of cryoprotective agent to the solution, and the solution inside and outside the cells has not frozen.
When it is cold to between -5~-15°C, the extracellular solution first freezes while the cells remain unfreezed. Unfreezing water molecules in cells will have higher chemical energy than water molecules in the freezing solution of the extracellular part. As a result, in order to maintain the balance of chemical energy with extracellular water molecules, the water molecules in the cell flows out of the cell. When the cooling rate is too slow, the risk of intracellular ice crystal formation is lower, which also indicates that cell dehydration is maximized; when the cooling rate is faster, intracellular dehydration will not be severe, and the cells will be exposed to a non-physiological environment for a shorter time; when the cooling rate is too fast and the dehydration is not sufficient, the formation of intracellular ice nuclei cannot be prevented.
is visible. It is not what we hope for when it is too fast or too slow, but what needs to be more focused on optimization is the "faster" situation. We need to choose different freezing methods and equipment according to the freezing scale, so as to control the cooling rate to decrease by 1~3℃ per minute.
Evolution of frozen storage method
Figure 3 Traditional cotton wrap frozen storage
Under relatively simple conditions in the early experimental environment, we would wrap cells into a large ball of cotton or gauze and put them into a 4℃ refrigerator, then put them in a -20℃ refrigerator after about half an hour, and then put them in a -80℃ refrigerator for about two hours overnight, but this method cannot stably control the actual cooling rate and has been gradually eliminated by the market.
Figure 4 Program cooling box freeze
Nowadays, commercial program cooling box solves the problem of controlling the cooling rate, and can achieve a slow cooling rate of about 1℃ per minute. The program cooling box is controlled by filling 100% isopropanol in the interlayer. We can put the cell freezing tube into the freezing box and then directly put it in the -80℃ refrigerator. This method is simple and easy to operate, but the frozen storage scale is small and cannot meet the needs of large-scale production.
Figure 5 Program cooler freeze
For cell lines to be marketed for production or some relatively fragile cells, they need a more accurate cooling rate to achieve the cooling rate required by cells in different cooling intervals. Many commercial cellular program cooling equipment can achieve this operation, which can manually design and edit the cooling interval, cooling time, and cooling rate, so it can better meet the needs of drug production and biological research.
We will design a series of screening experiments for the target cell line. On the basis of conventional protective agents, trehalose , albumin , etc. can also be added according to the cell type, and the cooling procedure is optimized. By adjusting the cooling rate, time, and interval, the difference between sample temperature and cavity temperature is balanced to obtain a smooth and suitable temperature curve for the cell. This method can control the temperature of the entire cooling process, and can develop differentiated cell freezing processes for different types of cells, while achieving the goal of large-scale cell freezing.
Figure 6 Cooling curve of the program cooler
(the upper curve is the sample temperature curve, and the lower curve is the cavity temperature curve)
From "freezing" to "storing"
What I said before is how to better freeze cells, so how can frozen cells be preserved for a long time? The most important thing about
is the cryopreservation temperature. Cryo-preservation temperature refers to the ultra-low temperature that can preserve cells for a long time. At this temperature, the cell biochemical reaction is extremely slow or even stopped. After long-term preservation, it can still maintain normal structure and function after recovery. Different cells and organisms and different cryopreservation methods should achieve the same cryopreservation effect. The cryopreservation temperature can be different, but from the perspective of reality and benefits, the low temperature environment of liquid nitrogen (-196℃) is the best cryopreservation temperature at present. At -196°C, the cell's life activities almost completely stopped, but the structure and function of the cell is intact after resuscitation. If the freezing process is correct, the general biological samples can be stored at -196°C for more than ten years. Cells are preserved using -70℃~-80℃, which has no significant impact on cell activity in the short term, but as the freezing time is extended, the cell survival rate is significantly reduced.
Generally speaking, cell freezing is an important part of ensuring the sustained and stable progress of cell culture. The development of standardized large-scale cell freezing platform technology is the top priority for establishing a cell bank with excellent quality and uniform and stable production.
In biological cytology studies, it is sometimes necessary to culture and analyze cancer cells in vitro. To keep cells well, you must use serum, such as Ausbian® special imported fetal bovine serum , its endotoxin is less than 3Eu/ml, making the cells healthier and making the experiments smoother for customers.
Article source: Tang Hao Cell World
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