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0～-60 0 C is the crystallization temperature of water in tissues and cells. When the temperature enters this range, the water in the tissue begins to crystallize and damage the cell and tissue microstructure. Various freezer refrigerators The stability of the purified biomacromolecules can be maintained in the medium and long term, but the biomacromolecule stability, cell activity and tissue microstructure in the tissue cannot be maintained. Ultra-low temperature refrigerator It can maintain the stability of biomacromolecules in tissues in the medium term; maintain cell viability and tissue microstructure in the short term. -136 0 C is the glass transition temperature of water. The crystallization of water no longer causes significant damage to cells and tissues. The various biochemical reactions in the sample are almost stopped. Liquid nitrogen tank / tank gas phase; It can maintain the stability of microscopic structure of biological macromolecules, cells and tissues in the medium and long term, and is recommended for long-term frozen tissues for many sample banks. -196 0 C is the temperature at which liquid nitrogen evaporates, which is the lowest temperature that can be achieved by conventional methods. The various biochemical reactions in the sample can be considered to stop, and the damage of the crystals of water to the cells and microstructure can be neglected. Liquid nitrogen tank / tank liquid phase It can maintain the stability of biological macromolecules, cell activity and tissue microstructure in tissues for a long time, but has high requirements for storage consumables. 
In the 1950s and 1970s, Basil Luyet, James Lovelock, Peter Mazur and other scholars conducted in-depth research on the effects of freezing on cells and tissues, and found that the damage mainly originated from ice crystal damage and solution osmotic pressure damage. As the temperature drops, the water inside and outside the cell freezes, and the formed ice crystals cause damage to the cell membrane and cell wall, leading to cell death. This cell damage caused by icing inside the cell is intracellular ice damage. Ice crystal damage is caused by the cooling rate being too fast, and the faster the cooling rate, the greater the ice crystal damage. At the same time, as the temperature drops, the water outside the cell will freeze first, so that the electrolyte concentration in the unfrozen solution will increase, and the lipid on the cell membrane will be damaged by exposure to the high solute solution for a long time. The cells undergo dehydration and leakage, resulting in a large amount of water infiltrating into the cells during rewarming, causing cell death. This cell damage caused by an increase in the concentration of the solute in the preservation solution is called solution damage. Solution damage is caused by the cooling rate being too slow, causing the cells to be exposed to a high concentration of solution for too long, and the slower the cooling rate, the more serious the damage. 
Ice crystal damage and solution damage can be minimized by controlling the cooling rate of the sample. The control of the cooling rate can be achieved by stepwise freezing or programmed freezing at different temperatures. Compared with step-by-step cooling, the program desuperheater can more accurately control the cooling rate of the sample itself by adjusting the flow rate of liquid nitrogen to the freezer space. For example, when a 0--5 0 C liquid sample is converted into a solid phase change, heat is generated, causing the sample to return to temperature, causing additional damage to the sample. The program desuperheater can increase the cooling intensity during the phase change to avoid this damage. The program desuperheater has been widely used in frozen sperm, eggs, fertilized eggs, stem cells, skin and other samples. This method is used by about 300,000 to 400,000 in vitro fertilized infants worldwide. A cryopreservation program usually includes several stages, such as slow freezing and quick freezing. In the presence of cryoprotectant, the common freezing rate is 0.3-1.5 0 C/min, and fast freezing is 5-8 0 C/min. In the interval of 0 to -40 0 C, many programs use slow freezing. However, the specific cooling program may have a large difference depending on the frozen sample. 
The theory of vitrification was first proposed by Luyet. The solidification of liquid can be divided into two forms: one is crystallization, the molecules in the solution are arranged in an orderly manner; the other is amorphous or vitrified, the molecules in the liquid are disordered, and the state before unsolidification is maintained. . In normal freezing, water easily forms crystals in the cells to cause crystal damage, and crystals are formed outside the cells to cause solution damage. However, under the conditions of ultra-fast vitrification freezing, the cells are vitrified and solidified inside and outside, no ice crystals are formed or only small ice crystals are formed, and the cell membrane and the organelle are not damaged, and the cells are not in the high concentration of the solute for a long time. Exposed and damaged. However, the ultra-high temperature drop required for vitrification is difficult to achieve under normal experimental conditions. In 1981, Fahy proposed to use a high concentration of cryoprotectant (vitrification solution) to greatly reduce the cooling rate requirements, and in 1985, Rail and Fahy completed the vitrification of the mouse embryo to achieve the breakthrough in the use of this technology. .
Biological sample cryopreservation
1 storage temperature
Long-term storage of biological samples typically uses as low a temperature as possible to reduce biochemical reactions within the sample to increase the stability of the various components within the sample. Common storage temperatures for biomacromolecules, cells, tissues and organs are -80 0 C (ultra-low temperature refrigerator), -140 0 C (liquid nitrogen phase or cryogenic refrigerator) and -196 0 C (liquid nitrogen liquid phase). The longer the stable storage time of the low sample. 0 to -60 0 C is the crystallization temperature of water, which easily damages the microstructure of cells and tissues. Generally, this temperature is not used to preserve tissues and cells. Some purified biomacromolecules can be stably stored at 0--60 0 C for a certain period of time, but in the tissue sample, the biomacromolecules are affected by various factors in the cell tissue, and the stability may be significantly reduced, so usually the sample bank does not Use 0 to -60 0 C as the storage temperature.
-80 0 C is a safe temperature below the crystallization temperature of water, and the biochemical reaction in the sample is significantly weakened. It is also the lowest temperature that can be used in automated storage devices today.
Deep cold refrigerator
-80 0 C sample storage
-80 0 C is lower than the crystallization temperature range of the more hazardous water. It is also the temperature that can be achieved by the ultra-low temperature refrigerator of common equipment. It is considered based on factors such as ease of operation, storage and cost. This temperature is also the current preservation of the sample. Common temperatures for macromolecular activity. However, it is still inconclusive as to how long the temperature of different biomacromolecules can be maintained. The stability of DNA in tissues can be maintained for several years or longer at -80 0 C. However, for RNA, it is easy to be gradually degraded by RNase widely distributed in cells and various tissues. In different cells and tissues, the length of stable storage of RNA is also quite different, but generally it is less than 5 years. In some sensitive experiments, RNA has been degraded in less than one year at -80 0 C, so for long-term preservation of RNA activity, it is recommended to use a lower temperature, or use a small portion of the sample to extract RNA and the remaining Samples are stored synchronously. Biomolecules such as proteins and lipids in other samples can be preserved in the sample at -80 0 C, but the duration is different and the stability is gradually attenuated. If it is to protect a specific biological macromolecule known in the sample, a stabilizer for the molecule can also be added. If the biomacromolecules to be preserved in the sample are not determined, it is recommended to use a lower temperature storage. In addition, the current large-scale automated sample access equipment can only be used with -80 0 C ultra-low temperature equipment, and can not be used with liquid nitrogen equipment. UK Biobank in the UK is storing a copy of some samples (~9.5 million) in a working sample at -80 0 C, using automated equipment; another copy (~5.5 million) is stored in a liquid nitrogen phase in another place. Secure backup, manual access to samples.
-140 0 C sample storage
-140 0 C is the glass transition temperature below water (~-136 0 C), which is also the temperature that can be achieved by liquid nitrogen phase and cryogenic refrigerator. The biological activity of the sample is greatly reduced in this temperature range and is preserved. The ideal temperature for cell activity in the sample. The mixture of ice and water can maintain a phase transition temperature similar to 0 0 C. The liquid and vapor phase nitrogen in the insulated liquid nitrogen container should be maintained at a phase transition temperature of -196 0 C. However, in practice, the liquid nitrogen container lid is not sufficiently sealed, resulting in a temperature gradient between the liquid nitrogen level and the liquid nitrogen container can. The National Cancer Institute recommends that the temperature at the mouth of the liquid nitrogen container should be kept below -140 0 C. Samples that are not determined for future use should be stored in liquid nitrogen phase mode to protect cell activity in the tissue.
The cryogenic refrigerator is electrically cooled and does not require liquid nitrogen. The stable temperature after filling the sample is usually below -140. Compared with the use of liquid nitrogen, the advantage is that it does not require frequent addition of liquid nitrogen and is easy to maintain. However, the cooling rate of electric refrigeration is lower than that of liquid nitrogen. Once the container is opened and sampled, it is easy to cause a large range of temperature fluctuations, and the temperature recovery time is relatively long, so it is more suitable for less open and take samples. In addition, the electric refrigeration refrigerator must guarantee power supply. It is recommended to use liquid nitrogen equipment in the event of a power outage to provide backup storage.
-196 0 C sample storage
-196 0 C is the temperature at which liquid nitrogen volatilizes, so only liquid nitrogen liquid storage technology can reach this temperature. The life activities in the sample are basically stopped at this temperature, and the stability of the sample can be preserved for a long time. It is the most effective method for long-term preservation of cell activity, complex structure and activity of tissues and organs in samples, and is widely recognized. Liquid storage of liquid nitrogen containers requires further protection against cross-contamination between samples compared to other different temperature freezing modes. The National Cancer Institute recommends using a cryotube with a spiral to encapsulate the sample. However, during the cooling process, the sample suddenly drops from the ultra-low temperature refrigerator (-80 0 C) to liquid nitrogen, causing the shrinkage of the frozen cap and the tube body to be inconsistent, which easily leads to the penetration of liquid nitrogen into the cryotube and thus increases the cross-contamination between samples. risks of. One solution is to heat-shrink each of the cryotubes using a special cryotube sleeve, or use a sealing film to wrap around the interface between the cryotube cap and the body for 2-3 turns. The former method will lengthen the tube and require a higher freezer box. The second method will make the tube body slightly thicker. It is recommended to use a conventional frozen box of 10x10 size at the bottom.
2 refrigeration technology
Programmed freezing/stepwise freezing
Vitrification
Vitrification is a relatively new technology and is suitable for use in samples that are difficult to handle with conventional routines, such as complex tissues and organs. However, this technique has not shown a particular advantage for samples that are better processed by conventional procedures (such as cells and small tissue). Vitrification of normal water requires extremely fast speeds that are difficult to achieve in conventional laboratories. In order to reduce the glass cooling requirements of the tissue, it is necessary to add a high concentration of cryoprotectant to the sample, and the commonly used concentration is about 40% to 60% (W/V). Even a few minutes can cause significant damage to cells and tissues. Therefore, although vitrification reduces the ice crystal damage and solution damage in the freezing process, it does not show better results in some comparative tests. On the contrary, its complicated operation brings inconvenience. Currently in vitrification, the method of reducing the damage of the cryoprotectant is mainly to use a mixture of different cryoprotectants and ice blockers. Through this improvement, Twenty-First Century Medicine successfully transplanted frozen and thawed rabbit kidneys into rabbits and maintained normal function.
Snap freezing
The freezing method is usually used to protect purified biomacromolecules, or to extract tissue samples of biomacromolecules in the future. This is done by placing the sample directly into liquid nitrogen and transferring it to a cryogenic refrigerator or a lower temperature environment for short periods of time. A more common example is freezing tissue for future extraction of nucleic acids. This practice can damage most cells and fine tissue structures and cannot be used to preserve the activity of cells and tissues within a sample.
3 cryoprotectant
A cryoprotectant is a substance that protects the cell and tissue microstructure from freezing damage and is usually formulated as a solution of a certain concentration. The addition of a cryoprotectant to the cell suspension protects the cells from solution damage and ice crystal damage. The cryoprotectant combines with the water molecules in the solution to cause hydration. The crystallization process of the weakened water increases the viscosity of the solution and reduces the formation of ice crystals. At the same time, the cryoprotectant can protect the cells from solute damage by maintaining a certain molar concentration inside and outside the cell, reducing the concentration of electrolyte in the un-freezing solution inside and outside the cell. Usually only red blood cells, most microorganisms, and a very small number of nucleated mammalian cells are suspended in water or a simple salt solution without a cryoprotectant, and frozen at an optimum freezing rate to obtain a live cryopreservation. However, for most nucleated mammalian cells, there is no optimum freezing rate and no live frozen material can be obtained without the addition of a cryoprotectant. For example, the mouse bone marrow cells are suspended in a balanced salt solution without a cryoprotectant, and are frozen at a freezing rate of 0.3-600 0 C/min, and more than 98% of the cells are killed; and a glycerol cryoprotectant is added for freezing. When stored, more than 98% of the cells can survive.
Cryoprotectants can be classified into two types, permeable and non-permeable, depending on whether they penetrate the cell membrane. Osmotic cryoprotectants are mostly small molecules that penetrate into the cells through the cell membrane. Such protective agents mainly include Dimethyl Sulfoxide (DMSO), glycerin, ethylene glycol, propylene glycol, acetamide, methanol and the like. The protection mechanism is to infiltrate into the cells before the cell cryopreservation completely solidifies, and to produce a certain molar concentration inside and outside the cell, and to reduce the concentration of electrolyte in the un-freezing solution inside and outside the cell, thereby protecting the cells from the damage of the high concentration electrolyte. The intracellular water will not be excessively extravasated, avoiding excessive dehydration and shrinkage of the cells. Currently used are DMSO, glycerin, ethylene glycol and propylene glycol. DMSO penetrates into cells faster, and the protection effect is better during the cryopreservation process. It is commonly used, and the commonly used concentration is 5%-10%. DMSO itself but significant damage to the cells at a temperature of 4 0 C even worse damage is not recommended because DMSO was added after the sample is placed for a long time at this temperature, cooling device or program generally gradient cooling temperature achieved with the use of a continuous box The uniform speed is reduced.
Non-permeable cryoprotectants cannot penetrate into cells, usually macromolecules, mainly including polyvinylpyrrolidone (PVP), sucrose, polyethylene glycol, dextran, albumin, and hydroxyethyl starch. There are many hypotheses about its protection mechanism. One of them may be that macromolecular substances such as polyvinylpyrrolidone can preferentially combine with water molecules in solution, reduce the content of free water in the solution, reduce the freezing point and reduce the formation of ice crystals; The large molecular weight reduces the electrolyte concentration in the solution, thereby reducing solute damage.
Different cryoprotectants have different advantages and disadvantages. The current trend is to use a combination of two or more cryoprotectants in combination. Since many cryoprotectants protect cells under low temperature conditions, they are harmful to cells at normal temperatures. Therefore, the cryoprotectant should be removed in time after the cells are rewarmed.